Fish Physiology: The Physiology of Developing Fish, Part A : Eggs and Larvae

FISH PHYSIOLOGY PHYSIOLOGY FISH VOLUME XI XI VOLUME The Physiology Physiology of of Developing Developing Fish Fish The...

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FISH PHYSIOLOGY PHYSIOLOGY FISH VOLUME XI XI VOLUME The Physiology Physiology of of Developing Developing Fish Fish The Part A A Part Eggs and and Larvae Larvae Eggs

CONTRIBUTORS CONTRIBUTORS D. D. F. F. ALDERDICE

THOMAS P. P. MOMMSEN J. PETER J. ROMBOUGH J. H. S. S. BLAXTER BLAXTER H. VON WESTERNHAGEN RANDAL K. BUDDINGTON PATRICKJ. THOMAS A. HEMING PATRICK J. WALSH KENJIRO KENJIRO YAMAGAMI

FISH PHYSIOLOGY PHYSIOLOGY FISH Edited by by Edited

W . SS.. H HO OA R W. DEPARTMENT OF OF ZOOLOGY ZOOLOGY DEPARTMENT UNIVERSITY OF OF BRITISH BRITISH COLUMBIA COLUMBIA UNIVERSITY VANCOUVER, BRITISH BRITISH COLUMBIA, COLUMBIA, CANADA CANADA VANCOUVER,

D.. J. J. RANDA AL LL L D DEPARTMENT OF ZOOLOGY ZOOLOGY DEPARTMENT OF BRITISH BRITISH COLUMBIA COLUMBIA UNIVERSITY OF VANCOUVER, BRITISH BRITISH COLUMBIA, COLUMBIA, CANADA VANCOUVER,

VOLUME XI XI VOLUME

The Physiology Physiology of of Developing Developing Fish Fish The Part A A Part Eggs and and Larvae Larvae

ACADEMIC ACADEMIC PRESS, PRESS, INC. INC.

Harcourt Harcourt Brace Brace Jovanovich, Jovanovich, Publishers Publishers

San San Diego Diego New New York York Berkeley Berkeley Boston Boston London London Sydney Sydney Tokyo Tokyo Toronto Toronto

COPYRIGHT CJ COPYRIGHT @ 1988 1988 BY BY ACADEMIC ACADEMICPRESS. PRESS. INC. INC. ALL ALL RIGHTS RIGHTS RESERVED. RESERVED. NO PART PART O F THIS PUBLICATION PUBLICATION MAY MAY BE BE REPRODUCED REPRODUCED OR OR NO OF TRANSMITTED IN IN ANY ANY FORM FORM OR BY ANY ANY MEANS. MEANS, ELECTRONIC ELECTRONIC OR BY OR MECHANICAL, INCLUDING INCLUDING PHOTOCOPY. PHOTOCOPY, RECORDING. RECORDING, OR OR MECHANICAL. OR ANY ANY INFORMATION INFORMATION STORAGE STORAGE AND AND RETRIEVAL RETRIEVAL SYSTEM. SYSTEM, WITHOUT WITHOUT PERMISSION IN IN WRITING WRITING FROM FROM TTHE H E PUBLISHER. PUBLISHER PERMISSION

ACADEMIC PRESS, ACADEMIC PRESS, INC. INC.

1250 Sixth Sixth Avenue. Avenue. San San Diego. Dieeo. California California 92101 92101 1250

United Kingdom Edition published by ACADEMIC PRESS INC. (LONDON) LTD. 24-28 Oval Road. 24-28 Oval Road. London London NW NWII 7DX 7DX

United Kin dom Edition ublished by ACADEME PRESS I&. (LONDON) LTD.

(Revised for (Revised for vol. vol. 11) 1 1)

Library Library of of Congress Congress Cataloging Cataloging in i n Publication Publication Data Data

Hoar, Hoar, William William Stewart, Date Date

Fish physiology. Fish Vols.8Vols. 8-

edited edited by by W. S. S. Hoar Hoar let [ e t al.J al.]

Includes Includes bibliographies bibliographies and and inpexes. indexes.

v. 1. 1. Excretion, ionic regulation, and Contents: v.

-

-

metabolism metabolism - v. v. 2. 2. The The endocrine endocrine system system -

-

v. 11. 11 - v.

developing fish. pt. A. A. Eggs Eggs and The physiology of developing larvae. juveniles larvae. pt. pt. B. B. Viviparity Viviparity and and posthatching juveniles (2 (2 v.) v.1

1 . Fishes-Physiology-Collected Fishes-Physiology-Collected works. works. 1.

I. I. Randall, Randall, D. D. J. J.

II. I I . Conte, Conte, Frank P., P., Date Date

III. I l l . Title. Title.

QL639.1 .H6 QL639.1.H6

597'.01 597l.01

76-84233 76-84233

ISBN 0-12-350433-3 0-12-350433-3 (v. 11, 11, pt. A) (alk. paper) ISBN (v. PRINTED STATES Of PRINTED IN IN THE THE UNITED UNITEDSTATES OF "MERle" AMERICA 88 89 90

91

987654321

CONTENTS CONTENTS

CONTRIBUTORS CONTRIBUTORS PREFACE PREFACE CONTENTS OF OF OTHER OTHER VOLUMES VOLUMES CONTENTS

ix ix

xi xi xiii xiii

1. Pattern Pattern and and Variety Variety in in Development Development 1. ]. H. S. S. Blaxter J. I. I. II. 11. III. 111. IV. IV. V. VI. VI. VII. VII. VIII. VIII. IX. IX. X. X. XI. XI. XII. XII. XIII. XIII.

2. 2.

Introduction Introduction Rearing Rearing Techniques Techniques Progress and Diversity Diversity of Development Progress and of Development Terminology Terminology of of Early Early Life Life History History Stages Stages Egg Egg Size Size and and Egg Egg Quality Quality The The Effect Effect of of Starvation Starvation The The Effect Effect of of Captivity Captivity The The Effect Effect of of Fixation Fixation on on Shrinkage Shrinkage Rate Rate of of Development Development Organ Organ Systems Systems Structure Structure and and Function Function Critical Critical Periods Periods Conclusions Conclusions References References

1 2 4 15 15 17 23 27 30 31 31 33 33 41 41 44 44 47 47 48 48

Respiratory Respiratory Gas Gas Exchange, Exchange, Aerobic Aerobic Metabolism, Metabolism, and and Effects Effects of of Hypoxia Hypoxia during during Early Early Life Life

Peter PeterJ. J.Rombough Rornbough I.I. Introduction Introduction II. 11. Respiratory Respiratory Gas Gas Exchange Exchange III. 111. Aerobic Aerobic Metabolism Metabolism IV. IV. Effect Effectof of Hypoxia Hypoxia V. V. Conclusions Conclusions References References

59 59 60 60 82 82 123 123 143 143 144 144 vV

vi vi

CONTENTS CONTENTS

Osmotic and and Ionic Ionic Regulation Regulation in in Teleost Teleost Eggs 3. Osmotic 3. and Larvae and D. FF.. Alderdice Alderdice D. Introduction I. Introduction

II. Oogenesis 1 1. Oogenesis

III. Fertilization Fertilization 111. Development IV. Development V. Conclusions References References

163 167 176 183 236 242

of Pollutants on on Fish Eggs 4. Sublethal Effects of 4.

and Larvae H.. uon von Westernhagen Westernhagen H Introduction I. Introduction

II. Sublethal Sublethal Effects Effects during during Development Development 11. III. Sublethal Sublethal Effects Effects Displayed Displayed by by Larvae Larvae Hatched Hatched 111.

from Treated Treated Eggs Eggs from IV. Sublethal Sublethal Effects Effects on on Larvae Larvae Not Not Exposed Exposed as as Eggs Eggs IV. V. Discussion, Discussion, Problems, Problems, and and the the Future V. Future References References

253 258 296 3 15 319 330

55.. Vitellogenesis Vitellogenesis and and Oocyte Oocyte Assembly Assembly Thomas P. P . Mommsen and Patrick J. J. Walsh Walsh I. I. Introduction Introduction II. 11. Vitellogenesis Vitellogenesis III. 111. Oocyte Oocyte Assembly Assembly

IV. IV. Epilogue Epilogue References References

6. 6.

348 349 377 391 391 395

Yolk Absorption in Embryonic and Larval Fishes

Thomas A. A . Heming and Randal K. K . Buddington I. I. Introduction Introduction II. 11. Structural Structural Aspects Aspects of of Yolk Yolk Absorption Absorption III. 111. Yolk Yolk Composition Composition during during Development Development IV. of Yolk Yolk Absorption Absorption IV. Rate Rate of V. V. Efficiency Efficiency of of Yolk Yolk Utilization Utilization VI. VI. Nonyolk Nonyolk Nutrient Nutrient Sources Sources during during Early Early Development Development VII. of Embryos Embryos and and Larvae Larvae VII. Nutrition Nutrition of References References

408 408 410 410 414 414 424 424 430 430 434 434 437 437 438 438

CONTENTS CONTENTS

vii vii

7. Mechanisms of Hatching in Fish 7. Kenjiro Yamagami I. I. 11. II. 111. III. IV. IV. V.

Introduction-Early Studies Studies on Fish Hatching Introduction-Early Hatching-Gland Cells Cells Hatching-Gland Hatching Enzyme and Choriolysis Hatching Hatching in Fish Physiology of Hatching Epilogue-Problems to Be Solved in the Future Epilogue-Problems References References

447 447 449 449 459 459 480 480 489 489 490 490

AUTHORINDEX INDEX AUTHOR

501 501

SYSTEMATIC INDEX SYSTEMATIC INDEX

525 525

SUBJECTINDEX INDEX SUBJECT

537 537

This Page Intentionally Left Blank

CONTRIBUTORS CONTRIBUTORS

Numbers contributions begin. Numbers in parentheses indicate the pages on which the authors’ authors' contributions

D. F. ALDERDICE (163), Department of of Fisheries and Oceans, Fish FishF. ALDERDICE (163), Paci$c Biological Biological Station, Nanaimo, Brit Briteries Research Branch, Pacific V 9 R 5K6 ish Columbia, Canada V9R

J.

( l ) ,Dunstaf Dunstaffnage SS.. BLAXTER BLAXTER (1), fnage Marine Research Laboratory, Oban, Scotland Oban, Argyll PA34 4AD, Scotland

H. J. H.

RANDALK. BUDDINGTON BUDDINGTON (407), Department RANDAL (407),

of Physiology, Physiology, University of of California, Los Angeles, California 90024 of

THOMAS HEMING(407), (407), Pulmonary THOMAS A. HEMING

of Inter InterDivision, Department of of Texas Medical Branch, Galveston, Galveston, nal Medicine, University of Texas 77550-2780 77550-2780

THOMAS P. (347), THOMAS P. MOMMSEN MOMMSEN (347), Department

of of Zoology, University of of British Columbia, Columbia, Vancouver, Vancouver, British Columbia, Columbia, Canada V6T 2A9

PETER]. (59), PETERJ. ROMBOUGH ROMBOUGH (59), Zoology

Department, Brandon University, University, Brandon, Manitoba, Canada R7A 6A9

H . VON VON WESTERNHAGEN WESTERNHAGEN (253), H. (253),

(ZenBiologische Anstalt Helgoland (Zen trale), 52, Federal Republic of 0-2000 Hamburg 52, of Germany trale), D-2000

PATRICK ]. PATRICK J . WALSH WALSH(347), (347),

Rosenstiel School of of Marine and Atmo Atmoof Miami, Miami, Florida 33149 spheric Science, University of

KENJIRO YAMAGAMI YAMAGAMI(447), (447), Life KENJIRO

Science Institute, Sophia University, University, Chiyoda-ku, Chiyoda-ku, Tokyo 102, 102, Japan

ix


PREFACE

Dramatic changes occur in the physiology of most animals during their their development. development. Among Among the the vertebrates, vertebrates, birds birds are are entirely entirely ovipa oviparous, live for variable periods in a cleidoic egg, and show fundamental hatchalterations in excretion, nutrition, and respiration at the time of hatch ing. In contrast, contrast, the eutherian mammals mammals are all viviparous, depending ing. on on the the maternal maternal circulation circulation and and aa specialized placenta placenta to to provide provide food, food, exchange gases, and exchange gases, and discharge discharge wastes. wastes. The The physiology physiology of of both both mother mother and is highly and fetus fetus is highly specialized specialized during during gestation gestation and and changes changes funda fundamentally time of birth. Fishes oviparous and mentally at at the the time of birth. Fishes exemplify exemplify both both the the oviparous and the the viviparous viviparous modes modes of of development, development, with with some examples examples that that are are intermediate two. In intermediate between between the the two. In these these two two volumes, volumes, we we present present reviews of many, but not all, aspects of development. The chapters in Part A relate to the physiology of eggs and larvae: different patterns of larval eflarval development development osmotic osmotic and and ionic ionic regulation, regulation, gas gas exchange, exchange, ef fects fects of of pollutants, pollutants, vitellogenesis, vitellogenesis, the absorption absorption of of yolk, yolk, and the the mechanisms hatching. Chapters Chapters in in Part Part B deal with with maternal-fetal maternal-fetal mechanisms of hatching. relations, meristic variation, smolting salmonids, the ontogeny of of be behavior, havior, and and the the development development of of sensory sensory systems. systems. The editors editors wish wish to to thank thank the the authors authors for their their cooperation cooperation and and dedication to this project and also to express express their deep appreciation to the many reviewers whose careful readings and constructive criti criticisms cisms have have greatly greatly improved improved the the final final presentations. presentations. W. W. S. S. HOAR

D. J. RANDALL D.

xi xi


CONTENTS OF OTHER VOLUMES CONTENTS

Volume I The Compartments and and the of Electrolytes Electrolytes The Body Body Compartments the Distribution Distribution of

W. W. N. N . Holmes and Edward M. M . Donaldson

The Kidney Cleveland P. P . Hickman, Hickman, Jr., Jr., and BetYamin Benjamin F. F . Trump Salt Salt Secretion Secretion

Frank P. P . Conte The The Effects Effects of Salinity on the Eggs Eggs and Larvae Larvae of Teleosts

F. F . G. G . T. T . Holliday

Formation Formation of of Excretory Excretory Products Products

Roy P. Forster and Leon Goldstein Intermediary Intermediary Metabolism Metabolism in in Fishes Fishes

P. P . W. W. Hochachka Nutrition, Nutrition, Digestion, Digestion, and and Energy Energy Utilization Utilization

Arthur M. M. Phillips, Jr. Jr.

AUTHOR INDEX-SUBJECT AUTHOR INDEX-SYSTEMATIC INDEX-SYSTEMATIC INDEX-SUBJECTINDEX INDEX

Volume Volume II 11 The The Pituitary Pituitary Gland: Gland: Anatomy Anatomy and and Histophysiology Histophysiology ]. J. N. Ball and Bridget 1. 1. Baker

The The Neurohypophysis Neurohypophysis

A. A . M. M. Perks Prolactin Paralactin) and Prolactin (Fish (Fish Prolactin Prolactin or or Paralactin) and Growth Growth Hormone Hormone ]. J. N. N. Ball Ball Thyroid Function Function and Its Control in Fishes Fishes

Aubrey Gorbman Gorbmun xiii xiii

xiv xiv

CONTENTS CONTENTS OF OF OTHER OTHER VOLUMES VOLUMES

Endocrine Pancreas The Endocrine August Epple The Adrenocortical Adrenocortical Steroids, Steroids, Adrenocorticotropin Adrenocorticotropin and and the the Corpuscles Corpuscles The of Stannius Stannius of I. ChesterJones, Chester Jones, D D.. K K.. 0. o. Clzan, Chan, 1. I. W W.. Henderson, aand]. N.. Ball Ball 1. nd]. N The Ultiinobranchial Ultimobranchial Glands Glands and and Calcium Calcium Regulation Regulation The D.. Harold Copp D Neurosecretory System Urophysis and Caudal Neurosecretory Howard A.. Bern Howard A AUTHOR INDEX-SYSTEMATIC INDEX-SYSTEMATIC INDEX-SUBJECT INDEX-SUBJECT INDEX INDEX AUTHOR

III Volume 1 11 Reproduction Reproduction William S. Williaiit S . Hoar

Hormones and Reproductive Reproductive Behavior in in Fishes Fishes N. N . R. R . Liley Sex Sex Differentiation Differentiation

Toki-o Toki-o Yamamoto Yainainoto Development: Development: Eggs Eggs and and Larvae Larvae ]. 1. H. H . S. S . Blaxter Fish Cell and Tissue Tissue Culture Culture

Ken Wolf Wolf and M. M . C. C . Quimby Quiinby Chromatophores and Pigments

Ryozo Fujii Bioluminescence Bioluminescence

]. 1.A. A. C. C . Nicol Poisons Poisons and and Venoms Venoms

Findlay Findla y E. E. Russell AUTHOR AUTHOR INDEX-SYSTEMATIC INDEX-SYSTEMATIC INDEX-SUBJECT INDEX-SUBJECT INDEX INDEX

Volume Volume IV IV Anatomy Anatomy and Physiology Physiology of of the the Central Central Nervous Nervous System System

Jerald Jerald J. 1.Bernstein Bernstein The The Pineal Pineal Organ Organ

James Jaines Clarke Clarke Fenwick Fenwick

CONTENTS OF OF OTHER OTHER VOLUMES VOLUMES CONTENTS

Autonomic Nervous Nervous System Systems Autonoinic Graeme Campbell Campbell Craeine The Circulatory Circulatory System System The D. ]. Randall D. J . Randall Acid -Base Balance Balance Acid-Base C.. Albers Albers C Properties of of Fish Fish Heinoglobins Hemoglobins Properties Austen Riggs Riggs Austen Gas Exchange Exchange in in Fish Fish Gas D. ]. Randall D. J . Randall The Regulation Regulation of of Breathing Breathing The G. Shelton Shelton G. Air Breathing Breathing in in Fishes Fishes Air Kjell Johansen Kjell Johansen The Swiin Swim Bladder Bladder as as aa Hydrostatic Hydrostatic Organ Organ The Johan B. Steen Steen Johan Hydrostatic Pressure Pressure Hydrostatic Malcolm S. Gordon Gordon Malcolm S. Immunology of Fish Fish Immunology of

John Cushing John E. E. Cushing

AUTHOR AUTHOR INDEX-SYSTEMATIC INDEX-SYSTEMATIC INDEX-SUBJECT INDEX-SUBJECT INDEX INDEX

Volume Volume V V Vision: Vision: Visual Visual Pigments Pigments

F. F . W. W. Munz Munz Vision: Vision: Electrophysiology Electrophysiology of of the the Retina Retina

T. T . Tomita Tomita

Vision: of Visual Visual Behavior Behavior Vision: The The Experimental Experimental Analysis Analysis of

David David Ingle lngle Chemoreception Cheinoreception

Toshiaki]. Toshiaki J . Hara Hara Temperature Temperature Receptors Receptors

R. R . W. W . Murray Murray

Sound Sound Production Production and and Detection Detection

William Williain N. N. Tavolga Taoolga

xv xv

xvi xvi


The Labyrinth Labyrinth The O. Lowenstein Lowenstein 0, The Lateral Lateral Organ Organ Mechanoreceptors Mechanoreceptors The A e Flock l kke Flock The Mauthner Mauthner Cell Cell The ]. Diamond'. J. Diamond Electric Organs Organs Electric M. V. L. Bennett Bennett M . V . L. Electroreception Electroreception M.. V V.. L. Bennett Bennett M AUTHOR INDEX-SYSTEMATIC INDEX-SUBJECT INDEX-SUBJECT INDEX AUTHOR

Volume VI VI Volume The Effect Effect of of Environmental Environmental Factors Factors on on the the Physiology Physiology of of Fish Fish The F. E.]. Fry F . E. J. F r y Biochemical Environment Biochemical Adaptation Adaptation to to the the Environment

P. P . W. W. Hochachka and G. G . N. N. Somero Freezing Resistance in Freezing Resistance in Fishes Fishes

Arthur L. L. DeVries Learning Learning and and Memory Memory

Henry Henry Gleitman Gleitrnun and Paul Rozin Rozin The The Ethological Ethological Analysis Analysis of of Fish Fish Behavior Behavior

Gerard Gerard P. P . Baerends Baerends Biological Biological Rhythms Rhythms

Horst Horst O. 0. Schwassmann Schwassmunn Orientation Orientation and and Fish Fish Migration Migration

Arthur Arthur D. D . Hasler Hasler Special SpecialTechniques Techniques D. D .].J. Randall Randall and and W. W . S. S . Hoar Hoar AUTHOR AUTHORINDEX-SYSTEMATIC INDEX-SYSTEMATIC INDEX-SUBJECT INDEX-SUBJECT INDEX INDEX

Volume Volume VII VII Form, Form, Function, Function, and and Locomotory Locornotory Habits Habits in in Fish Fish

C. C.C. C.Lindsey Lindsey


Swimming Capacity W.. H. H. Beamish Beamish FF.. W Hydrodynamics: Nonscombroid Nonscombroid Fish Fish Hydrodynamics: W.. Webb Paul W Hydromechanics, Morphology, Locomotion by Scombrid Fishes: Hydromechanics, and Behavior Behavior and John]. Magnuson John J . Magnuson Especially Skipjack Body Temperature Relations of Tunas, Especially William H H.. Neil1 Neill EE.. Don Stevens and William

Locomotor Muscle Quentin Bone The Respiratory and Circulatory Systems Systems during Exercise David R. Jones Jones and David J. J . Randall Metabolism Metabolism in in Fish during Exercise

William R. Driedzic and P. W W.. Hochachka William AUTHOR AUTHORINDEX-SYSTEMATIC INDEX-SUBJECT INDEX-SUBJECT INDEX INDEX

Volume VIII VIII Nutrition Nutrition

C. C . B. B. Cowey and]. and]. R. Sargent Feeding Feeding Strategy Strategy

Kim D. D. Hyatt The The Brain Brain and and Feeding Feeding Behavior Behavior

Richard E. Peter Digestion Digestion

Ragnar Fiinge Fange and David Grove Grove Metabolism Metabolism and and Energy Energy Conversion Conversion during during Early Early Development Development

Charles Charles Terner Terner Physiological Physiological Energetics Energetics

]. J . R. R . Brett Brett and and T. T . D. D. D. D. Groves Groves Cytogenetics Cytogenetics

]. J . R. R. Gold Gold Population Population Genetics Genetics

Fred W .Allendorf Allendorf and and Fred Fred M. M . Utter Utter Fred W.

xvii xvii

xviii xviii


Enhancement of of Growth Hormonal Enhancement M.. Donaldson, Ulf Ulf H H.. M M.. Fagerlund, David David A. Higgs, Edward M J. R. McBride and J.

Environmental Factors and Growth R.. Brett J}.. R Growth Rates and Models

W. E. Ricker W. AUTHOR INDEX-SYSTEMATIC INDEX-SYSTEMATIC INDEX-SUBJECT INDEX-SUBJECT INDEX AUTHOR

Volume IXA Reproduction Reproduction in Cyclostome Fishes and Its Regulation

Gorbman Aubrey Gorbmun

Reproduction in Cartilaginous Fishes (Chondrichthyes) (Chondrichthyes) M.. Dodd }. J,M Reproduction The Brain and Neurohormones Neurohormones in Teleost Reproduction Richard EE.. Peter The Cellular Origin of of Pituitary Gonadotropins in Teleosts P. G. W. J. }. van Oordt and J. J. Peute G. W. Teleost Gonadotropins: Isolation, Biochemistry, and Function

T. Bun N Ng g David R. Idler ldler and T. The Functional Functional Morphology of of Teleost Gonads

Yoshitaka Nagahama Nagahntna The Gonadal Steroids

A. Fostier, B. B.Jalabert, Jalabert, R. Billard, B. B . Breton, and Y. Y. Zohar

Differentiation in Teleost Fishes Yolk Formation and Differentiation T. T . Bun Ng N g and David R. Idler ldler .'An An Introduction to Gonadotropin Receptor Studies in Fish

Glen Van Van Der Kraak AUTHOR INDEX-SUBJECT AUTHORINDEX-SYSTEMATIC INDEX-SYSTEMATIC INDEX-SUBJECTINDEX INDEX

Volume Volume IXB IXB Hormones, Pheromones, and Reproductive Behavior in Fish

N . R. R . Liley and N. N . E. E . Stacey N.


xix xix

Environinenta~Influences on Gonadal Activity in Fish Environmental T . J . Lam T.}. Hormonal Control of Oocyte Final Maturation in Fishes Maturation and Ovulation in

Frederick W. Goetz Frederick Sex Control and Sex Reversal in Fish under Natural Natural Conditions

S . T. T . H. H . Chan Chan and W. S. S. B. B . Yeung S. Hormonal Horinonal Sex Control and Its Application Application to Fish Culture

M . Donaldson George A. Hunter and Edward M. Fish Gamete Preservation and Spermatozoan Spermatozoan Physiology

Joachim Joachiin Stoss Stoss Induced Final Maturation, Ovulation, and Spermiation in Cultured Fish

Edward M. Donaldson and George A. Hunter Chromosome Set Manipulation Manipulation and Sex Control in Fish

Gary G a y H. H . Thorgaard AUTHOR INDEX-SYSTEMATIC INDEX-SUBJECT INDEX-SYSTEMATIC INDEX-SUBJECTINDEX INDEX

Volume XA XA General Anatomy of of the Gills

George Hughes Gill Internal Morphology

Pierre Laurent Innervation and Pharmacology of the Gills

Stefan Nilsson Model Analysis of Gas Transfer in Fish Gills

Johannes Piiper and Peter Scheid Oxygen and Carbon Dioxide Transfer across Fish Gills

David Datiid Randall and Charles Ch.arle.9Daxboeck Daxboeck

Acid-Base Regulation in Fishes Acid -Base Regulation Norbert Heisler Physicochemical Physicochemical Parameters for Use Use in Fish Respiratory Respiratory Physiology

Robert G. G . Boutilier, Boutilier, Thomas A. A. Heming, and George K. K . lwama lwaina Robert AUTHOR AUTHORINDEX-SYSTEMATIC INDEX-SYSTEMATICINDEX-SUBJECT INDEX-SUBJECTINDEX INDEX

xx xx


Volume XB Volume

Permeation Water and Nonelectrolyte Permeation Isaia Jacques Zsaia Teleosts : The Roles of of Respiratory Branchial Ion Movements in Teleosts: and Chloride Cells and N N.. Mayer-Gostan P. Payan, JJ.. P. Girard, and ATPases Ion Transport and Gill ATPases Guy de Renzis and and Michel Bornancin

in Fish Gills Transepithelial Potentials in W.. T. T. W W.. Potts W of Chloride C hloride The Chloride Cell: The Active Transport of and the Paracellular Pathways J. A. A. Zadunaisky

Hormonal Control of o f Water Movement across the Gills J. J . C. Rankin and Liana Bolis Metabolism of o f the Fish Gill

Thomas P. Mommsen

The Roles ooff Gill Permeability and Transport Mechanisms in Euryhalinity David H. Etians Evans David H. The Pseudobranch: Pseudobranch: Morphology and Function

Pierre Laurent and Suzanne Dunel-Erb Perfusion Methods for the Study of of Gill Physiology

S. FF.. Perry, P. SS.. Davie, C C.. Daxboeck, A. A. G. G. Ellis, S. and D. G. G. Smith AUTHOR INDEX-SUBJECT INDEX AUTHORINDEX-SYSTEMATIC INDEX-SYSTEMATIC INDEX-SUBJECT INDEX

Volume Volume XIB

The Maternal-Embryonic Maternal-Embryonic Relationship in Viviparous Fishes

Wourms, Bryon D. Grove, Grooe, and Julian Lombardi John P. Wourms, First Metamorphosis Metamorphosis

John H. H . Youson Factors Controlling Meristic Variation

C. C.C. C. Lindsey


The Physiology of Smolting Smoking Salmonids

W. SS.. Hoar Ontogeny of Behavior and Concurrent Developmental Changes in Sensory Systems in Teleost Fishes

L. G. G. Noakes and ]ean-Guy Jean-Guy ]. J . Godin David L. AUTHORINDEX-SYSTEMATIC INDEX-SYSTEMATIC INDEX-SUBJECT INDEX INDEX AUTHOR INDEX-SUBJECT

xxi xxi


1 PATTERN AND VARIETY IN DEVELOPMENT ]. J.

H. S. BLAXTER H.

Dunstaffnage Marine Marine Research Research Laboratory Laboratory Dunstaffnage Oban, Argyll Argyll PA34 PA34 4AD, 4AD, Scotland Scotland Oban, I. Introduction Introduction I. II. Rearing Rearing Techniques Techniques 11. III. Progress and Diversity of of Development 111.

IV. Terminology Terminology of of Early Early Life Life History History Stages Stages IV. V. Egg Egg Size and and Egg Quality A. Egg Egg Size A. Size B. Egg Quality Quality B. Egg The Effect Effect of of Starvation Starvation VI. The VII. The Effect VII. The Effect of of Captivity Captivity VIII. VIII. The The Effect Effect of of Fixation Fixation on on Shrinkage Shrinkage IX. Rate Rate of of Development Development X. Organ X. Organ Systems Systems A. A. Alimentary Alimentary System System B. B. Respiratory Respiratory System System C. C. Locomotor Locomotor System System D. D. Sense Sense Organs Organs Xl. Structure XI. Structure and and Function Function XII. Critical XII. Critical Periods Periods XIII. XIII. Conclusions Conclusions References References

I. I. INTRODUCTION INTRODUCTION

Present Present interest interest in in the the development development of of fish fish has has aa twofold twofold basis. basis. First, First, the the factors factors that that determine determine brood brood strength strength and and so so recruitment recruitment to to commercial commercial fisheries fisheries may may well well operate operate during during the the early early life life history history stages. stages. Second, Second, the the practice practice of of aquaculture aquaculture and and the the range range of of species species used used are are expanding expanding rapidly. rapidly. Improvements Improvements in in hatchery hatchery techniques techniques during two decades decades have have made made it it possible possible to to rear rear almost almost any any during the the last last two species, even the halibut Hippoglossus hippoglossus (V. 0iestad, species, even the halibut Hippoglossus hippoglossus (V. Oiestad, per personal sonal communication) communication) if if fertile fertile eggs eggs are are available. available. Manipulation Manipulation of of 1 FISH FISH PHYSIOLOGY. VOL. VOL. XIA XIA

PHYSIOLOGY,

1 CopyrightIt)0 1988 1988by by Academic Academic Press, Press, Inc. Inc. Copyright All All rights rights of of reproduction reproductionin in any any form form reserved. reserved.

2

J. S. BLAXTER J. H. S.

spawning time has also meant that a steady supply of of larvae-for larvae-for example, of turbot Scophthalmus Scophthalmus maximus and northern anchovy En Engraulis m ordax can be maintained throughout the year, even with mordar-can temperate species. species. Experimental material is thus readily available for the study of optimum rearing conditions, nutrition and growth, critical periods in development, toxicity testing, and the like, all germane to the assessments of the fishery biologist and the practices of the fish farmer. This material is also available to experimental embryologists farmer. and to physiologists interested in the ontogeny of organ systems, or to behaviorists interested in the ontogeny of behavior. Great insight into function is possible in larval stages that lack certain organs or have them only partially developed. In many species, it is the transparency of the larvae, their lack of hemoglobin, their simple intestinal tracts, undifferentiated skeleton, and incompletely developed nervous sys system and sense organs that can make them especially useful as experi experimental animals animals.. fish The progress of work oon n the developmental biology ooff fi s h since 1970s can be traced in a series of symposia and other meet meetthe early 1970s ings. International Early Life History Symposia were held in 1973, 1973, 1979, and 1984 1984 in Scotland, the United States, and Canada (Blaxter, (Blaxter, 1979, 1974; 1 ; Marliave, 1974; Lasker and Sherman, 198 1981; Marliave, 1985). 1985). A symposium on the "The “The Ontogeny and Systematics of Fishes," Fishes,” dedicated to the 1983 (Moser (Moser et memory of E. E. H. H. Ahlstrom, Ahlstrom, was held in California in 1983 al., 1984), he Ameri 1984),and larval fish conferences are held annually by tthe Ameri(e.g., Hubbs, 1986). 1986). can Fisheries Society (e.g., of “Fish Physiology,” a brief brief By way of introduction to this volume of "Fish Physiology," account will first be given of recent advances in techniques of rearing, variageneral life history stages, and terminology. It is intended that varia tion of experimental material and the sources of variation will be an paunderlying but continuing theme. Thus diversity of development, pa rental effects on the young, and the effects of captivity, starvation, and fixation will be discussed. FinaIly, Finally, the development of structure will be related to the development of function with particular reference to possible critical periods in ontogeny. ontogeny. -

11. II. REARING TECHNIQUES Over the past few years improvements in techniques for rearing fish marine fi sh have increased the number of species available for experiexperi ment and for aquaculture. These techniques are summarized by

11..

PATTERN PATTERN AND AND VARIETY VARIETY IN IN DEVELOPMENT DEVELOPMENT

3

Kinne ((1977), (1984). The greatest advance 1977), Blaxter ((1981), 1981), and Hunter (1984). of the rotifer has been the use of very small food items, especially of G ymnodinium Brachionus plicatilis, but also the naked dinoflagellate Gymnodinium splendens and other organisms such as Mytilus trochophores and sieved natural zooplankton, as a food source in the very young stages 34). Interest is now when the size size of the mouth is limiting ((see see page 34). “green-water” culture, where the larvae are increasing in the use of "green-water" of algae such as Chlorella, Chlorella, which maintained in fairly high densities of may damp out metabolite fluctuations, perhaps improve oxygenation, and provide a secondary food source for the larvae (Houde, 1977; 1977; Morita, 1985). 1985). In the future the use of compounded diets, especially in the form of microcapsules small enough to be eaten whole, may pro provide a further breakthrough. Appelbaum ((1985) 1985) reared Dover sole Solea solea entirely on compounded diets and cited other similar suc successful work on plaice Pleuronectes platessa, vendace Coregonus al alhula, labrax, turbot, catfish Clarias ga gabula, sea bass Dicentrarchus lahrax, riepinus, and the Atlantic silverside Menidia menidia. The best survival rate of sole on an artificial diet was obtained when live brine shrimp Anemia Artemia nauplii were provided provided for the first 10 10 days of of feeding. feeding. Artemia is certainly still the staple live food, food, both in experimental and Anemia (Sorgeloos, 1980). 1980). It has become increasingly ob obapplied fish culture (Sorgeloos, vious that Anemia Artemia from different sources can vary in quality-for quality-for example, in fatty acid "profi le"-and success and failure in the past example, “profile”-and unappreciated factor (van (van Ballaer et may have hinged on this hitherto unappreciated Kanazawa, 1985). 1985; Kanazawa, 1985). Dabrowski ((1984) 1 984) reviewed work on some al., 1985; of of the nutritional aspects of rearing fish larvae and the relevance of digestive processes. Other factors in rearing, such as optimum food density, stocking density, type of tank, light, and other environmental conditions, have now been established. Production of eggs out of season by the use of of artificial photoperiods, photoperiods, temperature, and hormone injections have also greatly improved the availability oflarvae (see Lam, 1982). of larvae year-round (see 1982). One of the most striking advances has been the improvement in survival and growth when larvae are reared in the absence of preda predators in large-scale facilities, or "mesocosms," “mesocosms,” in the form of of large on onshore tanks, large plastic-walled cylinders sited in sheltered coastal orjmpounded (Kvenseth and 0iestad, Oiestad, waters, or .impounded coastal bays or lagoons lagoons (Kvenseth 1984; 0iestad 1984; Oiestad et al., 1985; 1985; Gamble et al., 1985; 1985; Morita, 1985; 1985; Paulsen et al., al., 1985; ai., 1985). 1985; Sturmer Stunner et al., 1985). Atlantic cod Gadus morhua, turbot, and red drum Sciaenops ocellatus have been reared with unprece unprecedented success.

4

J. J. H. H. S. S. BLAXTER BLAXTER

PROGRESS AND D DIVERSITY III. PROGRESS IVERSITY OF DEVELOPMENT 111. Some Some comprehensive keys have recently appeared in the literature that give a good insight into the variety of eggs and larvae and their development. 1976) describes the eggs and planktonic stages development. Russell ((1976) (1983)the ichthyoplankton of the west westof British marine fishes, Fahay (1983) ern North Atlantic, and Auer ((1982) 1982) that of the Lake Michigan region of the Great Lakes Basin. Basin. The early life history stages of fish and their characteristics are discussed more generally by Blaxter (1969), (1969), Hem Hemcharacteristics pel ((1979), 1979), and Kendall et al. al. (1984). (1984). Great variety exists from species to species and, in particular, the size and extent of differentiation signifiwhen the young fish first becomes free-living is of considerable signifi cance for its chance of survival. final fish.such During the fi nal ovarian maturation of the eggs of marine fish. such MeZanoas the Atlantic cod, whiting Merlangius merlangus, haddock Melano grammus aeglefinus, aeglefinus, and plaice, there is a massive uptake of water and concomitant reduction in protein phosphate (Craik (Craik and Harvey, 1984a).This influx influx of water, such that the water content may reach as 1984a). high as 92% of the egg weight, weight, is an adaptation to pelagic life because the egg fluids are hypotonic and make the eggs buoyant. Freshwater fish with demersal eggs, like the rainbow trout Salmo gairdneri, po fish polauarcticus, and pike Esox lucius, do not show these wan Coregonus lavarcticus, changes. changes. initiaI buoyancy of pelagic fi fish eggs, like those of of the flounder sh eggs, The initial P. P . jlesus, depends on the salinity in which the female is kept before (Solemdal, 1973). 1973). Females from low salinities tend to pro prospawning (Solemdal, duce eggs that are neutrally buoyant at lower salinities, buoyancy, of osmocourse, not only being affected by water content but also by the osmo fluid. larity of the egg fluid. There is great variety in the reproductive styles of fish (Table I). I). In most species the eggs develop independently, but there are many (Breder and Rosen, 1966). In littoral species instances of parental care (Breder Rosen, 1966). gobies, this takes the form of guarding such as cottids, blennies, and gobies, the eggs, eggs, but nests may be built with one or other parent guarding and is found often ventilating the nest. Mouth brooding of eggs and larvae is in cichlids such as tilapia and in ariid catfish. catfish. Other species have evolved ovoviviparity or viviparity, the former where the eggs de develop within the female, female, the latter where nourishment is provided via “placental” structures (trophotaenia) (trophotaenia) within the female (see (see Wourms "placental" XIB). Recently Ridley (1978) (1978) and Blumer (1979) (1979) and Grove, volume XIB). significance. While summarized parental care and its evolutionary significance. (in 61 families), care by the care is more common by the male parent (in 61 families),

1. PATTERN AND VARIETY IN DEVELOPMENT 1. DEVELOPMENT

5

41 female occurs in 4 1 families. Care by the male is clearly linked to the prevalence of external fertilization in fish and generally to polygi\my polygamy and male territoriality. The morphological characteristics of of fish eggs are described by (1980), and Matarese and Russell ((1976), 1976), Ahlstrom and Moser (1980), Sandknop ((1984). 1984). Typically marine eggs are single, buoyant, and with of about 11mm (although the range is from 0.6 to 4.0 a modal diameter of mm) sh lay demersal eggs with a modal diameter mm).. Most freshwater fi fish somewhat greater than 11 mm. mm. The eggs may merely rest on the subTable Table II Classifi cation of Classification of Reproductive Styles· Styles" A.

Nonguarders N on guarders 1. Open 1. Open and and substratum substratum spawners spawners a. Pelagic spawners b. Rock and gravel spawners with pelagic larvae c. Rock and gravel spawners with benthic larvae c. d. Nonobligatory plant spawners d. e. e. Obligatory plant spawners f. Sand spawners g. g. Terrestrial spawners, in damp conditions 2. Brood hiders 2. a. b. c. d. d. e. e.

Beach spawners; above waterline at high tides Annual spawners; eggs estivate Rock and gravel gravel spawners spawners Cave Cave spawners spawners Spawners in live invertebrates

B. B. Guarders 1. Substratum spawners 1. a. a. Pelagic spawners; at surface of hypoxic waters Above-water spawners; male splashes clutch b. Above-water b. c. Rock spawners d. Plant spawners 2. Nest spawners 2. a. a. Froth Froth nesters nesters b. b. Miscellaneous substratum and materials nesters c. Rock and gravel nesters d. Glue-making Glue-making nesters e. e. Plant material nesters f. Sand nesters g. Hole nesters g. of host h. Anemone nesters; at base of

(continued) (continued)

S. BLAXTER J. H. S.

6 Table Table 11 (Continued) (Continued)

C. C. Bearers 1. 1. External bearers a. Transfer brooders; brooders; eggs carried before deposition b. Auxiliary brooders; adhesive eggs carried on skin under fins etc. c. Mouth brooders d. Gill-chamber Gill-chamber brooders e. Pouch brooders e. 2.

a

Internal bearers a. Facultative internal bearers; occasional internal fertilization of normally oviparous fish, fish, eggs rarely retained long b. Obligate lecithotrophic lecithotrophic live bearers; no maternal-embryonic maternal-embryonic nutrient transfer c. M atrotrophous oophages and adelphophages or a few eggs develop developMatrotrophous adelphophages;; one or ing at expense of other eggs or embryos d. Viviparous trophoderms; trophoderms; nutrition partially or entirely from female via "placental" “placental” structures structures

Adapted from Balon (1981a). (1981a).

stratum, or have some means of attachment such as adhesive threads or a supporting pedestal. In species such as salmonids salmonids the eggs are buried in the gravel, and the grunion Leuresthes tenuis lays its eggs intertidally in the sand. Other types of of demersal eggs are found in some littoral marine species and, in the more offshore Atlantic her herring, capelin Mallotus villosus and Pacific cod Gadus macrocephalus. While While teleosts teleosts usually usually have have round round eggs, eggs, most most engraulids engraulids have have eggs eggs that are ellipsoidal, thought to be an adaptation to reduce cannibalism by the filter-feeding parents after spawning. spawning. Other families like the gobies gobies have have slightly slightly flattened flattened eggs, and and demersal eggs eggs are are sometimes sometimes irregular in shape. Oviparous elasmobranchs have eggs of unusual shapes (the "mermaid's purse") with tendrils for attachment. Tendrils “mermaid’s purse”) are also found in the silverside Atherinopsis and the gar Relone, Belone, while the flying fish Oxyporhamphus has spines (Boehlert, (Boehlert, 1984). 1984). It is easy speto understand the adaptive value of tendrils in distantly related spe cies, but it is much more difficult to explain the ornamentations of of the chorion. Most teleosts have a smooth surface to the chorion, but the chorion. unrelated inshore dragonet Callionymus and bathypelagic gonostogonosto matid Maurolicus muelleri have chorions with hexagonal facets, and the fl atfish Pleuronichthys coenosus has a chorion with very many flatfish small facets. small The yolk is usually translucent, unpigmented, and homogeneous in texture, but may be segmented in primitive species like the pil-

11.. PATTERN AND VARIETY IIN N

DEVELOPMENT

7

chard Sardina pilchardus and sprat Sprattus sprattus. sprattus. In some soleids the segmentation is confined to the periphery of the yolk, and in other species like the jack mackerel Trachurus symmetricus segmentation appears progressively during early development. pe development. Most commonly, pelagic fi sh eggs have a single oil globule in the yolk. yolk. Of a total of 515 fish species checked by Ahlstrom and Moser ((1980), 1980), 60% 60% had one oil glob glob15% had multiple oil globules. The ule, 25% had no oil globule, and 15% oil globule, when single, usually lies at the vegetal pole in Marine fi sh larvae. It is generally thought that the oil globules are a spespe fish cialized form of nourishment and have a minimal effect on buoy buoyancy. Following activation or fertilization the egg absorbs water, the perivitelline space forms, forms, and the chorion hardens. The perivitelline space is usually narrow but is wide in some "primitive" “primitive” species such as the pilchard, in some eels, eels, and in unrelated species like the striped bass Morone labrax and long rough dab Hippoglossoides platessoides. Cleavage is meroblastic in hagfish, hagfish, elasmobranchs, elasmobranchs, and teleosts, al although in the lampreys it is holoblastic but with the formation of of micro- and macromeres. n Amia, gar macromeres. In primitive groups like the bowfi bowfin Lepisosteus, and sturgeon Acipenser, cleavage is is intermediate or semiholoblastic. semiholoblastic. The embryo develops as a blastodisc at the animal pole. The periphery of the blastodisc overgrows the yolk (epiboly), (epiboly), eventually enclosing it to form a gastrula but leaving an opening, the blastopore. The embryonic axis axis forms forms by a process of convergence and concentration in relation to the dorsal lip lip of the blastopore at the neurula stage but the quantity of uences the timing of such of yolk infl influences events. The head and eye eye cups are soon soon identifiable and the trunk lengthens and separates from the yolk sac. sac. The heart functions well before hatching, and in some demersal eggs a vitelline circulation can sac. Examples of the development of a marine be seen within the yolk sac. (dab)and a freshwater egg (rainbow (rainbow trout) are given in Figs. 11 and egg (dab) 2. 2. Before hatching, the embryo becomes very active and the chorion is ssoftened is oftened as a result of enzymes secreted by hatching glands. The larva depends very degree of differentiation of the newly hatched larva much on the species and egg size, size, and the incubation period depends factors and on temperature (see (see Fig. 18). 18). In many many marine on these factors species the mouth and jaws are are not formed, the eye eye is is not pelagic species pigmented, the yolk yolk sac sac is is huge, and a primordial finfold finfold runs around pigmented, position. Apart Apart from a few melanophores, the the trunk in the median position. larva is is very transparent. All newly hatched larvae larvae have free neuro neurolarva masts on the head and trunk, and otoliths are present in the otic capcapmasts

8

J. J. H. H. S. BLAXTER BLAXTER

Fig. 1. 1. Development of of the dab Limanda Limanda limanda, using Apstein’s Fig. Apstein's stages. stages. [From of Academic Press.) Press.] Russell (1976), with permission of

sule. sule. Other marine species hatch with the alimentary system nearly Some larvae are very advanced, functional and with pigmented eyes. Some and in loricariids the dorsal and caudal fin are partly developed at hatching (Fuiman, 1984); in flying fish, flexion of the notochord (Fuiman, 1984); (which (which precedes caudal fin formation) actually occurs before hatching. In the salmonids-for (Fig. 2)-although salmonids-for example, rainbow trout (Fig. 2)-although the yolk sac is still large, the larva (alevin) (alevin) is better developed and espe especially the vascular system and vitelline circulation are conspicuous with the blood containing hemoglobin. The young of cichlid and ariid also further developed and adapted to early life mouth brooders are also within the parental mouth. In ovoviviparous ovoviviparous and viviparous species

11.. PATTERN

AND VARIETY IN DEVELOPMENT

9

� :. ,

0

...,

'

-"

.

�

•

Fig. 2. Development of the rainbow trout Salmo Salmo gairdneri, gairdneri. (A) (A) 8-Blastomeres. (B) (B) one-third epiboly. (C) (C) 0-5 0-5 Somites, one-half one-half epiboly. (D) (D) Otic Early embryo apparent, one-third 10-20 somites, total somites placodes, three-fourths three-fourths epiboly. placodes, epiboly. (E) (E) Caudal bud with 10-20 somites 5158, heart beating. (F) (F) Posterior Posterior cardinal veins formed, choroid of of eye pigmented. (G) (G) Near hatching, pelvic fins fins develop. (H) (H) Hatched alevin, first anal and dorsal fin rays. (1.1) (1,J) Later alevin stages as yolk yolk is resorbed. Scale bars 2 mm long. [Redrawn from Vernier (1969).] (1969).]

(Amoroso, (Amoroso, 1960) 1960) the the young young may may hatch hatch effectively effectively as as postmetamorphic postmetamorphic juveniles. juveniles. mackerel, Examples of early life history stages, those of the jack mackerel, northern Figs. 3 and northern anchovy, anchovy, and and Pacific Pacific hake, hake, are are illustrated illustrated in in Figs. and 4. 4. The The changing changing shape shape of the the larvae larvae is is clearly clearly shown, shown, with with the the imporimpor-

J. H. S. BLAXTER J. H. S.

10 10

EGGS

YOLK SAC

PRE FLEXION

]

]

FLEXION

POST FLEXION

JUVENILE

Fig. 3. 3. Early Early life history history stages stages of the thejack mackerel mackerel Trachurus symmetricus. symmetricus. [From [From Fig. the original original drawings drawings of Ahlstrom Ahlstrom and and Ball Ball in Kendall Kendall et al. al. ((1984), with permission of 1984), with the American Society Society of Ichthyologists Ichthyologists and and Herpetologists.] Herpetologists.] the

11.. PATTERN AND

VARIETY IN DEVELOPMENT

1 1 11

F

Fig. 4. Northern anchovy Engraulis 4. Development of teleost larvae. larvae. (A-E) (A-E) Northern Engruulis mordax, mordux, 2.5,7.5,11.5,18.4,31.0 [Redrawn from Kramer and Ahlstrom ((1968).] (F-H) Pacifi Pacific 2.5, 7.5, 1 1.5, 18.4, 31.0 mm. mm. [Redrawn 1968).] (F-H) c 4.3, 7.7, 7.7, 111.0 1.0 mm. hake Merluccius productus, 4.3, mm. [Redrawn [Redrawn from Ahlstrom and Counts ((1955).] 1955).]

tance of exion of of fl flexion of the notochord notochord and development of of the caudal fin being emphasized, with implications for improved swimming. The duration of of the yolk-sac yolk-sac period depends on both species and temperature but als o on egg size (see also (see also p. p. 17). 17). The argentine Argentina silus and the halibut have egg diameters of of 3.0-3.5 3.0-3.5 mm. Unexpectedly, the newly hatched larvae are very undeveloped but the halibut takes 50 days to resorb its yolk (at 5.3°C) 5.3"C) and reaches a length of 1. 5 mm (Blaxter et ai., 983a) and the argentine reaches a of 111.5 al., 11983a) prodigious length of 17 mm on of 17 on its yolk supply (Russell, (Russell, 1976). 1976). During the yolk-sac period the mouth and gut and the eyes be period become functional to allow the larva to switch from endogenous to exo exogenous nutrition. The subsequent larval period ranges from a few days to some months (and even 2-3 2-3 years in eels), eels), depending on tempera-

12 12

H. S. S. BLAXTER BLAXTER J. H.

ture and species. species. During During this this time time the the larva is likely likely at at least least to to double double ture and larva is its length and to increase its weight by 10 to 100 times. Transient its length and to increase its weight by 10 to 100 times. Transient characters, such such as as spines, spines, may may appear appear (Fig. (Fig. 5), 5), which are presumably presumably characters, which are antipredator Other bizarre bizarre structures, structures, such as eyestalks, eyestalks, antipredator adaptations. adaptations. Other such as elongated elongated fin fin rays, rays, or or tentacles, tentacles, may may also also appear, appear, often often as as larval larval charac characters ters (Fig. (Fig. 5), 5), to to be be lost lost later later in in development. development. The The cobitid Misgurnus fossilis laments for time (Fuiman, fossilis even even has has external external gill gill fi filaments for aa time (Fuiman, 1984). 1984). The The importance importance of of allometric allometric growth growth during during larval larval development development has 1983) and and Fuiman Fuiman has been been emphasized emphasized by by Fuiman Fuiman ((1983) and Strauss Straws and ((1985). 1985). In some some species relative relative growth growth intensity intensity follows follows aa U-shaped U-shaped gradient gradient along along the the body body with with fastest fastest growth growth in in the the caudal caudal region, region,

f

Fig. 5. 5. Teleost Teleost larvae larvae showing showing spines spines and and other other processes. processes. (A) (A) Holocentrus vexilla uexillaFig. rius 5.0 5.0 mm. mm. (B) (B) Sebastes macdonaldi tnacdonaldi 9.0 9.0 mm. mm. (C) (C)Lophius piscatorius piscatorius 26 26 mm. mm. (D) (D) rius Acanthurid 77 mm. mm. (E) (E)Ranzania Ranzania laevis laeois 2.8 2.8mm. mm. (F) (F)Myctophum aurolaternatum 26 26 mm. Acanthurid (G)Campus Carupus aC1l8 ucus 3.8 3.8 mm. mm. (H) (H) Trachipterus Trachipterus sp. sp. 7.6 7.6 mm. (l) (I) Zu cristatu8 cristatus 6.5 6.5 mm. mm. [Re [Re(G) drawn drawn from from Moser Moser (1981).] (1981).]

11. . PATTERN PATTERN AND AND VARIETY VARIETY IN IN

DEVELOPMENT DEVELOPMENT

13 13

of the body. Growth is also linked to an increase in the propulsive area of of feeding and respiratory fast in the head region, where elaboration of functions may be taking place. IIn n sculpins, however, the caudal rere gion grows grows more slowly than the rest of of the body and growth is fastest in the head region. of adult characters (such (such as fi fin Progressive differentiation of n rays and skeleton) occurs. The larvae eventually pass through a process of metamorphosis to the juvenile stage. stage. This process may be rather be prolonged. Typically the blood becomes pig pigabrupt or it may he mented, scales and pigment appear on the body surface, the meristic characters such as fin rays are complete, and the body shape becomes like the adult. The juvenile appears as a small adult. In fl atfish, meta flatfish, metamorphosis is a remarkable process as the fish starts to change from the bilaterally symmetrical larva to an asymmetrical juvenile lying on on one (abocular or blind) side side (Fig. 6). Changes take place to the skull and (Fig. 6). sense organs and, in particular, particular, the eye of the abocular side migrates across the top of of the skull. By way of of summary, summary, Table II I1 lists some of of the characteristics of of a few species to emphasize the diversity that is is to be found. The young of many elasmobranchs, with a long incubation period, effectively hatch as juveniles, albeit with a yolk sac. sac. In species with parental care the early larvae may also be advanced or "precocial." “precocial.” This variety makes it difficult to categorize early life histories in a neat and con convincing way.

c

Fig. 6. platessa. (A) 6. Development Development and and metamorphosis metamorphosis in in the the plaice plaice Pleuronectes Pleuronectes platessa. (A) Yolk-sac 6.6 mm. 7.3 mm. (C-F) Stages morpho Yolk-sac larva, larva, 6.6 mm. (B) Larva Larva at at first first feeding, feeding, 7.3 mm. (C-F) Stages of of meta metamorphosis with scale bar 2 mm. mm. [Redrawn [Redrawn from from Ryland Ryland (1966).] (1966).1 sis with eye eye migration; migration; scale bar 2

Table II Early Life History Characteristics·

Species

Common name

Egg diameter (mm)

Hatching length (mm)

Hatch

First feed

Metamorphosis

Temperature range (0C)

3-4 6-7 3-4 2-3 2-3

1.4-2.9 2.5 0.8-1.5 0.6-0.9 0.4-0.5

1.9-3.6 4.0 1.3-2.0 1 .1-l.7 0.9- 1 . 1

4.2-5.0 10-12 1 1-13 5-6 1 1-15

4-12 7-11 9-15 13-18 13-15

5-8 6-7 1.7

1.0-3.0 2.0-3.0 0.15

2.0-4.5 9.0-10.0 0.7

12�24 ?15-16 ?

6-14 4-7 26

4 4-5 15-25 100 240-310

0.6-0.7 1.5-2.0 20-22 24-32 -104

1.7-2.6 2.0-2.4 26-28 28-36 -104

? Not clearcut Not clearcut 28-36 < 104

28 20-25 1-7 4-12 4-12

Gadus morhua Pleuronectes platessa Scomber scombrus Scophthalmus maximus Engraulis mordax

Cod Plaice Mackerel Turbot Northern anchovy

Clupea harengus Hippoglossus hippoglossus Acanthurus triostegus Oreochromis (=Tilapia) mossambicus Oryzias latipes Salmo salar Scyliorhinus caniculus Squalus acanthias

Herring Halibut Convict surgeonfish

1.1-1.9 1 .7-2.2 1.0-1.4 0.9-1.2 0.6-0.7 x 1.3-1.4b 0.9-1.7 3.0-3.2 0.7

Tilapia Medaka Salmon Spotted dogfish Spur dogfish

1.7-2.2 1.0-1.3 5-6 65 (long) 24-32

Weeks from fertilization to

Data from Blaxter (1969), Blaxter et al. (1983a), Howell (1979), J. R. Hunter and C. Kimbrell (personal communication), Iversen and Danielssen ( 1 984), Jones ( 1972), Kuhlmann et al. (1981), Rana (1985), and Russell ( 1976). b Eggs are ellipsoidal, minor and major axes given. •

11.. PATTERN PATTERN AND AND VARIETY VARIETY IIN N DEVELOPMENT DEVELOPMENT

15 15

IV. TERMINOLOGY TERMINOLOGY OF EARLY LIFE HISTORY STAGES A terminology is important both for understanding the literature and for brevity in describing development. A good terminology should so easily should be be as as simple simple as as possible possible (and (and so easily remembered) remembered) and and linked to both form and function. The production of of a generally ac accepted is aa current cepted terminology terminology is current issue issue in in ichthyology, ichthyology, and and some some of of the the varied attempts attempts to to produce produce standardization standardization are are shown in in Fig. 7. 7. The problems problems are are discussed discussed by Snyder Snyder and and Holt Holt (1983). (1983). The The diffi difficulties lie in producing a terminology that embraces all species and all patterns fish, and patterns of of development development in in fish, and it it almost becomes an an intellectual intellectual challenge challenge to to achieve this. this. Some Some workers favor favor aa large large number of of stages, stages, others very very few; few; one point point of view suggests suggests terminology terminology based based on size alone, on size alone, another another that that ecological ecological considerations considerations should should be para paramount. Some Some workers (e.g., (e.g., Balon, Balon, 1984; 1984; see see Fig. 22) 22) use the term "embryo" cover the “embryo” to to cover the period period from from fertilization fertilization to to first first feeding feeding and and consider consider hatching to to be be aa relatively relatively insignificant insignificant process. process. While While it it is is certainly certainly true true that that the the change change from from endogenous endogenous to to exogenous exogenous food food supply supply is is aa major major hurdle hurdle for for the the organism organism to to overcome, overcome, it it should should not not be be forgotten forgotten that that eggs eggs cannot cannot avoid avoid predators predators although although hatched hatched larvae larvae can. can. Many Many species species of of fish fish hatch hatch in in aa very very well developed developed state, state, espe especially cially where where ovoviviparity, ovoviviparity, viviparity, viviparity, or or other other parental parental care care is is in involved, volved, or or where where the the incubation incubation period period within within the the egg egg is is long; long; other other species development. It species hatch hatch in in aa much much earlier earlier state state of of development. It is is difficult difficult to to resolve such aa wide resolve aa nomenclature nomenclature to to cover cover such wide variation variation in in ontogeny. ontogeny. The The present present author author prefers prefers to to use use the the term term "embryo" “embryo” only only to to the the point of point of hatching, hatching, does does not not accept accept the the terms terms "prelarva" “prelarva” and and "post “postlarva," larva,” which which suggest suggest stages stages before before and and after after aa larval larval stage, stage, and and uses uses the the term term "larva" “larva” to to cover cover development development from from hatching hatching to to metamorpho metamorphosis and sis and the term term "juvenile" “juvenile” from from metamorphosis metamorphosis to to first first spawning. spawning. Terms such as "fingerling" “fingerling” or "young-of-the-year" “young-of-the-year” are unsatisfactory: the former former can can hardly hardly be applied applied to to very very short short fish fish or or the the latter latter to to species species with with aa short short generation generation time. time. A simplistic simplistic approach approach to to termi terminology may well well require require additional qualification to be given, given, such such as as “ "yolk-sac" yolk-sac” larva, larva, or or it it may may have have to to be be made made clear clear that that some some species species hatch in an an advanced state state of development. development. This is is in broad agree agreement 1984), who also ment with with Kendall Kendall et al. al. ((1984), also favor dividing the larval stage stage into "preflexion," “preflexion,” "flexion," “flexion,” and "postflexion" “postflexion” substages, substages, refer referring ring to to the the turning turning up up of of the the notochord notochord tip tip during during the the first first stages stages of of development of the caudal fin fin (Fig. (Fig. 3). 3). Since flexion flexion is is accompanied by n rays by rather rather rapid rapid development development of of other other characters characters such such as as the the fi fin rays and and change change of of body body shape, shape, as as well well as as aa dramatic dramatic improvement improvement in in

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Fig. 8, 8. (A) (A)Percent Percent survival survival of of Atlantic Atlantic salmon salmon parr parr Salrno Salmo saZar salar from from eggs eggs of of differ differFig, ent diameter. diameter. Each Each point point refers refers to to aa different different female. female.There There isis no no significant significant correlation correlation ent et al. al. (1979).] (1979).1(B) (B)The Thetime time to to50% 50% between egg egg size size and and survival. survival. [Redrawn [Redrawnfrom from Glebe Glebe et between survival from from hatching hatching of of unfed unfed tilapia tilapia Oreochrornis Oreochromis (Tilapia) (Tilapia)mossarnbicus mossambicus fry fry related related survival of the the eggs. eggs. Each Each point point refers refers to to aa different differentclutch clutch of of eggs. eggs. The The to the the mean mean dry dry weight weight of to (r = = 0.923, 0.923, d.f. d.f. == 23, 23, pp < < 0.01). 0.01). [Redrawn [Redrawn from from Rana Rana (1985).] (1985).] regression is is significant significant (r regression

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o�----�-------,----,--, 82 97 61 21 AYS DDAYS Fig. 9. 9. The The weight weight (mean (mean ±fS.E.) S.E.)of of rainbow rainbow trout trout Salrna Salmogairdneri gairdnedfry fryderived derivedfrom from Fig. full rations. rations. Day Day 00 refers refers to to fifirst feeding, so so that that the the eggs of of females females held held on on half half and and full eggs rst feeding, difference in in weight weight isis still still detectable detectable 97 97 days days later. later. [Redrawn [Redrawn from from Springate Springate et et al. al. difference (1985).] (1985).]

20

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Fig. n subsequent length ooff fry iin n four New Bruns Fig. 10. 10. The effect of egg diameter oon Brunswick (Eastern (Eastern Canada) Canada) Atlantic salmon Salrno Salmo salar S Q Z U ~ stocks. stocks. The four stocks stocks are shown by different symbols. symbols. Each point refers to a different female. female. [Redrawn [Redrawn from Glebe et et refers to al. (1979).] (1979).]

4. 4. Diet. The way in which the diet of the female affects fecundity and egg size needs clarification. clarification. Much may depend on the phase of the egg maturation cycle during which an experimen experimengiven. Diets may delay or accelerate spawning, so tal diet is given. allowing more or less time for material to be laid down in the comegg, or diets may affect the processes of atresia. The most com mon effect of starvation or overcrowding is to reduce fecun fecundity, and for good feeding or low stocking density, to increase fecundity (Wooton, (Wooton, 1979). 1979). Concomitant changes of of egg size often, but not always, occur; with poor feeding, egg size is sometimes increased (Wooton, (Wooton, 1979) 1979) and sometimes de deai., 1978f and creased, for example, in haddock (see (see Hislop et al., 1978jand ai., 1985). in rainbow trout (Springate et al., 1985). Despite the extensive experimental work on egg size, it is not clear the extent to which changes of fecundity or egg size have adaptive value in the wild and whether egg survival can be enhanced or not.

11..

PATTERN AND AND VARIETY VARIETY IN DEVELOPMENT DEVELOPMENT PATTERN

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Fig. Fig. 16. 16. Size hierarchies. Increase of of length range with age in northern anchovy Engraulis mordax, mordar, Atlantic herring Clupea harengus, turbot turbot Scophthalmus maximus, and ayu Plecoglossus Plecoglossus altiuelis reared in the laboratory. [Redrawn from Blaxter (1981) altivelis reared ( 1981) and Tanaka (1973).] (1973).]

S. BLAXTER H. S. BLAXTER JJ.. H.

28

((1974) 1974) found a reduction in the size hierarchy of of the zebrafish Danio ((= = Brachydanio) rerio fed an adequate diet. After 32 days, larvae fed Brachydanio) on a low ration ranged from 5 to 12.5 mm (mean 9.5 mm), and after 39 12.5 (mean 9.5 mm), days another group fed on a high ration ranged from 7.5 mm days another group fed on a high ration ranged from 16.0 16.0 to to 117.5 mm (mean mm). (mean 16.9 16.9 mm). Rearing in captivity also tends to produce shorter, fatter fish, fish, with high (Fig. 17) 17) and high condition condition factors factors (Fig. and growth growth abnormalities abnormalities such such as as fore foreshortened snouts, neoplasms of the head, and failure of of eye migration and fin development in flatfish. 1980) found that the hearts of of flatfish. Arthur ((1980) northern anchovy larvae, as determined by length of of the ventricle,

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“wild” fish fish as as they grow, grow, based mainly on Atlantic herring; see see text for reared and "wild" details. The change of condition factor (shown (shown bottom right) in the the sea results from from details. loss of yolk, causing a fall, fall, followed by progressive ossification, causing a rise. initial loss Condition factors of reared starved larvae tend tend to to be higher than "wild" “wild” larvae, showing Condition that starvation criteria criteria cannot cannot be obtained obtained satisfactorily from from captive larvae. larvae. that

1. 1. PATTERN AND VARIETY

IN DEVELOPMENT

29

were as much as 40% longer in laboratory-reared laboratory-reared compared with sea seacaught individuals. Excessive contact with the walls of the tank, other forms of stress, and social factors factors can be implicated in causing these forms abnormalities. of the volume of of the rearing container was investigated The effect of by Theilacker ((1980b) 1 980b) using jack mackerel larvae reared at the same density in 10-1 10-1 and 100-1 100-1circular tanks. The beneficial effect of the larger volume was apparent after only 4-5 4-5 days of of feeding, when the larvae were larger and in better better nutritional condition. The dramatic effect of using rearing facilities on the mesocosm scale was shown by Morita ((1985) 1985) and Paulsen et al. al. ((19851, 1 985), who reported excellent growth and survival of Pacific herring larvae and turbot larvae respec respec3 on-shore tanks. Sturmer et al. tively in 20-m al. ((1985) 1985) found similar 20-m3 benefi cial effects using red drum larvae in large on-shore beneficial on-shore tanks and Kvenseth and 0iestad 1 984) and 0iestad al. ((1985) 1985) using cod larvae giestad ((1984) Oiestad et al. 3• in a coastal impoundment of 60,000 60,000 m m3. High density of larvae may also create problems other than those associated with physical interactions. Crowded conditions may cause the production of inhibitory substances. Although originally demon demonstrated in amphibians, such substances may be implicated in popula populawhite cloud tion control of of the guppy Lebistes (Poecilia) (Poecilia) reticulatus, whitecloud mountain minnow Tanichthys albonubes, tiger barb Barbus tetra tetrazona, sp. (Rose, (Rose, 1960). 1960). Laale xona, rainbow trout, and ciscoes Leucichthys sp. and McCallion ((1968) 1 968) found that the development of zebrafish could be arrested before gastrulation by the use of of supernatant homogenates produced from other zebrafish embryos embryos.. Such effects are likely to be relevant in the wild only to species that live in crowded conditions or in stagnant water. In biochemical terms, hatchery fish fish often have a higher percentage fat content than their wild counterparts but lower percentage protein and ash (Table 11 in Blaxter, (Fig. 17). Blaxter, 1976) 1976) (Fig. 17). The biochemical and morphological changes occurring in hatchery fi sh can be refl ected in fish reflected their behavior and especially in their locomotor performance. Crowd Crowding induces stress and aggression that can be partly alleviated by adequate food, for example, in the medaka Oryzias latipes (Magnu (Magnuson, 1962) 1962) and Atlantic salmon (Symons, 1967) found (Symons, 1968). 1968). Barns Bams ((1967) that the fry fry of of wild migrant sockeye salmon Oncorhynchus nerka could stem a water current and avoid predation better than hatchery fry, fry, although mainly this was due to the fact that they were bigger. crowding, sensory deprivation may be As the inverse of stress and crowding, implicated as a feature of hatcheries or rearing tanks (Blaxter, (Blaxter, 1970). 1970). Developing fi sh may not be subjected to the normal interplay of fish of light and shade, nor to the very high light intensities appertaining in the

30

J. J. H. H. S. S. BLAXTER

wild. They may not be able to practice avoidance or other responses in the absence of a natural substratum, typical water currents, or preda predators. Such deprivation will be particularly harmful where learning is involved in the development of behavior patterns. The problems are well demonstrated by attempts to establish hatchery-reared plaice in the sea (Blaxter, (Blaxter, 1976). 1976). Survival was negligible, and it is is likely that inadequately developed predator-avoidance, burying behavior, and feeding mechanisms were to blame. A number of workers have assessed the effect of of blinding, or rear rearing in darkness, on the eye and optic tectum of fish larvae. PHugfelder Pflugfelder (1952) (1952) found that unilateral blinding of of newly hatched swordtails swordtails Xiphophorus and guppy Lebistes caused a reduction in the develop development of of the contralateral optic tectum, mainly by a decrease in volume of the ganglion cells. Experiments in darkness confirmed that the effect was caused by lack of of visual input rather than by degeneration products released from dying axons 1 970) axons of the optic tract. Blaxter ((1970) could find no retinal degeneration in dark-reared Atlantic herring lar larvae. Zeutzius et al. al. (1984) apia Sarothero (1984) found that dark-rearing of til tilapia Sarotherodon ((= = Tilapia) Tilapia) mossambicus did not affect the normal outgrowth of the nerve fibers of of the optic tectum into the retina. It did, however, reduce the optic layer and the differentiation of the synapses, where the number of synaptic vesicles increased. Dark-rearing can affect behavior. Blaxter ((1970) 1970) found that newly hatched Atlantic herring larvae were very inactive when returned to the light after rearing in the dark and subsequently showed a high mortality. Zeutzius and Rahmann (1984) (1984)found that dark-reared larvae apia failed to swim up after yolk resorption; visual acuity was of til tilapia impaired after 20-30 20-30 days in the dark, and after 50 days no optokinetic mininystagmus was present. Effects on body weight and length were mini mal, although the increase in body depth was substantial compared with control fish. Sensory deprivation may operate at aa more subtle level. For exam example, both Breder and Halpern (1946) 1960, 1961) (1946) and Shaw ((1960, 1961) had diffi diffiof zebrafish and Menidia. culty in rearing isolated individuals of Menidia. SurSur vival was poor, and in Menidia schooling was retarded when isolates were brought together.

VIII. SHRINKAGE VIII. THE EFFECT OF FIXATION ON SHRINKAGE The interpretation of developmental events related to size, and factor, will depend on whether estimates of growth rate and condition factor, ossification, or those with live or fixed material is used. Larvae lacking ossification,

1. PATTERN 1. PATTERN AND AND VARIETY VARIETY IN IN DEVELOPMENT DEVELOPMENT

3 311

form, will be especially prone to shrinkage, shrinkage, not only a long thin body form, fixatives, but also as a result of capture by plankton net if sam samfrom fixatives, pled from the wild. This problem has been addressed in the larvae of of (1963), in Atlantic Californian sardine Sardinops caerulea by Farris (1963), 1971), in Pacifi c herring by Schnack and Rosenthal herring by Blaxter ((1971), Pacific ((1977-1978) 1977-1978) and Hay ((1981) 1981) and northern anchovy, Pacific Pacific mackerel PaScomber japonicus, jack mackerel Trachurus symmetricus, and Pa cific barracuda Sphyraena argentea by Theilacker ((1980a) 1980a) and Theilacker and Dorsey ((1980), 1980), and in southern fl ounder Paralichthys flounder lethostigma 1 984). The degree of of shrinkage Zethostigma by Tucker and Chester ((1984). depends on the concentration and osmolality of the fixative, the pe period of fi xation, and the age of fixation, of the larvae. A shrinkage of of 5-10% 5-10% is normally experienced in long thin larvae, especially when young, but may be as little as 2% 2%in older stages. stages. Capture by net, either simulated (Blaxter, 11971; (Blaxter, 97 1 ; Theilacker, 1980a) 1980a) or after release of larvae in the path of a net (Hay, xation, may cause shrinkage as great (Hay, 1981), 1981),followed by fi fixation, as 20%, fixation is delayed. The effect is much less 20%, or even more if if fixation serious in older larvae with ossified skeletons. skeletons. Theilacker and Dorsey ((1980) 1980) also mention the loss of of dry weight following fixation. Formalin fixation caused a 30% loss in weight of fixation. Fonnalin of larval Pacifi c sardines and fi xation in ethyl alcohol a 30-80% loss in Pacific fixation 30-80% loss Pacifi c mackerel larvae. Pacific larvae. Methods of of preservation and curation are discussed by Lavenberg et al. ((1984), 1 984), who recommend final preservation in 70% ethanol to fixatives Probobviate problems of buffering acid fi xatives such as formalin. Prob lems of buffering are, however, trivial compared with the amount of shrinkage in formalin or alcohol. IX. RATE OF DEVELOPMENT IX. DEVELOPMENT

The rate of development is clearly under genetical control. Within a species it is most strongly influenced influenced by temperature, and this is exemplified by data on days to hatch at different temperatures in 13 13 species (Fig. 1 986) give further data on three (Fig. 18). 18).Herzig and Winkler ((1986) cyprinids. Blaxter ((1969) 1969) and Herzig and Winkler ((1986) 1986) discuss the mathematical relationship between temperature and incubation and the use of QlO Q l o and other temperature coefficients. In particular Q Q llOo is found to vary with range of temperature, and it may be that optimum temperatures for development, where hatching rates are highest, take place where the QlO 9 1 0 is between 2 and 3. Temperature can also influence size at hatching, efficiency of of yolk utilization, growth, feeding rate, time to metamorphose, behavior and

J. J. H. H. S. S. BLAXTER

32 32 200 200

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Fig. lB. 18. The time from fertilization to hatching for 111 of teleost related to Fig. 1 species of (1) Desert temperature. Data originally cited in Blaxter ((1969) 1969) except where stated. (1) pup fish Cyprinodon Cyprinodon macularius, macularius, (2) ( 2 )brook trout Salvelinus Salvelinus fontinalis, fontinalis, (3) ( 3 )rainbow trout pupfish Salmo gairdneri, galrdneri, (4) (4) smelt Osmerus eperlanus, (5) (5)Atlantic Atlantic herring Clupea Clupea harengus, (6) (6) plaice Pleuronectes platessa, platessa, (7) (7) Pacifi Pacific Gadus macrocephalus, (8) (8)rockling rockling Enche Enchec cod Gadus lyopus cimbrius, cimbrius, (9) (9) mackerel mackerel Scomber scombrus, (10) (10) grey mullet Mugil cephalus (Nash 1 1) striped bass Morone (Nash and Kuo, Kuo, 1975), 1975), and ((11) Morone saxatilis.

rates, and metabolic swimming speed, digestion and gut evacuation rates, demand. Temperature Temperature also has indirect indirect effects on larvae through the demand. viscosity, and phytoplankton blooms oxygen capacity of water, its viscosity, fish (Theilacker and Dorsey, 11980). 980). Much of the data on marine fi sh are summarized by these authors and in more general terms by Kinne Brett (1970). In particular, Brett (pp. (pp. 524-527) 524-527) gives a com com((1963) 1963) and B rett (1970). prehensive table of upper and lower lethal temperatures of embryonic fish. and postembryonic stages of marine and estuarine fish. is discussed by Garside The structural response to temperature is

11.. PATTERN PATTERN AND AND VARIETY VARIETY IN IN

DEVELOPMENT DEVELOPMENT

33

((1970) 1 970) with particular reference to the effect of of temperature on the interaction of differentiation and growth. Salmonids Salmon ids and clupeids inin cubated at high temperatures tend to weigh less at hatching. The best known effect is on meristic characters-counts characters-counts of serial structures scales, and gill rakers, which are labile such as vertebrae, fin rays, scales, (within limits) and susceptible to various environmental factors factors (Tan (Tining, 1952; 1952; Barlow, 1961; 1961; Blaxter, 1969; 1969; also see Lindsey, volume XIB). Until recently it has not been clear what adaptive advantage might exist for varying numbers of meristic characters. It has been shown that individuals of a particular species with more vertebrae are often longer, and it seems plausible that they might also be more flexible and able to swim faster. 1984) have recently faster. Swain and Lindsey ((1984) shown that there was selective predation for vertebral number in young sticklebacks sticklebacks Gasterosteus aculeatus preyed on by sunfish Le Lepomis gibbosus. The survival of8.2-mm-Iong 1.7 of 8.2-mm-long sticklebacks was 1.31.3-1.7 fish 31 times greater for fi s h with 3 1 vertebrae than with 32. This effect was 8.9-mm-long sticklebacks, nor was there any influence not found with 8.9-mm-Iong of temperature. Early development can be enhanced hormonally. Dales and Hoar of chum salmon 0. ((1954) 1954) treated the eggs of O. keta with thyroxine and thiourea, an antithyroid compound. Thyroxine accelerated growth of of the body wall and pectoral fi n s, increased guanine deposition, de fins, decreased pigmentation, caused exophthalmia, exophthalmia, but reduced the rate of increase of body length. Thiourea decreased guanine deposition and also decreased the rate of growth in length. length. More recently, it has been shown that immersion in thyroxine accelerates growth in larval til apia tilapia Sarotherodon ((= = Tilapia) Tilapia) mossambicus, carp Cyprinus carpio, and milkfish Chanos chanos chan os and enhances survival in tilapia and carp (Lam, 1980; (Lam, 1980; Lam and Sharma, 1985; 1985; Lam et al., al., 1985). 1985). Further infor information-for example, on the influence of hormones on smoltification mation-for salmonids-will be found in Hoar (volume XIB XIS). of salmonids-will ). X X.. ORGAN SYSTEMS

A. Alimentary System A. Many species hatch without a mouth, but this develops rapidly to allow for the transfer from endogenous yolk to exogenous food. food. Feed Feeding in many species is a predatory act requiring vision, and feeding does not occur in the dark, especially in the very young stages (Blax(Blax-

34

J. H. H. S. S. BLAXTER J. BLAXTER

ter, 1981, 1986; Hunter, 1980, 1981,1986; 1980, 1981). 1981). The size or gape of the mouth at first feeding, and therefore the size of food that can be taken, is crucial first for survival at the end of the yolk-sac yolk-sac stage (Fig. (Fig. 19A,B). 19A,B).Experiments show that the size of of food taken is is related to the gape of the jaw and that both increase with age of the larvae. Length and complexity of the alimentary tract increase as the lar larvae grow. 1984) describes three groups of fish larvae grow. Dabrowski ((1984) based on the morphology of the alimentary tract and gut enzymes. Most species have early larvae without a functional stomach or gastric beglands; salmonids, on the other hand, have a functional stomach be fore changing to external feeding. Tanaka ((1973) 1973) gives a full account of of the development of the alimentary tract in 21 21 Japanese marine and freshwater species. species. At first feeding there are no pharyngeal teeth and few, if mu if any, taste buds. The esophagus has longitudinal folds and mucous cells; the intestine and rectum are lined with columnar epitheepithe lium, and it is likely that most digestion occurs here. Cilia are present in the gut of early clupeoid and salangid larvae and may help to pass food along the gut (Iwai (Iwai and Rosenthal, 1981). 1981). The liver, gallbladder, and pancreas are also formed early. In many species, but not in salmosalmo nids like the rainbow trout, the stomach and pyloric caeca develop late in larval life as the pattern and quantity of feeding changes. These subsequent processes are well described by O'Connell 1981) in the O'Connell ((1981) northern anchovy and Govoni Govoni ((1980) 1980) in the spot Leiostomus xan xan-

thurus. thurus. The length of the gut influences the passage time for food. food. For example, in roach Rutilus rutilus larvae the food is is retained for only 2.5 2.5 h at 20°C, 20°C, whereas in the adult it is 6 h (Hofer, (Hofer, 1985). 1985). The time for digestion and resorption and for the recovery of digestive enzymes is therefore reduced in the larval stages. Dabrowski ((1984) 1984) reviewed work on the appearance of digestive enzymes during development. Clark et al. al. ((1985) 1985) found an increase in protease activity of Dover sole stage. Similarly, Similarly, Lauff and from the age of 24 days up to the adult stage. Hofer ((1984) 1984) found a progressive increase in activity and number of of proteolytic enzymes with age in whitefish Coregonus hybrids, in rain rainbow trout, and in roach. Higher proteolytic activity in the roach could be correlated with the lack of a stomach. stomach. In the rainbow trout the well welldeveloped Ideveloped digestive tract, which is differentiated into a stomach, py pyloric caeca, and a short intestine at first feeding, may compensate for a lower level of proteolytic activity. The relatively underdeveloped state of the alimentary system may explain why most species have carnivorous larvae, although later, when the gut lengthens, they may become herbivorous. It may also

PATTERN AND AND VARIETY VARIETY IN IN DEVELOPMENT DEVELOPMENT 11. . PATTERN

35

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Fig. of the mouth in the Fig. 119. 9 . (A) (A) The relationship between body length and width of larvae of Pacific mackerel Scomber Scomber of hake Merluccius merluccius, cod Gadus morhua, Pacific (1980).] (B) “Profile” "Profile" japonicus, and anchovy Engraulis ringens. [Redrawn from Hunter (1980).] of of the body length: vertical jaw gape relationship in the larvae of of 19 teleost teleost species. species. [Redrawn [Redrawn from Shirota (1970). (1970).]J

J. H. S. S. BLAXTER BLAXTER J. H.

36

explain why why artificial artificial food food is is less less satisfactory satisfactory for for young young larvae, larvae, since since explain live food food contains contains exogenous exogenous enzymes enzymes that that may may aid aid digestion digestion (Da(Da live browski and and Glogowski, Glogowski, 1977). 1977). browski B. Respiratory Respiratory System System B. By first first feeding feeding most most species species have have larvae larvae with gill slits slits and gill By with gill and gill arches (Tanaka, (Tanaka, 1973), 1973), but but gill gill filaments filaments develop develop later. later. The The cobitid cobitid arches Misgurnusfossilis fossilis has has external external gill gill filaments filaments for for aa time time (Fuiman, (Fuiman, 1984). 1984). Misgurnus Harder (1954) ( 1954) gave gave aa particularly particularly thorough thorough account account for for the the herring, herring, Harder where the the filaments filaments first first appear appear at at aa body body length length of of 20 mm mm several several where weeks after after hatching. hatching. De De Silva Silva (1974) ( 1974) measured measured the the gill gill area area of of both both weeks herring and plaice larvae during early development and related them herring and plaice larvae during early development and related them the surface surface area area of of the the body body (Fig. (Fig. 20). 20). In In the the early early stages stages it it is is clear clear to the to respiration is cutaneous. The larval heart is present even before that that respiration is cutaneous. The larval heart is present even before hatching and and pumps pumps aa colorless colorless body body fluid fluid around around an an as as yet yet unknown unknown hatching A

10

40

30

10

0>

""

E

�E E

3

1

00,

·1

oj :t

20

-;..

..." 0 a.

2

ON 10

·oI +----r---,..--==:;;....,r----r-�-r__-__r--.__-..,..-__+_ O o0 20 110 0 20 30 40 30 40

:2 Ol E

�

�

0

I "

o

Length l e n g t h (mm) (mm)

Fig. 20. 20. The effect of of size on on oxygen oxygen uptake uptake (ILl (p1 O�), O a ) ,Q0 QO,2 (ILl (pl Odmg Odmg dry dry weight/h), weighvh), Fig.

2/mg wet gill area area (mm (mm2/mg wet weight) weight) and and body body area area (mm2/mg (mm2/mgwet wet weight) weight) in in (A) (A) Atlantic Atlantic gill Clupea harengus harengus and and (B) (B) plaice plaice Pleuronectes Pleuronectes platessa platessa larvae. larvae. [Data [Data from from De De herring Clupea herring Silva and and TytIer Tytler (1973) (1973) and and De De Silva Silva (1974).] (1974).] Silva

1. 1.

100 lo0j

B 6

J

\

40 40

l a ii cc e e PP la

0: 110

0 330 Ol"ElQ

E E

3

"'/

= 5a :l. t

E r . L

37 37

I N DEVELOPMENT PATTERN AND VARIETY IN

2!O 0 .,(y

1:

2 -" 0 0

:

2

a a. L

3 :>

�

�

0 0

n

"

/

0 10

· o 1 +-----�-=�----_r--�--_+ 0 o 20 10 30 40 m m) Ll eenn g g tt h h ((mrn)

Fig. 20. 20. Fig.

:;: Ci E N

1

0 C

o

(Continued) (Continued)

system. Weihs ((1980b) vascular system. 1980b) found that yolk-sac larvae of northern oxygen saturation of the water anchovy were more active when the oxygen ambidropped below 60%, suggesting the requirement to renew the ambi limiting. ent water close to the body when oxygen is limiting. conclusions In relating size to respiration the following theoretical conclusions may be drawn: 2 a body length length2 Total body area for cutaneous respiration Q( 3 = body length length3 Body weight Q( a body lengthlength-'I Respiratory area per unit weight Q( 0 22 requirement requirement Q( body weightD'8 weighP8 or body length l e ~ ~ 2g'4 t(see (see h ~ 'Winberg, ~ O 1960) 1960) 002 or body a body weightweight-0'2 (QO2) Specific oxygen requirement (Q0 2) Q( OoB 1ength-O"j lengthThus the body surface area per unit weight declines as the body (QO,) delength-',I , whereas the oxygen requirement per unit weight (Q0 length2 ) de ooB. This would lead to a critical length-0,6. clines less rapidly, as body length-

38 38

J. H. H. S. S. BLAXTER BLAXTER J.

situation situation without without the the development development of of gills gills to to increase increase the the respiratory respiratory surface surface area area (Fig. (Fig. 20). 20). Although Although the the blood blood of of herring herring and and plaice plaice and and many many other other species species does does not not appear appear pink pink until until metamorphosis, metamorphosis, its its precursors, precursors, or or related related substances substances such such as as myoglobin, myoglobin, can can be identified identified histochemically histochemically soon soon after after hatching. hatching. The The circulating circulating body body fluids fluids were were reported reported to to be be acellu acellular lar until until about about 16 16 mm mm in in the the herring herring and and 10 10 mm mm length length in in the the plaice plaice (De 1 982) found 1974), but but Hickey Hickey ((1982) found unidentified unidentified cells cells 8-14 8-14 JLm pm (De Silva, Silva, 1974), in in diameter diameter in in newly newly hatched hatched larvae larvae of of both both species species and and an an efficient efficient wound-healing wound-healing mechanism mechanism in in herring, herring, plaice, plaice, and and Atlantic Atlantic salmon salmon lar larvae. vae. In system has In the the walleye walleye Stizostedion vitreum vitreum the the circulatory circulatory system has been 1 979). The The blood blood becomes becomes been described described by by McElman McElman and and Balon Balon ((1979). red red before before hatching, hatching, although although the the development development is is not not precocial. precocial. In In the the precocial hemoglobin may may be be precocial larvae larvae of of salmonids salmonids and and elasmobranchs, elasmobranchs,hemoglobin present, present, and and the the blood blood pink, pink, at at hatching. hatching. In In these these species species the the body body size so size is is large large enough enough for for cutaneous cutaneous respiration respiration to to be be inadequate inadequate and and so increase increase in in oxygen-carrying oxygen-carrying capacity capacity of of the the blood blood is is essential, essential, even even if if the the gills gills are are in in aa rudimentary rudimentary state. state.

C. Locomotor System Most species hatch with V-shaped myotomes acting against the notochord as a hydrostatic skeleton. Additional myotomes myotomes may be added posteriorly, but the final number is attained during the early larval period. Generally the myotomes, myotomes, which comprise white muscle, become progressively more complex in shape and interdigitate with adjacent myotomes. myotomes . The red muscle develops initially as a myotube, a superficial cylindrical sheath around the body, e.g., in northern anan chovy (O’Connell, (O'Connell, 1981), 1981), zebrafish (van Raamsdonk et al., 1982), 1982), herher ring (Batty, 1984), 1984), and red sea bream Pagrms Pagrus major (Matsuoka and Iwai, 1984). 1984). In Coregonus sp. (Forstner et al., 1983) 1983) the red muscle extends as a thin layer dorsally and ventrally from the lateral line. In all these species it later concentrates in a strip in the midlateral posiposi 21). tion on the flank (Fig. tion (Fig. 21). Larvae usually hatch with a primordial primordial median median finfold; the median fins often first appear appear as a discontinuity in the margin margin of of the finfold; a few fin rays then appear, which gradually increase in number number and size. In species with a homocercal tail the caudal fin develops after the tip of of the notochord notochord turns up (flexion, (flexion, see p. 15). 15). Lateral fins, used for stability, maneuvering, and sometimes for propulsion, develop differdiffer ently. Pectoral buds, fin-like structures that that lack lack rays, are often present present at hatching. Pelvic Pelvic buds buds and fin rays develop later.

ch

DEVELOPMENT 1. PATTERN 1. N DEVELOPMENT PATTERN AND AND VARIETY VARIETY IIN

L�,

Reynolds R eynolds number n u m b e r R= R= du

R R

RR

10 -- 200 200 10

20 0 0

cD= Co = k k

Inertial I nertia l forces forces ii rn m portanf portant

Intermediate Intermediate z one zone

n develops, caudal develops c a udal ffiin

red muscle mu sc l e changes

>

� a

C a ra n g i f o r m Carangiform

’00 lateral lateral line

Fig. 21. Changes in hydrodynamic considerations considerations as fish larvae grow related to the Reynolds number (R), (R), where L is body length, U U is swimming speed, and V is the kinematic viscosity (see Webb, 1975). 1975). An inertial regime exists where R > > 200 and a viscous regime where R < 10, 10, with an intermediate zone between. The drag coefficient also changes. At the same time, the larvae change from a a serpentine to a carangicarangi (CD) (Gn) also form swimming mode as the tail flexion occurs and the red muscle develops from a myotube surrounding the whole body in a thin sheet (TS) (TS) to a strip situated along the midlateral position position on the flank. flank.

A number of workers (Webb, (Webb, 1975; 1975; Weihs, 1980a; 1980a; Batty, 1984; 1984; Webb Webb and and Weihs, Weihs, 1986; 1986; Blaxter, Blaxter, 1986) 1986) have have considered considered the the "locomo “locomotor regime" of of larvae of different size (Fig. (Fig. 21). 21). Where tor regime” larvae of Where the Reynolds number 10 (in very small larvae), viscous forces are parapara number (R) is below 10 effi mount and continuous high-speed swimming is energetically efficient. Where R is greater than 200, inertial forces are more important and beat-and-glide swimming is more efficient. Since R depends on as the body length and velocity, the hydrodynamic regime changes as larvae grow or alter their swimming speed. Often they may occupy an intermediate intermediate zone between between the the viscous viscous and and inertial inertial regime. During During later later development development there there are, are, in in many many species, increases increases in in the the surface surface area for propulsion as a result of of the appearance of of the the caudal caudal fin fin and and allometric allometric growth growth (Fuiman, (Fuiman, 1983). 1983). Linked to swimming is the buoyancy of of larvae. larvae. Although unim unimportant portant for for demersal demersal larvae, larvae, pelagic pelagic larvae larvae can can potentially potentially waste waste much much energy energy in maintaining their position in the water column. As larvae grow the skeleton they become grow and and the skeleton ossifies ossifies they become heavier heavier (Fig. (Fig. 17) 17) and and tend tend

40

BLAXTER H. SS.. BLAXTER JJ.. H.

more and more to negative buoyancy. Conversely, Conversely, if if they starve, they will tend to neutral buoyancy (Fig. (Fig. 15). 15).The larvae of many species fill the the swimbladder soon after hatching, or at the end of of the yolk resorp resorption, probably by swallowing air at the surface (Doroshev et al., al., 1981). 1981). This stage, sometimes called "swim-up," “swim-up,” can be critical for successful later development, e.g., e.g., in the turbot. The time of appearance of of the swimbladder varies widely between species. For example, the her herring fi lls its swimbladder at a length of about 30 mm, while the north fills northern anchovy inflates its swimbladder at about 10 10 mm (Hunter and Sanchez, 1976) and the menhaden Brevoortia tyrannus Sanchez, 1976) tyrunnus at 13 13 mm (Hoss 1982). In the physostomatous northern anchovy (Hoss and Blaxter, Blaxter, 1982). and menhaden there is is a diel die1 rhythm, the larvae filling their swimbladders by swallowing air at the surface at night (Hunter and Sanchez, 1976; 1976; Hoss and Phonlor, 1984). 1984). Sense Organs D. Sense 1. YE 1. THE THEE EYE In some species the eyes are free of of pigment at hatching and histo histological examination confirms confirms that they must be nonfunctional. By fi rst first feeding the eyes eyes are pigmented, and vision plays a major part in feed feeding. Of 10 1970), 10 teleost families examined by Blaxter and Staines ((1970), eight had larvae at first feeding with a pure-cone retina, and only an anguillid and a macrourid, caught in deep water, had a pure-rod ret retina. A pure-cone retina at first feeding was also found in the northern ina. anchovy by O'Connell 1981) and in the goldfish by Johns ((1982). 1982). In O’Connell ((1981) zebrafish, rods can be identified identified 9 days after hatching (Branchek and Bremiller, 1984), 1984), and at hatching in Pacific salmon, Oncorhynchus Pacific salmon, spp. (Ali, (Ali, 1959). spp. 1959).In the advanced young of the viviparous guppy Poeci Poecilia al. ((1983) 1 983) found a well-differentiated duplex Zia reticulata, Kunz et aZ. retina even before birth. Retinomotor movements of the masking pig pigment and the photoreceptors usually develop concomitantly with the rods (Ali, 1959; Blaxter and Staines, 1970; Kunz and Ennis, 1983; (Ali, 1959; Staines, 1970; 1983; Neave, 1984) 1984) so that the process of light-and-dark adaptation is linked to the establishment of a duplex retina. The development of of visual performance is described by Blaxter ((1986), 1986), improvements in acuity being the most noticeable feature. 2. MECHANORECEPTORS 2. MECHANORECEPTORS

Free neuromasts are present at hatching in all species examined (Iwai, 1967, 1967, 1980), (Iwai, 1980), usually on the head and sometimes on the trunk. of the development of of the Disler ((1971) 1971) gives a very detailed account of

11.. PATTERN PATTERN AND AND

VARIETY VARIETY IN IN DEVELOPMENT DEVELOPMENT

41

free neuromasts and lateral line of of many freshwater species, including the sturgeon Acipenser Acipenser stellatus, stellatus, Pacific salmon O. 0 . keta, and some percids and cyprinids. Generally the initial number of of free neuro neuromasts is low but they proliferate and may become regularly arranged along the flank. flank. In marine species similar systems are found in gadids (Fridgeirsson, (Fridgeirsson, 1978), 1978), northern anchovy (O'Connell, (O’Connell, 1981), 1981), Atlantic herring (Blaxter et al., al., 1983b), 1983b), halibut (Blaxter (Blaxter et al., al., 1983a), 1983a), plaice and turbot (Neave, 1986), 1986), and spotted bass Micropterus punctatus (Kokkala (Kokkala and Hoyt, 1985). 1985). The lateral line canals almost invariably develop some time after hatching: at 17 17 mm in menhaden, 18-20 18-20 mm in northern anchovy, 242410 mm in the plaice, 26 mm in Atlantic herring, 8 mm in the turbot, 10 and 12 12 mm in spotted bass bass.. Thus young larvae have very incomplete mechanoreceptors mechanoreceptors.. The development of of the inner ear is not well known, except that larvae have one or more pairs of of otoliths at hatch hatching, which must give them a basic perception of of posture. 3. 3. CHEMORECEPTORS CHEMORECEPTORS Olfactory pits are described in the early larval stages of of Atlantic 1 978), tilapia by Iwai (1980), herring by Dempsey ((1978), (1980), northern anchovy by O'Connell 1981) walleye Stizostedion O’Connell ((1981) Stixostedion vitreum by Elston et al. al. ((1981), 1981), carp by Appelbaum ((1981), 1981), and striped bass Morone saxatilis by Bodammer ((1985). 1985). Kokkala and Hoyt ((1985) 1985) described taste buds in larval 1 980) found them between 11and 14 14 days larva1 spotted bass and Iwai ((1980) posthatching in tilapia, pond smelt Hypomesus transpacijicus, transpacijkus, gold goldfish, sea bass Lateolabrax japonicus, puffer Fugu niphobles, flatfish Kareius bicoloratus, and red sea bream Pagrus major. major. XI. STRUCTURE XI. STRUCTURE AND FUNCTION FUNCTION

Clearly most larvae, apart from some highly developed ovovivipa ovoviviparous or viviparous species, or species with very large eggs, go through struca massive increase in complexity while free-swimming. Since struc tures are often absent or incompletely developed, the associated be behaviors are also absent or poorly developed. When relating structure to function, mention should be made of the theory propounded by 1981b, 1984) Balon ((1981b, 1984) that ontogeny is saltatory, meaning that develop development proceeds by a series of rather rapid changes in both structure and function with relatively prolonged intervals in between, during which a more-or-Iess more-or-less steady state exists as the organism prepares itself itself for the next rapid change. change. Thus development does not proceed by a

42

J. S. BLAXTER J. H. H. S. BLAXTER

continuous continuous accumulation accumulation of of small small changes. changes. As As an an example, example, Balon Balon cites cites aa cyprinid, cyprinid, the the bream bream Abramis ballerus (Fig. (Fig. 22). 22). A A number number of of studies studies link link the the development development of of structure structure with with func function. best examples tion. One One of of the the best examples is is found found in in the the work work of of Hunter Hunter and and Coyne 1982) on Coyne ((1982) on northern northern anchovy anchovy (Fig. (Fig. 23), 23), where where age age and and length length are are related related to to the the development development of of the the sensory, sensory, respiratory, respiratory, digestive, digestive, and and locomotor systems and locomotor systems and associated associated behavior behavior and and ability ability to to withstand withstand starvation. 1985) gives starvation. Fukuhara Fukuhara ((1985) gives aa similarly similarly comprehensive comprehensive account account of sea bream of the the functional functional morphology morphology of of the the red red sea bream Pagrus major. major. Allen et al. al. ((1976) 1 976) related development of swallowing swallowing behav Allen related the the development behavior, avoidance avoidance responses, responses, and and shoaling shoaling specifically specifically to to the the develop development pro-otic bullae bullae and and swimbladder swimbladder of of Atlantic Atlantic herring herring larvae larvae ment of of the pro-otic (Fig. 1 985) (Fig. 24). 24). In aa somewhat somewhat similar similar fashion, fashion, Kawamura Kawamura and and Ishida Ishida ((1985) compared compared the development development of the the whole whole sensory sensory system system of the the floun flounder der Paralichthys olivaceus to to primary primary orientation, orientation, feeding, feeding, migration, migration, and and activity activity (Fig. (Fig. 25). 25). Considerable Considerable differences differences in in morphological morphological and and

Itep 4

slep 3

L A R VA E M8 R 'tO

$lep 2

,'.p 1 AGE

consecutive steps in the ontogeny of of the Danubian bream Fig. 22. A scheme of consecutive Abrarnis ballerus, demonstrating saltatory development. During each step various Abramis structures structures grow and differentiate at different rates but are completed and become func functional at the same time, at the end of of the step, thus enabling the larvae to make substan substanauthor’s terminology that the tial and rapid changes in behavior. Note according to the author's embryo changes to the larva at fi rst feeding, after step 3. ed and redrawn from 3. [Modifi [Modified first (1984).] Balon (1984).]

1. PATTERN 1. PATTERN AND AND VARIETY VARIETY IN IN DEVELOPMENT DEVELOPMENT

43 43

species; the behavioral events can be seen in these two unrelated species; herring, with an extended larval period metamorphosing into a pe peflatfish lagic schooling species, the flounder, a flatfi sh with a shorter larval period ending in settlement. (1980)gives a detailed but similar style of summary of of de deBalon (1980) Salvelinus. Despite their five velopmental events in fi ve species of charr Salvelinus. of close relationship, relationship, they they show show substantial substantial differences differences in in the the timing timing of the the appearance appearance of of both both morphological morphological and and behavioral behavioral features features such such as as fi ns, melanophores, fins, melanophores, branchial branchial respiration respiration and and swimbladder swimbladder filling. filling. The ontogeny of behavior, especially in salmonids and cichlids, is (1978) and Noakes and Godin (volume (volume XIB). XIB). discussed by Noakes (1978) of the early life These groups are of particular interest because much of history may be passed in gravel beds or under the care of a parent. Such a lifestyle may enhance the protection of im of the young but it imposes other problems, such as the avoidance of abrasion or cannibal-

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Fig Fig.. 23. 23. Developmental Developmental events events during during the the early early life life history history of of the the northern northernanchovy anchovy Engraulis Engruulis mordax. mordax. RBC, RBC, Red blood blood cells; cells; time time to to 50% 50%starvation starvation is is the the number number of days days after after which which 50% 50% of of unfed unfed larvae larvae died died (equivalent (equivalent to to point-of-no-return). point-of-no-return).For For further further discussion 1982).] discussion of of Reynolds Reynolds numbers numbers see see Fig. Fig. 21. 21. [From [From Hunter Hunter and and Coyne Coyne ((1982).1

• eo

J. J. H. H. SS.. BLAXTER

44

SENSE SENSE ORGANS ORGANS

BEHAVIOR BEHAVIOR Length Length

(mm) (mm)

-

Hatch--Hatch

Feeding starts starts

-

10

Eyes we w ellll pigmented pigmented Pure

cone r retina e t i n a present present 30-40 free uromasts 30-40 f r e e ne neuromasts Ol pits O lfactory factory p i t s present present

Respond to t o probes probes

Otic bulla Swallowing behavior behavior

Startle S t a r t l e response r e s p o n s e to to auditory a u d i t o r y stimuli stimuli

-

20

2o

30

-

30

Start Start o off metamorphosis metamorphosis Start S t a r t of of

shoaling shoaling

t -

i

-

-

-

develops

B Buullll a starts s t a r t s to t o fi f i ll l l with w i t h gas gas Lateral L a t e r a l line line

Rods Rods sstart tart

ccanals a n a l s sstart t a r t on head

to t o recruit recruit

Bulla B u l l a usually u s u a l l y gasg a s -fi f i llled led Swimbladder develops develops

Twin

cones c o n e s appear

Pure

cone

area d e v e l o p s area

develops

Retinomotor movements movements commence

40

-

Metamorphosis complete complete --

-

-

Head lateral lateral

line line

complete

Fig. 24. 24. The The development development of of parts parts of of the acoustic acoustic system system and and swimbladder swimbladder of of Atlan Atlantic al. ((1976).] 1976).] tic herring herring Clupea Clupea harengus harengus in in relation relation to to behavior. behavior. [Adapted [Adapted from Allen Allen et et al.

ism, which are quite different from free-living fish larvae. Both the behaviorist and the physiologist need to be aware of the ecology and reproductive habits of their experimental material before making evaluations of their data. XII. MI. CRITICAL PERIODS

The high fecundity of most fish and the low survival rate of of their offspring imply a high mortality from a number of possible causes: inherited defects, egg quality, starvation, disease, predation. Whether these sources of mortality occur continuously or sporadically or

1. 1.

45

PATTERN PATTERN AND AND VARIETY VARIETY IN IN DEVELOPMENT DEVELOPMENT BEHAVIOR BEHAVIOR

SENSE ORGANS SENSE Time Time Hatch

First F i r s t horizontal horizontal orientation orientation Avoi d obstacles Avoid o b s t a c l e s by mechanoreception mechanoreception Positive P o s i t i v e phototaxis phototaxis First F i r s t feeding feeding

S e l e c t food food ii n sea sea Select

Migrate

coastward coastward

S e t t l e on on substratum substratum Settle

d)) ((d

''> i I

T

Body length length

2.5 2.5

112 2

115 5

16 16

f

Eyes poorly loped p o o r l y deve developed

O l f actory a c t o r y p ii tt ci c i lliated iated Olf P a i r of of Pair

f r e e neuromasts neuromasts on on head free

Otic O t i c hair h a i r ce c ellll ss c ii ll iiaated ted

0 . 38 0.38 33

(mm) (mm)

Two Two pairs pairs o off

free sts f r e e neuroma neuromasts

o n h e a d and t on head t rrunk unk

33.5 .5

w ellll pigmented pigmented Eyes we morphology of of eye Gross morphology

complete

7 .0 7.0

F i r s t taste t a s t e buds on g ii ll ll arches arches First

8 4 8 .. 4

i n oral oral T a s t e buds in Taste

cavity cavity

8.8 B .B

223 3

112.5 2.5

113.3 3.3

225 5

First F i r s t retinal r e t i n a l rods rods

F i r s t head lateral lateral First

l i n e canal canal line

Nares formed Nares

229 9

First F i r s t twin twin

cones cones

Taste buds on on lips lips T a s t e buds Pelagic P e l a g i c tt o benthic b e n t h i c ii n sea sea Positive P o s i t i v e phototaxis phototaxis di i ssaappe p p ears ars Nocturnally N o c t u r n a l l y active active

30 30

333 3

553 3

7 No chemi cal prey chemical p r e y de d etection t e c t i o n 667 Bury Bury entirely e n t i r e l y ii n sand sand

N e g a t i v e l y phototactic phototactic Negatively

7 1 71

1

Complete Complete head late l a t e ral ral

Complete Comp lete

t rrunk u n k lateral lateral t

line line

line line

Fig. 25. The development of of the sense sense organs organs and and behavior behavior in the the Hounder· flounderPamFig. Para oliuaceus. [Adapted [Adapted from from Kawamura and Ishida (1985).] (1985).] Kawamura and lichthys olivaceus.

whether there are particularly "critical “critical periods" periods” of high mortality is is often uncertain. The previous sections sections and and Figs. Figs. 22-25 22-25 illustrate illustrate well the the accretion of new structures and functions functions as as larvae grow grow until the (apart from from reproductive behavior) behavior) is is achieved at full adult repertoire (apart stage. At the present time time it is is not possible to the onset of the juvenile stage. conclude the extent to which developmerit 98 1b, development is is saltatory (Balon, (Balon, 11981b, 1984)or gradual. gradual. Such Such a conclusion requires a thorough study study of phys phys1984) iology and and behavior behavior as as well as as anatomy. anatomy. iology Because Because the larvae lack certain behavioral responses, and because

46


they are going going through through aa massive massive morphogenesis, morphogenesis, it it seems seems almost almost they are inevitable that critical periods arise through which larvae have to pass inevitable that critical periods arise through which larvae have to pass to allow development to proceed. These critical periods are especially to allow development to proceed. These critical periods are especially related to feeding and predation but but also also to to respiration. respiration. Some Some potenpoten related to feeding and predation tially critical periods may be listed as follows : listed as follows: tially critical periods may 11.. Hatching. This depends hatching en Hatching. This depends on on the the production production of of hatching enzymes to zymes to break break down down the the tough tough chorion chorion that that protects protects the the em embryo from the of wave wave action action or or the pressures pressures and abra and abrabryo from the rigors rigors of sion within within or or on on aa spawning spawning substratum substratum (see (see Blaxter, Blaxter, 1969, 1969, for for sion discussion). aa discussion). 2 . First-feeding. Both high mortality at first-feeding first-feeding in rearing 2. of brood survival under natunatu experiments and and considerations of ral conditions conditions have have suggested suggested in in the the past past that that one one of of the the main main ral phases of larvae change from phases of high high mortality mortality occurs occurs when when the the larvae change from endogenous (sometimes called endogenous to to exogenous exogenous sources sources of of food food (sometimes called the this thesis thesis is the mixed mixed feeding feeding stage). stage). Obviously Obviously relevant relevant to to this is the (see Fig. 19) in relation relation to to the the size size of of prey prey the gape gape of of the the jaw jaw (see Fig. 19) available and quantity and quality of of the May (1974), ( 1974), available and the the quantity and quality the prey. prey. May Vladimirov (1975), ( 1975), and and Blaxter Blaxter (1984) (1984) discuss discuss this and Vladimirov this problem problem and conclude it is is likely likely that mortality often often takes takes place place rather rather conclude that that it that mortality steadily over over the the early early life life history. history. Mortality Mortality rates rates are are somesome steadily times as as high high in in the the egg egg stage stage of of pelagic marine fish fish as as in in the the times pelagic marine larval stage per day), predation can can day), suggesting suggesting that that predation Iarval stage (5-20% per predominate over starvation on some occasions. Furthermore, rearing rates rearing in in the the absence absence of of predators predators can can lead lead to to excellent excellent rates of the major major critical pe 3). First-feeding First-feeding as as the critical peof survival survival (see (see page page 3). riod in development, while sometimes being applicable, should treated with caution as as aa general general concept. concept. with caution should thus thus be treated Respiration. The The disadvantageous disadvantageous decline decline in in body body surface surface area area 3. Respiration. with size in relation to oxygen requirements has already been discussed (Fig. (Fig. 20). 20). There There is is some evidence of of aa high some evidence high phase phase of of discussed mortality associated associated with with early early development development of of the gill filafila mortality the gill ments, for for example, example, in in herring herring (De (De Silva, Silva, 1974). 1974). ments, S wim-up. The The first-filling of the swimbladder (and of the bulla 4. Swim-up. first-filling of the swimbladder (and of the bulla system buoyancy and system in in clupeoids) clupeoids) is is essential essential for for maintaining maintaining buoyancy and other functions such as hearing associated with a gas-filled swimbladder. species, such swimbladder. In In many many species, such as as sea sea bass bass and and sea bream bream (Chatain, 1986), 1986), the the swimbladder swimbladder appears appears soon soon after after hatching. hatching. (Chatain, In the physostomatous salmonids, salmonids, such as brown brown trout, trout, rainrain In the physostomatous such as bow trout, and whitefish whitefish C C.. clupea jormis, the the swimbladder swimbladder is is clupeuformis, bow trout, and by swallowing swallowing air air at the surface surface at the end yolk resorpresorpfilled by at the at the end of of yolk

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tion (Tait, 1960). If access to the surface is prevented, the (Tait, 1960). pneumatic duct remains open and the swim bladder can be swimbladder filled later. filled later. The The motivation motivation to to fill the the swimbladder swimbladder is is very very strong; Salvelinus (Cristivomer) (Cristivomer) namaycush strong; in in experiments experiments Saiveiinus fill the will swim up for at least 270 m without fatigue to fill swimbladder is empty. swimbladder if if it it is empty. In In physoclists physoclists failure failure to to fill the the swimbladder can lead to abnormal behavior and sometimes to death (e.g., al., (e.g., in mullet, sea bass, and turbot) (Doroshev et ai., 1981). 1981). In clupeoids (Blaxter (Blaxter and Batty, 1985) 1985) avoidance re responses sponses to to sound sound stimuli stimuli fail fail to to develop unless unless the the bulla bulla con contains tains gas. gas. 5. Metamorphosis. 5. Metamorphosis. In In those those species species where where aa marked marked metamor metamorphosis the larval larval stage, stage, aa number number of of major major phosis occurs at at the the end end of the changes changes occur. occur. Increasing Increasing conspicuousness, conspicuousness, as as the the transparent transparent larval is lost, it essential develop other lost, makes makes it essential for for fish fish to to develop other larval form form is protective schooling or or burying burying behavior, behavior, protective mechanisms, mechanisms, such such as as schooling in in order order to to avoid avoid predation. predation. Protective Protective coloration coloration mechanisms mechanisms also also develop develop as as the the scales, scales, pigment, pigment, and and new new chromatophores chromatophores appear. must be vulnerable vulnerable at at this this time, time, especially especially appear. Single Single fish fish must in pelagic habitat, habitat, and and survival survival in in aa schooling schooling species species may may in the pelagic well depend well depend on on early early successful successful aggregation aggregation with with conspecifics. conspecifics. At At or or near near metamorphosis metamorphosis many many marine marine species species move move inshore inshore to need to nursery grounds grounds and and flatfish flatfish need to settle settle on on an an appro approto seek nursery priate such as priate substratum substratum such as sand. sand. XIII. C ONCLU S IONS CONCLUSIONS

The aim of this chapter is to give the less initiated reader, espe especially cially potential potential experimentalists, experimentalists, an an account account of of the the variety variety of of material material now within the This variety now available available within the early early life life history history stages stages of of fish. fish. This variety has has its its attractions attractions and and its its disadvantages. disadvantages. Fish Fish eggs eggs and and larvae larvae present present aa wealth of new new and and interesting interesting problems problems related related to to development. development. In In wealth the the case case of of marine marine fish fish larvae, larvae, new new sources sources of of material, material, hitherto hitherto ne neglected glected because because of of difficulties difficulties in in rearing, rearing, are are now now available. available. The The re research aware of of the pace pace at at which which search worker worker needs, needs, however, however, to to be aware events events are are occurring. occurring. Body Body shape shape is is changing changing rapidly, rapidly, new new external external structures modified, and new internal or modified, and new internal organs organs are are structures are are being being added added or appearing For example, or being being elaborated. elaborated. For example, aa sensory sensory physiologist physiologist appearing or needs some insight needs to to have have some insight into into the the state state of of development development of of the the sense sense organ and its connections to the central nervous system, a student of osmoregulation needs to osmoregulation needs to know know the the state state of of development development of of the the kidney, kidney,

S. BLAXTER BLAXTER H. S. J, J . H.

48

and aa nutritionist nutritionist requires requires some some knowledge knowledge of of the the repertoire repertoire and and and activity of the digestive enzymes . Major changes in organs can occur activity of the digestive enzymes. Major changes in organs can occur within the duration of of an experiment. Staging of of experimental material is therefore paramount. material is therefore paramount. Within the material can Within aa species, species, the the viability viability of ofthematerial can depend depend on on parental parental effects size and effects such such as as egg egg size and egg egg quality. quality. Successful Successful experimentation experimentation often requires some some expertise often requires expertise in in husbandry husbandry and and handling handling to to ensure is in in good ensure that that the the material material is good condition condition and and performing performing optimally. optimally. Small size may Small size may demand demand sensitive sensitive and and delicate delicate techniques. techniques. One One of of the the main main rewards rewards for for perseverence perseverence is is that that behavioral behavioral and and physiological physiological experiments experiments can can be be done done in in the the absence absence of of certain certain organs organs or or structures structures and and again again as as such such organs organs and and structures structures develop. develop. Invasive or or ablation ablation techniques techniques can can thus thus be be avoided avoided and and experiments experiments can on intact can be be done done on intact animals. animals.

ACKNOWLEDGMENTS I am am extremely grateful to Dr. L. A. Fuiman, at present present on a National Science Foundation Post Doctoral Fellowship at the Scottish Marine Biological Biological Association, Oban, Oban, for for reading reading and and commenting commenting on on this this chapter chapter in in draft draft form form and and for for making making many many constructive constructive and and useful useful comments comments to to improve improve it. it. The following have been most helpful in correspondence, correspondence, allowing me to use draw drawings, or Balon, Dr. Dr. B. D. D. Glebe, Glebe, Prof. Prof. R. ings, or in in other ways ways providing providing information: information: Prof. Prof. E. K. Balon, D. D. Hoyt, Dr. J. R. Hunter, Dr. A. A. W. Kendall, Jr., Jr., Dr. H. G. G. Moser, Moser, Dr. K. Rana, Prof. Prof. J. S. S. Ryland, Dr. Dr. D. E. E. Snyder, Snyder, Dr. Dr. J. E. E. Thorpe, Thorpe, and Dr. Dr. J.-M. Vernier. II am also grateful to Catriona Stewart, Stewart, who who drew several of the figures. figures.

REFERENCES

Eggs and and larvae of the Pacific Pacific hake Merluc MerlucAhlstrom, E. E. H., and and Counts, R. C. ((1955). Ahlstrom, 1955). Eggs Fish. Bull. Bull. 56, 56, 295-329. cius productus. Fish. Ahlstrom, E. E. H., and Moser, H. H. G. G. ((1980). Characters useful in identification of pelagic Ahlstrom, 1980). Characters Rep. Calif CaZSf. Coop. Coop. Oceanogr. Oceanogr. Fish. Invest. Inuest. 21, 121-131. 121-131. fish eggs. Rep. marine fish Ali, M. A. (1959). (1959).The The ocular structure, structure, retinomotor and photobehavioral responses of Ali, Can.J. J. Zool. Zool. 37, 37, 965-996. 965-996. juvenile Pacific Pacific salmon. salmon. Can. juvenile Allen, J. J. M., M., Blaxter, Blaxter, J. H. H. S., S., and and Denton, Denton, E. E. J. J. (1976). (1976).The The functional anatomy and Allen, of the the swimbladder-inner swimbladder-inner ear-lateral ear-lateral line system system in in herring herring and development of development J. Mar. Biol. Biol. Assoc. Assoc. U.K. U.K.56, 56, 471-486. 471-486. sprat. ]. sprat. Amoroso, E. C. C. ((1960). Viviparity in in fishes. fishes. Symp. Syrnp. Zool. 2001.Soc. Soc. London 1, 1, 153-181. Amoros 1960). Viviparity �, E. Appelbaum, S. S. ((1981). Zum Geruchsorgan der der Karpfenlarven Karpfenlarven (Cyprinus (Cypdnus carpio). carpio).Zool. Zool. Appelbaum, 1981). Zum Anz. 227-233. Anz. 206, 206,227-233.

Appelbaum, S. S. ((1985). Rearing of of the the Dover sole sole Solea Solea solea (L) ( L )through its its larval larval stages Appelbaum, 1985). Rearing using artifi artificial 49,209-221. 209-221 . cial diets. Aquaculture 49, (1980). Differences Differences iin heart size size between between ocean-caught and and laboratorylaboratoryArthur, D. D. K K.. (1980). Arthur, n heart

11.. PATTERN PATTERN AND AND VARIETY VARIETY IN IN DEVELOPMENT DEVELOPMENT

49 49

E x p . Mar. Bioi. Biol. grown larvae of the northern anchovy Engraulis mordax Girard. JJ.. Exp. E d . 43, 99-106. 99-106. Ecol. ed. (1982). (1982). "Identifi “Identification of Larval Fishes of of the Great Lakes Basin with Auer, N. A., ed. cation of Drainage,” Great Lakes Fish. Comm. Spec. Pub!. Publ. Emphasis on the Lake Michigan Drainage," 82-3. University University of Michigan: Ann Arbor. NO. 82-3. No. of the size of of fish eggs, the date of of spawning Bagenal, T. B. ((1971). 1971). The interrelationship of J. Fish Biol. 3,207-219. and the production cycle. J. Bioi. 3, 207-219. (1980).Comparative ontogeny of ofcharrs. “Charrs: Salmonid Salmonid Fishes of ofthe the Balon, E. K. (1980). charrs. In "Charrs: Saluelinus” (E. (E. K. Balon, ed.), ed.), pp. 703-720. 703-720. Junk, The Hague. Genus Salvelinus" of reproductive Balon, E. K. ((1981a). 1981a). Additions and amendments to the classification of Environ. Biol. 6,377-389. Bioi. Fishes 6, 377-389. styles in fishes. Environ. Balon, E. K. (1981b). (1981b).Saltatory processes and altricial to precocial forms in the ontogeny ontogeny Zool. 21,573-596. 21, 573-596. of fishes. Am. Zool. ections on some decisive events in the early life of Balon, E. K. (1984). (1984).Refl Reflections of fishes. Trans. Trans. 113, 178-185. Am. Soc. 113, Am. Fish. Fish. SOC. 178-185. Bams, R. A. (1967). (1967). Differences in performance of of naturally and artificially propagated propagated Barns, sockeye salmon migrant fry as measured with swimming and predation tests. J. ]. sockeye Fish. Res. Res. Board 1 1 17-1 153. Fish. Board Can. Can. 24, 24,1117-1153. Barlow, G. ((1961). 1961). Causes and significance of of morphological variation in fishes. Syst. Syst. 17. Zool. 2001.10, 105-1 105-117. Batty, R. S. (1984). (1984). Development of swimming movements and musculature of larval (Clupea harengus). Exp. Bioi. 10, 217-229. harengus).]. J . Exp. Biol. 1110,217-229. herring (Clupea Gillet, C. C. (1981). Billard, R., Bry, C., and Gillet, (1981). Stress, environment and reproduction reproduction in “Stress and Fish" Fish” (A. D. Pickering, ed.), ed.), pp. 185-208. Academic teleost fish. fish. In "Stress Press, London. Blaxter, J. H. H. S. S. ((1969). 1969). Development: Eggs and larvae. In In "Fish “Fish Physiology" Physiology” (W. (W. S. S. Hoar and D D.. J. Randall, eds), eds), Vol. Vol. 3, 3, pp. 177-252. Academic Press, New York. Blaxter, J. H. S. s. (1970). Sensory deprivation and sensory input in rearing experiments. experiments. (1970). Sensory Helgol. Helgol. Wiss. Wiss. Meeresunters. Meeresunters. 20, 642-654. 642-654. BIaxter, J. H. S. S. ((1971). 1971). Feeding and condition of Rapp. P.-V. Blaxter, J. of Clyde herring larvae. Rupp. Reunions Cons. Int. Int. Explor. Reunions Cons. Explor. Mer Mer 160, 129-136. Blaxter, J. H. S., S., ed. Fish." Springer-Verlag, Berlin ed. (1974). (1974). "The “The Early Life History of of Fish.” and New York. s. (1976). Blaxter, J. H. H. S. (1976). Reared and wild fish-How fish-How do they compare? In In "Proceedings “Proceedings of the Tenth European Symposium on Marine Biology" E. Jas Biology” (G. Persoone and E. Jas1, pp. 111-26. 1-26. Inst. Mar. Sci. Sci. Res., Bredene, Belgium. pers, eds.), eds.), Vol. Vol. 1, Blaxter, J. H. S. (1981). H. S. (1981). The rearing of larval fish. In In "Aquarium “Aquarium Systems" Systems” (A. D. Hawkins, ed.), ed.), pp. 303-323. 303-323. Academic Press, London. BIaxter, J. H. S. s. (1984). System (1984).Ontogeny, Ontogeny, systematics and fisheries. In In "Ontogeny “Ontogeny and SystemBlaxter, J. atics of C. Moser et al., eds.), Soc. Ichthyol. Herpetol., Spec. Pub et al., eds.), Am. Am. SOC. Herpetol., Spec. Publ.I. of Fishes" Fishes” (H. (H. G. No. 1, 1, pp. 1-6. 1-6. Allen Press, Lawrence, Kansas. BIaxter, H. S. S. (1986). (1986).The development of of sense organs and behavior behabior in teleost larvae Blaxter, J. H. with special reference to feeding and predator Soc. Trans. Am. Am. Fish. Fish. SOC. predator avoidance. Trans. 1115,98-114. 15, 98- 1 14. BIaxter, (1985).The development of of startle responses in herring Blaxter, }. J. H H.. S., and Batty, R. SS.. (1985). Bioi. Assoc. larvae. ]. J. Mar. Mar. Biol. Assoc. U.K. U.K.65, 65, 737-750. 737-750. Blaxter, J. H. S., S., and Ehrlich, K. F. F. (1974). (1974). Change in behavior behavior during starvation of of In "The “The Early Life History ofFish" of Fish” (J. (J. H. S. BIaxter, Blaxter, ed.), herring and plaice larvae. In pp. 575-588. 575-588. Springer-Verlag, Berlin and New York. BIaxter, H. S., uence of S., and Hempel, G. (1963). (1963). The infl influence of egg size on herring larvae Blaxter, J. H. (Clupea Cons. Int. Int. Explor. 211-240. (Clupea harengus harengus L). ]. J . Cons., Cons., Cons. Explor. Mer Mer 28, 28,211-240.

50

S. BLAXTER BLAXTER JJ.. H. S.

Blaxter, fishes. Adv. Blaxter, J. H. S., and Hunter, J. R. (1982). (1982). The biology of of the ciupeoid clupeoid fishes. Adu. Mar. Mar. Bioi. Biol. 20, 20, 1-223. 1-223. Blaxter, H. S., and Staines, 1970). Pure-cone retinae and retinomotor responses in Blaxter, J. J. H. Shines, M. ((1970). 449-460. larval teleosts. ]. J . Mar. Mar. Bioi. Biol. Assoc. Assoc. U.K. U.K. 50, 50,449-460. Blaxter, H. S., Danielssen, D., Moksness, Blaxter, J. J. H. Moksness, E., and 0iestad, Oiestad, V. (1983a). (1983a). Description of of the early development of the halibut Hippoglossus Hippoglossus hippoglossus hippoglossus and attempts to Bioi. (Berlin) 99-107. rear the larvae past first feeding. Mar. Mar. Biol. (Berlin)73, 73,99-107. Blaxter, S., Gray, J. A. B., and Best, Blaxter, J. J . H. S., Best, A. A. C. G. G . (1983b). (1983b). Structure and development of of the free neuromasts and lateral line system of Bioi. Assoc. U.K. of the herring. ]. J . Mar. Mar. Biol. Assoc. U.K. 63, 247-260. 63,247-260. Bioi. 54, 149-161. Blumer, L. S. (1979). fishes. Q. Blumer, (1979). Male parental care in bOIlY bony fishes. Q. Rev. Rev. Biol. 149-161. Bodammer, Bodammer, J. E E.. (1985). (1985). A morphological morphological study on the olfactory olfactory orgall organ of of striped bass (M orone saxatilis) (Morone saxatilis) larvae at the time of of yolk sac absorption. Abstr. Abstr. 9th 9th Ann. Ann. Larval Laroal 985. Fish Am. Fish. Fish. Soc. Fish Con! ConJ Am. SOC. 11985. Boehlert, G. G. W. W. (1984). (1984). Scanning electron microscopy. microscopy. In In "Ontogeny “Ontogeny and Systematics Systematics of Am. Soc. Fishes" et al., eds.), eds.), Am. SOC.Ichthyol. Herpetol., Spec. Spec. Publ. No. 1, 1, Fishes” (H. (H. G. Moser et pp. 43-48. 43-48. Allen Press, Lawrence, Lawrence, Kansas. Kansas. (1984). The development of photoreceptors in the Branchek, T., Branchek, T., and Bremiller, R. (1984). 4, 107-1 15. zebrafish Brachydanio Brachydanio reTio. rerio. I. I. Structure. ]' J . Compo Comp. Neural. Neurol. 22 224, 107-115. Breder, C. C. M., Jr., and Halpern, HaIpern, F. (1946). (1946). Innate and acquired behavior affecting the Physiol. 2001. Zool. 19, aggregation aggregation of of fishes. fishes. Physiol. 19, 154-190. Breder, C. C. M M.,. , Jr., and Rosen, Rosen, D. R. (1966). (1966). "Modes “Modes of of Reproduction in Fishes." Fishes.” Natural History Press, Garden City, New York. York. Brett, J. R. (1970). (1970). 3. Temperature 3.3. 3.3. Animals 3.32. Fishes. Functional responses. In In Brett, J. “Marine Ecology" Ecology” (0. (0. ed.), Vol. I, 1, Part 1, 1, pp. 515-560. 515-560. Wiley (Interscience), (Interscience), "Marine Kinne, ed.), London. Buckley, J. (1981). (1981). Biochemical Biochemical changes during ontogenesis of cod (Gadus (Gadus morhua morhua Buckley, L. J. L.) and winter flounder (Pseudopleuronectes Rapp. P.-V. P.-V. Reun, L.) (Pseudopleuronectes americanus) americanus) larvae. larvae. Rapp. Reun, Cons. Int. 178,547-552. 547-552. Cons. Int. Explor. Mer 178, Chatain, B. (1986). (1986). La vessie natatoire chez Dicentrarchus Dicentrarchus labrax labrax et Sparus Sparus auratus. auratus. Aspects 303-311. Aspects morphologiques du d6veloppement. dhveloppement. Aquaculture Aquaculture 53, 53,303-311. Chenoweth, S. S. B B.. (1970). (1970). Seasonal Seasonal variations variations in condition of of larval herring in Boothbay Can. 27, Area of J . Fish. Fish. Res. Res. Board Board Can. 27, 1875-1879. 1875-1879. of the Maine Coast. Coast. ]. Clark, J., MacDonald, N. L., and Stark, (1985). Development of prot eases and an Clark, J,, Stark, J. R. (1985). proteases examination (Solea examination of of procedures for analysis of of elastase activity in Dover sole (Solea solea). Fish" (C. (C. B. Cowey, J. G. “Nutrition and Feeding of Fish” Cowey, A. M. Mackie, Mackie, and J. solea). In In "Nutrition Bell, eds.), pp. 217-221. 217-221. Academic Press, London. Clemmesen, C. M. (1987). (1987). Laboratory studies on RNNDNA ratios of of starved and fed Clemmesen, C. herring (Clupea (Clupea harengus) harengus) and turbot (Scophthalmus (Scophthalmus maximus) maximus) larvae. larvae. ]. J . Cons., Cons., 122-128. Cons. Int. Explor. Mer 43, 122-128. Cons. Craik, J. C. C. A. (1985). (1985). Egg quality and egg pigment content in salmonid fish. fish. Aquacul Aquacul61-88. ture 47,61-88. ture 47, Craik, Craik, J. J. C. A., and Harvey, Harvey, S. M. (1984a). (1984a). Biochemical Biochemical chan" c h a w ,.;curring .;curring during final maturation of J . Fish Fish Bioi. Biol. 24, 24, 599599of eggs of of some marine and freshwater teleo�.s. teleo5,s. ]. 610. 610. Craik, Craik, J. C. C. A., and Harvey, Harvey, S. S. M. (1984b). (198413).Biochemical Biochemical changes associated with over overof rainbow trout Salmo Salmo gairdneri gairdneri Richardson. Richardson. Aquaculture Aquaculture 37, 37, ripening of the eggs of 347-357. 347-357. Craik, S. M. (1984c). Craik, J. J. C. C. A., and Harvey, Harvey, S. (1984~).Egg quality in rainbow trout: trout: The relation between egg viability, selected aspects of stripping, of egg composition, composition, and time of stripping. Aquaculture 15-134. Aquaculture 40, 1115-134.

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PATTERN AND AND VARIETY VARIETY IN IN DEVELOPMENT DEVELOPMENT PATTERN

51 51

Dabrowski, K. (1984). (1984).The feeding of of fish larvae: Present "state “state of of the art" art” and perspec perspec807-833. Reprod. Nutr. Nutr. Dev. 24, 24,807-833. tives. Reprod. (1984). Mouth size and recommendation recommendation of feed size Dabrowski, K., and Bardega, R. (1984). 40,41-46. fish. Aquaculture 40, preferences in three cyprinid fish. 41-46. Dabrowski, K., and Glogowski, Clogowski, J. J. (1077). (1077).Studies on the role of of exogenous proteolytic fish. Hydrobioiogia Hydrobiologia 54, 129-134. enzymes in digestion processes in fish. Dales, S., S., and Hoar, W. S. (1954). (1954).Effects of of thyroxine and thiourea thiourea on the early develop. (Oncorhynchus keta). keta). Can.l. Can. J . Zool. 32, 32,244-251. of chum salmon (Oncorhynchus 244-251. ment of Dempsey, C. C. H 1978). Chemical stimuli stimuli as a factor in feeding and intraspecifi H.. ((1978). intraspecificc behav behaviour of herring larvae. ]. Bioi. Assoc. Assoc. U.K. 739-747. J . Mar. Biol. U.K. 58, 58,739-747. De Silva, C. (1974). (1974).Development of of the respiratory system in herring and plaice larvae. “The Early Life History of of Fish” 465-485. Springer In "The Fish" (J. 0. H. H. S. S. Blaxter ed.), pp. 465-485. Verlag, Berlin and New York. De Silva, C., and TytIer, Tytler, P. (1973). (1973).The influence of of reduced environmental environmental oxygen on the metabolism Sea Res. 345metabolism and survival of herring and plaice larvae. Neth. Neth. ]. J. Sea Res. 7, 7,345362. 362. Disler, N. N. N. (1971). (1971)."Lateral “Lateral Line Sense Organs and Their Importance Importance in Fish Behav Behavior." ior.” Israel Program for Scientifi Scientificc Translations, Jerusalem. Doroshev, S. I., J. W., and Hogan, K. ((1981). 1981). Initial swim bladder infl ation I., Cornacchia, J. inflation P.culture. Rapp. Rapp. P. in the larvae of physoclistous fishes and its importance for larval culture. V. Reun., Reun., Cons. Cons. Int. Int. Explor. Erplor. Mer Mer 178, 495-500. Eaton, R. C., and Farley, Farley, R. D. (1974). (1974). Growth and reduction reduction of of depensation of of zebra Copeia pp. 204-209. Brachydanio reno rerio reared in the laboratory. Copeia fish Brachydanio Ehrlich, K. F. F. (1974a). (1974a). Chemical changes during growth and starvation starvation of of larval Pleuronectes platessa. 39-48. Pleuronectes platessa. Mar. Mar. Bioi. Biol. (Berlin) (Berlin)24, 24,39-48. Ehrlich, K. F. ((1974b). 1974b). Chemical changes during growth and starvation of of herring lar lar"The Early Life H istory of 0. H. S. Blaxter, ed.), pp. 301-323. vae. In In “The History of Fish" Fish” (J. 301-323. Springer-Verlag, Berlin and New York. York. Ehrlich, K. F., Blaxter, J. H. S., and Pemberton, R. (1976). (1976).Morphological Morphological and histologi histological changes during the growth and starvation of Mar. of herring and plaice larvae. Mar. 18. Bioi. (Berlin) Biol. (Berlin)35, 105-1 105-118. Elston, R., Corazza, L., 1981). Morphology and development of L., and Nickum, J. J. G. ((1981). of the olfactory organ in larval walleye Stizostedion Stizostedion vitreum. oitreurn. Copeia Copeia pp. 890-893. 890-893. Fahay, M M.. P. (1983). (1983).Guide to the early stages of of marine fishes occurring in the western Atl. Fish. North Atlantic, Cape Hatteras to the southern Scotian shelf.]. shelf. J . Northwest. Northwest. Atl. Fish. Sci. 4, 1-423. Sci. Farris, D. D. A. (1963). (1963).Shrinkage of of sardine (Sardinops (Sardinopscaerulea) caerulea) larvae upon preservation preservation in buffered Copeia pp. 185-186. 185-186. buffered formalin. Copeia Forstner, H., Hinterleitner, S., Mahr, K., and Wieser, W. (1983). (1983).Towards a better defini definition of Coregonus sp.: Biochemical, histological and physio physioof "metamorphosis" “metamorphosis” in Coregonus Can.]. J. Fish. Aquat. Sci. Sci. 40, 1224-1232. 1224-1232. logical data. Can. Fowler, L. G. ((1972). 1972). Growth and mortality of of fingerling chinook salmon as affected by Prog. Fish-Cult. Fish-Cult. 34, 66-69. 66-69. egg size. Prog. Fridgeirsson, E. (1978). Fridgeirsson, E. (1978). Embryonic development deveIopment of five species of of gadoid fishes in Icelandic waters. Rit Rit Fiskideildar Fiskideildar 5, 1-68. 17-123. Fuiman, L. A. ((1983). 1983). Growth gradients in fish larvae. ]. Fish 1arvae.J. Fish Bioi. Biol. 23, 1117-123. Fuiman, L. A. (1984). (1984).Ostariophysi: Development and relationships. In In "Ontogeny “Ontogeny and Systematics of G. Moser et Soc. Ichthyoi. (H. G. et ai., al., eds.), eds.), Am. SOC. Ichthyol. Herpetoi., Herpetol., of Fishes" Fishes” (H. Spec. Pub!. 1, pp. 126-137. Allen Allen Press, Lawrence, Kansas. Kansas. Publ. No. 1, Fukuhara, O. 0. (1985). (1985).Functional morphology and behavior behavior of of early life stages of of the red sea bream. Bull. ]pn. Soc. Bull. Jpn. SOC. Sci. Sci. Fish. Fish. 51, 731-743.

52

J. H. S. S. BLAXTER BLAXTER J . H.

E . (1974). (1974). Influence Influence of of size of of eggs and age of of female on hatchability hatchability and Gall, G. A. E. 60, 26-35. 26-35. of rainbow rainbow trout. Calif. Fish Game 60, growth of Gamble, J. J. C., MacLachlan, P., P., and Seaton, D. D. D. (1985). (1985). Comparative Comparative growth growth and of autumn autumn and spring spawned Atlantic Atlantic herring larvae reared in large development of Prog. Ser. 26, 26, 19-33. 19-33. experimental ecosystems. Mar. Ecol.: Prog. experimental ( 1970). 3. Temperature. 3.3. 3.3. Animals. 3.32. Fishes. Fishes. Structural responses. Garside, E. T. (1970). "Marine Ecology” Ecology" (0. (0. Kinne, ed.), Vol. 1, Part 561-573. Wiley (Inters(Inters In “Marine Part 1, pp. 561-573. dence), London. London. cience), ( 1979). “Variation "Variation in Atlantic Salmon Glebe, B. D., Appy, T. D., and Saunders, R. L. (1979). (Salmo salar) Reproductive Traits Programs," (Salmo salar) Reproductive Traits and their their Implications in Breeding Breeding Programs,” Pap. M:23 M :23 (mimeo). Anadromous and Ex and Catadromous Fish Comm., Int. Counc. Expi or. Sea, Copenhagen. plor. ( 1980). Morphological, histological of alimentary alimentary histological and functional aspects of Govoni, J. J. (1980). canal and associated organ development in larval Leiostomus xanthurus. Rev. Can. Can. 69-80. Bioi. 39, 69-80. Biol. Harder, (1954). Die Enhvicklung Entwicklung der Respirationsorgane beim beim Hering. Z. Anat. Harder, W. (1954). U8, 102-123. 102-123. Entwicklungsgesch. 118, Entwicklungsgesch. ( 1981). The effects of of capture and fixation fixation on gut contents and body of Hay, D. E. (1981). body size of Pacific herring larvae. Rapp. Rapp. P.-V. P.-V. Reun., Reun., Cons. Cons. lnt. Int. Explor. Explor. Mer Mer 178, 395-400. 395-400. 1979). "Early of Marine Fish: The Egg Stage.” Stage." Univ. of of Hempel, G. ((1979). “Early Life History of Washington Press, Seattle. (Clupea ( 1967). Egg weight weight in Atlantic herring (Clupea Hempel, G., and Blaxter, J. H. S. (1967). harengus L.). J].. Cons., Cons. Int. Explor. Mer 31, 170-195. 170-195. Cons., Cons. Herzig, A., and Winkler, H. (1986). (1986). The influence of of temperature on the embryonic chalcoides development of Abramis brama, brama, Chalcalburnus Chalcalburnus chalcoides of three cyprinid fishes, Abramis i d . 28, 171-181. mento and Vimba vimba. J].. Fish B menta Bioi. 171,... 1 81. Exp. Mar. Biol. Bioi. Ecol. Ecol. 57,149-168. 57, 149-168. Hickey, G. M. M . (1982). ( 1982). Wound healing in fish 1arvae.J. larvae.]. E x p . Mar. Hirose, K., Machida, Y., ( 1979). Induced ovulation of of Japanese Y., and Donaldson, E. M. (1979). yokohamae) with human chorionic gonadotrophin and salmon flounder (Limanda (Limanda yokohamae) gonadotropin, with special reference reference to changes in quality of of eggs retained in the ovarian cavity after ovulation. Bull. ]pn. Jpn. Soc. Sci. Fish. 45, 4 5 , 31-36. 3146. G., Robb, A. P., and Gauld, J. A. Hislop, J. R. G., Gauld, J. A. (1978). (1978).Observations on effects of of feeding level on growth and reproduction in haddock Melanogrammus (L.) in Melanogrammus aeglefinus aeglefinus (L.) aptivity. ]. ccaptivity. J . Fish. Fish. Bioi. B i d . 13, 85-98. 85-98. (1985). E Effects of artificial diets on the digestive processes of of fi fish Hofer, R. (1985). ffects of sh larvae. In "Nutrition (C. B. Cowey, A. M. Mackie, and J. G. Bell, eds.), “Nutrition and Feeding in Fish" Fish” (C. pp. 213-216. 213-216. Academic Press, London. Hoss, D. E., and Blaxter, J. H. S. S. ((1982). 1982). Development and function of IIoss, of the swimblad swimbladder-inner Brevoortia tyrannus tyrannus der-inner ear-lateral ear-lateral line system in the Atlantic menhaden Brevoortia (Latrobe). Bioi. (Latrobe). ]. J . Fish B i d . 20, 20, 131-142. 131-142. Hoss, D. D. E., and Phonlor, G. (1984). (1984). Field and laboratory observations on diurnal swimbladder inflation-deflation in larvae of of gulf gulf menhaden Brevoortia patronus. Fish. Fish. Bull. Bull. 82, 82, 513-517. 513-517. Houde, Houde, E. E. (1977). (1977). Food concentrations and stocking density effects on survival and growth of laboratory-reared larvae of bay anchovy Anchoa Anchoa mitchilli mitchilli and lined sole Achirus lineatus. 333-341 . lineatus. Mar. Bioi. B i d . (Berlin) (Berlin)43, 43,333-341. Howell, B 1979). Experiments on the rearing of larval turbot, Scophthalmus B.. R R.. ((1979). Scophthalmus maxi maximus L. 215-225. L.Aquaculture 18, 18,215-225. Hubbs, 1986). American sh conference. Trans. Hubbs, C. C. ((1986). American Fisheries Society. Ninth larval larval fi fish Trans.Am. Am. Fish. 98- 171. Fish. Soc. SOC. US, 115,98-171.

11..

PATTERN PATTERN AND AND VARIETY VARIETY IN IN DEVELOPMENT DEVELOPMENT

53

Hunter, J. R. ((1980). 1980). The feeding behavior and ecology of of marine fish larvae. In In "Fish “Fish Behavior and its Use in the Capture and Culture of Fishes" 0. E. E. Bardach, J. J. Fishes” (J. Magnuson, Magnuson, R. C. May, and J. M M.. Reinhart, eds.), eds.), pp. 287-330. 287-330. ICLARM, Manila, Philippines. Philippines. Hunter, J. R. ((1981). 1981). Feeding ecology and predation of marine fi sh larvae. In fish In "Marine “Marine Fish Larvae, (R. Lasker, ed.), Larvae, Morphology, Ecology and Relation of Fisheries" Fisheries” (R. Washington Sea Grant Program, pp. 34-87. 34-87. Univ. of of Washington Press, Press, Seattle. Seattle. Hunter, J. R. ((1984). 1984). Synopsis of of culture methods for marine fish larvae. In In "Ontogeny “Ontogeny and Systematics of al., eds.), Am. Soc. Ichthyo!. Herpeto!., (H. G. Moser et et al., eds.), Am. SOC.Ichthyol. Herpetol., of Fishes" Fishes” (H. Spec. Pub!. 1, pp. 24-27. 24-27. Allen Allen Press, Lawrence, Kansas. Publ. No. 1, Hunter, J. R., and Coyne, K. M. (1982). (1982). The onset of of schooling in northern anchovy Rep. Calif. Invest. 23, 246-251 . larvae Engraulis mordax. mordax. Rep. Calif. Coop. Coop. Oceanogr. Fish. Znoest. 23,246-251. Hunter, J. R ation ooff the R.,. , and Sanchez, Sanchez, C. (1976). (1976). Diel Die1 changes in swim bladder infl inflation larvae of Engraulis mordax. mordax. Fish. Fish. Bull. Bull. 74, 74, 847-855. 847-855. of the northern anchovy Engraulis Huse, I., and Skiftesvik, A. B. B. (1985). (1985). "Qualitative “Qualitative and Quantitative Quantitative Behaviour Behaviour Studies in Starving and Feeding Turbot (Scophthalmus (Scophthalmus maximus marimus L.) Larvae," Larvae,” Pap. F:38 F:38 (mimeo). Maricult. Comm., Int. Counc. Counc. Explor. Sea, Copenhagen. (mimeo). Iversen, S. S. A., and Danielssen, Danielssen, D. S. S. (1984). (1984). Development and mortality of cod (Gadus (Gadus morhua temperatures. In In "The “The Propagation of Cod rnorhua L.) eggs and larvae in different temperatures. Gadus Gadus morhua morhua L." L.” (E. (E. Dahl, D. D. S. S. Danielssen, E. E. Moksness, Moksness, and P. P. Solemdal, Solemdal, eds.), 49-65. Inst. Mar. Mar. Res., Flj1Jdevigen Flfidevigen Bio!. Biol. Stn., Arendal, Arendal, Norway. eds.), Part Part 1, pp. 49-65. Iwai, T. T. (1967). (1967). Structure Sbucture and development development of lateral line cupulae in teleost larvae. In In "Lateral (P. H. Cahn, “Lateral Line Detectors" Detectors” (P. Cahn, ed.), ed.), pp. 27-44. 27-44. Indiana Univ. Press, Bloomington. Iwai, T. (1980). (1980). Sensory anatomy and feeding of fish larvae. larvae. In In "Fish “Fish Behavior Behavior and its Iwai, T. Use J. J. Magnuson, R. C. C. Use in the Capture and Culture Culture of Fishes" Fishes” 0. (J. E. Bardach, J. May, and J. eds.), pp. 124-145. ICLARM, Manila, Philippines. J. M. M. Reinhart, Reinhart, eds.), Iwai, Rosenthal, H. H. (1981). (1981). Ciliary Ciliary movements movements in guts guts of early early ciupeoid clupeoid and Iwai, T., and Rosenthal, 365-367. salangid larvae. Mar. Mar. Ecol.: Ecol.: Prog. Prog. Ser. Ser. 4, 4,365-367. Johns, P. 1982). Formation of photoreceptors in larval and adult goldfish.]. P. R. ((1982). ofphotoreceptors goldfish.]. Neurosci. Neurosci. 2, 178-198. Jones, A. (1972). turbot Scophthalmus (1972).Studies Studies on egg development and larval rearing of ofturbot Scophthalmus maximus marimus L., L., and brill Scophthalmus Scophthalmus rhombus rhombus L., in the laboratory. ]. 1.Mar. Mar. Bioi. Biol. 965-986. Assoc. U.K. ASSOC. U.K.52, 52,965-986. Kanazawa, Kanazawa, A. (1985). (1985). Essential fatty acid and lipid requirement of fish. In In "Nutrition “Nutrition and Feeding MacKie, and J. J. G. Bell, Bell, eds.), eds.), pp. 281281Feeding in Fish" Fish” (C. (C. B. B. Cowey, Cowey, A. M. MacKie, 298. 298. Academic Press, London. Kawamura, G., (1985).Changes in sense organ morphology morphology and behav behavG., and Ishida, K. (1985). 1, iour with growth Paralichthys olivacus. oliuacus. Bull.]pn. BuJl.]pn. Soc. SOC. Sci. Sci. Fish. Fish. 551, growth in the flounder Paralichthys 155-165. Kazakov, 1981). The effect of the size Kazakov, R. V. V. ((1981). size of of Atlantic salmon Salmo Salmo salar salar L. eggs on embryos I.Fish Fish Bioi. Biol. 19, 19, 353-360. embryos and alevins. alevins. ]. Kendall, A. A. W., W., Jr., Jr., Ahlstrom, Ahlstrom, E. E. H., and Moser, H. H. G. G. (1984). (1984).Early life history stages of fishes In "Ontogeny “Ontogeny and Systematics of Fishes" Fishes” (H. (H. G. G. Moser fishes and their characters. In et 1-22. Allen Press, et al., al., eds.), eds.), Am. Am. Soc. SOC.Ichthyo!. Ichthyol. HerpetoJ., Herpetol., Spec. Spec. Pub!. Publ. No, No. 1, 1, pp. 111-22. Lawrence, Kansas. Kansas. Kinne, O. 0.(1963). (1963).The effects of temperature and and salinity on on marine and and brackish water animals. I. 301-340. I. Temperature. Oceanogr. Oceanogr. Mar. Mar. Bioi. Biol. 1, 1,301-340. Kinne, O. 1977). Cultivation 0 .((1977). Cultivation of animals. animals. Pisces. In In "Marine “Marine Ecology" Ecology” (0. (0.Kinne, ed.), ed.), Vo!' Vol. 3, 3, Part 2, 2, pp. 968-1004. 968-1004. Wiley Wiley (Interscience), (Interscience), London.

54


Kj�rsvik, S. (1983). Kjgrsvik, E., and L�nning, Lgnning, S. (1983).Effects of of egg quality on nonnal normal fertilization and early development of Bioi. 23, 1-12. morhua L. ]. J . Fish Fish Biol. 23,l-12. of the cod Gadus morhua Kj�svik, Kjgisvik, E., Stene, A., and L�nning, Lginning, S. (1984). (1984).Morphological, Morphological, physiological physiological and geneti genetical studies of egg quality in cod (Gadus (Gadus morhua L.). L.).In "The “The Propagation of of Cod Gadus L." ((E. D. S. Danielssen, Danielssen, E. Moksness, Gadus morhua morhua L.” E . Dahl, D. Moksness, and P. Solemdal, eds.), 1, pp. 67-86. 67-86.Inst. Mar. Res., Res., Fl�devigen Flgidevigen BioI. Biol. Stn., Stn.,Arendal, Arendal, Norway. eds.), Part 1, Kokkala, I., and Hoyt, R. R. D. (1985). (1985).The development of of anatomical anatomical features and the early ecology ecology of Ann. Larval Am. Fish. Abstr. 9th 9thAnn. Larval Fish Fish Conf Conf. Am. Fish. Soc., SOC., of the spotted bass. Abstr.

1985. 1985.

Kramer, D., and Ahlstrom, Kramer, D., Ahlstrom, E. H. (1968). (1968).Distributional atlas offish of fish larvae larvae in the Califor California Current region: Northern anchovy Engaulis Engaulis mordax 1951 through 1965. mordax Girard, 1951 1965. Calif Coop. Oceanogr. CaliJ Coop. Oceanogr. Fish. Fish. Invest. Invest. Atlas Atlas 9, 9,1-269. 1-269. u. (1981). Kuhlmann, Kuhlmann, D., Quant, G., and Witt, U. (1981).Rearing of of turbot larvae (Scophthalmus (Scophthalmus maximus L.) on cultured food organisms organisms and post-metamorphosis post-metamorphosis growth on natural marimus L.) and artificial artificial food. Aquaculture Aquaculture 23, 23, 183-196. 183-196. Kunz, S. (1983). Kunz, Y. Y.W., and Ennis, S. (1983).Ultrastructural diurnal changes of the retinal photore photoreceptors in the embryo of P.). Cell Cell Dif fer. 13, 13, (Poecilia reticulata reticulata P.). Dqfer. of a viviparous teleost (Poecilia 115-123. 115-123. Kunz, Y. W., Ennis, S., and Wise, C. (1983). (1983).Ontogeny of the photoreceptors in the embryonic retina of Poecilia reticulata reticulata (Teleostei). (Teleostei). Cell Cell Tis Tisof the viviparous guppy, Poecilia sue Res. 230, 469-486. sue Res. 230,469-486. Kvenseth, P. G., and 0iestad, Oiestad, V. (1984). (1984).Large scale rearing of cod fry on the natural food production in an enclosed pond. In "The Propagation of In “The of Cod Gadus morhua morhua L.” (E. Dahl, D. Moksness, and P. Solemdal eds.), eds.), Part 2, 2,pp. L," (E. D. S. S. Danielssen, E. E. Moksness, 645-655. 645-655. Inst. Mar. Res., Fl�devigen Flgidevigen BioI. Biol. Stn., Stn., Arendal, Norway. Laale, H. W., and McCallion, McCallion, D. J. J. (1968). (1968).Reversible developmental arrest in the embryo of sh Brachydanio Brachydanio reTio. Exp. Zool. 167, redo. J. J . Erp. 167, 117-123. 117-123. of the zebra fi fish Lam, Lam, T. J. J. (1980). (1980).Thyroxine enhances larval development and survival survival in Sarothero Sarotherodon 21, 287-291. (Tilapia) mossambicus don (Tilapia) mossambicus Ruppell. Aquaculture Aquaculture 21,287-291. Lam, sh culture. Can.]. Lam, T. T.J. J. (1982). (1982).Applications Applications of of endocrinology to fi fish Can.J.Fish. Fish. Aquat. Aquat. Sci. Sci. 39, 1 1 1- 137. 39,111-137. Lam, Lam, T. J., and Shanna, Sharma, R. (1985). (1985).Effects of salinity and thyroxine on larval survival, growth 201-212. Cyprhus carpio. carpio. Aquaculture Aquaculture 44, 44,201-212. growth and development in the carp Cyprinus Lam, Lam, T. J., Juario, J. V., and Banno, J. (1985). (1985).Effect of of thyroxine on growth growth and develop develop179-184. ment in post-yolk-sac larvae in milkfish milkfish Chanos Chanos chanos. chanos. Aquaculture Aquaculture 46, 46,179-184. Lasker, R., and Shennan, Sherman, K., eds. (1981). (1981)."The “The Early Life History of of Fish: Recent Studies," 178.Int. Counc. Explor. Studies,” Rapp. P.-V. Reun., Cons. Int. Explor. Explor. Mer, Vo!' Vol. 178. Sea, Sea, Copenhagen. Lauff, 1984). Proteolytic enzymes in fish development and the impor R.((1984). imporLauff, M., and Hofer, R. 335-346. tance of Aquaculture 37, 37,335-346. of dietary enzymes. Aquaculture Lavenberg, R. J., McGowen, Woodsum, R. E. (1984). McGowen, G. E., and Woodsum, (1984).Preservation and cura curaSystematics of (H. G. Moser et et al., al., eds.), Am. Soc. tion. In In "Ontogeny “Ontogeny and Systematics of Fishes" Fishes” (H. SOC. Ichthyo!. 1,pp. 57-59. 57-59. Allen Press, Lawrence, Kansas. Kansas. Ichthyol. Herpeto!., Herpetol., Spec. Spec. Pub!. Publ. No. 1, McElman, J. F., and and Balon, Balon, E. K. (1979). (1979).Early Early ontogeny ontogeny of of walleye, walleye, Stizostedion Stizostedion vi viMcElman, Environ. Biol. Bioi. Fishes treum, treum, with steps of saltatory saltatory development. development. Environ. Fishes 4, 4,290-348. 290-348. McGurk, McGurk, M. D. D.(1984). (1984).Effects Effects of of delayed feeding and temperature on the age of of irre irreversible starvation and on the rates ofgrowth and mortality of of Pacific Pacific herring larvae. Mar. Bioi. (Berlin) (Berlin) 84, Mar. Biol. 84, 13-26. 13-26. Magnuson, (1962). An analysis of Magnuson, J. J. J. (1962). of aggressive behaVior, behavior, growth and competition for food and space in the medaka Oryzias Can. J. Zool. 40, 313-363. Oryzias latipes. latipes. Can. J . Zool. 40,313-363.

l. 1.

PATTERN IN DEVELOPMENT DEVELOPMENT PATTERN AND AND VARIETY VARIETY IN

55

Mann, R. H C. A. (1985). H.. K., and Mills, C. (1985).Variations in the sizes of of gonads, eggs and larvae of the dace Leuciscus 13, 277-287. Leuciscus leuciscus. leuciscus. Environ. Environ. Bioi. Biol. Fishes Fishes 13,277-287. J. B., ed. ed. (1985). Marliave, J. (1985). International International Symposium on the Early Life History of of Fish 445and 8th Larval Fish Conference, 1984. Trans. Trans.Am. Fish. Soc. SOC. 114, 114,445Conference, Vancouver Vancouver 1984. 621. 621. (1986). Effects of of egg size on offspring fitness and maternal fecundity in the Marsh, E. (1986). 18-30. orangethroat darter Etheostoma spectabile (Pisces: Percidae). Copeia pp, pp. 18-30. orangethroat Matarese, C., and Sandknop, E, E. M 1984). Identifi cation offish M.. ((1984). Identification of fish eggs. In "Ontogeny “Ontogeny and Systematics of (H. G. Moser et al., a1., qds.), �ds.), Am. Soc. of Fishes" Fishes” (H. SOC.Ichthyo!. Ichthyol. Herpeto!., Herpetol., Spec. Pub!. 1, pp. 27-31 27-31.. Allen Press, Lawrence, Kansas. Kansas. Publ. No. 1, Matsuoka, M., and Iwai, T. (1984). (1984). Development of of the myotomal musculature musculature in the Red Sea bream. Bulllpn. Bull ]pn. Soc. Sci. Fish. Fish. 50, 29-35. SOC. Sci. 50,29-35. May, R. C. ((1974). 1974). Larval mortality mortality in marine fishes and the critical period period concept. In "The H. S. Blaxter, ed.), pp. 3-19. 3-19. Springer-Verlag, “The Early Life History of of Fish" Fish” (J. (J.H. Berlin and New York. Mollah, M. F. s h (Clarias F. A., A,, and Tan, E. S. P. (1983). (1983).Viability of of catfi catfish (Clarias macrocephalus Gunther) eggs fertilized BioI. 22, 563-566. fertilized at varying post-ovulation post-ovulation times. times.].]. Fish. Biol. 22,563-566. Morita, S. S. (1985). shery and review of (1985). History of the herring fi fishery of artificial propagation propagation Aquat. Sci. Sci. 42, Supp!., Can. ]. 1. Fish. Aquat. Suppl., 222-229. techniques for herring in Japan. Can. Moser, H. G. (1981). (1981). Morphological and functional aspects of of marine fish larvae. In "Marine “Marine Fish Larvae, Morphology, Ecology and Relation to Fisheries" Fisheries” (R. (R. Lasker of Washington Press, Seat Seated.), Washington Washington Sea Grant Program, pp. 90-131. Univ. of tle. Moser, H. G., Richards, W. J., Cohen, D. M., Fahay, M. P., Kendall, A. W., Jr., and Richardson, S., eds. ((1984). 1984). "Ontogeny “Ontogeny and Systematics of of Fishes," Fishes,” Am. Soc. SOC.lch Ichthyo!. Herpeto!., Pub!. No. 1. Allen Press, Lawrence, Kansas. thyol. Herpetol., Spec. Spec. Publ. Nash, C. E., and Kuo, C.-M. (1975). (1975).Hypotheses for problems problems impeding the mass propa propagation of grey mullet and other finfish. Aquaculture 5, 119-133. 119-133. finfish. Aquaculture Neave, D. A. ((1984). 1 984). The development of of the retinomotor reactions in larval plaice (Pleuronectes platessa L.) L.) and turbot (Scophthalmus Exp. (Scophthalmus maximus marimus L.). L.). ]. J. E r p . Mar. Mar. Bioi. Ecol. 76, Biol. Ecol. 76, 167-175. Neave, D. A. (1986). (1986).The development of the lateral line system in plaice plaice (Pleuronectes (Pleuronectes Assoc. U.K. U.K. 66, platessa L.) and turbot (Scovhthalmus (Scophthalmus maximus maximus L.). ]. J . Mar. Mar. Bioi. Biol. Assoc. 66, platessa 683-693. 683-693. D. L. G. G. (1978). Noakes, D. (1978).Ontogeny of behavior in fishes: A survey and suggestions. In "The (G. M. “The Development of Behavior, Comparative and Evolutionary Aspects" Aspects” (G. Burghardt and M. Bekoff, Bekoff*eds.), eds.), pp. 103-125. Garland STPM Press, New York. O'Connell, P. ((1976). 1976). Histological criteria for diagnosing the starving condition condition in OConnell, C. P. early post yolk sac larvae of the northern anchovy Engraulis Engraulis mordax mordax Girard. Girard.].]. Exp. Exp. 285-312. Mar. Bioi. Ecol. Ecol. 25, Mar. Biol. 25,285-312. O'Connell, P. (1981). O’Connell, C. P. (1981). Development of of organ systems in the northern northern anchovy En Engraulis mordax 429-446. Zool. 21, 21,429-446. mordar and other teleosts. Am. Zool. 0iestad, G., and Folkvord, A. ((1985). 1985). Mass production Oiestad, V., Kvenseth, P. G., production of of Atlantic cod juveniles Gadus morhua in a Norwegian saltwater pond. Trans. Am. Fish. Soc. 14, Trans.Am. SOC. 1114, 590-595. Paulsen, H., Munk, P., P., and Ki!1lrboe, Ki6rboe, T. (1985). (1985). "Extensive “Extensive Rearing of of Turbot Larvae (Scophthalmus L.) on Low Concentrations of (Scophthalmus maximus marimus L.) of Natural Plankton," Plankton,” Pap. F:33 (mimeo). (mimeo). Maricult. Comm., Int. Counc. Explor. Sea, Copenhagen. F:33 Pflugfelder, O. (1952). Weitere 0.(1952). Weitere volumetrische volumetrische Untersuchungen Untersuchungen tiber iiber die Wirkung der Augenexstirpation und der Dunkelhaltung auf auf das Mesencephalon und die

56

H. S. S. BLAXTER BLAXTER JJ.. H.

Pseudobranchien von Fischen. Fischen. Wilhelm Wilhelm Raux' Rour’ Arch. Arch. Entwicklungsmech. Entwicklungsmech. Org. Org. 145, 145, 549-560. 549-560. Rana, 1985). Influence Rana, K K. J. J. ((1985). Influence of of egg egg size size on on the the growth, growth, onset onset of of feeding, feeding, point-of-no point-of-no1 19return, of unfed unfed Oreochromis Oreochromis mossambicus mossambicus fry. fry. Aquaculture Aquaculture 46, 46,119return, and and survival survival of 131. 131. Ridley, M 1978). Paternal care. M.. ((1978). care. Anim. Anim. Behav. Behav. 26, 26, 904-932. 904-932. Rose, 1960). A 1, Rose, S. S. M. M. ((1960). A feedback feedback mechanism mechanism of of growth growth control control in in tadpoles. tadpoles. Ecology Ecology 441, 188-199. 188-199. Russell, 1976). "Eggs Russell, F. F. S. S. ((1976). “Eggs and and Planktonic Planktonic Stages Stages of of Marine Marine Fishes." Fishes.” Academic Academic Press, Press, London. London. 1966). Observations Ryland, S. ((1966). Observations on on the the development development of of larvae larvae of of the the plaice plaice Ryland, J. J. S. Pleuronectes Cons., Cons. Cons. Int. Pleuronectes platessa platessa L. L. in in aquaria. aquaria. ]. J. Cons., Int. Explor. Erplor. Mer Mer 30, 30, 177-195. 177-195. Schnack, Schnack, D., D., and and Rosenthal, Rosenthal, H. H. (1977/1978). (1977/1978). Shrinkage Shrinkage of of Pacific Pacific herring herring larvae larvae due due to to Ber. Dtsch. 222formalin Dtsch. Wiss. Wiss.Komm. Komm. Meeresforsch. Meeresforsch. 26, 26,222formalin fixation fixation and and preservation. preservation. Ber. 226. 226. Shaw, E. ((1960). 1960). The Zool. 33, Shaw, E. The development development of of schooling behavior in in fishes. fishes. Physiol. Physiol. 2001. 33, 79-86. 79-86. Shaw, 1961). The fishes. II. 34, 263-272. Shaw, E. ((1961). The development of of schooling in in fishes. 11. Physiol. Physiol. Zool. Zool.34,263-272. Shirota, A. sh larvae (in A. (1970). (1970). Studies on the mouth size of fi fish (in Japanese). Japanese). Bull.]pn. Bull.Jpn.Soc. SOC. Sci. 353-368. Sci. Fish. Fish. 36, 36,353-368. Snyder, D. D. E., and Holt, J. J. G. ((1983). 1983). Terminology Workshop. (Mimeo) (Mimeo) Univ. Texas Mar. Sci. Sci. Inst. Port Aransas, Texas. Texas. Solemdal, P. ((1973). 1973). Transfer of of Baltic flatfish to a marine environment and the long term effects on reproduction. 15, 268-276. Oikos 15, 268-276. reproduction. Oikos Sorgeloos, P. (1980). (1980). The use of of brine shrimp Artemia Artemia in aquaculture. aquaculture. In In "The “The Brine Shrimp Artemia" Artemia” (G. (G. Persoone, P. Sorgeloos, O. 0. Roels, and E. Jaspers, eds.), eds.), Vol. 3, 3, pp. 25-46. 25-46. Universa Universa Press, Wetteren, Belgium. 1985). Effects of Springate, J. R. C., and Bromage, N. R R. ((1985). of egg size on early growth and survival in rainbow trout (Salmo (Salmo gairdneri Richardson). Aquaculture 47, 47, 163-172. 163-172. Springate, J. 1984). The J. R. C., Bromage, N. R, R., Elliott, J. J. A. K, K., and Hudson, D. L. ((1984). timing of ovulation and stripping and their effects on the rates of of fertilization and survival to eyeing, hatch and swim-up in the rainbow trout (Salmo R). (Salmo gairdneri R.). Aquaculture 313-322. Aquaculture 43, 43,313-322. Springate, J. R. C., Bromage, N. R, R., and Cumaranatunga, Cumaranatunga, P. R R. T. (1985). (1985). The effects of different fecundity and egg quality in the rainbow trout gairdneri). different ration on fecundity trout (Salmo gairdneri). G.. Bell, “Nutrition and Feeding in Fish" Fish” (C. B. Cowey, A. M. Mackie, and J. G In "Nutrition eds.), pp. 371-393. 371-393. Academic Press, London. eds.), Strauss, R. R E., and Fuiman, L. A. (1985). (1835). Quantitative Quantitative comparisons of of body form and Straws, Can.]. 2001.63, Zool. 63, (Teleostei: Cottidae). Cottidae). Can.]. allometry in larval and adult Pacific sculpins (Teleostei: 1582-1589. Stunner, L. N., Sturmer, N., McCarthy, C. E., and Rutledge, W. P. (1985). ( 1985). Hatchery production production of of red Larval Fish Con$ Conf Am. Fish. SOC., Soc., drum fingerlings in Texas. Abstr. 9th Annu. Larval 1985. 1985. Swain, D. P., and Lindsey, C. C. (1984). ( 1984). Selective predation for vertebral of vertebral number of Gasterosteus aculeatus. aculeatus. Can. Can. J].. Fish. Fish. Aquat. Aquat. Sci. Sci. 41, 41, 12311231young sticklebacks Gasterosteus 1233. 1233. Symons, P. E. E. K. K ((1968). 1968). Increase in aggression and in strength of of the social hierarchy among of food. J].. Fish. Fish. Res. Res. Board Board Can. Can. 25, 25, among juvenile Atlantic Atlantic salmon deprived of 2387-2401. 2387-240 1. Can . J].. Zool. Zool. 38, 38, Tait, J. S. S. (1960). (1960). The first filling ooff the swim bladder iinn salmonids. Can. 179-187. 179187. -

_

1. 1.

PATTERN AND AND VARIETY VARIETY IIN N DEVELOPMENT DEVELOPMENT PATTERN

57

Tanaka, M. (1973). (1973). Studies in the structure and function of the digestive system of of teleost larvae. D. Agric. Thesis, Kyoto University, Japan. Taning, Bioi. Rev. (1952). Experimental study of of meristic characters in fishes. Biol. Rev. T h i n g , A. V. (1952). Cambridge Philos. Soc. SOC. 27, 169-193. Theilacker, C. 1978). Effect G. H. ((1978). Effect of of starvation on the histological and morphological Fish. Bull. Bull. 76, 403characteristics Trachurus symmetricus symmetricus larvae. Fish. 76,403characteristics ofjack mackerel Trachurus 414. Theilacker, C. G. H H.. (1980a). (1980a). Changes in body measurements of of larval northern anchovy Engraulis Bull. 78, Engraulis mordax mordax and other fishes due to handling and preservation. Fish. Fish. Bull. 685-692. 685-692. Theilacker, C. H. (1980b). Theilacker, G. H. (1980b). Rearing container size affects morphology and nutritional condition of larval jack mackerel Trachurus Bull. 78, 789-791 . Trachurus symmetricus. symmetricus. Fish. Fish. Bull. 78,789-791. 1981). Effect o Theilacker, C. G. H H.. ((1981). off feeding history and egg size oonn the morphology of of Rapp. P.-V. Cons. Int. jack mackerel, mackerel, Trachurus Trachurus symmetricus. symmetricus. larvae. Rapp. P.-V.Reun. Reun. Cons. Znt. Explor. Explor. Mer 432-440. Mer 178, 178,432-440. Dorsey, K. (1980). Theilacker, C. (1980). Larval fish diversity, a summary of of laboratory G. H., and Dorsey, and field research. Intergov. (IOC) Workshop 105-142. Intergou. Oceanogr. Oceanogr. Comm. Comm. (ZOC) Workshop Rep. Rep. 28, 28,105-142. Thorpe, 1984). Development rate, fecundity and egg J. E., Miles, Miles, M M.. S., and Keay, Keay, D. D. S. S. ((1984). Thorpe, J. size in Atlantic salmon Salmo 289-305. Salmo salar salar L. Aquaculture Aquaculture 43, 43,289-305. Tucker, J. Chester, A. J. J, W., Jr., and Chester, J. (1984). (1984). Effects of salinity, formalin concentration concentration and buffer on quality of preservation of southern flounder (Paralichthys (Paralichthys lethos Zethostigma) Copeia, pp. 981-988. 981-988. tigma) larvae. Copeia, F., Leger, LBger, P., P., and Sorgeloos, P. (1985). (1985). Preliminary Amat, F., Hontoria, F., van Ballaer, E., Amat, results on the nutritional Artemia nauplii for nutritional evaluation of w3-HUFA-enriched o3-HUFA-enriched Artemia 49, 223-229. larvae of the the sea bass Dicentrarchus labrax. labrax. Aquaculture 49,223-229. van tin, C., and Pool, C. van Raamsdonk, W., W., van't van’t Veer, L., L., Veeken, K., Hey Heytin, C. W. W. (1982). (1982). Differentiation Brachydanio rerio, rerio, the the zebrafish. Differentiation of muscle fiber types types in the teleost Brachydanio Anat. 51-62. Anat. Embryol. Embryol. 164, 164,51-62. Vernier, J.-M. ((1969). chronologique du developpement embryonnaire de la Vernier, 1969). Table chronologique truite arc-en-ciel, Salmo Salmo gairdneri gairdneri Rich. 1836. 1836. Ann. Ann. Embryol. Embryol. Morphog. Morphog. 2, 495495520. 520. Vladimirov, V. I. 1975). Critical periods in the development of fishes (translated I. ((1975). (translated from Russian). ]. J . Ichthyol. Ichthyol. 15(6), 15(6), 851-868. 851-868. Wallace, J. C., C., and Aasjord, D. (1984). (1984).An investigation investigation of the the consequences of egg size Wallace, J. Arctic chaIT, cham, Salvelinus Saluelinus alpinus (L.). (L.).I. J. Fish. Fish. Bioi. Biol. 24, 24,427-435. for the culture of Arctic 427-435. T. ((1985). nutrition ffor develor further devel Watanabe, T. 1985). Importance of the study of broodstock nutrition opment of aquaculture. In "Nutrition “Nutrition and Feeding in Fish" Fish” (C. (C. B B.. Cowey, A. M M.. G. Bell, eds.), ppp. 394-414. Academic Press, London. London. p . 394-414. MacKie, and J. C. Watanabe, Y. 1985). Histological changes Y. ((1985). changes in the liver and intestine of freshwater goby larvae during short-term starvation. Bull. Bull. lpn. Jpn. Soc. SOC. Sci. Sci. Fish. Fish. 51, 707-709. 707-709. Watanabe, T., T., Kitajima, Kitajima, C., C., and Fujita, S. S. (1984). (1984). Effect of of nutritional nutritional T., Arakawa, T., quality on reproduction reproduction of of red red sea sea bream. bream. Bull. Bull. lpn. Jpn. Soc. S O C . Sci. Sci. quality of of broodstock broodstock diets diets on Fish. 50, 495-501 495-501. . Fish. 50, Webb, W. (1975). (1975). Hydrodynamics Hydrodynamics and and energetics energetics ooff fish fish propulsion. propulsion. Bull. Bull. Fish. Fish. Res. Res. Webb, PP.. W. Board Can. Can. 190, 190, 1-158. Webb, Webb, P. P. W., W., and and Weihs, Weihs, D. D. (1986). (1986). Functional Functional locomotor locomotor morphology morphology of of early early life life history 15-127. Trans. Am. Am. Fish. Fish. Soc. SOC. 115, 115, 1115-127. history stages. stages. Trans. Weihs, D. (1980a). (1980a). Energetic significance of �hanges changes in swimming swimming modes modes during Weihs, D. growth 597-604. Engraulis mordax. mordax. Fish. Fish. Bull. Bull. 77, 77,597-604. growth of larval anchovy Engraulis Weihs, 1980b). Respiration Weihs, D. D. ((1980b). Respiration and and depth depth control control as as possible possible reasons reasons for for swimming swimming of of northern anchovy Engraulis 17. Engraulis mordax mordax yolk-sac larvae. Fish. Fish. Bull. Bull. 78, 78, 109-1 109-117.

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58

Whipple, ]., J., Eldridge, M., M., Benville, P., Bowers, M., Jarvis, Jarvis, B., and Stapp, N. (1981). (1981). The condition and viability in striped bass effect of inherent parental factors on gamete condition Morone. Rapp. P.-V. Reun., 93-94. Reun., Cons. Cons. Int. Znt. Explor. Erplor. Mer 178, 178,93-94. Winberg, G. G. (1960). (1960). Rate of of metabolism metabolism and food requirements of of fishes. Fish. Fish. Res. Res.

Board Can. Transl. Board Can. Transl. 194. 194.

J. (1979). (1979). Energy costs of of egg production production and environmental determinants of of Wooton, R. ]. fecundity in teleost fish. Symp. Zool. Soc. Soc. London 44, 133-159. 133-159. Zeutzius, I., I., and Rahmann, H. ((1984). 1984). Infl uence of dark-rearing on the ontogenetic Influence (Cichlidae Teleostei). I. Effects of Sarotherodon mossambicus mossambicus (Cichlidae Teleostei). I. of development of Sarotherodon Biol. body weight, body growth pattern, swimming activity and visual acuity. Exp. Bioi. 43, 77-85. 43,77-85. Zeutzius, t, 1984). Influence of Zeutzius, I., Probst, W., and Rahmann, H. ((1984). of dark-rearing on the (Cichlidae, Teleostei). ontogenetic Sarotherodon mossambicus mossambicus (Cichlidae, Teleostei). II. 11. ontogenetic development of of Sarotherodon Effects on allometric allometric and growth relations and differentiation differentiation of of the optic tectum. Exp. Erp. Bioi. Biol. 43, 87-96. 87-96.

2 RESPIRATORY GAS EXCHANGE, AEROBIC METABOLISM, AND EFFECTS OF HYPOXIA DURING EARLY LIFE

ROMBOUGH PETER J].. ROMBOUGH Department Zoology Department Brandon Brandon University University Brandon, anitoba, Canada R7 A 6A9 Brandon, M Manitoba, R7A 6A9 11.. Introduction II. Exchange 11. Respiratory Respiratory Gas Gas Exchange A. A. The The Boundary Boundary Layer Layer B. The B. The Egg Egg Capsule Capsule C. C. The The Perivitelline Perivitelline Fluid Fluid D. Cutaneous Gas Exchange E. Respiratory Pigments F. F. Branchia Branchiall Gas Gas Exchange III. 111. Aerobic Aerobic Metabolism Metabolism A. A. Measurement Techniques B. B. Biotic Biotic Factors C. C. Abiotic Abiotic Factors Factors IV. IV. Effect Effect of of Hypoxia Hypoxia A. A. Environmental Environmental Hypoxia Hypoxia B. Physiological Physiological Hypoxia Hypoxia V. V. Conclusions References References

I. INTRODUCTION The The basic basic mechanisms mechanisms involved involved in in respiratory respiratory gas gas exchange exchange in in juvenile juvenile and and adult adult fish fish are are fairly fairly well well established established (see (see reviews reviews by by Jones Jones and and Randall, Randall, 1978; 1978; Randall, Randall, 1982; 1982; Randall Randall et al., al., 1982; 1982; Randall Randall and and Daxboeck, Daxboeck, 1984). 1984). The The study study of of oxygen oxygen metabolism metabolism in older older fish, fish, similarly, 1 ; Beamish, similarly, is is well well advanced advanced (reviewed (reviewed by by Fry, Fry, 1957, 1957, 197 1971; Beamish, 1978; 1978; Brett Brett and and Groves Groves,, 1979; 1979; Tytler Tytler and and Calow, Calow, 1985). 1985). In In contrast, contrast, relatively relatively little little is is known known of of respiratory respiratory gas gas exchange exchange and and energy energy usage usage during during early early life. life. This This arises arises not not so so much much from from aa lack lack of of effort effort on on the the FISH FISH PHYSIOLOGY. PHYSIOLOGY, VOL. VOL. XIA XIA

59 59

Copyright Copyright © 0 1988 1988 by by Academic Academic Press, Press, Inc. Inc. All orm reserved. All rights rights of of reproduction reproduction in in any any fform reserved.

60

PETER JJ.. ROMBOUGH

part of of researchers-in researchers-in excess of 500 papers dealing with various as aspects of oxygen supply and demand during early life have been pub published in the last 20 years-but years-but rather from the lack of of a systematic approach approach to to the the problem. problem. The The aim aim of of this this review review is is to to collate collate the the large large amounts data that that are are currently currently available available and and to to fi fitt it it into into aa concep concepamounts of data tual framework that can be used as the basis for future investigations. investigations.

II. 11. RESPIRATORY GAS EXCHANGE

Analytical models provide a useful framework for the study of of re reexchange. The cascade model, in particular, has been spiratory gas exchange. used widely to describe various aspects of vertebrate respiratory func func(Dejours, 198 1981; 1982; Weibel, 1984; 1984; DiPrampero, 1985). 1985). tion (Dejours, 1 ; Piiper, 1982; In this model, respiratory gases are viewed as passing through a series specific of resistances, each of which is correlated with a specifi c process or structure. indi of the indistructure. The overall resistance of of the system is the sum of steady-state conditions, overall flow vidual resistances and, under steady-state through through the the system system is is equal equal to to the the flow flow through through each each of of the the elements. elements. The model is especially useful in helping to define the nature and magnitude of the various resistances in the respiratory pathway and the partial pressure gradients necessary to overcome them. Such analanal ysis is complicated for early life stages because of changes in respira respiratory rate and the nature and relative importance of of resistances during development. development. These problems, while formidable, formidable, are are not not insurmount insurmountable, of the cascade model able, as evidenced by the successful application of to the study of gas transport in mammalian (Dejours, (Dejours, 1981) 1981) and avian embryos (Dejours, (Dejours, 198 1981; 1984). Unfortunately, 1 ; Piiper and Scheid, 1984). insufficient information to apply the model rigor rigorthere is currently insufRcient ontogeny. However, ously to the study of of gas exchange during teleost ontogeny. enough enough is known known of of gas gas exchange exchange in in developing fish to to use use the the cascade cascade model in a more general way, that is, as a guide to help identify and major resistances resistances during development. development. describe the major In many ways gas exchange is very similar in embryos and larvae, particularly once organogenesis is complete. In both stages gas exex change is primarily cutaneous. Branchial exchange typically becomes dominant only near the end of the larval period. However, in spite of of sepathis similarity, it is often convenient to treat embryos and larvae sepa of the major impact the egg capsule (zona radiata) radiata) has rately because of on gas exchange during embryonic life. The egg capsule, in addition significant to acting as a signifi cant barrier in its own right, creates two other barriers, uid, that barriers, the the external external boundary boundary layer layer and and the the perivitelline perivitelline fl fluid, that together ). together have have an an equal equal or or greater greater impact (Fig. (Fig. 11).

2. 2. RESPIRATORY RESPIRATORY GAS GAS EXCHANGE, EXCHANGE, AEROBIC AEROBIC METABOLISM METABOLISM

61 61

boundary l ayer

t:..I,....\-- pore canal

vitell ine membrane

perivite l l i ne space

Fig. Fig. 1. 1. Schematic Schematic diagram diagram showing showing the the major major resistances resistances to to respiratory respiratory gas gas ex exchange change for for fish fish embryos embryos (not in proportion). proportion). The heavy arrow indicates indicates the direction of of water water How. flow. The The actual actual shape shape of of the the trailing edge edge of of the the boundary boundary layer layer will will depend depend on on water water velocity velocity and and egg egg size. size.

A. A. The Boundary Layer The laminar boundary layer is is a semistagnant region of water adja adjacent to the egg urface where oxygen egg ssurface oxygen is depleted and metabolic wastes accumulate. The The boundary layer actually has no outer limit but for s ually defined as for practical purposes is uusually as the distance from from the egg surface where local local conditions are equivalent to 99% 99% of free-stream conditions conditions (Vogel, (Vogel, 1981). 1981). For the laminar flow flow regimes that eggs are typically exposed to (Reynolds (Reynolds numbers < < 75; 75; Johnson, 1980), 1980), the thickness of ize and in of the boundary layer is proportional to egg ssize in1965) indicated that versely proportional to water water velocity. velocity. Daykin ((1965) the the thicknes thicknesss of the solute boundary layer, layer, d" d,, is is less than the thick thickness of the velocity boundary layer and can be estimated from from the Sherwood Sherwood number (Sh) (Sh) and egg diameter (d) (d) using the equation

d,

=

d(Sh-')

(1) (1)

PETER PETER J. ROMBOUGH ROMBOUGH

62

The Sherwood number number is number dependent dependent on The Sherwood is aa dimensionless dimensionless number on two other other dimensionless dimensionless numbers, numbers, the the Reynolds number (Re) (Re) and and the the Schmidt number ((Sc). Sc). For spherical eggs, Sh can be estimated as

+

l3 · Re) lIZ Sc1 = 2.0 + 0.8(B 0.8(B.Re)1'2 Sclm Sh =

(2) (2)

dv 1 , Sc where l , and B where Re = = p, pdu-l, Sc = = VDOD-', B is is the the ratio ratio of of the the interstitial interstitial and and bulk bulk velocities velocities (Daykin, (Daykin, 1965; 1965; Johnson, Johnson, 1980). 1980). Here Here p, p is is bulk bulk water water velocity, velocity, d is is egg egg diameter, diameter, vu is is kinematic kinematic viscosity, viscosity, and and D D is is the the diffusion diffusion coefficient. coefficient. Interstitial Interstitial velocity velocity is is equal equal to to bulk bulk velocity velocity for for isolated eggs. eggs. According to Eq. (2), (2),the thickness of the oxygen bound boundary layer would be about 0.02 0.02 and 0.05 0.05 cm, respectively, for single eggs with diameters of 1 and 0.5 cm in a 100 of 0. 0.1 100cm h-1 h-l current. Increas Increasing current velocity to 1000 1000 cm h-1 h-' would reduce boundary layer thickness to approximately 0.0008 0.0008 and 0.02 cm, respectively. The higher metabolic (Winnicki, 1968; DiMichele and Powers, 1984a) (Winnicki, 1968; 1984a) and growth (Silver et al., 1963; Shumway et al., al., 1963; al., 1964) 1964) rates reported at higher water velocities are probably due to reductions in boundary boundarylayer thickness (Daykin, 1965; Wickett, 1975). 1975). Estimating interstitial (Daykin, 1965; velocities is a problem for eggs laid in masses or in substrate. If If a hexagonal array is assumed, the interstitial velocity in an egg mass averages about 9. 1 times the bulk velocity through the mass (Daykin, 9.1 (Daykin, 1965; Wickett, 1975). esti 1965; 1975). Interstitial velocities in substrate can be esti1 mated from porous bed theory as P,i = p,e, where P,i is interstitial pi = ~ E - I , pi velocity, p, p is bulk velocity, and eE is the empirically determined poros porosity (Johnson, (Johnson, 1980). 1980). The driving force required to overcome the resistance imposed by the boundary layer can be predicted by rearranging the Fick's equa equation for diffusion through a plane to yield -

C c11 -- C coo == YOzds(47TrZD)-1 b02dS(4~r2D)-l

(3) (3)

where C C11 is the free-stream oxygen concentration, C Coo is the oxygen concentration at the egg surface, YOz VO2 is the rate of oxygen consump consumpuivalent to the more tion, and rr is the egg radius. This equation is eq equivalent widely used mass transport equation (Daykin, 1975; (Daykin, 1965; 1965; Wickett, 1975; Johnson, 1980) 1980) in which the reciprocal of the mass transport constant, 1 , replaces dsD-l. kk-l, d,D-'. The value of k is estimated as k = = (Sh)Dd-1• (Sh)Dd-'. E quation (3) Equation (3) indicates that the driving force required to meet meta metabolic demands is directly proportional to the rate of oxygen consump consumption and thus increases more or less steadily throughout embryonic development. Daykin ((1965) 1965) estimated that a partial pressure gradient across across the boundary layer of about 52 mm Hg was required to meet the oxygen requirements of chum (Oncorhynchus 0.37 cm) (Oncorhynchus keta) keta) eggs (r (T = = 0.37 cm) near hatch at 10°C 10°C and a flow rate of of 85 cm h-1• h-l. Smaller pressure

2. 2.

RESPIRATORY AEROBIC METABOLISM RESPIRATORY GAS GAS EXCHANGE, EXCHANGE, AEROBIC METABOLISM

63

differences are required for smaller eggs because of generally lower metabolic rates and thinner boundary layers. layers. For example, Wickett (1975) 0.05 cm) (1975) estimated that cod (Gadus (Gadus macrocephalus) eggs (r (T = = 0.05 cm) incubated at 5°C - 1 would require a pressure 5°C in a current of 170 170 cm h h-’ difference across the boundary layer of only about 17 17 mm Hg to fully satisfy satisfy their oxygen requirements. Until recently it was assumed that forced convection (i.e., bulk water flow) flow) was the major means of of supplying oxygen to eggs. It now appears that under certain circumstances natural (free) (free) convection may be important as well. Embryonic metabolism gives rise to solute concentration gradients across the boundary layer. O'Brien O’Brien et al. al. ((1978) 1978) have shown that the oxygen-depleted, carbon dioxide-rich wa water immediately adjacent to the egg is is denser than the well oxygen oxygenated, low C O2 water in the free stream. COZ stream. In still water, this sets up a toroidal flow as the denser solution adjacent to the egg sinks (Fig. (Fig. 2). 2). Water velocities in the toroid can be relatively high. For example, O'Brien al. ((1978) 1 978) observed an average velocity of 72 cm h-l h-1 in the O’Brien et al. toroid set up by eyed eggs (400 (400 degree-days, 100C) 10°C)of coho salmon (0. (0. kisutch). kisutch).At this velocity, natural convection would be about 150 times as eeffective ffective as simple diffusion in supplying oxygen under "“static” static" conditions. The effectiveness of of natural convection can be expected to increase as metabolic rate increases, due to greater depletion of of oxy oxygen and accumulation of carbon dioxide in the boundary layer. Thus, natural convection may act as a homeostatic mechanism helping to balance oxygen supply and demand. Whether natural convection plays a significant role in nature will depend on bulk water velocity and the orientation of the egg mass. Analysis vecAnalysis of mixed regimes is complex but, in general, natural con convec-

.... , .."--- ,,, /' J A' " I , "

Fig. 2. Stylized depiction of the toroidal toroidal fl flow around a respiring ow of water set up around respiring egg as a result of natural convection in the absence of bulk water movements. [From [From 1978).] O'Brien O’Brien et al. al. ((1978).]

64

PETER J. ROMBOUGH PETER J. ROMBOUGH

tion will play a greater role than forced convection at low bulk veloci velocities [see [see Vogel ((1981, 198 1, pp. 178-195) on how the ratio of of the Grashof number and the square of of the Reynolds number can be used to esti estimate the relative importance of ] . In nature, bulk veloci of the two forces forces]. velocities are often low. For example, the bulk flow in many spawning beds is less than the toroidal velocity set up by coho eggs in still water (O'Brien (O’Brien et al., 1978). 1978). Similarly, interstitial velocities in egg masses of species such as lumpfish (Cyclopterus (Cyclopterus lumpus) lumpus) and lingcod (Ophiodon (Ophiodon elongatus) elongatus) can be expected to be rather low. low. It has been suggested, but not demonstrated, that voids in such masses may pro provide avenues for natural convection to supply oxygenated water to the interior (O'Brien (O’Brien et al., 1978; 1978; Giorgi and Congleton, 1984). 1984). Natural convection will be most effective if eggs are oriented so that the heavier oxygen-depleted water can sink and its movement is is not op opal., 1978; 1978; Johnson, 1980). posed by forced convection (O'Brien (O’Brien et al., 1980).

B. The Egg Capsule The egg capsule traditionally has been viewed as the major barrier to diffusive gas exchange during embryonic life. This may seem obvi obvious, but the empirical evidence supporting this viewpoint is is actually rather scanty. Strongest support comes from observations that incipi incipient limiting oxygen tensions (Pc) (P,)drop significantly on removal of of the capsule (Hayes (Hayes et al., 1951; 1951; Rombough, 1986, 1986, 1987). 1987). If If metabolic rate and the drop in Pc P, are known, the diffusion coefficient of the capsule (Dc) Fick's equation for diffusion through a (DJ can be calculated using Fick’s plane [Eq. [Eq. (3)]. (3)]. Several investigators have done this (Hayes (Hayes et al., 1951; 1951; Alderdice et al., al., 1958; 1958; Daykin, 1965; 1965; Wickett, 1975), 1975), but the 1 , is that worked out by 2 Sx 10-5 cm cm2 s-l, value most often cited, 0.18 x Wickett (first (first presented presented in Daykin, 1965) 1965) using data provided by Hayes et al. ((1951) 1951) for Atlantic salmon (Salmo 1975) (Salmo salar). salar). Wickett ((1975) recognized that his estimate was based on rather sketchy data and indicated that standard diffusion tests should be conducted to check its validity. Unfortunately, this has not been done for teleosts, al al(1987) recently used a Krogh-type Krogh-type diffu diffuthough Diez and Davenport (1987) sion chamber to estimate the oxygen oxygen diffusion coefficient of sion of the egg case of the dogfish, Scyliorhinus canicula. canicula. Interestingly, the value 1 , is 2 Sthey arrived at, 0.285 x Wickett's value for X 10-5 cm cm2 s-l, is similar to Wickett’s Atlantic salmon. However, given the structural differences between the dogfish egg case and the salmon capsule, this cannot be taken as confirmation of Wickett's Wickett’s value.

2. 2.

RESPIRATORY GAS EXCHANGE, AEROBIC METABOLISM RESPIRATORY GAS EXCHANGE, AEROBIC METABOLISM

65

Wickett’s value for D, of water. Wickett Wickett's Dc is about one-tenth that of pene((1975) 1975) pointed out that the surface area of the radial pores that pene

trate the capsule is similarly about one-tenth the total surface area of of the capsule and speculated that this may indicate that diffusion takes place primarily through the pore canals rather than through the cap capsule matrix. If this is true, true, doubt is cast on on the the validity of the the practice sule matrix. (e.g., Daykin, 1965; 1965; Wickett, 1975; 1975; Kamler, Kamler, of some investigators (e.g., 1976; 1984) applying diffusion coefficients, 1976; DiMichele and Powers, 1984) calculated for one species, to another unrelated species. There is con considerable (Lfbnning, siderable variation variation among among teleosts teleosts in in capsule capsule structure structure (Lqhning, 1972; Stehr 1979; Groot 1985). Even 1972; Stehr and and Hawkes, 1979; Groot and and Alderdice, Alderdice, 1985). Even in in closely related salmonids, salmonids, pore pore area can vary anywhere between 7% and Groot and 30% of of the the total total surface surface area area ((Groot and Alderdice, Alderdice, 1985). 1985). and 30% Recent evidence suggests that the capsule may not be as great a barrier to diffusive gas exchange Bereexchange as was supposed previously. Bere zovsky 1979) used microelectrodes zovsky et aI. al. ((1979) microelectrodes to to measure measure the the dissolved dissolved oxygen uid, and capsule, perivitelline perivitelline fl fluid, and vitelline oxygen profile profile across across the capsule, membrane fossilis). membrane of of the the recently recently fertilized fertilized loach loach eggs eggs (Misgurnis fossilis). Surprisingly, they recorded very little drop in oxygen concentration across across the the capsule. capsule. In contrast, contrast, there there was was aa gradual gradual decline decline in in oxygen oxygen tension across the perivitelline fluid and a sharp drop across the vitel vitel(Fig. 3). 3). Sushko ((1982) 1982) reported a similar oxygen pro proline membrane (Fig. fi le for loach eggs incubated in helium file helium-oxygen nitrogen-oxygen -oxygen and nitrogen-oxygen gas Alderdice et aI. al. (1984) (1984) may may pro progas mixtures mixtures.. Observations Observations made made by Alderdice vide an explanation for why the capsule appears to offer relatively little resistance to gas exchange. exchange. Alderdice et al. al. (1984) (1984) observed that the hydrostatic pressure exerted on the capsule of steelhead (S. (S. gairdneri) gairdneri) was considerably less than the osmotic pressure of the perivitelline fluid and calculated an perivitelline fluid and calculated an effective effective filtration filtration pressure pressure of of about -62 -62 mm Hg driving water into the perivitelline space. space. They reasoned reasoned that that according according to to Starling's Starling’s hypothesis hypothesis this this should should lead lead to to the the movement movement of of water water into into the the perivitelline perivitelline space. space. Since Since the the egg egg is is in in volume volume equilibrium, equilibrium, this this must must be be balanced balanced by by an an equal equal outflow outflow by by fi ltration. Exactly is not not clear, clear, since since filtration. Exactly how how this this would would be be accomplished accomplished is the the capsule is not a linear structure like a capillary. Alderdice et al. (1984) (1984) suggested that the capsule may act like a balloon with micro microsieves accompanied sieves in in its its wall. wall. Increasing Increasing internal internal pressure pressure would would be accompanied by volume expansion. As the capsule expanded the pores would enen large and more water would fl ow out. Volume and tension increases flow thus (1984) proposed proposed that that this this thus would would be be self-limiting. self-limiting. Alderdice Alderdice et al. (1984) process process would would tend tend to to facilitate facilitate respiratory respiratory gas gas exchange exchange and and point point to to radiotracer and Rudy, (Potts and Rudy, 1969; 1969; Loeffler Loeffler and and Lovtrup, Lovtrup, 1970; 1970; radiotracer studies studies (Potts

PETER J. ROMBOUGH PETER J . ROMBOUGH

66

1 60

I

I

140 1 1 20 E E ...

I I I I I I I I I I I I I I I I I bl I I I

10

III > III ...

Z III c:I >)C o

40

yolk

pvf

: 2ow

20 r-

� I

__ __ __ __ __

v", lolll.& I mom� br� a.e� -r�� _� .3 0 ! 3 d.66 0 0.

�____

RA AD D II A AL L R

... capoule I� �� _

I

____

d.9 0.9

mm) DISTANCE DISTANCe ((mm)

Fig. Fig. 3. 3. Oxygen Oxygen profiles across across recently recently fertilized fertilized roach roach eggs measured using plati platinum microelectrodes. symbols, normal eggs; symbols, eggs in which rate eggs; closed symbols, microelectrodes. Open symbols, of oxygen oxygen uptake uptake was stimulated 3.3-fold 2,4-dinitrophenol;pvf, pvf, perivitel perivitel3.3-foldusing 10-4 M 2,4-dinitrophenol; layer. [After [After Berezovsky et al. al. (1979).] (1979).] fluid; line fl uid; bl, boundary layer.

Loeffler, LoeMler, 1971) 1971) indicating an exchange of water across the capsule equivalent to the volume of the perivitelline fluid every 1-4 1-4 min. A connective flux of this magnitude would add to the diffusive flux of of oxygen across the capsule but, perhaps more importantly, the currents generated would tend to prevent the establishment of of a large concenconcen tration gradient across the capsule. capsule. C. The Perivitelline Fluid As noted earlier, microelectrode studies (Berezowsky et al., al., 1979; 1979; Sushko, 1982) 1982) indicated that, at least for early embryos, much of the Sushko, acturesistance to gas exchange previously attributed to the capsule actu ally resides in the perivitelline fluid. The perivitelline fluid can be expected to have an oxygen diffusion coefficient similar to that for ~ 2 . 5x water ((=2.5 X 10-5 cm2 cm2 s-l) S - I ) (Dejours, (Dejours, 1981), 1981), but because diffusion of the perivitelline perivitelline fluid may distances are much larger the net impact of of the capsule. capsule. While capsule thicknesses in be greater than that of

2. 2.

RESPIRATORY GAS GAS EXCHANGE, EXCHANGE, AEROBIC AEROBIC METABOLISM METABOLISM RESPIRATORY

10 1 0-

-!

gI

IJJ w

....J

7

....J

6

> > IJJ z IJJ

§?

x o

Pc = 3.54 O.477(Vo2) •

5

fi1 4 � � :3 (j) B

l> _ 6°C 6'C 90C o 1 2°C • 15°C

9

0:.. 8 8

67

2

o

P50=0.968+ 0 . 2 3 7 ( V O ~ )

2 4 6 8 1 0 12 1 4 1 6 8 10 12 14 16 METABOLIC p g02 0 2 individual-' META BOLIC RATE ((�g Individual- I h-'1 h-I ) I

Fig. 4. Relationships between critical dissolved oxygen levels (P,) (Pc) and. and routine "02 = metabolic rate and Pso concentration at which V02 = 0.5 rV02) rV02) and Pa values (oxygen concentration routine routine metabolic rate for steelhead embryos embryos incubated incubated at constant constant temperatures. temperatures. Equa Equaportions of of the curves. curves. [From [From Rombough Rombough (1987).] (1987).] tions are for the linear portions

salmonids range between /Lm (Groot Alderdice, 1985), 1985), salmonids range between 15 and and 70 pm (Groot and and Alderdice, the across the perivitelline perivitelline space in excess excess of of 500 /Lm. the distance distance across space can can be in pm. Absolute distances distances tend tend to smaller in in pelagic pelagic eggs, eggs, but but because because of of aa Absolute to be smaller thinner capsule capsule and larger amount amount of of perivitelline perivitelline thinner and comparatively comparatively larger fluid, the the relative relative impact perivitelline fluid on gas exchange fluid, impact of of the the perivitelline fluid on gas exchange would be even larger in salmonids. would be even larger than than in salmonids. The impact the perivitelline uptake can impact of of the perivitelline fluid fluid on on oxygen oxygen uptake can be seen seen in P, values values during during early early development development (Rom (Romin the the rapid rapid increase increase in in Pc bough, bough, 1987). 1987). Critical Critical oxygen oxygen tensions tensions for for steelhead steelhead increase increase very very rapidly rapidly in in relation relation to to metabolic metabolic rate rate until until about about the the time time embryos embryos began move and uid. Thereafter, values P, values began to to move and stir stir the the perivitelline perivitelline fl fluid. Thereafter, Pc increase more increase more slowly slowly and and in in direct direct proportion proportion to to metabolic metabolic rate rate (Fig. (Fig. 4). sense, stirring 4). Thus Thus in in aa teleological teleological sense, stirring of of the the perivitelline perivitelline appears appears to to be if metabolic met. RezniReznibe necessary necessary if metabolic oxygen oxygen demands demands are are to to be met.

68

PETER PETER J. J. ROMBOUGH ROMBOUGH

chenko et al. al. (1977) polarographic electrode chenko (1977) used used aa modified modified polarographic electrode to to model model oxygen uid. H Hee oxygen exchange exchange across across the the egg egg capsule capsule and and perivitelline perivitelline fl fluid. found found that that when when the the analog analog of of the the perivitelline perivitelline fluid fluid was was stirred, stirred, the while that PO22 at at the the body body (electrode) (electrode) surface surface increased increased while that under under the the the P0 capsule decreased. This This had capsule (membrane) (membrane) decreased. had the the effect effect of of increasing increasing the the steepness en steepness of of the the concentration concentration gradient gradient across across the the capsule capsule and and enhancing 1983) hancing net net oxygen oxygen transport. transport. Peterson Peterson and and Martin-Robichaud Martin-Robichaud ((1983) observed observed that that Atlantic Atlantic salmon salmon embryos embryos began began to to stir stir the the perivitelline perivitelline fluid fluid fairly fairly early early in in development. development. Trunk Trunk movements movements began began abruptly abruptly at at about 200 degree-days with an of degree-days of of development development with an initial initial frequency frequency of about about 60-120 - I . Dye Dye studies exures 60-120 flexures flexures h h-l. studies indicated indicated that that trunk trunk fl flexures about resulted movement along resulted in in rapid rapid water water movement along the the trunk trunk and and from from one one side side of uid to of the the perivitelline perivitelline fl fluid to the the other. other. These These movements movements were were appar apparently of nature, since ently of aa respiratory respiratory nature, since an an unexpected unexpected water water failure failure lead leading hypoxia resulted ing to to hypoxia resulted in in an an increase increase in in frequency frequency of of trunk trunk flexures flexures.. Trunk Trunk movements movements normally normally decline decline rather rather abruptly abruptly to to aa frequency frequency of of only every 2 2-4 350-400 degree-days degree-days in in Atlantic Atlantic salmon salmon (Peter (Peteronly 11 every -4 h by 350-400 son and 1983). However, However, by by this this time time the the embryo embryo son and Martin-Robichaud, Martin-Robichaud, 1983). had begun to ns rapidly 40-150 min-I. min-l. had begun to move move its its pectoral pectoral fi fins rapidly at at aa rate rate of of 40-150 This This rate rate was was maintained maintained until until hatch. hatch. Dye Dye studies studies indicated indicated that that these these movements generated a rapid water fl ow (=300 (=300 cm h-1) h-l) in the imme immeflow diate ns but ex flexdiate area area of of the the pectoral pectoral fi fins but were were not not as as effective effective as as trunk trunk fl ures in completely mixing the perivitelline fluid. fluid. Complete Complete mixing was exures. periodic trunk trunk fl flexures. was accomplished accomplished by periodic Recent Recent studies studies of of amphibians amphibians suggest suggest that that stirring stirring of of the the perivitel perivitelline line fluid fluid may may facilitate facilitate oxygen oxygen transport transport within within an an egg egg mass mass as as well well as as individual eggs. Burggren ((1985) within individual 1985) noted that oxygen partial pressures were higher and carbon dioxide partial pressures were lower in the interior of the egg mass of the frog Rana palustris than simple diffusion diffusion was was the only process process involved. involved. would be b e expected expected if simple perivitelline fl fluid. (1985) Frog embryos use cilia to stir the perivitelline uid. Burggren (1985) cilia could suggested that the currents generated by movement of of the cilia lead to oxygen being transported to the interior of the egg mass by capconvection as well as diffusion. Oxygen would diffuse across the cap sule sule at the surface of the egg closest to the outside of the egg mass. of oxygen-rich water would then be moved by ciliary action This mass of to the opposite side side of the egg where oxygen then would diffuse of the egg mass. An outward across the capsule toward the center of oxygen molecule thus could be passed from egg to egg in a manner water being passed along a bucket bucket brigade. somewhat analogous to water Carbon dioxide would pass in the opposite direction. This This appears to be a plausible mechanism for supplying oxygen to

2. 2.

RESPIRATORY EXCHANGE, AEROBIC AEROBIC METABOLISM RESPIRATORY GAS GAS EXCHANGE, METABOLISM

69

the interior of teleost as well as amphibian egg masses. Many teleosts linglay large, compact masses of eggs. For example, the egg mass of ling 1984). At cod may be up to 5 liters in volume (Giorgi and Congleton, 1984). propresent, there is not enough information on oxygen and current pro files “bucket-brigade” fi les within such egg masses to adequately test the "bucket-brigade" prohypothesis. However, there is some circumstantial evidence that pro cesses in addition to bulk water flows flows may be involved. Giorgi and (1984) noted that while oxygen concentrations in the cen cenCongleton (1984) ter of a lingcod egg mass declined rather sharply following cessation of current flow, flow, levels did not decline to zero as expected but stabi stabiof 10%air saturation. saturation. Davenport ((1983) lized at about 10% 1983) similarly indicated lumpfish that oxygen levels in the egg mass of lumpfi sh declined more slowly intriguthan expected when aeration ceased. These observations are intrigu previing but say little about the mechanisms involved. As discussed previ ously, these observations can be explained equally as well by natural “bucket-brigade” hypothesis. They do, convection as by the "bucket-brigade" do, however, suggest that egg masses do not depend solely on forced convection to meet metabolic oxygen demands. (1973)noted that the deeper eggs in the egg mass of species Braum (1973) such as herring (Clupea (Clupea harengus) harengus) are threatened with asphyxia as a circulation. He suggested that the perivitelline result of poor water circulation. fluid fl uid could function as an oxygen reservoir to tide embryos over short of much significance. The periods of anoxia. anoxia. This is unlikely to be of fluid, 100%satu satuamount of oxygen in the perivitelline fl uid, assuming it is is 100% rated, rated, is only only sufficient sufficient to to meet meet the the oxygen oxygen requirements requirements of of advanced advanced embryos for 1-2 min. calculation assumes would not not min. This This calculation assumes that that oxygen oxygen would embryos for 1-2 diffuse course it would under under hy capsule-which of of course it would hydiffuse back back out out of of the capsule-which poxic conditions-and conditions-and that there is no convective exchange between fluid water-which is likely. the perivitelline fl uid and the surrounding water-which The perivitelline fluid provides the immediate environment for perivitelthe developing embryo, and it is the gas concentration in the perivitel line fluid-not of physiological signifi signififluid-not the surrounding water-that water-that is of cance. As predicted by the mass transport equation, equation, Eq. Eq. (3), (3), oxygen concentrations in the perivitelline fl uid decline progressively as de fluid development proceeds. Assuming relatively constant capsule conducconduc tance rising metabolic POZ, the the only only way way the the rising metabolic demands demands tance and and ambient ambient P02, associated associated with with tissue tissue growth growth can can be be met met is is by by an an increase increase in in the the driving capsule. This necessitates driving force force across across the the capsule. necessitates aa reduction reduction in in the the P02 of the PO2 of the perivitelline perivitelline fluid. fluid. Berezovsky Berezovsky et al. al. (1979) (1979) demonstrated demonstrated such aa drop fluid P02 PO2 when when the the metabolic metabolic rate rate of of loach loach such drop in in perivitelline perivitelline fluid of dinitrophenol (Fig. embryos was stimulated by low concentrations concentrations.of (Fig. 3). 3). A decline decline in in perivitelline perivitelline fluid fluid P02 PO2 is is also also implied implied by by the the gradual gradual

70


increase in Pc P, that was seen during the course of steelhead develop development (Fig. (Fig. 4; Rombough, 1987). 1987). Recently, Diez and Davenport (1987) (1987) showed that the P02 PO2 of of the fluid in the egg case of of the dogfish declined as development proceeded. Finally, similar declines in P02 PO2 have been well documented for reptilian and avian eggs (Dejours, (Dejours, 1981). 1981). Bird eggs in particular have been studied extensively, and since many of the structures in bird and fish eggs can be considered analogous, the type of relationships seen in bird eggs probably apply to fish eggs as well. For example in the hen egg, oxygen levels in the air space, fluid, which is analogous to the perivitelline fl uid, decrease as metabolic rate increases (Wangensteen, 1972). 1972).This increases the diffusion gradi gradient across the shell, which like the teleost capsule is pierced by tiny pores, and automatically ensures a greater rate of diffusive flux. flux. It does so, however, at the expense of arterial P02 PO2 levels which gradually decline as development proceeds. Blood gas relationships have not been examined in fish embryos, but if the analogy with bird eggs holds, they probably follow a similar pattern. A decrease in perivitelline fluid P02 PO2 late in embryonic develop development appears to be the trigger that initiates hatching in at least some teleosts. If If advanced embryos are placed in hypoxic water, premature hatching occurs (Yamagami, (Yamagami, 1981 1981;; DiMichele and Powers, 1984a; 1984a; Ishida, 1985). 1985).Conversely, hatching can be delayed more or less indefi indefinitely under hyperoxic conditions (Taylor 1977; DiMichele and (Taylor et al., 1977; Taylor, 1980; 1980; Ishida, 1985). 1985). Low oxygen levels do not appear to act directly on the hatching glands. Studies involving various anesthetics suggest the response is mediated by the central nervous system (Ishida, (Ishida, 1985). 1985). The location of the oxygen sensor is not known. Hatching can be regarded as an adaptive response to physiological hypoxia. nes of the egg capsule reduces the hypoxia. Escape from the confi confines ambient oxygen level required to meet metabolic requirements by 30-50 30-50 mm Hg (Rombough, 1987). 1987). However, removal of the capsule does not alter the basic mechanisms involved in respiratory gas ex exchange.

D. Cutaneous Gas Exchange Respiratory gas exchange in fish, fish, and indeed in all vertebrates, is ininitially cutaneous. As development proceeds there is a gradual in crease in the relative importance of gills, although in many species the skin remains the major site of gas exchange throughout the embryonic periods.. Recent evidence indicates that even in adults the and larval periods skin may persist as an important site for respiratory gas exchange

2.


71 71

(Kirsch and Nonnotte, 1977; 1977; Lomholt and Johansen, 1979; 1979; Steffenson (Kirsch and Lomholt, 1985; 1985; Feder and Burggren, 1985). 1985). Studies of gas exchange during the early life stages of teleosts have tended to be descriptive. descriptive. As a result, most of what we know of respira respiraof tory mechanisms has been inferred from studies of the morphology of adaptawhat are assumed to be respiratory structures. Morphological adapta tions to facilitate gas exchange appear early in development. Boulekbache and Devillers ((1977) 1977) suggested that the function of the microvilli present on the outer surfaces of blastomeres of rainbow trout (S. (S. gairdneri) was to increase the surface area for respiratory gas exchange. In many species well-developed vascular networks form just under the skin during early organogenesis (Fig. (Fig. 5). 5). These capil capillary beds are often associated with specialized cutaneous structures, such as an enlarged yolk sac, expansive medial finfolds, finfolds, or enlarged pectoral fins fins,, that greatly increase the surface area available for gas exchange. Detailed descriptions of such specialized structures are provided by Taylor (1913), Sawaya (1942), 1942), Kry (1913), Sawaya (1942), Wu and Liu ((1942), Kryzanowsky ((1934), 1934), Smimov 1975), Lanzing Smirnov (1953), (1953), Soin (1966), (1966), Balon ((1975), (1976), McElman and Balon ((1979), (1981) al. (1976), 1979), Liem (198 1 ) and Hughes et al. ((1986), 1986), among others. The degree to which embryonic and larval larval respiratory structure are elaborated varies widely among species. species. Several authors have sugA

.'

B

f S I I u: e vs

,

s

I�

Fig. 5. 5. (A) Cutaneous gas exchange structures structures in in 5-day-old 5-day-old Tilapia mossambica. mossambica. (B) (B) Fig. Schematic Schematic diagram diagram showing showing blood blood flow flow through through caudal caudal and and rectal rectal vascular vascular systems: systems: cvs, cvs, caudal vascular system; system; h, system; vv, vitelline h, heart; heart; r, rectum; rectum; rvs, rvs, rectal vascular vascular system; vein; ym, yolk mass. mass. [[From From Lanzing ((1976).] 1976).]

72 72


gested that this refl ects variations in oxygen levels in spawning habi reflects habitat and can be used as the basis for a functional classification system (Kryzanowsky, 1934; Soin, 1966; 1966; Balon, 1975). 1975).It is beyond beyond the scope (Kryzanowsky, 1934; of of the the current current discussion to to examine examine the the merits merits of of such such systems, but but attention attention will will be be drawn drawn to to one one characteristic characteristic that that these these authors authors have have considered particularly important, lays considered particularly important, that that is, is, whether whether the the species species lays pelagic or pelagic or demersal demersal eggs. eggs. In In the the marine marine and and temperate temperate freshwater freshwater environments, environments, pelagic embryos tend tend to to have have relatively poorly devel developed oped capillary capillary plexes plexes near near the the body body surface. surface. Respiratory Respiratory pigments pigments (hemoglobin, myoglobin, myoglobin, perhaps (hemoglobin, perhaps carotenoids) carotenoids) usually usually do do not not appear appear until gills may until late late in in development, development, and and gills may not not become become functional functional until until near (Balon, 1975). near the the end end of of the the larval larval period period (Balon, 1975). Pelagic Pelagic eggs eggs tend tend to to be small and are normally found in well-oxygenated waters. As a result, it has been suggested that oxygen is not normally a limiting factor and thus thus specialized specialized respiratory respiratory structures structures are are not not necessary necessary (Hempel, (Hempel, 1979). ex1979). In contrast, demersal eggs are usually larger and often are ex posed to to relatively relatively low low oxygen oxygen concentrations concentrations for for extended extended periods. periods. According According to to Balon Balon (1975), (1975), this this has has resulted resulted in in selection selection for for extensive extensive vascularization vascularization of of the the body body surface surface and and the the elaboration elaboration of of specialized specialized cutaneous cutaneous gas gas exchange exchange structures. structures. Such Such structures structures tend tend to to develop develop early persist throughout early and and often often persist throughout the the larval larval period. period. Respiratory Respiratory pig pigments ments appear appear early, early, and and gills gills become become functional functional soon soon after after hatch. hatch. It should be recognized, though, that as with most generalizations generalizations in zoology, zoology, there there are are exceptions. exceptions. For For example, example, the the Indian Indian air-breather air-breather pelagic eggs, Anabas testudineus lays lays small small pelagic eggs, but but the the body body surface surface is is well vascularized, pigments appear well vascularized, and and respiratory respiratory pigments appear early early in in develop development 1986). These These adaptations adaptations are are not not particularly particularly ment (Hughes (Hughes et al., 1986). surprising realizes that surprising when when one one realizes that the the eggs eggs are are laid laid in in the the very very oxygen oxygenpoor waters of tropical swamps, and that it is only in the surface layer that levels are develop that oxygen oxygen levels are high high enough enough to to sustain sustain embryonic embryonic development. The effective surface area, the length of the diffusive pathway, the magnitude of the partial pressure gradient between the water and blood, the amount of blood perfusing the structure, and the convective movement of water past the structure are among the most important factors On factors influencing influencing the the performance performance of of respiratory respiratory gas gas exchangers. exchangers. On emthe basis of these characteristics, cutaneous gas exchange in fish em bryos highly efficient. efficient. As As mentioned mentioned bryos and and larvae larvae would would appear appear to to be highly previously, previously, specialized specialized exchange exchange structures structures comprise comprise aa relatively relatively large fraction of total body surface area in many species. For example, the well-vascularized (C. well-vascularized medial and paired fins of larval herring (C. harengus) harengus) and and plaice plaice (Pleuronectes (Pleuronectes platessa) platessa) account account for for about about 40% of of

2. 2.


73 73

total surface area at hatch (DeSilva (DeSilva and Tytler, 1973). 1973). In addition, the surface/volume ratio of most larvae is large because of their small surface/volume absolute size. size. Total surface area for a 11.6 .6 mg carp (Cyprinus (Cyprinus carpio) carpio) 2 glarvae is is in the order of 12,000 12,000 mm mm2 g-l.l. In contrast, total surface area 2 g-l of 1400 mm of a 350 350 mg juvenile is only about 1400 mm2 g-' (Oikawa (Oikawa and Itazawa, 1985). 1985). Cutaneous diffusion distances have been estimated for only a few species, but the available evidence indicates that distances are only slightly greater than lamellar diffusion distances in juveniles and adults. The skin is only two cells thick over most of the body surface in young larvae (Lasker, (Lasker, 1962; 1962; Jones et al., 1966; 1966; Roberts et al., 1973). 1973). Lasker ((1962) 1962) reported that the skin thickness of of larval sardine (Sar (Sardinops caerislea) . 7 f.Lm caerislea) ranged from 11.7 pm on the finfold to 3.0 tLm pm on the al. ((1966) 1 966) estimated a minimum lateral portion of the trunk. Jones et ul. skin thickness in larval herring (C. (C. harengus) 2.3 f.Lm. harengus) of about 2.3 pm. The actual length of the diffusive pathway is is somewhat greater. Many, particularly particularly pelagic, species have a relatively thick fluid layer be between the dermis and epidennis epidermis:: 5.0 5.0 tLm pm in the case oflarval of larval plaice (P. (P. platessa; Roberts et al., 1973). 1973). In addition, distances associated with diffusion across capillary walls and through the plasma should be taken into account. Even when this is is done, distances remain rela relatively small. Webb and Brett ((1972a) 1972a) measured a mean distance of 4.7 4.7 f.Lm pm from the surface of the skin to the center of of blood capillaries in embryos of of two species species of of viviparous seaperch (Rhacohilus (Rhacohilus vacca and Embioteca Iateralis). lateralis). Liem (1981) (1981) estimated 8-15 8-15 f.Lm pm for the total thickness of the water-blood water-blood barrier in larval Monopterus. Monopterms. These dis distances are considerably less than the cutaneous diffusion distances of of larval amphibians. amphibians. Burggren and Mwalukoma (1983) (1983) estimated a blood-water 20-50 f.Lm blood-water barrier of of 20-50 pm for larval bullfrog ((R. R . catesbeiana). catesbeiana). In this species up to 60% of of total gas exchange takes place across the skin (Burggren and West, 1982). 1982). The skin of of larval teleosts is probably at least as effective as an organ of gas exchange, given the shorter diffusion distances and, in many cases, more elaborate vasculariza vascularization. Cutaneous gas exchange in other vertebrates frequently suffers from a relatively small partial pressure gradient across the skin as a result of central mixing of oxygenated and deoxygenated blood prior to transit to the skin (Burggren, 1984). This problem appears to be (Burggren, 1984). minimized in many teleosts. Cutaneous gas exchange structures-for structures-for example, the caudal and rectal vascular systems of larval tilapia (Fig. (Fig. 5) 5) or the vitelline circulation of of salmonids-typically salmonids-typically receive blood that has already passed through at least a portion of of the systemic

74

PETER ROMBOUGH PETER J. ROMBOUGH

the made, the been made, have been measurements have situ measurements in situ no in Although no circulation. Although circulation. poor comparatively poor be comparatively should be structures should exchange structures the exchange entering the blood entering blood in oxygen thus maximizing maximizing the partial pressure gradient between the water. the water. and the blood and blood te of tephysiology of cardiovascular physiology the cardiovascular of the known of is known little is Extremely little Extremely effec more effecbe more would be obviously would exchange obviously Cutaneous exchange embryos. Cutaneous leost embryos. leost ( 1 979) Balon (1979) and Balon McElman and regulated. McElrnan be regulated. could be flow could blood flow if blood tive if tive capillary cutaneous capillary through cutaneous passing through blood passing of blood amount of the amount that the noted that noted vi walleye (Stizostedion (Stizostedion uiof walleye development of the development during the varied during beds varied beds shunting for shunting mechanism for implied aa mechanism this implied that this suggested that treum) and and suggested treum) result as aa result recruitment as gas exchange. optimize gas to optimize as to so as blood so blood exchange. Vascular Vascular recruitment dur rate durheart rate increased heart by increased blood pressure of higher of higher systemic systemic blood pressure caused caused by there However, there proposed. However, was proposed. hypoxia was physiological hypoxia of physiological periods of ing periods ing pat flow patblood flow in blood changes in ontogenetic changes between ontogenetic differences between be differences may be may oxygen in oxygen alterations in to transient due to changes due compensatory changes terns terns and and compensatory transient alterations perfusing blood perfusing of blood amount of the amount of the Reflex control demand. Reflex supply or supply or demand. control of recruit capillary recruitoccur, capillary well occur, may well structures may exchange structures cutaneous gas cutaneous gas exchange amphibian in amphibian demonstrated in been demonstrated has been hypoxia has to hypoxia response to in response ment in ment be demonstrated has yet 1984), but larvae larvae (Burggren, (Burggren, 1984), but it it has yet to to be demonstrated in in teleost teleost embryos embryos or or larvae. larvae. E. Respiratory Pigments E.

apAs mentioned previously, the stage at which hemoglobin first ap pears is highly variable. In many demersal species, such as salmonids, large numbers of pigmented erythrocytes are evident well before hatch, (Bahatch. This is thought to be an adaptation to hypoxic conditions (Ba 1975). In contrast, hemoglobin may not appear in the circulation lon, 1975). of pelagic species, such as herring, until after metamorphosis (De Silva, 1974). 1974). Lack of of hemoglobin has been proposed as a mechanism Silva, Howto limit predation by making pelagic larvae less conspicuous. How well-oxyever, it simply may be that hemoglobin is not required in well-oxy genated waters. Holeton ((1971) 1971) reported that rainbow trout larvae showed little distress when their hemoglobin was poisoned by carbon monoxide. Similarly, Iuchi (1985) (1985) reported that rainbow trout larvae survived to to the fry f.y stage after having their erythrocytes erythrocytes destroyed by treatment with phenylhydrazine. Indeed, it may not even be neces necessary for small larvae to have a functioning circulatory system. Burg Burggren (1984) (1984) points out that the so-called "cardiac “cardiac lethal" lethal” larval mutant of the amphibian Ambystoma, in which the heart forms forms but fails fails to beat, is able to survive after hatching for many days in well-oxygen well-oxygenated water.

2.


75

hemoglo The evidence to date indicates that embryonic and larval hemogloof juveniles bins are structurally and functionally distinct from those of and adults. Iuchi and Yamagami (1969) (1969) reported a gradual change in the electrophoretic banding pattern for the hemoglobins of of rainbow trout during the period between hatch and gravel emergence. Similar shifts in electrophoretic banding patterns have been observed in Ho Homasu salmon (0. (0.rhodurus) rhodurus) and brook trout (Salvelinus (Salvelinus fontenalis; Iuchi et al., 1975) 1975) and in coho salmon (0. (0. kisutch; Giles and Vanstone, 1976). Distinct embryonic and adult hemoglobins also have been rere 1976). Terwil liger, ported for several viviparous species (Ingermann and Tenvilliger, 1981a,b, 1982,1984; 1982, 1984; Ingermann et al., al., 1984; 1984; Weber and Hartvig, 1984; 1984; 1981a,b, 1984). Hartvig and Weber, 1984). ( 1973b) compared the chemicaI chemical and physiological properties Iuchi (197313) of larval and adult hemoglobins of of rainbow trout. Both were tetratetra of meric, but larval hemoglobin displayed a higher oxygen affinity, affinity, less of of a Bohr effect, virtually no Root effect, and greater cooperativity at physiological pH than adult hemoglobin. Larval and adult hemoglo hemogloof 31 31 mm Hg and 57.5 mm Hg, respectively, at pH bins had PSO P50 values of 25°C. The Bohr effect (A (a log PSdpH) P5o/pH) was 0.023 for larval hemohemo 7.2 and 25°C. of larval globin but 0.64 for adult hemoglobin. The oxygen capacity of hemoglobin was virtually unaffected by pH, while a drop to pH 6.5 of adult hemoglobins to 50% of of that at pH reduced the oxygen capacity of 8.0. Slopes S lopes of of Hill plots were n = 2.33 and n = 1.62, 1 .62, respectively, at 8.0. pH 7.2 for larval and adult hemoglobins. The high oxygen affinity and pH independence of larval hemoglobins are clearly advantageous to embryos and larvae exposed to the oxygen-poor, oxygen-poor, low-pH and high highCO2 C02 environments of of the perivitelline fluid and interstices of of the redd. The shift in electrophoretic banding patterns suggests that the of embryonic and larval blood is due primarily greater oxygen affinity of of the hemoglobins rather than to intrinsic differences in the structure of of modulators of of hemoglobin (Hb) (Hb) affinaffin to changes in concentrations of ity. This was shown to be the case for the viviparous eelpout Zoarces 1984; Hartvig and Weber, 1984). 1984). WeWe viviparous (Weber and Hartvig, 1984; (1984) reported that fetal hemoglobin had a higher 0O22 ber and Hartvig (1984) affinity affinity (P50 (Pm values of9 of 9 mm Hg and 23 mm Hg, respectively, at pH 7.5 and lOOC), 10°C), reduced Bohr effect, and greater cooperativity than adult hemoglobin in nucleoside triphosphate-free preparations. Measure Measureof intraerythrocyte nucleoside triphosphate triphosphate (NTP) (NTP) concentraconcentra ment of tions revealed no significant difference in the NTP/Hb ratios of fetal and adult blood. Differences in modulator concentrations, though, do appear to be important in some species. Ingermann and Terwilliger =

=

76

PETER PETER JJ.. ROMBOUGH

((1981b, 1981b, 1982, 1982, 1984) 1984) reported that part of the reason for the higher O2 0 2 affinity of the hemoglobin of fetal seaperch E E.. lateralis was a lower NTP/Hb ratio. ATP was the most abundant modulator modulator (82% (82% total NTP), 18% of total NTP), but there was also a significant amount of GTP ((18% evidence suggests NTP) present within fetal erythrocytes. Indirect evidence cofactors may modulate the O2 0 2 affinity of larval hemoglobins in some oviparous species species as well. DiMichele and Powers (1982) (1982) attributed of different lactate dehydrogenase dehydrogenase differences in hatching times of (LDH) (LDH) genotypes of Fundulus heteroclitus to the ability to deliver oxygen to tissues. The LDH genotype (LDH BaBa) that hatched (LDH B"B") hatched earli earliest was also the genotype that had the highest concentration of ATP in their erythrocytes as adults, and presumably as embryos. Increased ATP concentrations would lower hemoglobin O 0 22 affinity and thus re reperivi duce O 0 22 delivery to tissues near hatch when P02 PO2 levels in the perivitelline fluid are low. This would trigger release of the hatching en enzyme and lead to early hatch. Iuchi and Yamamoto (1983) em (1983) demonstrated that the shift from embryonic-larval bryonic-larval hemoglobins to juvenile hemoglobins in rainbow trout was the result of erythrocyte replacement. Erythrocytes containing embryonic hemoglobins were formed in the extraembryonic blood intermediate cell mass beginning about one-third through islands and intermediate producembryonic development. development. These hemopoietic centers ceased produc tion shortly after hatch, and centers in the kidney and spleen began to produce morphologically and antigenically distinct erythrocytes con containing juvenile-type hemo juvenile-type hemoglobins. Similar shifts in the site of of hemopoiesis have been reported for Atlantic salmon (Vernidub, 1966) 1966) and anglefish Pterophyllum scalare (AI-Adhami (Al-Adhami and Kunz, 1976). 1976). Hemo Hemoglobin switching based on erythrocyte replacement replacement may turn out to be widespread in lower vertebrates. Kobel Kobe1 and Wolff (1983) (1983) recently re reported that the shift from embryonic embryonic to larval type hemoglobins in the amphibian Xenopus borealis also was associated with a shift in the site of erythropoiesis. It has been suggested that pigments other than hemoglobin may be involved in respiratory gas exchange. De Silva ((1974) 1974) noted that myoglobin was formed prior to hemoglobin in herring larvae and indi indicated that this may refl ect a respiratory role during early development. reflect This hypothesis has not been tested. tested. Other authors have proposed a respiratory role for the carotenoid pigments (Smirnov, 1953; 1953; Volodin, 1956; Balon, 1975, 1975, 1984; 1984; Mikulin and Soin, Czec1956; Soin, 1975; 1975; Soin, 1977; 1977; Czec zuga, 1979). 1979). This hypothesis is is based largely on circumstantial evi evidence, namely, that eggs of species containing high concentrations of carotenoids are often relatively large and, thus, are faced with rela-

2.


77

tively large diffusion distances gas exchange is com distances.. The problem of gas compounded for some of these species by the fact that they must develop in low oxygen in comparatively comparatively low oxygen environments. environments. Unfortunately, Unfortunately, there there is is little experimental evidence to indicate that carotenoids actually aid in oxygen transport under such conditions. In fact, con fact, there is now considerable evidence indicating indicating that that low carotenoid carotenoid levels in in eggs, eggs, nor normally rich cantly reduce mally rich in in carotenoids, carotenoids, do do not not signifi significantly reduce survival survival (Steven, (Steven, 1949; 1949; Craik, 1985; 1985; Craik and Harvey, 1986; 1986; Tveranger, 1986). 1986). Craik (1985) (1985) speculates that carotenoids may play some minor, as yet unde undefined, fined, role role in in oxygen oxygen transport transport but but that that present present evidence evidence does does not not warrant warrant acceptance acceptance of of carotenoid-based carotenoid-based respiration respiration as as an an established established fact, 1975, 1984) fact, as as Balon Balon ((1975, 1984) would would suggest. suggest. We We have have seen seen how how the the movement movement of of water water past past the the egg egg and and stirring stirring of embryonic gas of the the perivitelline perivitelline fluid fluid enhance enhance embryonic gas exchange. exchange. Adequate Adequate ventilation of body surfaces is similarly necessary for efficient gas exchange after hatch. Fish have adopted a number of tactics to ensure that this occurs. Some, such as nest-fanning, mouth-brooding, and occurs. Some, wriggler-hanging, involve the parents. Others involve behavioral and physiological adaptations on the part of the larvae. Hunter 1972) noted noted that (Engrulis mor morHunter ((1972) that newly newly hatched hatched anchovy anchovy (Engralis dax) dux) exhibited regular bouts of swimming that did not appear to be associated with feeding or predator avoidance. avoidance. He suggested that enswimming might have a respiratory significance, presumably by en hancing hancing ventilation ventilation of of the the body body surface. surface. This This hypothesis hypothesis was was exam examined in some detail Weihs (1980) pointed out Weihs (1980, (1980, 1981). 1981). Weihs (1980) pointed out ined in some detail by Weihs that that because of of their their small small size, size, anchovy anchovy larvae larvae existed existed in in aa viscous viscous environment Re). Consequently, environment (Le. (i.e.,, low low Reynolds Reynolds number, number, Re). Consequently, both both the the larva tend to transported together larva and and its its immediate immediate surroundings surroundings tend to be be transported together by oceanic would remain oceanic currents currents.. A nonswimming nonswimming larva larva thus thus would remain in in the the same oxygen. Weihs same mass of of water water and and gradually gradually deplete deplete the the available available oxygen. Weihs (1980) (1980) developed developed aa mathematical mathematical model model for for diffusive diffusive oxygen oxygen uptake uptake by the the larvae larvae and and estimated estimated that that oxygen oxygen would would become become limiting limiting for for aa stationary saturation stationary day-old day-old larva larva at at concentrations concentrations below below 63% 63% air air saturation (ASV). (ASV). He He then then tested tested this this prediction prediction by by observing observing larval larval swimming swimming behavior As predicted, predicted, both both the the fre frebehavior at at various various oxygen oxygen concentrations. concentrations. As quency quency and and duration duration of of swimming swimming significantly significantly increased increased at at oxygen oxygen levels (Fig. 6). 6). levels below below 60% 60% ASV (Fig. Weihs' Weihs’ (1980) (1980) study study indicates indicates that that the the limiting limiting step step in in cutaneous cutaneous gas least in gas exchange, exchange, at at least in anchovy anchovy larvae, larvae, is is the the convective convective flow flow of of water remembered that that an anwater past past the the exchange exchange surface. surface. It It should should be remembered chovy chovy larvae larvae live live in in aa comparatively comparatively oxygen-rich oxygen-rich environment. environment. It It would even more more important important for for larvae larvae inhabiting inhabiting would thus thus appear appear to to be even

78


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Fig. Fig, 6. 6. Fraction Fraction of of time time spent actively actively swimming as as aa function function of ambient oxygen oxygen 1) northern anchovy concentration concentrationfor newly hatched (day (day 0) 0) and 24-h-old 24-h-old (day (day 1) anchovy larvae. larvae. [From [From Weihs (1980).] (1980).1

hypoxic environments to be able to generate large convective flows. This is accomplished in larvae of the Austrailian lungfish (Neocera (Neocerutodes forsteri) by means of cilia that direct a water current posteriorly todesforsteri) of many along the body surface (Whiting and Bone, 1980). 1980). Larvae of warm-water teleosts use their pectoral fins to create a similar water flow. Liem ((1981) 1981) reported that larvae of the air-breathing fish Monop flow. Monopterus albus use large, well-vascularized pectoral fins to direct a flow of of relatively oxygen-rich surface water backward along their body sur surface. face. The yolk sac and caudal region of Monopterus larvae are also extensively vascularized. vascularized. Microscopic observations indicated that sur surficial blood flow in these regions ran in the opposite direction to the flow of water generated by the pectoral fins. fins. When larvae were placed in a tube with water fl owing in the normally anterior to posterior flowing 41%.When the direction the oxygen extraction efficiency was about 41%. direction of water fl ow was reversed, extraction efficiency dropped to flow (Fig. 7). about 20% (Fig. 7). Liem ((1981) 1981) pointed out that on this basis the whole larva could be considered a functional analog of the gill iamel lamelfish. He noted that similar elaborate vascular networks and lae of adult fish. mobile pectoral fi ns were common in larvae of other species inhabit fins inhabiting hypoxic waters and speculated that such countercurrent flow mechanisms might be widespread. It should be noted, though, that pectoral fin movements are not always always associated with ventilating cutaneous gas exchange surfaces. Peterson (1975) (1975)found that, contrary to his expectations, the rapid pectoral fin movements of Atlantic salmon alevins did not direct water over the well-vascularized yolk sac. Instead, they appeared to be involved in drawing water over the sac. gills. gills.

2.

RESPIRATORY RESPIRATORY GAS GAS EXCHANGE, EXCHANGE, AEROBIC AEROBIC METABOLISM METABOLISM

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Fig. Fig. 7. (A) Larval Monopterus albus (4 days old) old) showing (1) (1) large vascular pectoral pectoral (2) well-developed network in the yolk sac, and (3) fins, (2) well-developed capillary network (3) well-vascularized well-vascularized generated by moveunpaired medial fin. (B) (B) Schematic representation of of water currents generated move ment of of pectoral pectoral fins. The stippled area shows the region from which water is drawn by water flowing the pectoral fins. (C) (C) The effectiveness of of oxygen extraction from water flowing in an anterior to posterior direction (countercurrent to blood flow) flow) or in a posterior to anterior direction (concurrent to blood flow). 130 ml I-’. I-I. (Reprinted with flow). The flow rate was 130 1981; copyright 0 © AAAS.) permission from Liem, 1981;

F. Branchial Gas Exchange The larval period is characterized by a shift in the site of respira respiratory gas exchange from the skin to the gills. Unfortunately, relatively little is is known of the physiology of this transition. It is known that species vary in the stage at which gills first appear and the speed with which they are elaborated. For example, gill arches begin to form in rainbow trout shortly after gastrulation and by hatch are complete with functional filaments and secondary lamellae (Morgan, (Morgan, 1974a,b). 1974a,b). Arctic char (Salvelinus alpinus) laments at hatch but alpinus) also possess fi filaments secondary lamellae do not begin to form until about 8 days posthatch (at 6.5°C; 6.5”C; McDonald and McMahon, 1977). 1977). In smallmouth bass (Mi (Micopterus dolomieu), dolomieu), filaments do not appear until about 7 days post posthatch (at (at 16°C; 16°C; Coughlan and Gloss, Gloss, 1984). 1984). Lamellae begin to form about 4 4 days days later, about the time the larvae larvae becomes free-swimming� free-swimming. Filaments and secondary lamellae first appear on gill arches of herring and plaice larvae about midway through the larval period, at body lengths of 10 10 and 8 mm, respectively (De (De Silva, Silva, 1974). 1974). Such description provides relatively little information about the relative importance of the gills in larval gas gas exchange. This question

PETER PETER J. J. ROMBOUGH ROMBOUGH

80

can be answered answered best best by by directly directly measuring measuring gas fluxes across across the the gills gills can gas fluxes and skin. This This has has been been done done for for amphibian amphibian larvae larvae (Burggren (Burggren and and skin. and West, 1982; Burggren, Burggren, 1984) 1984) but but not not for for teleost teleost larvae, larvae, even even though though West, 1982; the techniques techniques developed developed for for amphibians amphibians would would be relatively easy to to relatively easy the apply the absence parti apply to to some some of of the the larger larger fish fish larvae. larvae. In In the absence of of such such partitioning gills and and skin skin in in fish fish larvae larvae tioning studies, studies, the the relative relative importance importance of of gills must be inferred amount of of information information availavail inferred from from the the rather rather limited limited amount must able morphometrics of the two mentioned previ previable on on the the morphometrics of the two structures. structures. As mentioned ously, the the factors factors of of particular particular importance importance in in determining determining the the effieffi ously, ciency of of gas exchange structures structures are are total total surface surface area, area, diffusion diffusion ciency gas exchange distance, partial partial pressure pressure gradients, gradients, and and the convective supplies of distance, the convective supplies of blood and and water. water. blood (1974) reported reported that, at hatch, the gills of of both herring and De Silva (1974) plaice accounted accounted for for an an insignificant insignificant portion portion (IE

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Fig. (Oncorhynchus tshawytscha) Fig. 8. 8. Metabolic Metabolic intensities intensities of chinook chinook (Oncorhynchus tshawytscha) embryos embryos and and larvae constant temperatures. larvae incubated at constant temperatures.

PETER PETER JJ.. ROMBOUGH ROMBOUGH

92

existing embryos compensate existing tissues tissues and and that that embryos compensate for for the fact fact that that the the capsule 1957, 1958) 1958) capsule limits limits oxygen oxygen uptake uptake by by reducing reducing growth. growth. Smith Smith ((1957, demonstrated demonstrated that that in in both both brown brown trout trout and and Atlantic Atlantic salmon salmon metabolic metabolic intensity intensity was was directly directly correlated correlated with with specific specific growth growth rate. rate. There sharp increase increase in in metabolic metabolic intensity intensity on on hatch hatchThere is is typically typically aa sharp this increase increase can ing. As case for V 02, 0 2 , this can be attributed attributed to to removal removal ing. As was was the the case for V of the the capsule capsule and and perivitelline perivitelline fluid fluid as as barriers barriers to to diffusion diffusion and and to to 1964; Davenport enhanced enhanced activity activity (Holliday (Holliday et al., 1964; Davenport and and L�nning, Lgnning, 1980; 1983; Davenport, Davenport, 1983). 1983). Patterns Patterns after after hatch hatch 1980; Gruber Gruber and and Wieser, Wieser, 1983; 021M (routine) are V02/M (routine) are variable. variable. Wieser Wieser and and Forstner Forstner (1986) (1986) reported reported that that V began decline exponentially species of of began to to decline exponentially almost almost immediately immediately in in three three species cyprinids cyprinids (Rutilis (Rutilis rutilis, Scardinius erythrophthalmus, Leuciscus decline in cephaIus). DeSilva et al. al. (1986) (1986) noted noted aa similar similar decline in the the nile nile cephalus). DeSilva tilapia (Oreochromis niloticus). niloticus).A A more more common common pattern pattern is is for for specific specific tilapia (Oreochromis oxygen oxygen consumption consumption to to continue continue to to increase increase until until about about midway midway through period of endogenous endogenous feeding. feeding. This This pattern pattern has has been been through the the period reported for aa number number of of sal�onids salmonids (Smith, (Smith, 1957, 1957, 1958; 1958; Gruber Gruber and and reported for Wieser, Wieser, 1983; 1983; Wieser Wieser and and Forstner, Forstner, 1986; 1986; Rombough, Rombough, 1987), 1987), sardine sardine (Lasker 1964), cod cod (Lasker and and Theilacker, Theilacker, 1962), 1962), herring herring (Holliday (Holliday et al., 1964), (Davenport 1983).In In (Davenport and and L�nning, LZnning, 1980), 1980), and and lumpfish lumpfish (Davenport, (Davenport, 1983). Oz/M declined most of studies the fed, so so that that V VOdM declined most of these these studies the larvae larvae were were not not fed, rapidly rapidly once once endogenous endogenous food food supplies supplies became became limiting. limiting. For For fed lar larvae, vae, specific specific oxygen oxygen consumption consumption may may continue continue to to rise rise until until metamor metamorphosis relatively stable 1983), remain remain relatively stable (Holeton, (Holeton, 1973), 1973), phosis (Forstner (Forstner et al., 1983), or or decline decline gradually gradually (DeSilva (DeSilva and and Tytler, Tytler, 1973), 1973), depending depending on on the the species. species.

2. MASS MASSRELATIONSHIPS RELATIONSHIPS Some investigators, rather than reporting mass-specifi mass-specificc rates of ox oxygen consumption, have chosen to express the relationship between V V 02 0 2 and body mass (M) ( M ) in terms of the allometric power functiOII functiorl

V V 02 0 2

b = aM aMb

(6) (6)

=

where a and b are constants. Such allometric relationships have long princiinterested physiologists, although the underlying biological princi ples remain rather rather obscure. Expressing oxygen consumption in this potentially important fluctuations in fashion has the disadvantage that potentially V V 02 0 2 tend to be obscured by the overall trend. Nonetheless, the rela relationship is widely used, particularly for larvae. [b,Eq. (6)] (6)] calculated for larvae are The metabolic mass exponents [b, often Winberg's (1956) often compared compared to to Winberg’s (1956) generalized generalized value value of b = 0.80. 0.80. =

2. 2.


93 93

However, However, Winberg's Winberg's value was based primarily on studies involving juvenile and adult fish, fish, and there is no reason to assume that the same exponent applies to exponent applies to earlier earlier life life stages stages.. Indeed, Indeed, ontogenic ontogenic variations variations in in the value of the metabolic mass exponent are widespread throughout the animal kingdom (Zeuthen, 1970; Adolph, 1983; 1950, 1970; 1983; Wieser, (Zeuthen, 1950, 1984). 1984). In virtually all cases, values decrease as development pro proceeds. ceeds. This generalization appears to hold for many fish species. Kamler (1976) (1976) recognized four periods during the ontogeny of carp on the basis of different metabolic mass exponents: embryos, prefeeding larvae, feeding larvae, and postlarvae. Values were not calculated for embryos and prefeeding larvae, but graphic presentation of the data indicates cantly greater .0. indicates that the the mass mass exponents exponents were were signifi significantly greater than than 11.0. The mass mass exponent exponent of of feeding feeding larvae larvae was was approximately approximately unity unity (b while that (b = = 0.97) 0.97) while that of of postlarvae postlarvae was was typical typical of of juvenile juvenile and and adult adult fi sh (b (b= = 0.80). 0.80). fish High High mass mass exponents exponents during during early early life life appear appear to to be widespread, widespread, 16 species for although not universal, among teleosts. Table I lists 16 which the larval mass exponent approached or exceeded unity. In addition, inferred for for larvae larvae of of addition, mass mass exponents exponents close close to to unity unity can can be inferred brown trout (Gray, 1926; Wood, 1932), 1932), Pacifi Pacificc sardine sardine (Sardinops (Gray, 1926; caerule; Lasker and Theilacker, 1962), 1962), cod (Davenport and L�nning, L@nning, caerule; 1980), Pacific mackerel (Scomber (Scomber japonicus; Hunter and Kimbrell, 1980), 1980), 1980), and and largemouth largemouth bass bass (Micropterus (Micropterus salmoides; Laurence, Laurence, 1969) 1969) from the way in which metabolic intensity varies during development or from graphs showing 02 and M. VOZ M . Many showing the relationship between \1' investigators, investigators, however, however, have have reported reported metabolic metabolic mass mass exponents exponents for for are not significantly different from those expected for juve juvelarvae that are I). Not all such values, though, can be taken (Table I). nile and adult fish (Table VOZ M dur duras accurately representing the relationship between V 02 and M differening much of early life. In most cases no attempt was made to differen tiate between data collected for prefeeding and feeding larvae. Cetta (1982) point out that this practice tends to mask any and Capuzzo (1982) stage-specific differences in the value of the mass exponent. It will actuprobably turn out that in many cases larval mass exponents are actu ally considerably higher prior to exogenous feeding feeding than literature ally suggest. For example, example, careful examination of data pre prereports would suggest. that V VOZ sented by Laurence ((1978) sented 1978) indicates that 02 tended to increase more rapidly with tissue mass for very young cod and and haddock haddock larvae than it larvae. In the one case case in which calculations were made did for older larvae. using only data data for small small larvae larvae (haddock, (haddock, 4°C), P C ) , it was found that the mass (b = = 1.267). 1.267). In In mass exponent exponent was was significantly significantly greater greater than than unity unity (b contrast, data for for all all larval larval stages stages were were combined, combined, mass mass contrast, when when the data

Table Table II Rate-Mass Relationships of of Fish Larvae" Larvae" Metabolic Rate-Mass V02 = aM*) aMb) Allometric Allometric relationship ((VO, =

Species Species

co .a;...

Temp. (0C) ("C)

-

Stage Stage

Size Size range

aa

V vo, 02 units

b

2 1.0 1.0 b� ILl pl h-1 h-'

Cyprinus carpio carpio Cyprinus

20

L

< 6674 mg 74m g

0.64

0.97

Cyprinus carpio carpio Cyprinus Cyprinus carpio carpio Cyprinus

20 20

F L L

Cyprinus carpioh Cyprinus carpiob Abramis Abramis brama brama Anabas testudiAnahas testudineus neus Channa punctatus punctatus Channa Coregonus Coregonus sp. sp. b Acipenser haeri baeri Acipenser

23 20 28

L L L

1.2-4.6 g 1.2-4.6 < ll g ' l lg g 2-70mg 2-70 mg 1.6-40 mg 1.6-40mg 5-50 5-50 mg

1.27 0.60 0.60 1.27 1.27 1.56 1.56 0.45 1.00 1.00

0.80 0.95 0.98 0.98 0.80 0.80 0.054 -0.054 0.93 0.95

28 10 10 20

Saloelinus alpinus alpinus Salvelinus Salmo gairdneri Salmo gairdneri

2 4

L L L F L L-F L-F

Salmo Salmo gairdneri gairdneri

12 12

L-F L-F

1-10 mg 1-10mg 6-65 mg 6-65mg 111-28 1-28 mg 111-390 1-390 mg 50-124mg 50-124 mg 80 mg-7 g 80mg-7g mg-7 g 80 mg-7 80 mg-7 g 80mg-7g

1.00 14.8 14.8 2.4 0.35 2.45 3.1 3.1 111.0 1.0 6.8 19.9 19.9

Clupea harengus harengus Clupea Clupea Clupea harengus harengus

8 8 8

0. 1-0.7 mg 0.1-0.7 dry dry

11.19 . 19

0.91 0.12 1.31 1.31 0.85 1.09 1.09 0.96 11.11 .11 0.93 11.14 . 14 11.1 .1 0.74

L L

M M units mg wet

Comments

Referencec Referencec

Slope signifi cantly different significantly for small and large larvae

11

ILl pl h-1 h-' pl h-1 h-' ILl

mg wet mg wet

mg g-'h-' g-1 h-1 ml hh-'1 h-' mg h-1

mg wet g wet g wet

V 02lM recalculated VO&f

h-' mg h-1 ILmol pmol h-1 h-I g-1 g-' h-' mg h-1

g wet mg wet g wet

mg hh-11 ILmol pmol h-1 h-'

g wet g wet

ILmol -1 pmol h h-1

g wet

p1l h-1 h-' IL ILl pl hh-'1

mg mg dry dry mg mg dry dry

Recalculated V VOdM, 02lM, to metamorphosis Prolarvae Feeding larvae Yolk-sac larvae V02 rrVO2 aV02 V02 rrVO2 V02 aVO, a Hatch to MLDW

Recalculated

avo,

11 2 3 3 3 4 5 5 6 7 8 9

10 10 111 1 111 1

12 12 13 13

13 13

L L

18 18

L L

88

L L

13 13

L

18 18

L

Pseudopleuroneetes Pseudopleuronectes amerieanus americanus

7 7

L L

Melanogrammus aeglefinus aeglefinus

4

L

7 7

L

9 9

L L

Anehoa mitehilli Anchoa mitchilli

26

L L

Aehirus lineatus Achirus lineatus

26

L L

HypophthalmiehHypophthalmichthys thys molitrix molitrix Sparus Sparus aurata aurata

20

L L

19 19

L L

24

L

20

L

Pleuroneetes Pleuronectes platessa platessa

CD tit

Stizostedion lucioperea perca

0.1-0.7 mg 0.1-0.7 dry dry 0.1-0.7 mg 0.1-0.7 dry dry 0.7- 1 .2 mg mg 0.7-1.2 dry dry 0.07-1.2 mg 0.07-1.2 dry dry 0.07-1.2 mg 0.07-1.2 dry dry 7-10 7-10 pg ILg protein protein (dry) (dry) 70-200 mg 70-200 dry dry 50-1000 mg 50-1000mg dry dry 50-1000 mg 50-1000mg dry dry 8.9-424 8.9-424 lL pgg dry dry 14.3-248 14.3-248 ILg pg dry dry 1-50 mg 1-50mg

3.63 3.63

1.33 1.33

3.33

0.87

1.21 1.21

0.78 0.78

2.20

0.96

3.29

0.74

0.006 0.006

28-1000 ILg 28-1OOOpg dry dry 28-1OOOpg 28-lOOO lLg dry dry

0.8-79 mg 0.8-79

ILl h-' h-1 pl

mg dry dry mg

13 13

1.03 1.03

p 1 h-' ILl h-1

pg protein ILg (dry) (dry)

14

0.0053

1.27 1 .27

p I h-' ILl h-1

pg dry ILg

Small larvae only

15 15

0.071 0.071

0.68

0.179

0.55 0.55

0.0077

0.98

ILl h-' h-1 pl

pg dry ILg

Feeding larvae

16 16

0.014

0.94

ILl pl h-1 h-'

pg dry ILg

Feeding larvae

16 16

0.33 0.33

LOS 1.05

ILl p1 h-1 h-'

mg wet

Recalculated Recalculated

17 17

7.09 7.09

1.00 1.00

ILg pg h-1 h-'

mg mg dry dry

18 18

6.47 6.47

11.03 .03

0.31 0.31

0.82

g wet

5

5 0.8 b :5 h-* ml h-1


Table I (Continued) (Continued) V02 = aMb) aMb) Allometric relationship ((VO, =

Species

Perca jluviatilis Perea Rutilis rutilis Heteropneustes H eteropneustes fossilis fossilis Morone saxatalis saxatalis Morone calbasu Lebeo ealbasu Oreoehromis Oreochromis niloticus nilotieus Clupea Clupea harengus harengus Pleuroneetes Pleuronectes platessa platessa Pseudopleuroneetes Pseudopleuronectes americanus amerieanus Gadus Gadus morhua morhua

Arehosargus Archosargus rhomboidalis rhomboidalis

Temp. OC) (("C)

Stage

Size range

aa

b

V 30, 02 units

M units

20 20 20 28

L L L

1.5-32 mg 1.5--32 1.6-62 1.6-62 mg 2-20 2-20 mg

0.29 0.29 0.29 1.00 1.00

0.78 0.82 0.88 0.88

h-I1 ml hh-'- I ml h mg h h-'-1

18 18 28 30

E-L E-L L F

50-1500 50-1500 p. pg 100-300 100-300 mg 1-10 1-10 mg?

0.028 0.028 0.50 9.68 9.68

0.72 0.84 0.42 0.42

p l hh-'I p. -I mg h h-' pl.l hh-'I p

g dry p. dry Pg g wet mg mg dry dry

10 10 10 10

L L

0. 1-10 mg 0.1-10 0.5-10 0.5-10 mg

1.88 1.88 1.67 1.67

0.82 0.65

pIl hh-'1 p. p1l h h-'- I p.

mg dry mg mg dry dry

7

F

0.016

0.78 0.78

pll h h-'- I p.

44 7 10 10 26

L L L L

8-150 p. pg g 8-150 protein 50-1000 p. 50-1000 pg 50-1000 50-1000 p. pgg 50-1000 pg 50-1000 p. 18-66 18-66 p. pg

0.018 0.018 0.017 0.017 0.054 0.054 0.018 0.018

0.71 0.71 0.78 0.69 0.84 0.84

pll h h-'- 1 p.

g protein pg p. dry dry p.g dry dry

-I pll h h-' p.

p.g dry

Comments

Referencec

5 5 19 19

g wet g wet g wet

Recalculated

Recalculated Recalculated

20 2211 22 22 23 23 23 23 14 14

15 15 115 5 15 15 16 16

Abbreviations: L, larvae; F, fry; fry; MLDW, maximum larval dry weight. wejght. VO�M) and tissue mass. b Regression describes relationship between metabolic metabolic intensity ((VOdM) References: (1) (1) Kamler, 1976; 1972; (4) (4) Kaushik and Dabrowski, 1983; 1976; (2) (2)Winberg and Khartova, 1953 1953(cited in Kamler, 1976) 1976) (3) (3) Kamler, 1972; 1983; (5) (5) Kudrinskaya, 1969; (9) Khakimullin, 1985; 10) Holeton, 1973; 1973; (11) (11) 1969; (6) (6) Mishra and Singh, 1979; 1979; (7) (7) Singh et et al., 1982; 1982; (8) (8)Forstner et et al., al., 1983; 1983; (9) 1985; ((10) 1985; ((12) 1966; ((13) 1984; ((14) 1978; ((16) Wieser, 1985; 12) Blaxter and Hempel, 1966; 13) Almatar, 1984; 14) Cetta and Capuzzo, 1982; 1982; ((15) 15) Laurence, 1978; 16) Houde and Schekter, 1983; ((17) 17) Mukhamedova, 1977; 18) Quantz and Tandler, 1982; 1982; ((19) 19) Sheel and Singh, 1981 (20) Eldridge et 1982; (21) (21) Durve and Sharma, 1983; 1977; ((18) 1981;; (20) et al., 1982; 1977; (22) al., 1986; 1986; (23) 1977; (22) DeSilva et et al., (23) De Silva and Tytler, 1973. 1973. a

e

2. 2.

RESPIRATORY GAS EXCHANGE, AEROBIC METABOLISM RESPIRATORY GAS EXCHANGE. AEROBIC METABOLISM

97

exponents were found to be typical of of those seen in much older fish (b (b = = 0.55-0.78). 0.55-0.78). It It should should be recognized, recognized, however, however, that that mass mass exponents exponents are not always high, even for very young larvae. For example, a careful (1973) showed the metabolic mass expo expostudy by DeSilva and Tytler (1973) nents of both herring and plaice larvae to be considerably less than (0.82and 0.65, respectively). The reason why some species have unity (0.82 such relatively low mass exponents as larvae has not been established. Several Several reasons reasons have have been been advanced advanced to to explain explain high high metabolic metabolic (1976) suggested mass exponents during early development. Kamler (1976) that the transition from the high larval value to the lower juvenile value in carp was the result of of morphological changes affecting gas exchange. Pauly (1981). (1981).Pauly Pauly (1981) (1981) exchange. This This idea idea was was expanded expanded upon upon by Pauly proposed proposed that that the the underlying underlying factor factor limiting limiting metabolic metabolic rate rate and and hence hence growth was the the availability availability of of oxygen, oxygen, rather rather than than food food or or some some growth in in fish was intrinsic intrinsic control control mechanism. mechanism. He He reasoned reasoned that that if if oxygen oxygen was was not not limit limiting, ing, metabolic metabolic rate rate should should be be directly directly proportional proportional to to body body mass. mass. To To support support his his contention contention that that oxygen oxygen is is not not limiting limiting during during early early life, life, he noted noted that that gill surface surface area area of of larval larval herring herring and and plaice plaice increased at at aa much faster body mass mass exponents and much faster rate rate than than body mass (gill (gill area area mass exponents of of 3.36 and 1.59, respectively; De Silva, Silva, 1974). 1974). This was rather an unfortunate 1.59, choice larval metabolic metabolic choice of of species, species, since since in in both both herring herring and and plaice the larval mass exponent is significantly less than unity (0.82 (0.82 and 0.65, 0.65, respec respectively; 1973). This tively; De De Silva Silva and and Tytler, Tytler, 1973). This does does not not necessarily necessarily disprove disprove Pauly's Pauly’s basic basic hypothesis hypothesis (i.e., (i.e., that that the the ability ability to to supply oxygen oxygen to to tissues metabolic rate) tissues limits limits metabolic rate) but but indicates indicates that that more more sophisticated sophisticated analysis of total exchange capacity, perhaps similar to that done by Ultsch 1973) for is required required to adequately test for lungless lungless salamanders, salamanders, is to adequately test Ultsch ((1973) the hypothesis. As discussed earlier, cutaneous gas exchange is ex extremely must be included included in in any any analysis analysis of of tremely important important in in larvae larvae and and must diffusing diffusing capacity. capacity. In In both both herring herring and and plaice plaice cutaneous cutaneous surface surface area area expands body mass mass (mass 0.58 and and expands at at aa slower slower rate rate than than body (mass exponents exponents of of 0.58 0.50, capacity 0.50, respectively; respectively; De De Silva, Silva, 1974). 1974). Thus, Thus, the the total total diffuSing diffusing capacity of these larvae, larvae, in about the of these in fact, fact, may may be be expanding expanding at at about the same same rate rate as as metabolism. must be pointed pointed out out that that not not all all investigators investigators agree agree metabolism. It It must with this In particular, Itazawa ((1985) 1985) re rewith this hypothesis. hypothesis. In particular, Oikawa Oikawa and and Itazawa cently show no cently presented presented data data for for carp carp that that they they contend contend show no direct direct rela relaresting metabolism. tionship tionship between between respiratory respiratory surface surface area area and and resting metabolism. Forstner (1983) attributed attributed the the high high mass mass exponent exponent of of corego coregoForstner et al. (1983) muscle and nid to aa preponderence preponderence of of red red muscle and concommitant concommitant high high nid larvae larvae to activity mass activity of of oxidative oxidative enzymes. enzymes. The The decline decline in in the the value value of of the the mass exponent exponent following following metamorphosis metamorphosis was was attributed attributed to to aa major major reorganireorgani-

98


zation of metabolism in which glycolytic activity becomes progresprogres sively more important (Forstner et al., 1983; 1983; Hinterleitner et al., 1987). 1987). There are, however, major differences among species in the developmental enzymes (Hin developmental trajectories trajectories of of oxidative oxidative and and glycolytic glycolytic enzymes (Hinterleitner et al., d.,1987), 1987), and it is difficult to believe that high oxidative enzyme enzyme activity activity per per se se is is sufficient to to explain explain high high mass mass exponents exponents early salmonids generally early in in life. life. For For example, example, salmonids generally display display high high mass mass ex exponents least the yolksac stage ponents up up to to at at least the end end of of the the yolksac stage (Table (Table I, Wieser Wieser and Forstner, 1986; 1986; Rombough, 1987), 1987), yet activity is borne almost entirely 1983), and and levels levels of of oxida oxidaentirely by by white white muscle muscle (Forstner (Forstner et al., 1983), tive tive enzymes are are very very low (Hinterleitner (Hinterleitner et al., 1987). 1987). Quantz and Tandler ((1982) 1982) attributed the high metabolic mass ex exponent of larval gil thead seabream (Sparus gilthead (Sparus aurata) aurata) to their high feed feeding rate. They speculated that since the larvae were feeding more or less continuously, they should have a high specific dynamic action (heat (heat increment). increment). Noting Noting that that juvenile juvenile and and adult adult fish had had aa mass expo exponent near unity for active metabolism (Brett and Groves, 1979), 1979), they suggested that it was not surprising that mass exponents for larvae were were of of aa similar similar magnitude. magnitude. The allometric equations relating \102 VO, and M M frequently have been used to compare metabolic levels of different species or ecological groupings. Konstantinov (1980) (1980) points out that such comparisons are only valid if weight exponents are equal. equal. This severely restricts the use of such equations. Metabolic weight exponents are seldom the same species, values for b vary same for different species. species. Even in the same during development uenced by such as during development and and are are infl influenced by factors factors such as tempera temperature (Laurence, (Laurence, 1978; 1978; Konstantinov, 1980; 1980; Almatar, Almatar, 1984) 1984) and activity (De Silva and Tytler, Tytler, 1973; 1973; Wieser, 1985). 1985).

ACTIVITY 3. ACTIVITY As for juveniles and adults (Brett, (Brett, 1970), 1970), activity is the single most important factor influencing the metabolic intensity of of larvae (Blaxter, (Blaxter, 1969). 1969).The relationship between activity and oxygen consumption has ' been inves tigated in some detail for juvenile and adult fi sh (Beamish, investigated fish (Beamish, 1964, 1964, 1978; 1978; Brett, 1964, 1964, 1970, 1970, 1972; 1972; Brett and Groves, 1979; 1979; Fry, 1971). 1971). Yet only recently has there been much interest in the earlier life stages, although there are compelling reasons to suspect that ener energetic relationships may be significantly different from those of of older fish. The body musculature and supporting metabolic machinery de fish. develop gradually during the embryonic and larval periods and in many species do not assume typical juvenile patterns until after metamor-

2. 2.


99

1982; Forstner et al., ai., 1983; 1983; Batty, 1984; 1984; Wieser et phosis (Johnston, 1982; ai., 1985). 1985). In addition, juveniles and larvae are exposed to different al., hydrodynamic regimes at routine swimming speeds. In larvae, routine activities occur in an intermediate hydrodynamic environment where both resistive and inertial forces are important (Webb (Webb and Weihs, 1986). 1986). As larvae grow or or during burst swimming they move into the adult hydrodynamic regime, where inertial forces dominate. In many yle species this transition is accompanied by a change in swimming st style from continuous movement of of body and tail to a beat and glide pattern (Hunter, (Hunter, 1972, 1972, 1981; 1981; Weihs, 1980; 1980; Batty, 1984). 1984). To further confound matters, there are species-specific differences in development of of mus muscle types and biochemical pathways (Wieser et al., 1985; 1985; Hinterleitner Hinterleitner et al., ai., 1987). 1987). The study of of the physiological energetics of of the early life stages has been hampered by the lack of clear defi nitions for the various definitions levels of metabolism associated with activity. In juvenile and adult fish, fish, energy expenditure is is normally described in terms of standard V0 2 ) and active (a V0 2), routine (r V02) metabolism (see (s (sVO~), (rVO2) (aVO2) (see Brett and 1979, for definitions). Groves, 1979, definitions). These terms have been applied to to the early stages, but it should be noted that conditions conditions are somewhat different during early life. In older fish fish standard metabolism refers to the postabsorptive state, a condition that is obviously not met during endogenous feeding. feeding. In addition, the standard metabolic rate of em embryos and larvae includes a sizeable sizeable growth component so that, unlike in older fish, fish, metabolic rates rates can be depressed considerably below the so-called standard level without affecting survival. survival. What most investi investigators gators consider standard metabolism during early early life is actually sim simply the metabolic metabolic rate under conditions of minimal neuromuscular activity. The vast majority of investigators have have attempted to measure the embryonic embryonic and larval larval equivalent of routine routine metabolism. This is a rather nebulous nebuIous term, term, since, since, as as Wieser (1985) (1985)points out, out, it can vary at least twofold in resporise response to a variety of intrinsic and extrinsic factors. factors. For embryos and larvae, routine metabolism probably can be defined best as the average rate of aerobic metabolism under normal rearing conditions. conditions. Active Active metabolism in older fish fish usually refers refers to sustained activity. activity. The The young of many species, species, however, however, are are not capable of sustained activity. activity. As a result, result, most estimates of active metabolism during early life are are based based on burst activity. activity. It is is a moot moot point point whether such results are comparable with values for juveniles and adults. adults. V02 Absolute aerobic aerobic scope, scope, defined as as the difference between between aaV02 Y02 , is and ssV02, is of particular interest to physiologists because it repre represents the amount of energy available to a fish fish to cover the the cost of

100 100

PETER PETER J. ROMBOUGH ROMBOUCH

various biological activities. Unfortunately, it has proved difficult to estimate absolute scope during early life, although a new type of of metabolic chamber recently described by Dabrowski ((1986) 1986) may make such determinations easier in the future. future. It has proved some somewhat easier to estimate selected portions of absolute scope, in particu particuV02 (termed V02 and lar between aaV02 lar the the difference difference between and rrV02 (termed the the relative relative scope; scope; V02 and Wieser, (termed the Wieser, 1985) 1985) and and the the difference difference between rrVO2 and ss V V 02 0 2 (termed the routine scope; scope; Beamish, Beamish, 1964). 1964). It It thus is possible possible to to obtain rough rough estimates of absolute scope indirectly by adding values reported for routine and relative scopes. scopes. Some Some investigators have used the ratios of metabolic rates at the various expressvarious activity levels, instead of their differences, as a way of express V02/s V02 are re V02/rV02, and V02/s V02, aaVOZ/rV02, ing ing scope. scope. The The ratios ratios aaV02hV02, and rrVOzlsV02 referred to, respectively, as absolute factorial scope, scope, relative factorial scope, scope, and routine factorial factorial scope. scope. This is convenient for comparative purposes but does not tell the whole tale, since it gives no indication of absolute absolute costs, which are of profound ecological importance. As will be discussed later, scopes and factorial factorial scopes can change in response to various intrinsic and extrinsic factors. factors. It should be recognized, how however, that because of the manner in which they are calculated, they V02 may not always change in the same direction. For instance, if aaVO2 V 0 2 decline in parallel, absolute scope will remain constant but and s V 02 absolute factorial scope will increase. V02-s V02) is routinely estimated for ju Absolute aerobic scope (a (aVOz-sVOZ) juvenile and adult fish by forcing them to swim against a current at progressively faster speeds. speeds. The oxygen uptake at maximum sustained V02, while extrapolation of swimming speed is taken to represent aaVO2, the power-performance power-performance curve back to zero swimming speed yields V02• Ivlev ((1960a) 1 960a) appears to have been the only investigator to have ssVO2. applied this technique to very young fish. fish. He reported that young Atlantic salmon (400 mass) were capable of sustaining a rate of (400 mg wet mass) oxygen uptake 22.5 22.5 times their standard rate. This value is sometimes cited as the degree of metabolic expansibility that can be expected for fish fish larvae (e.g., (e.g., Blaxter, Blaxter, 1969). 1969). However, more recent evidence sug suggests that this is a gross gross overestimation and that absolute factorial scopes actually range from about 2.5 to 10, 10, depending on species, fish size, and temperature (Table (Table II). 11). of the standard Dabrowski ((1986) 1 986) recently used a modification of power-performance power-performance procedure to estimate aerobic scope for salmon fry. The fish were induced to swim at progres progresalevins and coregonid fry. sively faster speeds by making use of their optomotor reaction to a background. Swimming speeds were monitored and correlmoving background.

2.


101 101

ated with rates of of oxygen uptake. For young Atlantic salmon, the maxi maximum V 02 recorded was about 3.3 3.3 times the value estimated for zero VOZ activity. scope of Coregonus schinizi fry was activity. The absolute factorial scope somewhat lower, about 2.5. A less common way of of estimating aerobic scope in juvenile and sh is to take the difference between the maximum and mini adult fi fish miniVOZ exmum V 02 recorded while fish are held in a respirometer for an ex tended period. The value obtained is is correctly termed the aerobic scope for spontaneous activity but, at least in some species, it appears scope (Ultsch et al., al., 1980). investigato approximate absolute scope (Ultsch 1980). Several investiga tors have applied this technique to fish larvae. Holliday et al. 1964) al. ((1964) indicated that there was approximately a 10-fold 10-fold difference between the minimum and maximum V 02 of herring larvae. For sardine larvae VOZ the maximum difference in V 02 recorded was about 3.5-fold (Lasker VOZ and Theilacker, 1962), 1962), while in winter founder (Pseudopleuronecter americanus) americanus) the maximum difference was only about 3.0-fold (Cetta and C apuzzo, 1982). Capuzzo, 1982). It is not clear why the apparent scope of herring should be so much greater than that of the other species, but it is interesting that larvae of Pacifi c herring (Clupea Pacific (Clupea pallasi) pallasi) also appear capable of increasing their metabolic rate about lO-fold 10-fold (Eldridge et al., 1977). 1977). In the case of of the Pacific herring, it was stress caused by the shaking of the respirometer, rather than spontaneous activity, that led to elevated metabolic rates. As mentioned previously, it has proved difficult to induce larvae to swim in a respirometer and, as a consequence, other techniques have V02 and aaVO2. V02. The most common had to be developed to estimate ssV02 V02 has been to anesthetize the larvae (Holli method for estimating ssV02 (Holliday et al., al., 1964; 1964; DeSilva and Tytler, Tytler, 1973; 1973; Davenport and L�nning, Lgnning, 1986). This technique indicates routine factorial 1980; 1980; DeSilva et al., 1986). V02/s V02) in the range of scopes (r (rVO2lsV02) of 1.4-3.3, 1.4-3.3, depending on species and stage of development (Table (Table II). 11). Another method has been to assume that the capsule severely restricts activity just before hatch (Daven (Davenport and Lgnning, L�nning, 1980; 1980; Davenport, 1983). 1983). Davenport and L�nning L9nning ((1980) 1980) demonstrated that, at least in in cod, the metabolic rate of of em embryos just before hatch was not signifi cantly different from that of significantly anaesthetized larvae shortly after hatch. Assuming that the metabolic rate of unanesthetized larvae shortly after hatch is is representative of V02, this method gives routine factorial scopes for cod (Davenport rrVOz, (Davenport and L�nning, 1980) and lumpfish (Davenport, 1983) of about 2.0. Lgnning, 1980) (Davenport, 1983) The evidence is is rather sketchy, sketchy, but it appears that the difference V02 (routine V02 and ssVOZ between rrVOz (routine scope) scope) tends to decrease as larvae mature (DeSilva (DeSilva and Tytler, 1973; 1973; Davenport and L�nning, L$nning, 1980; 1980; De-

Table II I1 Table Scope for Activity Scope ... 0 CI 0 �

te

Species

Clupea Clupea harengus harengus

Stage Stage

Temp. (“C) (0C)

Gadus Gadus morhua morhua Cyclopteros Cyclopterus lumpus lumpus Oreochromis Oreochromis niloticus niloticus Salmo salar salar

E L L L L L L L L L L

8 8 8 8 8 10 10 10 10 5 5 5 5 5 5 5 5 30 20

Salvelinus Salvelinus alpinus alpinus

L

4

Pleuronectes plattesa Pleuronectes plattesa Gadus Gadus morhua morhua

Scope

Technique

VOv's V02) Routine factorial factorial scope (r (rV0dsVOp) V 2.1 rrvodanesthetized Ov'anesthetized V 2.1 302 02 VOv'anesthetized V V02 1.4 02 rrV0daneseetized 2 .5 Posthatch V Ov'prehatch V 2.5 VOdprehatch V02 02 VOv'anesthetized V 1.9 VOp 1.9 rrVOdanesthetized 02 VOv'anesthetized V 1.6 rrVOdanesthetized VOp 02 VOv'anesthetized V 2.2 rrV0daneseetized 2.2 V02 02 VOdprehatch VO 2.0 Posthatch V Ov'prehatch V 02p V tN02 in dark , 11.6 .6 02 in ligh VOp ligh.hO2 02 .0 2.0 VOdprehatch VOz 2 Posthatch V Ov'prehatch V VOv'anesthetized V 3.5 rrvodanesthetized 3.5 VOp 02 1.5 Extrapolated from power-perf or1.5 power-performance curve Posthatch V Oprehatch V 2 .6 2.6 VOflrehatch V 02 Op

Reference

Holliday et al. (1964) et al. (1964) Holliday et al. ((1964) 1964) et al. ((1964) 1964) Holliday et (1973) De Silva and Tytler (1973) De Silva and Tytler ((1973) 1973) Davenport and Lonning (1980) (1980) Davenport and Lonning ((1980) 1980) Solberg and Tilseth (1984) (1984) Davenport ((1983) 1983) 1986) De Silva et et al. ((1986) Ivlev (1960a) (1960a) Gruber and Wieser ((1983) 1983)

V02!'rV02) (aVO$rVOz) Relative factorial scope (a

Alburnus alburnus Salmo Salmo gairdneri gairdneri

(3 species) species) Cyprinids (3

9.7" 9.P 2.7 1.9 1.9 1 .8 -1.8 2.4 2.4 2. 1 2.1

L L L L L

20 4 12 12 20 12 12 20

E L L

14 14 14 14 7

2.8 2.8 3.5 3.0

L L

8 12.5 12.5

10.4 10.4 10.0 10.0

E L

10.0 10.0 8

L L F

20 22 14

Power-perfonnance Power-performance relationship Forced bursts Forced bursts Forced bursts Forced bursts Forced bursts

Ivlev ((1960b) 1 960b) Wieser et al. ((1985) 1985) Wieser et al. (1985) (1985) Wieser et et al. (1985) (1985) Wieser and Forstner ((1986) 1986) Wieser and Forstner (1986) (1986)

Spontaneous factorial scope

Sardinops Sardinops caerulea caerulea

.... Q �

Pseudopleuronectes Pseudopleuronectes americanus americanus Cltlpea Clupea harengtls harengus harengus Clupea harengtls pallasi pallasi Salmo salar Salvelinus Salvelinus alpinus alpinus

Salmo salar Salmo salar Coregonus Coregonus sp. sp. a 0

Unrealistically high, see text.

Observed max. V VOdmin. 02!'min. 02!'min. Observed max. V VOdmin. Observed max. 02!'min. ma.V VOdmin.

V 02 VOZ V 02 VO Z V 02 VOZ

Lasker and Theilacker (1962) (1962) Lasker and Theilacker (1962) (1962) Cetta and Capuzzo Capuzzo ((1982) 1982)

Observed 02!'min. V 02 , Observed max. max. V VOdmin. VO2 Posthatch max. 02!'prehatch V 02 max. V VOdprehatch VOZ

.

Observed max. V 02!'min. V 02 VO2 VOdmin. Posthatch max. V VOdprehatch VOZ Posthatch 02!'prehatch V 02 V02!'s V02) Absolute factorial scope (a (aVOdsV0,) 3.0 5.0

22.5" 22.5" 3.3 3.3 2.5 2.5

Power-perfonnance Power-performance relationship Power-performance relationship relationship Power-perfonnance Power-performance relationship relationship Power-perfonnance

Holliday et 1964) et aZ. al. ((1964) aE. ((1977) 1977) Eldridge et al. 1951) et al. ((1951) Hayes et Gruber and Wieser Wieser (1983) (1983) 1960a). Ivlev ((1960a). Dabrowski ((1986) 1986) Dabrowski ((1986) Dabrowski 1986)

104 104

PETER PETER J. ROMBOUGH ROMBOUGH

Silva et al., 1986). 1986). For example, exampIe, the routine scope oflarval of larval herring and plaice at metamorphosis, measured as the difference between un unanaesthetized 02, was 25% of of that that at at anaesthetized and and anaesthetized anaesthetized V V02, was only only about about 25% hatch The decrease in routine 1973). The decrease in routine scope scope re rehatch (DeSilva (DeSilva and and TytIer, Tytler, 1973). V02. V02 than flected flected aa more more rapid rapid decline decline in in rrV02 than in in ssVOz. Wieser Wieser and and his his co-workers co-workers (Wieser, (Wieser, 1985; 1985; Wieser Wieser et al., 1985; 1985; Wieser Wieser and and Forstner, Forstner, 1986) 1986) have have attempted attempted to to obtain obtain estimates estimates of of ac active metabolism by forcing larvae (using electrical or mechanical stim stimulation) to swim at burst speeds speeds for short periods (30-60 (30-60 s). s). The main driving force behind such activity was shown to be anaerobic (Wieser et al., 1985), 1985), but it was felt that the maximum rate of oxygen uptake during activity activity or or the the first few few minutes minutes of of recovery recovery (the (the response response time of 02 was of the the system system was was not not fast fast enough enough to to say say precisely precisely when when V V02 V02. Using maximal) maximal) approached approached aaVO2. Using the the average average rate rate of of oxygen oxygen uptake uptake V02, this prior technique gave this technique gave estimates estimates of of 1.9-2.7 1.9-2.7 and and prior to to activity activity for rrVO2, V02/rV02) of young rainbow 2.4-2.9 for the relative factorial scope (a (aVO2lrVOz) trout (Wieser (Wieser et ai., (Wieser and Forstner, al., 1985) 1985) and larval cyprinids (Wieser V02 is V02, absolute 1986), 1986),respectively. respectively. Assuming Assuming that that aaV02 is about about twice twice ssV02, absolute factorial to 6. 6. factorial scopes scopes would appear to to range range from from about about 4 to In In juvenile juvenile and and adult adult fish, fish, aerobic aerobic scope scope tends tends to to increase increase as as the the 1973; Wieser, 1985). 1985). The pattern is is not as fish grows (Brett and Glass, 1973; obvious for younger fish. rainfish. The metabolic expansibility of young rain bow body mass, bow trout trout increased increased with with body mass, more more or or less less as as expected expected (Wieser, (Wieser, 1985; Wieser ai., 1985). 1985; Wieser et al., 1985). For For example example at at 4°C, 4"C,relative relative factorial factorial scope scope increased from about 2.7 for yolk-sac larvae (80-120 mg) mg) to to about about 5.2 5.2 increased from about 2.7 for yolk-sac larvae for fry weighing between 3 and 10 g (Wieser et al., 1985). 1985). On the other hand, in· i n cyprinids, relative factorial scope was independent of body mass between 1 and 400 mg (Wieser (Wieser and Forstner, 1986). 1986). Wieser and Forstner ((1986) 1986) suggested that this may reflect the need of very small larvae to avoid the constraints small size normally places on aerobic scope if if they are to escape predation. The influence of temperature on aerobic scope appears to vary depending on the species. As was the case involving the effect of body mass, young rainbow trout follow a pattern similar to that seen in 1985). Routine meta metajuveniles (Wieser, 1985; juveniles and adults (Wieser, 1985; Wieser et al., ai., 1985). bolic rate increases steadily with temperature 4°C and 20°C temperature between 4°C (Fig. 9). 9). Up to about 12°C, 12"C,active metabolism also increases with tem tem(Fig. perature, but at a faster rate than routine metabolism, so that relative 12"C, however, there is a descope increases. At temperatures above 12°C, de crease in the rate at which active metabolism expands so so that relative V02/rV02) remains remains constant scope (a scope (aVOzlrVO2) constant or or even even declines. declines. The The reasons reasons for for this decline decline have have not not been been demonstrated, demonstrated, but it it may may be that, that, as as in

2. 2.

RESPIRATORY RESPIRATORY GAS GAS EXCHANGE, EXCHANGE, AEROBIC AEROBIC METABOLISM METABOLISM

105 105

• yolk yolk sac sac • 80-200 80-200 m9 me

32

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mo--1000 mg mg • 200-1000 V 3-10 3-10 9 B ...

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w W I-

-301 - 30 -30-

2b 2 « a:: LL !l.

w W Z

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Br::1i

w

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r

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t

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LOG LOG

K'] o [[ K']

--30 30 -20.

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-40

....J J

11.

I

w

::1i

0 110

cC

f�Y T

(3))/

I

6mVI lOx

- 50

(I)

,. �-.-+---

6 mY/ I O ,

-60 --70 70

I

0.2 0.2

100 100

(m M ) (mM)

I I mV/lOx mVI I O , II

g 9

c IZ W I-

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--5500 -

>

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�I ( 2)

-40 -40-

-60 -60I 0.2 0.2

E .5

mV/ IOx 228 8m V/IO x

8

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« 4 Ii= z 2

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( 2)

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Fig. 1.

�

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--

LOG LOG

f' f�

0 110

[NO [No+]+]0 ,

((mM) mM)

(Continued) (Continued)

}-

100 100

179

180 180

ALDERDICE D. F. ALDERDICE

(d) phase of (d) Fast Fast phase of recovery recovery from from hyperpolarization; hyperpolarization; duration, duration, 155 155 ± f 18 18 s. s. (e) (e) Slower Slower phase phase of of recovery, recovery, reaching reaching aa steady, steady, postactivation postactivation resting potential of 19 ± 15 mV min after resting potential of -19 -C 15 mV by by 9.4 ± +. 11 min after activation. activation. 2. 2. Membrane Membrane resistance. resistance. A A lO-fold 10-fold decrease decrease in in resistance resistance from from 40 40 to MOa occurred occurred over min following to 33 M over the the first first 2 min following activation. activation. Thereafter, 8 min, min, Thereafter, there there was was aa slow slow recovery recovery over over the the next next 8 resistance resistance ultimately ultimately reaching reaching aa value value 30% 30% greater greater than than the the preactivation value. preactivation value. From electrical events, the time of activation (Fig. 1A) was deter deter(Fig. lA) mined to be about 5 s after a sperm entered the micropyle. Nuccitelli ((19804 1980a) also examined the ionic basis for changes in membrane poten potential. The resting potential showed a limited dependence on external 2+ K+ independence of external Ca K+ and Na+ Na+ ions (Figs. (Figs. IB, lB, lC), lC), but independence Ca2+ and CI-. C1-. The plasma membrane of the unfertilized egg depolarized 99 mV/decade increase in [K+]o [K+], (outside) (outside) and 6 mY/decade mV/decade increase in mY/decade [Na+]o. 1 963) obtained a value of 16 “a+],. Ito ((1963) 16 mY/decade mV/decade increase in [K+]o [K+], in mature oocytes (eggs) Oryzias. Nuccitelli concluded that the (eggs) of of Oryzias. small leaks at the membrane membranesmall ion dependencies were due in part to leaks K+ and Na+ Na+ membrane per perelectrode seal and in part to a significant K+ studies indicated that the small initial meability. Other, preliminary, studies 2 + and Na+ Ca2+ Na+ entry as well as K+ K+ depolarization pulse is carried by Ca efflux. The strong K+ K+ dependence (28 (28 mY/decade mV/decade [K+]o [K+], (Fig. (Fig. IB) 1B) and efflux. hyNa+ response (6 (6 mY/decade mV/decade [Na+]o “a+], (Fig. (Fig. 1C) much lower Na+ lC) during hy perpolarization suggest that the increase in conductance during this K+ membrane permeability. About 88 phase is due mainly to increased K+ K+ permeability fell to near zero min after peak hyperpolarization, K+ mV/decade [Na+]o). “a+],). Na+ permeability increased slightly ((11 while Na+ 1 1 mY/decade flux resultThe hyperpolarization and increased K+ fl ux are suggested as result ing K+ channels channels due due ing from two possible mechanisms: ((1) 1 ) an opening of K+ 2 +]j and (2) to a rise in [Ca (2) the addition of K+ K+ channels, present in the [Ca2+]i vesicuvesicular membranes, that fuse with the plasma membrane by vesicu lar exocytosis. K+ permeability increase exocytosis. The transient nature of the K+ 2 +]j after [Ca2+Ii may be associated with the concomitant decrease in free [Ca passage of the calcium wave over the cytoplasm at activation, and a K+ channels channels in the mem memcorresponding closing of newly inserted K+ brane through endocytosis endocytosis of the vesicular membrane material brane (Gilkey, 11981, 1983; Kobayashi, Kobayashi, 1985). 1985). (Gilkey, 98 1 , 1983; events that coincide with with or follow follow the activation Of these several events potential immediately, an important one is is the very large increase in

3.

OSMOTIC AND IONIC REGULATION REGULATION IN IN TELEOST EGGS AND LARVAE LARVAE OSMOTIC

181

of free calcium. the internal concentration of calcium. In the sea-urchin egg this increase likely approaches the 300-fold 300-fold increase seen in the medaka (Jaffe, 1985). egg (Jaffe, 1985). Second is the eversion of the cortical alveoli, which moves as a wave over the egg surface from the point of of sperm contact. With exocytosis, the cortical alveoli deliver their contents into the presumptive perivitelline space immediately above the plasma memmem brane. There, the alveoli contents imbibe water from the external medium, raising the fertilization membrane (echinoderms) (echinoderms) or zona radiata (teleosts) radiata (teleosts) away from the plasma membrane of of the egg proper as the perivitelline fluid forms 1983) injected buffered calcium forms.. Gilkey ((1983) eggs. He found the level of free calcium into unfertilized medaka eggs. required to elicit a transient increase in cytoplasmic free calcium to be 0.51 and 55.1 (7.0-7.5). The between 0.51 . 1 mM, depending on internal pH (7.0-7.5). desequestering of free internal calcium appears to be an autocatalytic 2 + release. This threshold, required to elicit the 2 + -stimulated Ca Ca2+-stimulated Ca2+ Ca transient calcium wave, is well below the 30 mM of of free calcium reached at the peak of the transient. He concluded that the calcium wave is necessary and sufficient to activate the medaka egg. In the fertilization, the internal calcium content begins sea-urchin egg, after fertilization, to fall; cytosol and into the external fall; it appears to be pumped out of the cytosol 1976). In some (protomedium (Azarnia (Azarnia and Chambers, 1976). some invertebrates (proto somes) egg activation requires calcium, calcium, but it appears to enter the somes) cytosol from the external medium through voltage-gated channels in response al., 1979). 1979). response to to aa shift shift in in membrane membrane potential potential (Jaffe (Jaffe et al., Internal le in egg activation-or in suppression of Internal pH pH may may play play aa rrqle in egg activation-or in suppression of q activation. M edaka eggs with an apparent internal pH of 77.1 . 1 (Jaffe, (Jaffe, Medaka 1985) 1985) show a slowing of the calcium wave at pH 6.9 6.9 and its accelera acceleration at pH 7.3. 7.3. Reduction of internal pH from 7.0 7.0 to 6.5 6.5 in the medaka egg increases threefold the calcium threshold required for initiation of the calcium surge. The ovarian eggs eggs of frogs, frogs, taken directly from the surge. The oviduct, oviduct, were were shown to be unfertilizable because of their high C0 C022 content. Hence, low low ovarian pH may act as as a brake to activation prior is an apparent apparent doubling of to egg deposition. In sea-urchin eggs there is ions shortly after activation; activation; this alkalinization of the cytosol [OH-] ions -H+ ion appears, in in part, part, to to be the result of an electrically electrically neutral Na+ Na+-H+ appears, transfer. trans fer What can can be inferred from the the available available data regarding ionoiono- and osmoregulation in the teleost oocyte and in the egg at fertilization? osmoregulation Few Few data data are are available available on on molecular molecular processes, processes, and and the the general general nature nature of of possible possible ionic ionic involvements involvements must must be be cautiously cautiously inferred inferred from from other other animal animal groups. groups. Basically, Basically, various various types types of of ion-transporting ion-transporting channels channels appear appear in in the the plasma plasma membrane membrane of of the the developing developing oocyte. oocyte. At At various various

.

182 182

D. D. F. F. ALDERDICE ALDERDICE

times these may become functional, cease times these may become functional, may may proliferate, proliferate, or or may may cease functioning; functioning; they they may may be be electrically electrically neutral, neutral, electrogenic, electrogenic, or or voltage voltagegated. blocked by pH levels gated. Some Some may may be be blocked by particular particular pH levels in in the the surround surrounding of the the plasma membrane membrane is is depen depening medium. The The resting potential potential of dent dent on on its its selective selective ion ion permeability permeability and and the the voltage voltage gradient gradient set set up up by by differences differences in in ion ion concentrations concentrations on on either either side of of the the membrane. membrane. Generally becomes more Generally it it becomes more negative negative as as ion ion concentration concentration decreases decreases in in the medium. Hence, the external external (freshwater) (freshwater) medium. Hence, the the resting resting potential potential may may change (such as change in in relation relation to to external external stimuli stimuli (such as sperm sperm contact) contact) or or inter internal ionic activity nal stimuli stimuli (Ca2+ (Ca2+release), release), and and to to the the level level of of ionic activity in in the the external medium. As ions move, so so too will water move across the membrane in relation to membrane permeability, and to differences in in molar molar concentrations concentrations of of solute solute particles particles on on the the two two sides sides of of the the membrane. membrane. Relatively speaking, speaking, the oocyte prior to release is subject to the protective regulative mechanisms of of the parent. The ovulated egg responds responds to to changes in in the the ovarian ovarian fluid fluid (Sower (Sower et al., 1982), 1982),the the ovar ovarian fluid osmotically is very similar to the blood plasma (Hirano aZ., (Hirano et al., 1978), 1978), and the blood plasma is in balance physiologically with the osmotic activity of the external environment (Sower (Sower and Schreck, 1982). However, when shed externally at spawning, mature eggs will 1982). be be subject subject to to major major changes changes in in osmotic osmotic and and ionic ionic composition composition of the the external medium; in general they will be hypotonic to seawater and hypertonic to fresh water. It would seem that the first major regulatory challenge will occur at spawning, adding to the large number of mo molecular activities set in motion by activation and fertilization. fertilization. In the newly fertilized egg the only structure available with regulatory ca capacity would be the plasma membrane. It seems reasonable to assume that the membrane should be well prepared, in terms of of permeation, ion-channel function, and electrical activity, to cope with the changes occurring when the eggs are shed and fertilized over a range and variety of external conditions, conditions, whose extent would be related to the normal normal habitat habitat of of the the species. species. In In terms terms of of tolerance, no no doubt doubt these these ranges will have limits related both to permeability characteristics of of the plasma membrane and to the tolerance of the initial cell and its successors in the newly fertilized egg. In summary, it appears that an understanding of regulative charac characteristics of teleost eggs at fertilization will be centered on the plasma membrane and will require, initially, the development of basic, de descriptive data on the electrical and ion permeation characteristics of of the teleost egg. Further, examples need to be developed for stenoplas stenoplastic and euryplastic species from the marine and estuarine environ environments, as well as from fresh water; patterns that may exist would be

3. 3.

REGULATION IN IN TELEOST AND LARVAE OSMOTIC AND IONIC REGULATION TELEOST EGGS AND

183 183

difficult to identify from the limited information currently available. available. One use of One may may also also plead plead for for aa greater greater use of normal normal external external media media during during such such studies studies to to make make the the physiological physiological results results obtained obtained ecologically ecologically more meaningful. Areas of interest in specific studies would include: Resting ion permeation Resting potentials, potentials, ion permeation characteristics, characteristics, and and electrical electrical activity activity in mature, unfertilized unfertilized eggs. eggs. The The activation activation potential, potential, and and membrane membrane resistance resistance and and conduc conduc2 +, K+, tance Na+, and l - in K+, Na+, and C C1in the the external external tance with with respect respect to to Ca Ca2+, medium. medium. Action they occur. occur. Action potentials, potentials, if if they Ion-channel function in preactivation to postfertilization postfertilization stages. stages. Internal Internal ion ion events events during during activation activation and and fertilization. fertilization. 2 + , K+, Influence low-ionic-strength fresh fresh water water (e.g., (e.g., low Ca Ca2+, K+, Influence of low-ionie-strength Na+) Na+) on activation. The timing of major events during activation and fertilization in relation relation to to temperature temperature and and species. species. IV. DEVELOPMENT

Of the three levels Of the three levels of of potential potential regulatory regulatory capacity capacity mentioned mentioned ear earlier-cellular, lier-cellular, tissue, tissue, and and neurosecretory neurosecretory involvement-it involvement-it is is assumed assumed the oocyte oocyte and and mature mature eggs eggs are are restricted restricted to to the the first first of of these, these, continu continuing, ing, in in the the fertilized fertilized egg, egg, for for aa period period extending extending into into very very early early cell cell division. division. It It is is assumed assumed also also that that the the second second level, level, proliferation proliferation of of tis tissues sues with with regulatory regulatory capacity, capacity, may may begin begin with with blastodermal blastodermal over overgrowth earlier, with yolk, and and possibly possibly earlier, with an an initial initial level level of of regula regulagrowth of of the yolk, tory plug closure tory capacity capacity achieved achieved by by yolk yolk plug closure (Holliday (Holliday and and Jones, Jones, 1965). 1965). It It would would seem seem that that neurosecretory neurosecretory involvement involvement would would of of ne necessity development of specialized tissues, yolk plug plug cessity await await the the development of specialized tissues, after after yolk closure, process of continuing continuing elaboration elaboration through through the the larval larval and and closure, in in aa process later viewpoint the later stages. stages. From From this this viewpoint the components components of of the the fertilized fertilized egg egg will be examined examined that that may may contribute contribute to to regulation. regulation. In In order order of of devel develwill opment these would included the plasma membrane, the perivitelline fl uid and fluid and zona zona radiata, radiata, tissues tissues of of the the blastoderm, blastoderm, the the embryonal embryonal epi epidermis, cells, and, dermis, chloride chloride cells, and, briefly, briefly, aa consideration consideration of of the the transition transition from from the the embryonic embryonic regulative regulative mechanisms mechanisms to to those those of of the the juvenile, including gills, gut, Possible responses responses to evolving, including the the gills, gut, and and kidney. kidney. Possible to evolving. regulatory regulatory capacity capacity could could involve involve changes changes in in egg egg volume, volume, water water con content, levels of tent, levels of tissue tissue osmolarity, osmolarity, internal internal ion ion concentration, concentration, hydro hydrostatic These will will be static pressure, pressure, and and buoyancy buoyancy and and specific specific gravity. gravity. These be touched briefly, but emphasis is is placed placed on on regulation, regulation, regulative regulative touched on on briefly, but emphasis processes, where and when these might occur. processes, and and where and when these might occur.

184

F. ALDERDICE ALDERDICE D. F.

Properties of of the the Plasma Plasma Membrane Membrane A. Properties of a lipid The plasma membrane of a cell generally has the form of bilayer matrix matrix in in which which islands islands of of protein protein are (Korenbrot, bilayer are interspersed interspersed (Korenbrot, 1977). The lipid matrix phos 1977). The lipid matrix comprises comprises some some 5-10 5-10 lipids, including phosphatidyl phatidyl derivatives, derivatives, sphingomyelin, sphingomyelin, cholesterol, cholesterol, neutral neutral lipids, lipids, and and glycolipids. protein derive their characteristics glycolipids. The islands islands of of protein derive some some of of their characteristics from the the composition composition of of the the lipid lipid matrix. They may extend across across the from matrix. They may extend the entire of the membrane and provide access to reactants at its entire thickness of oc two surfaces. Most water transport across a membrane appears appears to occur by aa solubility-diffusion solubility-diffusion process process through through the hydrophobic bilayer cur mem lipid matrix. On the other hand, hand, ions appear to move across the memof the islands brane, by active or passive transport, by way way of the protein islandsreferred to as channels. As most cells do not require high generally referred water permeability, permeability, plasma membrane channels devoted to water movement would seem redundant. Nevertheless, water molecules may may penetrate penetrate aa membrane via via channels channels and and do do so in relation to to chan channel diameter. diameter. In In such such instances, instances, flows flows through through channels channels may may involve involve nel water-water, water-ion, ion-ion interactions (Levitt, (Levitt, 1984). 1984). water-water, water-ion, or ion-ion Three of a membrane include its wawa Three important functional aspects of ter of ions, and its ter permeability, transfer of its electrical properties. Various techniques have been used, refined and redefined for measuring the of particles across a membrane. There is a potential movement of potential for confusion because in the the because of of the the various various terminologies terminologies and and measures measures in confusion literature, as pointed out by Potts and Parry (1964). ( 1 964). The flow of of soZute solute z/s), across across aa membrane membrane may may be be measured as as aa diffusion diffusion constant (cm (cm2/s), or or as as aa permeability permeability constant (cm/s). (cm/s). Permeability Permeability of of aa membrane membrane to to aa solvent (water) (water) is measured in terms of of permeability coefficients. coefficients. ( 1961) and As demonstrated by Kedem and and Katchalsky (1961) and Katchalsky ( 1 965), the of particles across a membrane is and Curran Curran (1965), the movement of of dependent on the flows and forces operating in the system. A set of phenomenological equations, relating these flows and forces, may be written as

h = Lll Xl + LIZ Xz + . 12 = LZI Xl + LZ2 X2 + .

= Jn Jn =

L,1 Lnl x1 Xl

+ L,z LnnX, X2 + . -. -. + LnnXn Ln2 xz *

of the XXkk forces; that is, where the JJii flows are linear functions of

3. 3.

OSMOTIC AND IONIC REGULATION IN TELEOST EGGS EGGS AND LARVAE

i J ii = =

n n

2: C LL iikXk kXk k= I

k= l

185 185

2, . .. .,, nn)) (i = = 1, 2, .

.

X ii forces will be linear functions of of the theJk Hence, the X h flows, n

X i = 2: R ik h k= l

(i = 1 , 2,

.

.

.

, n)

The L L iikk = = U;lXk)xi ( J i / X k ) X , coefficients coefficients are flows characflows per unit force and are charac Rik = (X/h)); (Xi/Jk)licoefficients terized as conductances or mobilities. The R ik = of force per unit flow and represent resistances or have dimensions of J ii or Xj X i equations may be converted frictions. In practice, either set ofi into the other by standard matrix algebra. algebra. Furthermore, the matrices are symmetric, so that Lik = Lki

(i ( i =1= # k) k)

Hence, in ow, two-force Hence, in aa two-fl two-flow, two-force system system (one (one solute, solute, one one solvent), solvent), there there are three coefficients; in a three by three system, there are six coeffi coefficients. In general,

ILI 2:: 2 0 0,, LiiLa 2 L i and ILl is advantageous to trans In considering membrane properties, it is transform the original (thermodynamic) (thermodynamic) forces into simpler quantities. For example, it follows, in a binary system, that

L Lii ii

2 2::

0, 0,

Jv = = Lp L p ilP AP + + LPD LPDil1T AT iv iD ]D = = LDP LDPilP AP + + LD LDil1T AT J v is the volume flow of solvent (e.g. (e.g. water); iD J D is the velocity of where iv AP is the hydro hydrosolute relative to solvent, similar to a diffusional flow; ilP static pressure difference AT is is the the osmotic osmotic static pressure difference across across the the membrane; membrane; il1T pressure membrane; Lp L p is is the the mechanical mechanical filtra filtrapressure difference difference across across the the membrane; tion coefficient, to ilP; L is the diffusional mobility coeffi AP; L D is diffusional mobility coeffition coefficient, relating relatingfv to D iv cient, J D to to il1T; AT; and and LP L pD D is is the the coefficient coefficient of of osmotic osmotic flow, flow, cient, relating relating iD which equals equals LDP, L D p , the the ultrafiltration ultrafiltration coefficient. coefficient. Several Several examples examples may may show value of show the the value of these these multiflow-multiforce multiflow-multiforce relationships. relationships.

1. If 1. If il1T AT = = 0: 0: iv Jv = = Lp L p ilP, AP,

D = LDP i JD L DilP AP ~ =

Thus, with will produce Thus, with il1T AT = 00,, hydrostatic hydrostatic pressure pressure will produce both both aa volume volume flow ultrafiltration. flow and and aa diffusional diffusional flow, the the latter latter by ultrafiltration. =

If ilP AP = = 0: 2. If J v = L p D AT,

D= i ]D = LD LDil1T AT

186

D. F. ALDERDICE

Hence, with AP tlP = = 0, the osmotic pressure gradient will produce an flow. There also is a relation between osmotic flow and a diffusional flow. osmotic flow flow and and ultrafiltration, ultrafiltration, as as osmotic

(h) tl1T -

IfJv = 0 0:: 3. If lv =

!J.p=o

�

Lpv = Lvp

(tlP),v-o

=

=

(tlP k) -

�1T=O

-

LpD - Lp tl1T

That [(tlP),v=o = equ_ilibrium [(AP)Jv=o = AT] a1T] occurs only if if -Lpg That is, true osmotic equilibrium = - LPD = = LD LD in in an an ideal ideal Semipermeable semipermeable membrane membrane where where the the solute solute flow flow Lp = is zero. zero. is of the solute: solute : 4. Where the membrane permits passage of LpD -LpD

_

Lp LP

< > PP dd .. Earlier it was same membrane system, system, it often occurs occurs that P the same this inequality resulted from the fact that diffusion was assumed that this flow, and that isotopic methods accelerated in the direction of the net flow, against the net fl flow. Poiseuille's law always measure diffusion against ow. Using Poiseuille's

188

D. D. F. F. ALDERDICE ALDERDICE

of volume volume flow, flow, the difference difference between between the the two two measures measures provided provided aa of means of of calculating calculating the width width of of pores pores in in aa membrane membrane through through which which means such flows flows could could occur. occur. The validity validity of of this this inequality, inequality, however, however, has has such been argued argued for for some some time time (Hansson (Hansson Mild and and Lgvtrup, Lf/lvtrup, 1985). 1985). AlAl been though the earlier earlier assumptions assumptions now now generally generally are considered unrealunreal though the are considered istic, comparison comparison of of the two two coefficients coefficients remains remains valid. valid. Hence, Hence; where where istic,

Pf

=

LP

Pd

b.P AP

= w,

W

then then

(!)

Pf l:P = Pd Vw

=

w

=

=

0, 0,

and

CT = = 1 1 u

!::L Vww

If one one wishes wishes to to examine examine the the influence influence of of the the variables variables on on the the ratio, ratio, If CT to to vary, vary, where where allowing b.P AP and and v allowing Lp Pf Vw - (b.P _

_

-

Jv CT

b.7T)VW

then then b.7T JJvv AT b.7T) +J Vw Pd (Vw b.P lvCTGs) (Js - lv eT (VW AP VW AT) US J VC CsS + V~CS) pd

f P =!1

-

Jv JV

VdAP AT)^ Vw(b.P - v CT b.7T)W

Friedman 1986) provides an excellent survey of these and associated Friedman ((1986) transrelationships, and their utilization in problems of biological trans port. Water Water permeability permeability coefficients coefficients measure measure the the volume volume of of water water pass passing across a unit area of membrane per unit time. Although Pr P f and Pd usually usually are are reported reported in in centimeters centimeters per per second, second, this this actually actually repre repre3 cmP2 sents cm-2 Salso may sents aa contraction contraction from from cm cm3 s - lI.. The latter latter (( x X 104) lo4)also may be 3 pm+? reported /Lm-2 Ss-I, - I , equivalent - I . Other also have reported as as /Lm pm3 equivalent to to /Lm pm Ss-l. Other units units also have been used (Potts 1964). Where Where possible, possible, the the original original units units been used (Potts and and Parry, Parry, 1964). 3 pm-2 J.tm-2 Sused converted to to /Lm pm3 s-l1 used in in articles articles reviewed reviewed will be converted I ). (J.tm (pm ss-l). conA long series of most informative investigations has been con ducted ducted by by Lf/lvtrup, Lgvtrup, Hansson Hansson Mild, Mild, and and their their associates associates that that deserves deserves particular particular mention; mention; an an excellent excellent summary summary of of these these studies studies is is found found in Lf/lvtrup 1981). Using an electromagnetic diver Lgvtrup and Hansson Mild ((1981). balance, balance recorded recorded aa composite composite response response balance, they they found found that that the balance involving involving both both cytoplasmic cytoplasmic diffusion diffusion and and plasma plasma membrane membrane permeperme-

3. 3.

OSMOTIC AND IONIC REGULATION IN IN TELEOST EGGS AND LARVAE IONIC REGULATION

189 189

ation. That is, the measure of membrane permeability for molecules passing into a cell was influenced by the rate of diffusion of those cytoplasm. They molecules moving away from the membrane in the cytoplasm. of isotopic water diffusion selected an approach providing an estimate of 2 ss-') - l ) in the cytoplasm (D,cm cm2 cytoplasm';l ; this allowed an independent independent estimate (D, of the exchange permeation coefficient ((E, E , cm S-I) s-l) for the plasma of conmembrane. Further, they found that the amphibian oocyte, in con trast to the mature egg, has no measurable barrier to water perme permeation; that there is a relation between cortical tension, tightness of the plasma membrane, and tonicity of the external medium (low (low tonicity increases cortical tension and tightness of the membrane); membrane); that diffu diffusion in the cytoplasm is a complex function of temperature; and that and cell density is a function of water content. Finally, they derived Pf Pfand Pd ( =E, = E , cm S-I) s-l) coefficients for the plasma membrane, and questioned the popular interpretation that Pf Pf > > PPdd is related to the presence of of membrane channels. Finkelstein ((1984) 1984) has examined the water permeability coeffi coefficients obtained for various bilayer membranes, and fi nds they range finds 2 /-Lm X 10lo-'1 to 11 XX 10 lo2 pm S-I. s-l. For plasma membranes examined, the from 2 X range extends from 0.96 0.96 x . 14 X 1.14 X 10- 2 /-Lm p n S-I s-l ~ for midgastrula X 10- 2 to 1 2 to 2 X 2 /-Lm eggs of Fundulus sp. sp. at 23°C (Dunham et al., 1970) 1970)2 x 10 lo2 pm S-I s-l for erythrocytes; most values are found to be around 2 X x 10-1 lo-' /-Lm pm S-I. s-l. Dunham et al. (1970) (1970)concluded from the low Pd values they obtained for for the the eggs eggs that that water water transport transport across across the the egg egg membrane membrane was was very very slow, suggesting that no special mechanisms are necessary for volume regulation of the Fundulus egg. egg. The unfertilized oocyte of of the plaice (Pleuronectes platessa) shed into seawater (Potts (Potts and Eddy, 1973) 1973) had 8.6 X x 1010-22 /-Lm pm S-I, s-1, reducing to 1.7 1.7 X x 10- 3 /-Lm pm Ss-1 an initial Pd of 8.6 - I after 11 day. 1969) estimated Pd for the ovarian egg of day. Potts and Rudy ((1969) of the Atlantic salmon (Salmo salar), prior to shedding, to be 6 Xx 10-2 (Salmo salar), pm S-I, s-l, falling to 1 (McIntyre, 1973). 1973). sperm to methylmercury at concentrations > While While the the latter latter author author used used seawater seawater of of higher higher salinity salinity for for the the incuba incubation 1980) were were tion of of herring herring eggs, eggs, the the experiments experiments of of Ojaveer Ojaveer et al. ((1980) conducted in seawater seawater of of only only 5.6-5.8%0 5.6-5.8%0 salinity. salinity. In In these these experi expericonducted in ments, ments, not only copper but also cadmium at concentrations higher than 0.005 mg/l mg/l affected affected fertilization fertilization negatively, negatively, ultimately ultimately yielding yielding than 0.005 only 1.0 mg only 62% 62% fertilized fertilized eggs eggs at at 1.0 mg copper/I, copper/l, and and 60% 60% at at 0.5 0.5 mg mg cad cadmium/I. mium/l. Due to the fact that the two experimental series were con conducted ducted in in different different salinities, salinities, they they are are difficult difficult to to compare compare in in terms terms of of metal efmetal effects effects on on fertilization, fertilization, since since we we know know that that particularly particularly the the ef fects of cadmium are salinity-infl salinity-influenced (von Westernhagen et al., fects uenced (von 1974). The origin of this sublethal response is not clear. clear. It might be 1974). caused by the interference of cadmium with the jelly coat of the egg, thus altering the site site of the penetration of the sperm into the egg, the micropyle. micropyle. A direct direct influence influence on on the the formation formation of of the the zygote zygote can can probably probably also also be excluded excluded as as the the mode mode of of action action of of pollutants pollutants such such as as aromatic aromatic hy hydrocarbons drocarbons (xylene), (xylene), which which prevent prevent the the formation formation of of aa fertilized fertilized egg egg and 10mg/l mg/l in in cod cod and early early cleavage cleavage stages stages at at concentrations concentrations higher higher than than 10 eggs 1982). The The action action of of aromatic aromatic hydrocarbons hydrocarbons such such eggs (Kj6rsvik (Kjorsvik et al., 1982). as as para-xylene para-xylene on on fertilization fertilization and and early early cleavage cleavage and and the generation generation of of the the characteristic characteristic small small cells cells as as described described by by Lanning Lonning (1977) (1977) in in the the reaction reaction of of plaice plaice (Pleuronectes (Pleuronectes platessa) eggs eggs to to xylene xylene and and benzene benzene

262

H. VON WESTERNHAGEN

are probably derived from their properties of causing membrane dam damage and increased membrane permeability (Roubal (Roubal and Collier, 1975; 1975; Morrow et al., 975). Mechanisms located in the cell surface are im al., 11975). important for the fonnation formation of the cleavage membrane, as shown by Rappaport (1977) (1977) in an investigation of of cleavage in eggs from different invertebrates invertebrates.. As Roubal and Collier (1975) (1975) pointed out, aromatic hy hydrocarbons attack the outer surface of of membranes and may thus influ influence the mechanism of of cleavage, as apparent in the photographic evidence of Kjarsvik 1982) in fertilized plaice (P. (P. platessa) eggs. Kjorsvik et al. al. ((1982) eggs. Similar effects are caused by the carcinogen benzo[a]pyrene (BAP) (BAP) in flatfish fladish embryos (sand sole, Psettichthys melanostichus; flathead sole, Hippoglossoides elassodon) al., 1982). 1982). elassodon) (Hose (Hose et al., hyStrong depression of fertilization is also caused by chlorinated hy drocarbons incorporated into the egg from parental sources such as DDT and dieldrin in eggs of of winterflounder (Pseudopleuronectes americanus (Smith 1973) or polychlorobiphenyl (PCB) (Smith and Cole, 1973) (PCB) (34 (34 ppm) ppm) in Atlantic salmon (S. ( S . salar) salar) (Jensen et al., al., 1971). 1971). In these cases, residues (4.6 ppm DDT; 1.2 1.2 ppm dieldrin) inside the egg give rise to considerable reduction of 12%, thus of fertilization rate down to 40% and 12%, suggesting direct cytogenetic effects. High DDT residues in the same range may be responsible for the failure of reproduction in seatrout Cynoscion nebulosus (Butler (Butler et al., al., 1972). 1972). Alderdice et al. 1979a) have shown that metals such as cadmium al. ((1979a) delay the water hardening and the process of water uptake in Pacific herring eggs, while at the same time the primary bursting pressures of exposed eggs are reduced from between 700 and 1300 1300 g to 200 and 350 g at cadmium concentrations near 11mg/I. mg/l. Brungs (1969) (1969) observed a similar effect in zinc-treated eggs of the fathead minnow, Pi Piggs in zinc concentrations higher than 0.18 mg/l mephales promelas. E Eggs remain in a fl accid condition resulting frequently in the rupturing of flaccid of the egg capsules during handling. Since the hardening of the egg chorion requires calcium (Kusa, (Kusa, 1949; 1949; Lanning Lonning et al., al., 1984) 1984) and the presence of an enzyme (Zotin, (Zotin, 1958), 1958), this process might very well be influenced by cadmium, a metal that is chemically closely related to calcium; a strong calcium/cadmium calciumlcadmium interaction could be assumed, since cadmium competes with calcium for binding sites in the egg (Maljkovic and Branica, 197 1971; capsule (Maljkovic 1 ; von Westernhagen et al., 1975), 1975), thus interfering with the hardening process. The bound cadmium might alter the physical properties of the capsule and its jelIy jelly coat, (Alderdice et al., al., 1979a) 1979a) and penneability permeability to salt reducing its strength (Alderdice and water. Although the cadmium effect on capsule strength may be explained, effects of of other metals and substances are subject of of prob problematic interpretation.

4. 4.

EFFECTS EGGS AND EFFECTS OF OF POLLUTANTS POLLUTANTS ON ON FISH FISH EGGS AND LARVAE LARVAE

263 263

Reduced water uptake by salmonid eggs is also known to be caused by low pH. Thus Peterson and Martin-Robichaud ((1982) 1982) and Eddy and Talbot ((1983) 1983) as well as Rombough and Jensen ((1985) 1985) report inhibited water uptake of newly fertilized eggs of Atlantic salmon and 5.5and lower. Together with the reduced rainbow trout in water of pH 5.5 water uptake goes a decrease in the ability to resist deformation when consubjected to mechanical loads. loads. Rombough and Jensen ((1985) 1985) con exocycluded that the low pH probably interferes with cortical vesicle exocy affects the osmotic activity of perivitelline colloids, for in intosis and affects (Rudy and stance through denaturation of the proteinaceous colloid (Rudy 1969), so active. The same mech mechPotts, 1969), so that it is no longer osmotically active. anism might apply in the action of heavy metals.

C. E Early C. arly Development Blockage Blockage of phosphorylation of ADP caused mainly by aromatic hydrocarbons and naphthalenes may lead to visible effects on early cleavage patterns. Initial irregular cleavages can be related to cytoge cytogenetic damage and can be traced through blastodisc and early gastrula formation by the appearance of opaque cell patches indicating irregu irregular cell cell sizes. sizes. In the meroblastic fi sh egg, the early cleavages on the surface of fish the yolk separate the clearly visible developing blastodisc from the yolk mass. Hence any deviation from the "typical" “typical” cleavage pattern is is easily recognized and has frequently been used to describe sublethal effects on early embryogenesis. In particular, effects effects of temperature and salinity (Lieder, (Lieder, 1964; 1964; Alderdice and Forrester, 1968; 1968; von Wes Wes1968, 1970, 1970, 1974) 1974) have attracted the attention of scientists ternhagen, 1968, (see (see Chapter 3, 3, this volume). volume). In general, sublethal effects effects of pollutants are seldom visible at the very early cleavage stages. stages. Experimentally only substances such as the aromatic compounds compounds benzene and xylene provided irregular cleavages cleavages in the two- to eight-cell stages of plaice platessa) (Lonning, (Lonning, 1977) 1977) and cod (G. morhua) eggs (Pleuronectes platessa) (G. morhua) (Kjorsvik (Kjorsvik et al., al., 1982). 1982). The appearance appearance of the early cleavage stages upon treatment with aromatic hydrocarbons clearly demonstrates the impairment of cell division. division. Similar disruptive early cleavage patterns in cod eggs have been reported by Dethlefsen ((1977) 1977) on treatment of incubated eggs with DDT and DDE. We may find other substances that have the same effects on fi sh eggs, fish eggs, since during our current re research (unpublished data) Baltic we have frequently found data) in the Baltic abnormal early cell stages in pelagic eggs (cod, (cod, plaice, flounder, flounder, sprat) sprat) (Fig. (Fig. 1). 1).The same observations have been made for cod eggs by Kjors-

264

H. VON WESTERNHAGEN H.

Fig. 1. Aberrant Fig. 1. Aberrant early early cleavages cleavages and and gastrula gastrula (arrows) (arrows) in in (a, (a, b, c) c) cod cod (Gadus (Gadus morhua) morhua) eggs, eggs, (d) (d)sprat sprat (Clupea (Clupea sprattus) sprattus) eggs, eggs, and and (e, (e, f) f) plaice plaice (Pleuronectes (Pleuronectes platessa) platessa) eggs, eggs, caught caught with with aa plankton plankton net net in in the the Baltic Baltic in in 1983. 1983. Horizontal Horizontal bars bars 200 200 /-Lm. pm.

vik al. ((1984) 1984) in by Dethlefsen 1985) vik et al. in Norwegian Norwegian waters waters and and by Dethlefsen et al. ((1985) for the eggs of fl ounder Platichthys fiesus, flounder ftesus, dab Limanda limanda, and whiting in the the German German Bight. Bight. whiting Merlangius merlangus in Sublethal effects on the developing embryo are more pronounced in blastodisc stage epiboly and in the the blastodisc stage and and during during beginning beginning epiboly and may may readily readily (1906) describes effects of of Liel LiCl be caused experimentally. Stockard (1906) solutions F . heteroclitus, provoking provoking unusual unusual enlargement enlargement of of the the solutions on on F. segmentation cavity under the blastodisc. Moreover, it is at the blasto blastodisc sh (B. ( B . rerio), rerio),produce produce proto protodisc stage stage that that zinc-treated zinc-treated eggs eggs of of zebrafi zebrafish plasmic protrusions projecting abnormally from the sides sides of the em embryo 1977). When When exposed to to naphthalenes, naphthalenes, bryo (Speranza (Speranza et al., 1977). methylnaphthalenes, methylnaphthalenes, and and aromatic aromatic hydrocarbons, hydrocarbons, eggs eggs of of several several ma marine species display retarded early cell division and differentiation into irregular blastodiscs with opaque patches, indicating different cell sizes 1984; Falk-Petersen et ai., al., 1982), 1982),prob probsizes (Stene (Stene and Lonning, 1984; brief treatment dur durably originating from the initial cleavages. Only brief ing early cleavage frequently permits the embryo to develop normally apparup to midgastrulation before abnormal development becomes appar ent. blastoent. Typical Typical effects effects during during epiboly epiboly are are irregular irregular margins margins of of the blasto cells giving rise to malformed gastrulae, the derm with different size size cells embryo being less distinct and often surrounded by irregular cells (Falk-Petersen 1982). These defects become particularly evi(Falk-Petersen et al., 1982).

4. 4.

EFFECTS OF POLLUTANTS ON FISH EGGS AND LARVAE

265

dent in experiments with aromatic hydrocarbons and DDT and DDE, (G. morhua) morhua) eggs and Smith as reported by Dethlefsen ((1977) 1977) for cod (G. and Cole ((1973) 1 973) for the eggs of winter Hounder flounder (Pseudopleuronectes (Pseudopleuronectes However, embryos with similar aberrations are found in americanus). americanus). the field field (Fig. (Fig. 1). 1). Investigations by Longwell and Hughes (1980) (1980) pro provide some evidence evidence for statistically significant associations between of the health of of cytological, cytogenetic, and embryological diagnoses of pelagic pelagic mackerel mackerel eggs eggs and and heavy heavy metal metal and and toxic toxic hydrocarbon hydrocarbon levels levels of some surface waters. Embryos from these areas (New York Bight) show increased incidence of chromosome and mitotic abnormalities, which to the the observed observed developmental developmental aberrations. aberrations, Par Parwhich probably probably led to ticularly high incidences (>50%) of chromosome bridging and translo translocations cations are are encountered. encountered. It It may may be be difficult difficult to to explain explain the the cause cause and and significance of these aberrations of of early development. development. There could be several reasons, but most of them are probably biochemical in origin, inhibiting metabolic processes responsible for differentiation and 1976) suggest suggest aa blockage blockage and maintenance. maintenance. Rosenthal Rosenthal and and Alderdice Alderdice ((1976) of phosphorylation of adenosine diphosphate (ADP), (ADP),thus inhibiting the formation of adenosine triphosphate (ATP), (ATP), which is a prerequi prerequisite for a multitude of metabolic processes necessary for differentia differentiation. tion. If the energy budget of the embryo is severely reduced either by direct an overload overload in in direct blockage blockage of of the the above above mentioned mentioned pathway pathway or by an metabolic metabolic work work required required for for detoxification detoxification of of hydrocarbons hydrocarbons through through enzymatic enzymatic degradation, degradation, no no coordinated coordinated differentiation differentiation takes takes place place and and development is retarded or arrested. Similar effects can be provoked by exposure exposure of of herring herring embryos embryos (C. (C.harengus) harengus) to to dinitrophenol dinitrophenol (DNP) (DNP) (Stelzer (Stelzer et al., al., 1971), 1971), aa decoupler decoupler of of oxidative oxidative phosphorylation. phosphorylation. Other Other causes the causes for for aberrations aberrations from from the the normal normal cleavage cleavage patterns patterns may may be the substances affecting cell cleavage directly, as does colchi colchiaction of substances cine through inactivation of the chromosome transport mechanism of hydrocarspindle apparatus apparatus.. Similar effects are exerted by cyclic hydrocar the spindle 1977), and the effects resemble those caused by DNP bons (Lanning, (Lonning, 1977), (Watennann, 1940). 1940).Yet short-term effects, when sublethal, might not (Watermann, necessarily be persistent. Particularly during the very early cleavage stages, irregularities caused by different stressors might be adjusted in stages, the course of development. development. If, If, for instance, irregular cell cell patterns undergo further cleavages, the previously noticed asymmetrical pat patterns terns may may disappear disappear in in the the morula morula stage stage and and macroscopically macroscopically no no traces traces (H. von Westernhagen et al., of the initial aberration can be noticed (H. unpublished). The only only remnants of unequal initial cleavages may may be unpublished). chromosome aberrations. Thus, Thus, particularly in early developmental developmental stages in situ, situ, investigations demonstrate relatively high rates of chrochrostages

266

H. VON WESTERNHAGEN H.

mosomal aberrations (anaphase (anaphase aberrations) aberrations) in pelagic fish eggs (Longwell 1980; Kjorsvik et al., 1984; (Longwell and Hughes, 1980; 1984; Dethlefsen et al., 1985). 1985). The significance of these anaphase aberrations for later em embryos is not clear, but Kjorsvik and co-workers suggest that this is is a sign of “bad "bad quality” quality" eggs, yielding low rates of of advanced embryos. embryos. D. Advanced and Late Development A multitude of observations describes the morphological reactions of advanced fish embryos to various pollutants. The most conspicious damages at this stage are abnormal development of the spinal column, abnormal head and eye development, and irregular proliferations from the main body over the yolk surface. surface. Neither of of the mentioned pollutants provokes typical, single-pollutant-specific reactions in the embryo. Morphological aberrations are not particularly pollutant-spe pollutant-specific cific and may be caused also by natural stressors. deThere exists relatively little experimental work on abnormal de velopment in fish embryos before neurulation and formation of the head and optic cups. cups. Most of the effects described are of of advanced and late development, although in nature the situation is different in the 10sense that the rate of malformed early embryos in the sea is 4- to 10(Kjorsvik et al., 1984; 1984; Dethelfsen et al., al., fold that of the late stages (Kjorsvik 1985).One of the reaons why most authors concentrate on later devel devel1985). opmental stages is probably because early aberrations are very incon inconspicious. Besides, in one group, the salmonids, the early stages are extremely delicate to handle and most investigations study the salmo salmonid only beginning at the eyed stage. In this context the term ad advanced development applies to embryos at or beyond stage II, 11, as described by von Westernhagen ((1970) 1970) for cod, flounder, flounder, and plaice, e y e formation, visible tail bud, that is, with the beginning of head and eye and yolk two-thirds surrounded by blastoderm. irreguThe most conspicious damages at and before this stage are irregu lar margins of the periblast on the yolk displaying a serrated appear appearance and emigrating groups of opaque cells in the blastoderm blastodenn layer or in the space between blastoderm and periblast. We have found these (Fig. lc), lc), but the period sorts of defects in live eggs taken in the Baltic (Fig. of epiboly prior to organ formation is rarely described in literature. literature. of Probably before this stage of organ differentiation a lot of early dam damif no more stress is age can be repaired and the embryo may recover if applied. At the same time, this period (gastrulation before closure of applied. of blastopore) blastopore) is considered especially sensitive, and stressed embryos

4.

EFFECTS OF POLLUTANTS ON FISH EGGS EGGS AND LARVAE

267

development. However, at die rather than compensate with aberrant development. the time of organ formation, effects become particularly pronounced in head and notochord, as described for effects of copper and zinc in (G. morhua) morhua) embryos (Swedmark (Swedmark and Granmo, 198 1981) cod (G. 1 ) and lead at B . rerio) rerio) 0.05 and 0.07 mg/l in dechorionated embryos of zebrafish ((B. (Ozoh, 1980). 1980). (Ozoh, These malformations are very similar to those caused by treatment of fish with petroleum hydrocarbons or derivates. Thus, offish Thus, Linden ((1974, 1974, 1976, 1978) reported on herring eggs, which, when treated with crude 1976,1978) oil and/or oil dispersants from 3. 1 to 59 mg/l water-soluble fraction 3.1 (WSF) of crude and number 11 fuel oil, (WSF) oil, display abnormal spinal columns, abnormal heads, and lack of of spinal column. column. In comparable 0.1 to 11.0 mgA, Lonexperiments with Ekofisk oil at concentrations of 0.1 .0 mg/l, Lan 1977) described the same effects on plaice (Pleuroneces ning ((1977) (Pleuroneces pla platessa) embryos. tessa) An embryo embryo damaged in such a manner is is called a typical "oil “oil larva," larva,” with poorly differentiated head, protruding eye lenses, and a bent notochord. Inhibited pigmentation also can be taken as a suble sublestresss (Kjarsvik (Kjorsvik et al., 1982). thal effect and reaction to oil stres 1982). Embryos with these malformations are frequently found after oil spills in the vicinity of oil slicks, slicks, an indication of the fast action of of the WSF of crude oil on fish embryos (Longwell, (Longwell, 1977). 1977). Figure 2 shows the differ different gross morphological abnormalities of cod eggs caused by treat treatment with Iranian crude oil. Available literature on teratogenic effects of of chlorinated hydrocar hydrocarbons on advanced and late fish embryos can be summarized as fol follows. lows. Cod embryos (G. (G. morhua) morhua) exposed to DDT concentrations of mg/l and more react with irregular proliferations at the yolk 0.025 mgtl zigzag-growing spinal surface, and the embryo develops a bent or zigzag-growing column (Dethlefsen, (Dethlefsen, 1977). 1977). Sheepshead minnow (Cyprinodon (Cyprinodon va variegatus) and killifish (F. ( F . heteroclitus) eggs, when subjected to DDT and malathion (organophosphate (organophosphate insecticide), insecticide), carbaryl, or parathion at 10 mg/l, 10 mg/l, display developmental arrest prior to the initiation of heart beat. Blood pigmentation does not occur. occur. Cyprinodon C yprinodon variegatus de develops a malformed spine (Weis (Weis and Weis, 1974, 1974, 1976). 1976). Experiments conducted by Kaur and Toor ((1977) 1977) with carp eggs (Cyprinus (Cyprinus carpio) carpio) and the insecticides diazinon, fenitrotion, carbaryl, malathion, and phosphamidon produce similar effects. Upon exposure to concentra concentrations around 0.008 0.008 (diazinon), (diazinon), 0.25 0.25 (fenitrothion), (fenitrothion), 1.0 1.0 (carbaryl) (carbaryl),, 2.5 (malathion), 12 (phosphamidon) (malathion), and 1112 (phosphamidon) mg/l the embryos show stunted growth, curving of the tail, deformed head regions, enlargement of the pericardial sac, sac, circulatory failure, deformed vertebral column, and

H. VON WESTERNHAGEN

268 268

1 . 5 mm

Fig. 2. Abnormalities in embryonic development of of cod (Gadus (Gadus morhua) rnorhua) eggs un unFig. der the influence influence of (WSF) of Iranian crude oil: (a) (a) early of the water-soluble water-soluble fraction (WSF) (d) twinning, (e) (e)axis deformation, and cleavage, (b) (b) gastrula, (c) (c) embryo without head, (d) (f) (f) microphthalmia. microphthalmia. [From Kuhnhold ((1974).] 1974).]

poorly poorly developed developed eye eye pigment pigment and and chromatophores. chromatophores. Virtually Virtually all all of of these sublethal effects can also be observed when fish embryos embryos de dethese sublethal effects can also be observed when Wesvelop under naturally stressed conditions, as described by von Wes of temperature and salin salinternhagen ((1970) 1970) for the effects of extremes of ity ity on on plaice plaice (P. (P. platessa) embryos embryos (Fig. (Fig. 3). 3). Grossly deformed embryos may be caused Grossly deformed embryos may caused by by still still other other factors, factors, for for (pH 4.5) of of the incubating water in fathead minnow instance, low pH (pH (Pimephales (Pimephales promelas) promelas) (Mount, 1973) 1973) and Atlantic salmon (S. (S. salar). salar). Yet the typical injuries to salmon embryos by low pH, such as altera alterations in vascular structures, cellular dysplasia, necrosis, and sloughing 1980),are similar to those (Daye and Garside, 1980), of superficial ectoderm (Daye caused by heavy metals, detergents, halogenated organic compounds, Thus, it is apparent that it might be and some petroleum fractions. Thus, difficult if not impossible to identify any particular substances respon responsible one sible for for one one or or several several sublethal sublethal morphological morphological effects. effects. On On the one hand, it is difficult to distinguish clearly between morphological, morphological, physiological, or behavioral abnormalities, since one may result from

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EFFECTS OF POLLUTANTS ON FISH FISH EGGS EGGS AND LARVAE

269

Fig. 3. 3. Plaice Plaice (Pleuronectes (Pleuronectes platessa) platessa) eggs eggs and and larvae larvae incubated incubated under under extreme extreme temperature aberrant development: development: (a) (a) temperature and and salinity salinity conditions. conditions. Arrows Arrows indicate indicate zones zones of aberrant salinity; (b) aberrant O T , 25%0 25% salinity; (b)gastrula gastrula with with irregular irregular cell proliferation, proliferation, aberrant early early cleavage, cleavage, ooe, lOoe, l O T , 25%0 25% salinity; salinity; (c) (c) embryo embryo not not able able to to close close blastopore, blastopore, lOoe, 10°C, 20%0 20% salinity; salinity; (d) (d) distorted n development, distorted notochord, notochord, failure failure in in pectoral pectoral fi fin development, 2°e, 2"C,33%0 33% salinity; salinity; (e, (e, f) f ) crip cripm. pled 15% salinity. salinity. Horizontal Horizontal bars bars 200 200 p.. pm. pled larvae, larvae, lOoe, IOT, 15%0

the other, as they are frequently related. n anomalies related. For example, example, fi fin may ed (reduced) may be be related related to to modifi modified (reduced) dermal dermal respiration respiration (Rosenthal (Rosenthal and and Alderdice, Alderdice, 1976). 1976). On On the the other other hand, hand, the the detrimental detrimental action action of aa metal · on embryogenesis may be indirect. For example, cadmium in high high concentrations concentrations may may alter alter the the properties properties of of the the egg egg membrane membrane and and its its "jelly '?jelly coat," as as known from from herring herring eggs eggs (Alderdice (Alderdice et al., 1979c) 1979c)or eggs eggs of garpike (von (von Westernhagen et al., 1975), 1975), ultimately impeding oxygen exchange. Thus, Thus, observed malformations malformations in these experiments experiments may may be attributed attributed simply simply to to lack lack of of oxygen, oxygen, as as described described by by Braum Braum for herring eggs eggs incubated experimentally under low low oxygen oxygen ((1973) 1973) for tension, rather than to direct effects of the the metal. metal. In In fact, fact, some some of of the the anomalies anomalies resemble resemble monstrosities monstrosities produced produced during during incubation incubation at at low low oxygen oxygen levels levels as as described described by by Alderdice Alderdice et aI. 1 958) for 1973) for al. ((1958) for salmon salmon (0. (0.keta) keta) and and Braum Braum ((1973) for herring herring eggs eggs (Clupea (Clupea harengus). harengus). The The general general retardation retardation or or arrest arrest of of development development is is also also aa phenomenon phenomenon occurring occurring at at low low oxygen oxygen levels levels (see (see also also Ham Hamdorf, dorf, 1961). 1961).Therefore, Therefore, in in general, general, one one may may say say that that the the major major morphomorpho-

270

H. VON VON WESTERNHAGEN

logical logical aberrations aberrations such such as as notochord notochord distortions distortions and and head head and and eye eye malformations in late embryos are malformations occurring occurring in late embryos are not not particularly particularly pollu pollutant-specific tant-specific but but are are the the expression expression of of an an embryo embryo in in aa stressed stressed condi condition. tion. E E.. Effects Other than Morphological Aberrations As nonmorphological aberrations, effects on egg shell (chorion), (chorion), embryo activity, and the hatching enzyme are prominent. Deviating from the nonspecific cause/effect relationship for gross morphological deformities and pollutants, reduction of chorion strength is mainly caused by heavy metals and at times by low pH. Low pH typically also depresses activity of the hatching enzyme, which results in low or retarded hatch. Petroleum hydrocarbons are extremely effective de depressors of embryo activity measured as heart beat, pectoral fin move movement, or body activity. Total embryo activity, though, is likewise re reduced by other pollutants. Aside from the occurrence of gross gross malformations, several func functions of the chorion and the embryo are drastically impaired during and after exposure to these pollutants. As already mentioned, cad cadmium severely impairs hardening of the egg membrane after fertiliza fertilization. Cadmium-exposed herring eggs never reach the maximum hard hardness attained by untreated eggs, and the egg capsules remain flaccid throughout embryogenesis (Rosenthal 1974; von WesWes (Rosenthal and Sperling, 1974; temhagen et al., 1974; 1974; Alderdice et al., 1979a). 1979a). In conjunction with this effect, herring eggs upon treatment with cadmium display smaller volumes than individuals incubated under uncontaminated condi conditions (Alderdice al., 1979a,b), (Alderdice et al., 1979a,b), leaving a smaller perivitelline perivitelline space. Also, zinc at a concentration of 6.0 mg/l causes the softening of of egg membranes in C. C. harengus eggs (Somasundaram et al., 1984b) 198413) and brook trout (Salvelinus (Holcombe et ai., 1979). In fathead (Salvelinusfontinalis) fontinalis) (Holcombe al., 1979). minnows (Pimephales (Pimephales promelas), promelas),at a concentration of 295 or 145 145 1Lg/1 pgIl touchand above, Zn reduces chorion strength so that eggs burst upon touch ing. Bursting pressure is only 15% 15% of the normal value at zinc concen concentrations of 1360 1360 ILg/I pgIl (Benoit and Holcombe, Holcombe, 1978). 1978). Chorion strength is also negatively influenced by low pH, as shown by the investigations of Mount ((1973) 1973) with fathead minnow eggs (P. 5.9. The same effects of ( P . promelas) exposed to pH lower than 5.9. low pH on capsule strength are known for eggs of of rainbow trout (S. (S. gairdneri) 1981) report gairdneri) (Kugel, (Kugel, 1984), 1984), although Haya and Waiwood ((1981) hardening of of Atlatnic salmon eggs in water of pH 4.5 due to a change

4. 4.

EFFECTS OF POLLUTANTS EGGS AND EFFECTS OF POLLUTANTS ON ON FISH FISH EGGS AND LARVAE LARVAE

271 271

in the physical structure of the outer mucopolysaccharide layer of the chorion. chorion. The significance of the softening of the egg shell to substrate spawning fish such as salmonids eggs buried salmonids is is evident since the soft eggs fact, in the gravel, when subjected to movement, may easily break. In fact, the eggs of the fathead minnow at low pH become so flaccid that the cleaning action of the male on the egg clutches breaks the egg shells (Mount, (Mount, 1973) 1973) and kills the embryos. embryos. Similar detrimental effects on eggs of the substrate spawning Pacific herring may be expected when high metal concentrations cause softening of the egg membranes. membranes. When the embryo starts to develop a heartbeat, this parameter has frequently been used as a measure of pollutant effects. effects. Typically, heartbeat frequency increases with age, age, and although subject to con considerable variation caused by by disturbances this increase is is consistant with ongoing development (Rosenthal, (Rosenthal, 1967). 1967). Metals such as cadmium reduce embryonic heart rate considera considerably. Thus in garpike Belone belone embryos, reduction in heart rate can be caused by incubation in water containing 1.0 1.0 mg/l or more of the metal, depending on the salinity of of the incubating medium (Fig. (Fig. 4). 4). This is true also for the heartbeat in the Japanese medaka Oryzias latipes when the embryos are reared in 60 p.g p g methylmercury/I, methylmercury/l, where heartbeat is reduced from 80-90 80-90 to 50 beats per minute (Dial, (Dial, 1978). 1978). Zinc at a concentration of of 2 mg/l cause a transient 2 :: 1 heart block (two (two beats of the atrium for every beat of of the ventricle) after 3-4 3-4 days of of exposure in fathead minnow P. P . promelas (Pickering and Vigor, Vigor, 1965). 1965). Linden (1974, 1976, 1978) and Kiihnhold (1974,1976,1978) Kuhnhold (1978) (1978) demonstrated that petroleum hydrocarbons are extremely effective in the impairment of the normal functioning of the heart. Crude oil, especially in conjunc conjunction with oil dispersants, reduces heart-beat of herring embryos by 50%. 50%.Also, killifish F. F . heteroclitus embryos show sublethal responses to the water-soluble fraction of crude oil, which reduces heartbeat and overall embryo activity (Sharp al., 1979). (Sharp et al., 1979). Other hydrocarbons, such as benzene, applied at concentrations of 177 and 45 mg/l to incubating jars with Pacifi c herring C. pallasi and anchovy Engraulis mordax also Pacific reduce heart-beat of late embryos (Struhsaker et al., al., 1974) 1974) or induce irregularities, as does toluene in the embryo of the Japanese medaka 0. (Stoss and Haines, 1979). 1979). O. latipes (Stoss Changes in embryonic heart rate have also been caused experi experimentally by other pollutants, including DNP (Rosenthal (Rosenthal and Stelzer, 1970: C. harengus), 1970: C. harengus), sulfuric acid from titanium dioxide production (Kinne (Kinne and Rosenthal, 1967: 1967: C. harengus), harengus), or the organophosphate insecticide malathion (Weis (Weis and Weis, 1976: 1976: Cyprinodon variegatus). variegatus). At a stage of of development where the regular heartbeat is well

272 272

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4.

EFFECTS OF OF POLLUTANTS POLLUTANTS ON ON FISH FISH EGGS EGGS AND AND LARVAE LARVAE EFFECTS

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is developed far enough to enable the established, the embryonic axis is movements . These movements, embryo to make the first wriggling movements. accomplished by slight, repetitive body flexure, flexure, are presumed to circir accomplished perivitelline fluid, fluid, thus improving the increasing oxygen culate the perivitelline of the late embryo. In some species that release very advanced needs of larvae from their eggs (i.e., (Le., B B.. belone, 0. O. latipes, F F.. heteroclitus, salmonids), the undulating movements of of the body axis are supported salmonids), flapping of of the pectoral fins and the opercula. opercula. As is the case for by flapping heartbeat, embryonic movements may be severely impaired either directly through through the stressors or indirectly whenever egg volumes are reduced imposing mechanical blockage to movements. The influence of cadmium, for instance, on embryonic movements in herring eggs of becomes apparent when the embryo has grown to encircle the yolk. "wriggling" then is is replaced by “trembling,” "trembling," a high-frehigh-fre The normal “wriggling” is maintained maintained even in more advanced embryos, quency shivering that is "somersaults" within the which are normally performing rotations or “somersaults” of activities are influenced influenced by egg shell. As Fig. 5 shows, both types of (von Westernhagen et al., al., 1974). 1 974). cadmium in the incubating water (von 13 mg/l exert strong paralyzing efef Copper concentrations around 0. 0.13 be fects on herring embryos, which, with progressing development, become more and more immobilized (von Westernhagen et d al.,. , 1979). 1979). Other sublethal effects of of cadmium on embryo activity are displayed by the reduced pectoral fin movements of of garpike ((B. B . belone) embryos al., 1975) concentra (von Westernhagen Westernhagen et al., 1975) when exposed to cadmium concentramg/1. Stoss and Haines (1979) ( 1979) as well as Leung tions higher than 1.0 mg/l. and Bulkley ((1979) 1979) report influence of toluene on the opercular move move0. latipes, which ment of the late embryo of the Japanese medaka O. becomes erratic, irregular, and shallow at concentrations of of 80-100 80-100 pgIl (WSF). (WSF). A general decrease of embryonic activity is known for JLg/1 salar) embryos when reared at low pH of 4.0-4.5 4.0-4.5 compared salmon (S. salar) to controls in pH 6.7 (Peterson (Peterson and Martin-Robichaud, 1983). 1983).A possi possible explanation for the reduced activity in cadmium-treated embryos Pacific herring may be the effects of cadmium on enzyme activity. In Pacific eggs, Mounib et al. al. ((1976) 1976) found that exposure to 10 10 mg cadmium/l cadmiumll decreased activity of four important carbon dioxide-fixing dioxide-fixing enzymes: enzymes: propionyl coenzyme A (CoA) (CoA) carboxylase, nicotinamide adenine di dinucleotide (NAD) (NAD) and NADP NADP malic enzymes, and phosphoenolpy phosphoenolpyruvate (PEP) eggs, PEP (PEP) carboxykinase. In control eggs, PEP carboxykinase activ activity ity increases by two two orders orders of magnitude in the period from the early blastodisc just prior to hatching. The increase indicates the impor importance tance of the enzyme metabolism. metabolism. In contrast, there is considerably less less increase increase in in PEP PEP carboxykinase carboxykinase activity activity in in cadmium-exposed cadmium-exposed eggs eggs

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erent cad Fig. Fig. 5. 5. Activity Activity of herring herring (Clupea (Clupea harengus) harengus)embryos embryos influenced by diff different cadmium mium concentrations concentrations in the incubating incubating water. water. [From [From von Westernhagen Westernhagen et et ai. al. (1974).] (1974).]

up to the beginning of embryo activity, while in the controls activity further. Final activity of of the enzyme is more than 25% less increases further. of the controls. Relative activity of of propionyl-CoA carboxyl carboxylthan that of ase in the cadmium exposed eggs is about 20% 20% of the control level closure 32% prior to hatching. Relative prior to blastopore clos ure and only 32% of NAD and NADP malic enzymes enzymes remains stable up to eye activity of reduced by about 20% 20%just prior to hatching, when pigmentation but is reduced the embryo has completed differentiation and growth is dominant. In of the important role played played by carbon dioxide-fixing dioxide-fixing enzymes view of effect of cadmium in depreSSing depressing en enin biosynthetic processes, the eff ect of developmental stages may result in lethargic zyme activity during the developmental larvae. embryos and small and inactive larvae.

4. 4.

EFFECTS EFFECTS OF OF POLLUTANTS POLLUTANTS ON ON FISH FISH EGGS EGGS AND AND LARVAE LARVAE

275 275

As a consequence of the lowered activity of the embryo, embryo, the hatch hatching process may be severely impaired-on impaired-on the one hand, because the hatching enzyme is is not distributed throughout the perivitelline fluid and on the other hand, after the digestion of the inner layer of the zona radiata by the hatching enzyme, the emerging larva cannot break the nondigested outer part of the egg shell (Hagenmaier, (Hagenmaier, 1974a), 1974a),and thus remains longer in the egg casing or never hatches. Even though the proteolytic hatching enzyme may be produced and distributed sufficiently, heavy metals in the external and internal medium may not allow the enzyme to display its proteolytic functions fully. Hagenmaier ((1974b) 1974b) reports that manganese, zinc, mercury, or copper inhibits the proteolytic functions of of the enzyme. From experi experiments with Atlantic salmon (S. salar), brown trout ((S. ( S . salar), S . trutta) trutta),, and rainbow trout (S. 1967) thinks that zinc affects ( S . gairdneri), gairdneri), Grande ((1967) affects the enzymatic processes that soften the egg capsule. capsule. Also, Also, the pH of of the incubating medium has aa strong strong bearing on the functioning of of the hatching enzyme, which has a maximum activity at pH 8.5 8.5 in eggs of of the rainbow trout (Hagenmaier, 1974c) and from 7.5 to 8.0 in the (Hagenmaier, 1974c) 7.5 to 8.0 Pacific salmon (0. (0. keta) 1969). Thus, in experiments with keta) (Bell (Bell et al., 1969). perch (Perca fluviatilis) eggs, Runn et al. 1977) found strong impair (Perca$uuiatilis) aE. ((1977) impairment of of the activity of the hatching enzyme at low pH resulting in reduced hatch. Similarly, at pH 4.5, 4.5, chorionase activity of of Atlantic salmon eggs reached only 49% of its activity at pH 6.5 (Haya (Haya and Waiwood, 1981). Waiwood, 1981). F. ncubation Time and the Process of Hatching F. IIncubation Alterations in incubation time may be caused by premature or delayed hatching. Metals either shorten shorten or or lengthen lengthen this this period, period, delayed hatching. Metals may may either and no unequivocal prediction is possible; petroleum hydrocarbons as prediction petroleum WSF usually retard hatching and development. The effect of of chlorinchlorin ated hydrocarbons on hatching hatching have not been looked into closely. While low pH retards development in general, the major effects of oflow low of chorionase (hatching enzyme) 5.5, pH are on inactivation of enzyme) below pH 5.5, or prevents it entirely. which delays hatching or Onset of of hatching in teleost eggs begins with the secretion of of pro proteolytic hatching enzyme from the hatching glands around the head region of of the enzyme is of the embryo/larva. The secretion and activity of F.. heteroclitus heteroclitus eggs (Kaighn, relatively rapid, as seen in studies with F (Kaighn, 1964). Chorion treated with chorionase dissolves within 5-10 5-10 min. 1964). Thus 1970), Thus,, with the strong movements of of the late embryo (Poy, (Poy, 1970), hatching in F. F . heteroclitus should be completed within a few minutes,

276

H. VON VON \\"ESTERNHAGEN U’ESTERNHAGEN

while it may take several hours in salmonids with a thicker egg shell and lower incubation temperatures (Hayes, al., 1969). 1969). (Hayes, 1942; 1942; Bell et al., Incubation time, the period between fertilization and 50% 50% hatch, is mainly dependent on temperature, higher ranges rirnges accelerating devel development. Low oxygen concentrations, when maintained throughout development, lengthen the total incubation period (Hamdorf, 1 ; S. development, (Hamdorf, 196 1961; S.

gairdneri). gairdneri). Changes in time to hatch are common in fish embryos subjected to sublethal effects of pollutants. Many xenobiotic substances shorten the incubation period or cause premature hatch. Others, however, lengthen the development period or delay hatching. Due to the differ different effects of metals on the late embryo, incubation may be either shortened or lengthened. In most cases that have come to the atten attention of the reviewer, incubation time is shortened and larvae hatch prematurely. Rainbow trout (S. (S. gairdneri) gairdneri) has frequently been used for these essays. essays. Thus, Shabalina (1964) (1964) notes that cobaltous chloride in concentrations of up to 5 mg/l shortens the incubation period; hatching larvae are viable. Also, Also, nickel and copper when applied in concentrations concentrations of 11 mg/l accelerate development of of rainbow trout eggs by about 45% 45%with copper and by 20% 20%with nickel (Shaw (Shaw and Brown, 1971). 1971).The same is true for vanadium (44 (44mg/l) (Giles (Giles and Klaverkamp, Klaverkamp, 1982). 1982). Brook trout (Salvelinus (Salvelinus fontinalis) fontinalis) eggs incubated at 32.5 32.5 p.. pgg copperll copper/l hatch prematurely (McKim (McKim and Benoit, 1971) 1971) as do herring at even lower concentrations (>0. 1 mg/l) (>0.1 mg/l) (Ojaveer et al., 1980). 1980). Other metals such as cadmium and zinc have the same effects on fish em embryos as shown by Vladimirov (1969) (1969) for carp (Cyprinus carpio) carpio) and by Rosenthal and Sperling ((1974), 1974), von Westernhagen et al. al. ((1974), 1974), and Somasundaram et al. 1984a) for herring (Clupea al. ((1984a) (Clupea harengus). harengus). However, the reverse, a prolonged incubation period, is is also known to be caused by metals such as zinc, cadmium, copper, and mercury (Grande, (Grande, 1967; 1967; Servizi and Martens, 1978; 1978; Swedmark and Granmo, 198 1 ; Weis, 1984; 1981; 1984; Somasundaram et al., al., 1984a,b). 1984a,b).In the case concenof zinc, concentrations below 2.0 2.0 mg/l accelerate while higher concen trations retard development of of herring eggs. eggs. The above does not imply that embryos hatching early developed faster or that the late-hatching ones displayed delayed embryogene embryogenesis. In fact, fact, changes in differentiation pattern, known to occur in trout sis. (S. eggs, developing at low partial O ( S . gairdneri) eggs, 0 2 pressure (Hamdorf, (Hamdorf, 2 1961), (in the 1961), are not reported. Rather, the immature embryo hatches (in case of "acceleration") “acceleration”) or an over-mature larva hatches with a small yolk reserve and advanced differentiation (a functional mouth in the case of cod G. G. morhua; morhua; Swedmark and Granmo, 1981). 1981).

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EFFECTS OF POLLUTANTS ON FISH EGGS AND LARVAE EFFECTS

277 277

ObThere may be no general explanation for this phenomenon. Ob effects depend on the application of the metal, its concentra concentraserved effects exposure, etc. Thus the rea reation, stage of development, duration of exposure, sons for early or late hatching are frequently found in the history of the egg. On the one hand, egg. hand, an early hatch might be caused by a beneficial low levels of, of, say, zinc, on the embryo (Somasundaram et al., effect of Iow 1984a; C. C . harengus), harengus), with truly accelerated development in the sense 1984a; “sufficient challenge" challenge” concept forwarded by Smyth (1967), (1967), or of the "sufficient 0.133 by the highly detrimental effects of copper at concentrations of 0. 133 mg/l, which immoblize herring embryos totally, so that the hatching (no distribution of glands produce a punctiform hole in the chorion (no enzyme), causing premature liberation of the embryo (von (von hatching enzyme), 1979) (Fig. (Fig. 6). 6). On the other hand, late hatch may Westernhagen et al., 1979) concenbe caused by a retarded development such as caused by high concen (>2 mg/I), mg/l), with resulting inability of the embryo to trations of zinc (>2 1981), or because of mal malbreak the chorion (Swedmark and Granmo, 1981), functioning of the hatching enzyme proper as suggested by Servizi (1978) for the delayed hatching of sockeye (Oncorhy (Oncorhyand Martens (1978) (0.gorbuscha) gorbuscha) exposed to copper con connerka) and pink salmon (0. nchus nerka) pgl1.. A similar explanation may apply for the centrations higher than 6 p,g/l other substances dealt with in this context and the sublethal effects of hydrocarbons, which may likewise lengthen the time from petroleum hydrocarbons, fertilization to hatching or may shorten it. In contrast to the common effects of heavy metals on incubation, most authors report a delayed effect of petroleum hydrocarbons hydrocarbons on hatching when applied as the water-soluble fraction (WSF). (WSF). At low concentrations ((12.5%) 12.5%) of the WSF of crude oil, development was F . grandis (Ernst (Ernst et al., 1977); the accelerated, with early hatch in F. al., 1977); early hatch of Japanese medaka Oryzias Iatipes latipes is likewise considered premature hatching resulting resulting from stimulation of the the hatching mech mecha premature anism by oil components (Leung (Leung and Bulkley, Bulkley, 1979). 1979). However, most authors report delayed hatch of larvae after treatment of developing (1978) eggs and embryos with petroleum hydrocarbons. Thus, Linden (1978) C. harengus mem memreported delayed hatching in Baltic herring larvae C. WSF of light fuel oil; oil; this is also also true when bras exposed to 54 mg/l WSF (24, 48, 96 h) h) to 40 40-45 concenherring are pulse-exposed (24, -45 mg/l initial concen trations of benzene (Struhsaker et al., 1974). 1974). Delay in hatching also occurs in eggs of winter flounder Pseudopleuronectes americanus if 100 p.g/l pgll WSF of number 2 fuel oil the parents have been exposed to 100 (Kiihnhold et al., 1978), 1978),and and in F. F . heterocli heterocliduring gonad maturation (Kiihnhold 25% WSF of this oil (Sharp (Sharp et al., 1979). 1979). Other reports tus exposed to 25% Kuhnhold of petroleum hydrocarbons delaying hatching are given by Kiihnhold

278

H. VON VON WESTERNHAGEN WESTERNHAGEN

a

Fig. 6. 6. Clupea harengus. harengus.(a) (a) Empty egg chorions with punctiform hole after incuba incubation -(133 (b) prematurely 133 p.g pg Cull); CuA); (b) prematurely hatching hatching crippled crippled tion in in copper-contaminated copper-contaminated water water .( herring mm. herring larva larva after after incubation incubation in in copper-contaminated copper-contaminatedwater. water. Horizontal Horizontal bar bar 11 mm.

4. 4.

EFFECTS EFFECTS OF POLLUTANTS POLLUTANTS ON ON FISH EGGS EGGS AND AND LARVAE LARVAE

279

((1974) 1974) (G. (G. morhua), morhua), Stoss Stoss and Haines (1979) (1979) (0. (0.latipes, Zatipes, toluene) and Carls and Rice (1984) (1984) (Theragra (Theragra chalcogramma). chalcogrumma). Further, the polycy polycyclic aromatic carcinogen, benzo[a]pyrene, is known to retard develop development and hatching in rainbow trout (S. ( S . gairdneri) (Kocan and Landolt, 1984; Hannah et al., 1982). 1982). The effect of high concentrations of petro 1984; petroleum hydrocarbons on fish embryos may sometimes be a narcotizing one that reduces metabolism, thus slowing down development (Struhsaker et al., 1974; 1974; Carls and Rice, 1984) 1984) as well as exerting narcotic effects on the late ready-to-hatch embryo. There is little information on the disturbance of pro of the hatching process by chlorinated hydrocarbons (mainly (mainly pesticides and PCB). PCB). No direct impairment of hatching has been reported. Reports of variations of time to hatch refer to abnormally shaped embryos that hatch late due to physical failure to break the egg shell (Dethlefsen, (Dethlefsen, 1977; 1977; G. morhua, morhua, DDT) DDT) or to premature hatch in coho salmon Oncorhynchus kisutsch eggs, eggs, treated with Arocolor 1254 1254 (PCB) (PCB) at 4.4 and 15.0 15.0 f'g/l pg/l (Halter and Johnson, 1974), 1974), where early hatching may be caused by an alteration of the chorion due to PCB treatment. Reduction in hatching time also occurs in eggs of minnow Phoxinus phoxinus when the pa parental fish have been administered PCB PCB orally and the eggs contained high amounts ((1.5-170 1.5-170 mg/kg mgkg fresh weight) of Clophen 50. 50. Hatching time in eggs containing > >15 mgkg fresh weight PCB is significantly 15 mg/kg reduced compared to controls (Bengtsson, (Bengtsson, 1980). 1980). In contrast to the effects of pesticides on the hatching process and hatching time, which are diffuse and probably relate to the general disturbed condition of the embryo, effects of pH on the hatching pro process are much better understood. Report of sublethal effects of of pH on fish eggs and larvae are mainly restricted on the effects of low pH related to acidification of of lakes in North America and northern northern Eu Europe. The effect of low pH on development and hatching is is very con consistent in all but one of the available reports. Only Trojnar ((1977b) 1 977b) in his experiments with brook trout (Salvelinus fontinalis) eggs reported (Sa2veZinusfontinaZis) faster development and hatching at pH 4.65 than at pH 8.07, 8.07, where hatching took place 12 12 days later at the higher pH. Early hatching larvae at pH 4.65 4.65 did not appear to be premature, but were fully developed. This report is contrary to all other information on suble sublethal effects of low pH on early developmental stages of fish, fish, where there is general agreement that low pH prolongs the period from fer fertilization to hatching. Thus, Peterson et al. ((1980a,b) 1980a,b) show that the eggs of Atlantic salmon, S . salar, salary exposed to water of pH 4.0-5.5, 4.0-5.5, following eye pigmentation, showed delayed hatch. The same effects are reported by Swarts et al. al. (1978) fontinalis) eggs (1978) for brook trout (S. (S.fontina1i.s)

280 280

H. VON H. VON WESTERNHAGEN WESTERNHAGEN

incubated at pH 4.75. The delay in hatching may be considerable, reaching 14 14 days for eyed eggs of rainbow trout ((S. S . gairdneri) gairdneri) sub subjected to pH 4.0-4.5 4.0-4.5 when compared to controls incubated in pH 7.87.8(Kugel, 1984). 1984). In yellow perch (Perea (Percajluviatilis), fluuiatilis), incubation time 8.0 (Kiigel, increased by 29% at pH 4.0 compared to pH 6.4 6.4 (Rask, (Rask, 1983). 1983). The prolongation of the period from fertilization to hatching at low pH occurs also in eggs of of zebrafish (B. ( B . reTio) rerio) (Johansson (Johansson et al., al., 1973) 1973) and fathead minnow (Pimephales (Pimephales promelas) (Mount, (Mount, 1973). 1973).Likewise, low pH retards development of Pacific herring (C. (C. pallasi) pallasi) embryos, as already observed by Kelley ((1946), 1 946), while high pH (pH (pH 10) 10) accelerates B.. rerio). development slightly (Johansson (Johansson et al., 1973; 1973; B rerio). The factors factors involved may be either a general retardation of development, or a delay in the process of hatching, or both. Evidence for retarded devel development is 1946), but in most cases the prolonged is provided by Kelley ((1946), incubation period is due to an impairment of the hatching process due to inhibition of enzyme (chorionase) (chorionase) activity at lower pH. Due to the permeability of the chorion for hydrogen ions (Peterson (Peterson et al., al., 1980a), 1980a), the perivitelline fluid rapidly adjusts to the pH of the incubating me medium, and the pH may be too low for maximum enzyme activity. Thus at pH 5.2, chorionase activity of rainbow trout SS.. gairdneri embryos, is reduced to 10% 10% of the optimal rate at a pH of of 8.5 8.5 (Hagenmaier, 1974a), 1974a), and the process of hatching takes several days rather than hours. In salmon SS.. salar, reduction of chorionase activity at low pH is less drastic, but still, at pH 4.5 only 49% of of the activity at pH 6.5 is ob observed (Haya (Haya and Waiwood, 1981). 1981). Alevins of of Atlantic salmon (S. ( S . sa salar) lar) delayed in hatching are thus larger than when hatched at the normal pH (Peterson et al., 1980a). 1980a). Other indications of of incomplete lysis of the chorion at low pH are tail-hatched larvae with the chorion around the yolk or partially hatched larvae with the head still inside the egg shell (Johansson 1 ; Kiigel, (Johansson et al., 1977; 1977; Brown and Lyman, Lyman, 198 1981; Kugel, 1984). 1984).

G . Hatchability and Viable Hatch G. effects of pollutants One of the main interests scientists had in the effects on fish eggs and larvae was the reduction in hatching success. Effects of pollutants on hatchability and viable hatch are dependent on the stage of development, and the species and type of pollutant. Exposure before closure of blastopore causes more severe reduction in hatching “Viable hatch" hatch” is a success than when advanced stages are exposed. "Viable effects than "hatchability." “hatchability.” more sensitive indicator of pollutant effects

4. 4.


281 281

SSince ince the total of all previously mentioned effects on eggs and embryos is expressed in the number of larvae emerging, it appears useful to present data on reduction of hatchability and viable hatch (percent) in a table (Table (Table I). (percent) I). This should enable the readers to deter determine readily the effects of pollutants on emergence of larvae. For the user's user’s convenience, zero hatch is included. Data in Table I show that hatchability as a parameter to assess sublethal effects is is limited in its use by the differences in toxicity of of the various pollutants. Thus, in saltwater species, cadmium exerts detrimental effects only at high (environmentally nonrelevant) nonrelevant) concentrations in the range of 100010002000 ILg/1 1975; Voyer et al., 1979) 1979) or pg/l (von (von Westernhagen et al., 1974, 1974, 1975; more (Rosenthal (Rosenthal and Sperling, 1974; 1974; von Westernhagen and Dethlef Dethlefsen, 1975). 1975). In freshwater species reactions toward cadmium are more sensitive (Pickering (Pickering and Gast, Gast, 1972; 1972; Spehar, Spehar, 1976; 1976; Rombough and Garside, 1982). 1982). The situation is similar regarding the effects of zinc. Copper, in tum, turn, even in saltwater, saltwater, causes substantial effects on hatch hatchability and viable hatch at concentrations between 30 and 90 ILg/l pg/l (Blaxter, (Blaxter, 1977; 1977; Servizi and Martens, 1978; 1978; Ojaveer et al., 1980; 1980; Cosson and Martin, 1981). 1981). These concentrations are frequently found in sur surface microlayers of polluted areas (Hardy et al., d., 1985), 1985), which have been found to cant reduction in hatchability of herring (C. to cause signifi significant (C. harengus) harengus) eggs (von Westernhagen et al., 1987). 1987). Sublethal effects on hatching caused by chlorinated hydrocarbons are dependent on the toxicity of of the pollutant but also on the mode of of application. Via a biomagnifi cation effect, DDT reduces hatching in biomagnification eggs from female fathead minnow (Pimephales promelas), promelas),kept in wa wap g DDT/I DDT/l until spawning, from 89% to 74%, 74%, and the PCB ter with 2 ILg Aroclor 1254 1254 prevents hatching entirely in eggs from female fathead minnows treated with 4.6 4.6 ILg/1 pg/l (Nebeker et al., 1974). 1974). When Aroclor 1254 1254 is is applied to Cypronodon variegatus eggs at 10 10 ILg/I, pg/l, hatchability is is only 57% (Schimmel et al., 1974). 1974). In general it is evident that hatchability of eggs increases when later developmental stages are exposed to pollutants. In early-stage exposures, effects on percent hatching are more severe. This is in line with Stockard's 192 1) ideas concerning effects of development arrest Stockard’s ((1921) in fish embryos through abiotic substances. Thus, in F. F . heteroclitus, egg development may be stopped safely shortly after gastrulation is completed. Critical stages are those before closure of completed. of the blastopore, during which marked inequalities of of cellular proliferations are taking place. Since body-axis body-axis formation takes place fairly early in develop development (beginning with neurulation and continuing throughout onto ontogenesis), genesis), impairment of this process has its ultimate bearing on hatch-

Table Table II Sublethal Effects of Hydrocarbons, Chlorinated Hydrocarbons, pH) of Pollutants (Heavy Metals, Petroleum Hydrocarbons, pH) on Hatchability and Viable Hatch of of Fish Eggs· Eggs"

Species

Pollutant

of Day of exposure

Concentration ((PdU lLgll)

11 11 11 11 11 11 11

C 100 100 500

Hatchability (W (%)

Viable hatch ((%) %)

"C °C

Salinity (%0) (W

Reference

Heavy metals

Belone belone Belone belone

IoQ N go 8 IoQ N

Cd Cd

Brachydanio Brachydunio reTio rerio

Cu+Pb Cu+Pb

Brachydanio Brachydunio reTio rerio

Cu cu Pb Zn Zn

Brachydanio Brachydanio reTio rerio

H g Hg

Cichlasoma Cichlasoma nigronigrofasciatum fascia tum

Pb Pb

11 11 11 11 P P P

p P

P P P

pc P' pc P" pc P"

pc F pc P" pc p"

1000 lo00

2000 5000 C C 36 72 72 72 C 5000 C C 11 0.2 C 50 100 100 300 400 500 600 600

73 73 62 68 73 73 2 1 21 0 86 86 38 38 56 47 42

100b lOOb

15 15

41 4 1

29 30 95 45 30 17 17 27 21 21 44

von von Westemhagen Westernhagen et al. al. ((1975) 1975)

100 100 90 65 0 26

63 63 11 44 44

25

2 7 27

25

Ozoh (1979a) (1979a)

Speranza et et al. al. ((1977) 1977) Kihlstrom eett al. ((1971) 1971)

Ozoh ((1979b) 1979b)

Clupea harengus

cCuu

Clupea harengus

Cu cu

Cd N Q!l w

Clupea harengus

Cd

Clupea harengus

Cd

Dicentrarchus labrar labrax

cCu u

4 4 4 4 4 4 11 11 11 11 11 11 11 11 11 11 11 11 11 11 1 11 11 11 11 11 11 11 11 11 11 11 11 11

C 30 90 90 300 900 C 30 90 300 C 55 10 10 100 100 C 3 5 50 500 500 C 100 100 500 1000 1000 5000 5000 C 100 100 1000 1000 5000 5000 10000 lo000 C C 55 10 10 50 50 100 100

45 45 44 44 0 0 0 0 0 0 25 22 12 12 0 0 96 96 93 93 98 98 98 98 97 97 91 91

89 89 95 95 84 75 14 14 100 100 74 79 79 18 18 11

9-10 9- 10

90 81 81 71 71 71 71 86 81 81 82 82 66 13 13 94 95 95 93 62 62 0 0 87 87 83 83 16 16 0 0 0 0

29-32 29-32

Blaxter ((1977) 1977)

Ojaveer Ojaveer et al

(1980) (1980)

10 10

16 16

von von Westemhagen Westemhagen

et 1974) et al. ((1974)

10 10

16 16

Rosenthal Rosenthal and Sperling Sperling (1974) (1974)

Casson Cosson and Martin

(( 198 198 1) 1)

I (continued) (continued)

Table II (Continued) (Continued) Table

Species Species

Pollutant Pollutant

Fundulus heteroclitus

Hg Hg

Jordanellafloridae jloridae Jordanella

Cd, Zn

Menidia menidia

Cd

saratilis Morone saxatilis

cu Cu

Oncorhynchus Oncorhynchus nerka

cu Cu

N Q!) lI>-

Day of exposure exposure

11 11 11 11 11 11 11 11 11 11 11 I1 11 11 5 5 5 5 2 2 2 2 2 2 2 22 2

Concentration Concentration

(p.g1I)

C

44 10 10 20 30 40 40 60 80

C, c, Cc

17,28 17, 28 4.1, 47 4.1, 8.1, 75 139 16, 139 31,267 31, 267 C

1170 70 390 750 C

10 10 100 100 500 2800 5000 18 18 37 78

HatchHatchability ability %) ((%)

Viable hatch hatch (%o) ) (%

"C °C

25 25

89 81 81 81 81 73 69 41 41 6 0 66,70 66, 70 66, 76 66,76 73, 72 66,59 66, 59 68, 73 68,73 0, 0, 0 0 90 100 100 94 74 100 100 45 27 45 0 0

Salinity (%0) (%)

20 20

Sharp Sharp and and Neff Neff

(1980) (1980)

Spehar (1976) Spehar (1976)

25

15-19 15-19

Reference Reference

20

al. (1979) (1979) Voyer et al.

O'Rear ((1972) 1972)

98 98 96 96 50 50

6-9 6-9

Servizi and Martens (1978) (1978)

Cd

Oncorhynchus Oncorhynchus nerka nerku

Hg

Oncorhynchus Oncorhy nchus tshawytscha tshaw ytscha

Cu cu

Oryzias latipes Oryzias latipes

Hg

Pimephales promelas melas

Cd

Platichthys flesus Platichthys flesus

Cd

I>:) 00 �

2 2 2 2 22 2 22 2 22 2 11 11 11 11 11 11 11 11 11 11 11 11 11 11

11 11 11 11 11 11 11

174 174

0 0 96 96 97 97 96 95 95 95 95 93 93 95 95 64 64 0 0

C

0.4 0.4 1.5 1.5 5.7 C 1.0 1.o 2.5 4.3 4.3 9.3 9.3 C 2211 40 40 80 80 C 10 10 15 15 20 30 C 7.8 7.8 14 14 27 57 57 C

100 100 500 500 1000 1000 2000 2000 3000 3000 5000 5000

80 76 82 82 78 78 47 47 58 58 21 21 00 00 95 95 97 97 95 95 94 94 78 78 87 83 83 84 84 81 81 80 80 67 67 22

6-9 6-9

6-9 6-9

Servizi and Martens ((1978) 1978)

1314 13-14

Hazel and Meith

(1970) (1970)

26

?

25 25

Heisinger and Green (1975) (1975)

Pickering and Gast

(1972) (1972)

5 5

32

Westernhagen von Westemhagen and Dethlefsen

(1975) (1975)



Species

Pollutant

Pseudopleuronectes Pseudopleuronectes americanus americanus

Cd, Cd, Ag Ag

Pseudopleuronectes Pseudopleuronectes americanus americanus

Cd

� N 0 Q!) �

m

Salmo gairdneri

Cr

Day of exposure

Concentration (/Lg!l) (Pd)

11 11 11 11 11 11 22 2 2 22 2 2 2 11 11

C, C c

11

Salmo Salmo salar

Cd

SalveZinus fontiSaloelinus fontinalis naZis

Zn

11 11 11 11 11 11 11 11 11 11 11

Hatchability

(96) (%)

c,

100, 100, 18 18 99 550, 550,99 1000,180 1000, 180 1000 lo00 1000, 18 1000,18 C 100 100 320 1000 lo00 11150 150 1550 1550 2100 2100 C 20 200 2000 2000 C 2.8 111 1 29 90 270 270 870 343 343 724 709

93 80 80 87 90 100 100 98 88 98 98 100 100 84 84

93 97 95 95

Viable hatch (96) (%)

100 100 100 100 100 100 90 36 55 55 90 74 74 881 1 19 19 84 84 78 49

76 82 70 80 80 76 47 0 0

°C “C

Salinity (6) %,;)

Reference

99

2211

Voyer eett al. 1982) al. ((1982)

10 10

20 20

Voyer et et al. (1977) (1977)

5 12 12

Van der Putte et et 1982), expo al. exp. al. ((1982), at pH 6.5 6.5

5

Rombough and (1982). Garside (1982).

9

Holcombe et et al. ~ l

((1979) 1979)

.

Salvelinus fontifontinalis naZis

Salvelinus Salve linus fontifontinalis

Pb

P

mHg

t.:l CD -t

Salvelinus fontifontiSalvelinus nalis nalis

1 1 1 1 1 1 1 1 1 P

ceu u

P P P P P P P P P P 1 P, 1 P, P, 1 1 P, P, 1 1 P, P, 1 1 P, P, 1 1 P, P, P, 11

1382 1382 1353 1353 2017 2099 2099 4336 4336 4363 4363 e C 3 ,4 3,4 58 58 119 119 235 235 474 e C 0.03 0.09 0.09 0.3 0.3 0.9 0.9 2.9 2.9 e C 3.4 5.7 9.5 17.4 17.4 32.5 32.5

90 90 79 67 67 73 73 3 3 1.5 1.5 96-100 96-100 57-95 57-95 79 79 85-86 85-86 60-73 60-73 28 28 97 87 87 99 99 98 0,84 0 ,m 0 81 81 99 99 85 85 98 98 95 95 26

9 9

et al. al. Holcombe et ( 1 976) (1976)

McKim et et al. al.

(1976) (1976)

5-14 514

McKim and Benoit

( 1971) (1971)

Petroleum hydrocarbons

Clupea harengus

Oil

(WSF)

1 1 11 1 1 1 1

1 1 1

e C 600 600 1900 1900 5400 5400 17500 17500 36000 36000

32 32 50 50 26 26 28 28 24 24 44

14 14

6

Vuorinen and and AxeII (1980) h e l l (1980)

~~



Species

� K2 co co

g

Pollutant Pollutant

Clupea harengus harengus Clupea membras membras

Oil (WSF) (WW

Clupea harengus harengus Clupea pallasi Clupea harengus harengus Clupea pallasi pallasi

Benzene Benzene

Fundulus heteroheteroFundulus clitus clitus

Fundulus Fundulus heteroheteroclitus clitus

Oil (WSF) (WW

Oil (WSF) (WSF)

Oil Oil (WSF) WSF)

Day of exposure

Concentration Concentration (PdU (,ug/l)

Hatchability (%) (%) 100 100

11 11 11 11 3 3 3 P P 5-6 5-6

C 50 500 5000 50 500 500 C C 800 C

4 0 99 9 1 91 0 93 67 53

5-6 5-6 5-6 5-6 5-6 5-6 5-6 5-6 11 11 11 11 11

680 680 680 680 C 10% 10% WSF 20% 20%WSF 25%WSF 25% 25% WSF

44 32 27 0 90 90 72 7 85

11

25% 25% WSF

40

11 11 11 11

C 25% 25%WSF 50% 50%WSF 100% WSF 100%

100 100 100 100 60 0

60 60

Viable hatch (%)

Salinity

(W (%0)

"C °C

Reference

9-14 9-14

6

Linden Linden (1978)

111-12 1- 12

22

Struhsaker (1977) Struhsaker

20

Smith and Cameron ((1979) 1979) 8h h only only exposed exposed 24 h only exposed 8h h only exposed exposed 6 days days exposed exposed Sharp 1979) Sharp et et al. al. ((1979)

8-9 8-9

22

21 2 1

20

4 days only exexposed posed 8 days only exexposed posed Anderson et et al. al. ((1977) 1977)

Cyprinodon vavaCyprinodon riega tus riegatus Gadus morhua morhua Gadus

ta t-:) 0 co (D

Oil (WSF)

Mallotus villosus

Oil (WSF)

Platichthysflesus Platichthys flesus luscus

Oil (WSF)

l1

C C

11 11 11 11 11 11 11 55 55 55 55 14-20 14-20 14-20 14-20 14-20 14-20 14-20 14-20 14-20 14-20

25% WSF WSF 25% 50% WSF WSF 50% 100% WSF WSF 100% C C 100 100 1000 1000 10000 10000

d d

25 50 100 100 200 400 11700 700 2500 C C 10 10 100 100 10 10 100 100

d d

d d

d d

d d d d

d d

d d

Pseudop.- uronectes Pseudopleuronectes americanus

Oil (WSF)

P p P p P p

11 11

C C

100 100 1000 1000 10000 10000

C C 10 10 25 50 100 100 C C

100 100 88 88 62 62 00 20 20 14 14 4 00 53 53 24 24 19 19 17 17 100 100 100 100 88 88 75 75 68 68 90 90 89 89 89 89 86 86 88 88 89 90 90

58 58 55 55 44 44 24 24 15 15 99 00 00 63 63 65 65 53 53 60 60 42 42

221 1

20 20

5-6 5-6

29-34 29-34

Kiihnhold 1974) Kuhnhold ((1974)

6-7 6-7

34-35 34-35

Johannessen Johannessen

9-12 9- 12

((1976) 1976)

Mazmanidi Mazmanidi and Bazhasvili Bazhasvili

((1975) 1975)

1-10 1-10

31 31

Kiihnhold Kiihnhold et L al. al.

((1978) 1 978)



Species

Pollutant


Concentration Concentration P d )) ((1Lg!l

HatchHatchability (%)

Viable hatch (YO) (%)

“C °C

Salinity ((%) %0)

Reference .Reference

Chlorinated hydrocarbons Chlorinated

Cyprinus Cypriflus carpio carpi0

lQ co 0

Cyprinodon vavaCyprinodon riegatus riegatus

Simazine

Gramaxone Gramaxone

11 11 11 11 11 11

Taficide

11

Aroclor hoclor 1254 1254

11

11 11 11 11

Cyprinodon vauaCyprinodon riega tus riegatus

Aroclor 1254 1254

11 11 P P P P P p P

20,000 30,000 40,000 60,000 80,000 80,000 40,000 60,000 80,000 80,000 90,000 25,000 30,000 40,000 50,000 50,000 100,000 100,000

79 68 50 9 0 86 63 9 0 92 68

C C 0.1 0.1 0.32 1.0 3.2 3.2 10.0 10.0 C 0.1 0.1 0.32 0.32 l1.0 .o 3.2 10 10

79 69 73 82 75 57 93 88 88 80 98 85 72

and Yadav Yadav Kapur and ((1982) 1982)

16 16

16 16

16 16

34 34

111 1 0 29

16-32 16-32

Schimmel et et al. ((1974) 1974)

30

10-27 10-27

al. Hansen et al. (1974) (1974)

Gadus morhua

%

I!O 10 c ,...

DDT DDT

phorinus Phoxinus phoxinus

Clophen 50 A 50

Pimphales proPimephales melas

Aroclor 1254 1254

Pimphales proPimephaies melas Oncorhynchus kisutch

Aroclor 1254 1254

I1 11 11 I1 11 P P

pe P" pe P' p e P"

P P p P

P P P P P P f f fJ fJ f f

80 80

C C

60 60

150 ISO

60 60 40 30 30 49 39 39 43 43 8 8 74 55 55 63 63 79 79 0 0 0 89 89

300 300 700 700 800 800 C 1.6 1.6 15 15 170 170 C 0.23 0.23 0.52 0.52 1.8 1.8 4.6 15 IS C 0.5 0.5 2.0 0 4.4 7.8 15.4 15.4 26.0 56.4 56.4

84 84 74 96 96 88 88 79

8

12-16 12-16

35 35

Dethlefsen (1977) (1977)

Bengtsson 1980) Bengtsson ((1980)

al. Nebeker et al. (1974) (1974)

Jarvinen et al. al. ((1977) 1977)

12-14 12-14

Halter and Johnson ((1974) 1974)

17-19 17-19

Trojnar ((1977a) 1977a)

47 63 63 pH

Catostomus commersoni

8.1 B.l

5.8 5.8 5.4 5.0 5.0 4.5 4.5 4.2

1 1 11 11 I1 I1

56 56 61 61

60 60

55 16 16 0



Species Species

Cyprinodon nevanevaCyprinodon densis densis

Jordanella Jordanella jloridae floridae

1:0 cc 1:0

to

Perea jluviatilis jluviatilis Perca

3

Pimephales pmPimephales promelas rnelas Rutilus rutilus Rutilus rutilus

Perea Perca jluviatilis jluviatilis

Pollutant

8.3 7.0 6.5 6.0 6.0 5.5 5.5 6.8 6.0 5.0 5.0 4.5 7.3 5.5 5.0 4.5 5.5 5.0 4.5 7.5 6.6 6.6 5.9 5.9 5.2 5.2 7.7 6.1 6.1 5.6 5.2 4.7 8.0 5.6 5.1 5.1


1 1 1 11 11 11 11 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 14 14 14 14 14 14 P, P, 11 P, P, 11 P, p, 11 P, p, 11 11 2 2 2 2 11 2 2 2 2

Concentration Concentration (JLg/l) (CLgIl)

Hatchability (%) (%)

Viable hatch hatch (%0) (%)

°C "C

Salinity (%) (%)

51 51 32 9 3

0 44 57 51 51 17 17 0 100 96 70 8 98 44 51 51 78 80 42 0 89 82 40 30 6 53 53 12 12 6

Reference

Lee and Gerking ( 1980) (1980)

26

Craig and Baksi ((1977) 1977)

14

Runn et et al. (1977) ( 1977)

20-25 20-25

16 16

16 16

Mount (1973) (1973)

Johansson and Johansson Milbrink 1976) Milbrink ((1976)

Perca flucjiatilis jluviatilis Perca Salmo salar salar Salmo

salarg Salma salarg Salmo

Salmo salar Salmo salar � M to w

E Salmo Salmo tmttai trutta'

Salvelinus fontiSaloelinus fontinalis nalis

4.6 4.0 4.0 6.4 4.0 3.5 6.8 5.0 4.5 4.2 4.0

3.7 4.9 4.8 4.7 4.55 4.55 6.0-6.8 6.0-6.8 5.5 5.0 4.5 4.5 4.0 8.0 5.5 5.5 4.7 4.2 4.2 8.3 8.3 4.75 4.75 4.4 8.3 8.3 4.75 4.4 8.3 8.3 4.75 4.75 4.4 4.4

22 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 h h

h h h

11 11 11

11

35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35

2 0 90 41 0 96 91 92 94 89 0 70 60 50 40 100 100 100 100 97 -55 -55 0 0 98 98 90 70 90 91 91 2 1 21 100 100 64 12 12 80 80 75 75 50 50

15 15

Rask (1983) (1983)

4

Daye and Garside

(1979) (1979)

-10 -10

Lacroix (1985) ( 1985)

8

Peterson et al. al.

(1980a) (1980a)

4-6 4-6

Johansson et a1 al. ((1977) 1977)

10 10

Swarts et al. Swarts (3 differdiffer((1978) 1978) (3 strains) ent strains)



Species

Salvelinus Salvelinus fonti fontinalis nalis

fontiSalvelinus fonti nalis nalis

Pollutant

of Day of exposure

Concentration ( P d u) (JLg/l

7.0 6.6 6.1 6.1 5.6 5.6 5.1 5.1 7.0 7.0 6.6 6.6 6.1 6.1 5.6 5.6 5.1 5.1 4.5 C 6.5 6.5 6.0 5.5 5.0 4.5

P P P P P 11 11 1 11 11 1 1 11 1 11 11 11

82 74 59

Hatchability (%) (%)

Viable hatch (L) (%0) 9

°C "C

Salinity (70) (%)

Reference

(1976) Menendez (1976)

54 54

26 74 65

54 54

47 44 44

0 99 95 98 82 65 65 48

95' 95j 911 91i 96i 9f3 701 70i 58i 581

Kwain and Rose (1985) (1985)

OJ 0'

percent of of successfully inseminated eggs; P, parental exposure prior to spawning; C, control. Hatchability and viable hatch expressed as percent of total hatch. Percent of As ppm per 100 100 g body weight (injected). (injected). d Exposure from gastrulation. e Orally dosed, concentration as mg kg-' kg-1 wet weight. f f Two weeks before hatching. g g Data extrapolated from graph. Fxposure at eyed stage. h Exposure j .A va strain. Avl j Survival to swim-up. •

b

c

4. 4.

EFFECTS EGGS AND EFFECTS OF O F POLLUTANTS POLLUTANTS ON ON FISH FISH EGGS AND LARVAE LARVAE

295

ability of the embryo. In the majority of the cases, whenever hatching affected, embryos experimentally liberated from the chorion remain is affected, developin a curled position unable to swim, swim, indicating failures in develop prement that prevented hatching. This is not the case in embryos pre vented from hatching in waters of low pH. Within a time limit, em embryos not hatching at low pH can be induced to hatch almost immediately after transfer to high pH (see (see also Rask, 1983). 1983). This indi indihatching-via inhibition of the cho chocates that only the process of hatching-via rionase-is affected, while the development of the embryo has pro prorionase-is normally-an indication also for a true effect of low pH and ceeded normally-an not the secondary effect of high metal concentrations in the water due to low pH, as frequently suggested. As a consequence, though, when eggs are left throughout development in low pH, hatching success may be drastically reduced, as shown for Atlantic salmon (S. (S. salar) salar) (Daye and Garside, 1977, Lacroix, 1985), (Daye 1977, 1979; 1979; Lacroix, 1985), other salmonids salmonids (Jo (Jo1977), and various other species [Le., hansson et al., 1977), [i.e., white sucker (Trojnar, (Trojnar, 1977a), 1977a), walleye (Hulsman (Hulsman et al., 1983), 1983), desert pupfish (Lee 1980), roach and yellow perch (Milbrink (Milbrink and Johansson, and Gerking, 1980), 1975); 1975); Johansson and Milbrink, 1976; 1976; Runn et al., 1977; 1977; Rask, 1983)] 1983)l.. polluEven though hatchability iiss usually considered a measure ooff pollu tant effects on ontogenesis, it should be remembered that this ob obscures the fact that within these data substantial numbers of nonviable larvae may be included. Thus reference to the rate of "viable “viable hatch" hatch” as a means of assessing sublethal effects of pollutants would be prefer preferred, since since only the normal and viable larvae are of concern for recruit recruitment. It should be added that species such as gar pike, Relone Belone belone, Dicentrarchus (Paflitschek, 1979) 1979)even as larvae Dicentrurchus labrax, or the tilapias (Paflitschek, are so so vigorous that they live with major damage to their skeletal system. Yet when assessing effects on the frail clupeoid and “viable hatch" hatch” is is the more sensitive parameter, pleuronectid larvae, larvae, "viable (Rosenthal and Sperling, 1974; 1974; Maz Mazas can be seen from Table I (Rosenthal Bazhashvili, 1975; 1975; Voyer et al., 1977; Ojaveer et al., 1980). 1980). manidi and Bazhashvili, al., 1977; simultaWhenever values for viable hatch and hatchability are given simulta neously, viable hatch is substantially lower. lower. In many experiments, even the value for viable hatch overestimates the real fi gure for viable figure of effects may not be de delarvae, since, since, as already noted, a number of tected with the naked eye, eye, but require histological examination-for examination-for example, example, the effects of zinc on brain and muscle tissues of herring (C. (C. harengus) harengus) (Somasundaram (Somasundaram et al., 1984a,b; 1984a,b; Somasundaram, 1985). 1985). Other metabolic effects might appear after the young fish has passed the larval period. For instance, hatchability of rainbow trout (S. (S. gairdneri) gairdneri) is not affected by cromium at concentrations concentrations at 0.2 0.2 mg/l

296


(van alevins is (van der der Putte Putte et al., 1982), 1982), yet yet the the survival survival of of alevins is affected affected after after 32 weeks, pH of weeks, depending depending on on the the pH of the the rearing rearing water. water. The inability inability for for complete and caused by cadmium cadmium treatment complete and early early calcification calcification caused treatment is is an another shows only only later alevin's life salmon (S. (S. other effect effect that that shows later in in the the alevin’s life in in salmon salar) salar) (Rombough (Rombough and and Garside, Garside, 1984). 1984). Similar Similar hidden hidden effects effects that that become become apparent apparent only only after after histological histological examination (Ca examination are are known known from from fish larvae larvae treated treated with with crude crude oil oil (Cameron Smith, 1980; 1980; Hawkes meron and and Smith, Hawkes and and Stehr, Stehr, 1982), 1982),and and it it is is likely that that aa large large number number of of hitherto hitherto undetected undetected effects effects of of pollutants pollutants have have consid considerable erable bearing bearing on on the the percentage percentage of of viable viable larvae. larvae. III. SUBLETHAL ISPLAYED BY LARVAE 111. SUBLETHAL EFFECTS D DISPLAYED HATCHED FROM FROM TREATED EGGS

The larval larval stage stage of a fish, fish, although very different from the egg in outer outer appearance, appearance, is is not not totally totally different different in in its its physiological physiological state. state. Any Any impairment of functions functions or organs afflicted in the embryo is carried over to the free-living larva. larva. Hatching is is a rather arbitrarily deter determined since it mined point, point, since it may may occur occur at at aa variety variety of of ontogenetic ontogenetic stages. stages. Many Many larvae, larvae, after after emerging emerging from from the the egg egg shell, shell, are are still still incapable incapable of of feeding, due to the fact that the mouth is not yet functioning (clupeids, (clupeids, gadids, gadids, pleuronectids, pleuronectids, cyprinids, cyprinids, and and others), others), or or because because they they are are not not (salmonids) or at atcapable of swimming and remain on the bottom (salmonids) tached in the tached to to plants plants in in the the water water (pike), (pike). Species Species such such as as found found in the family family Belonidae, Belonidae, Cyprinodontidae, Cyprinodontidae, or or members members of of the the mouth-breed mouth-breeding may rely rely for for aa longer longer ing cichlidae cichlidae are are able able to to feed feed upon upon hatching. hatching. All may or or shorter shorter period period on on yolk yolk reserves reserves for for metabolism. metabolism. However, However, for for the the sake of convenience, the newly hatched larvae are treated together in sake section. this section.

A. Larval Length A. larvae from from eggs eggs incubated Reduction of length in newly hatched larvae (notably heavy metals, petroleum under the influence of pollutants (notably commonly observed hydrocarbons) is a commonly hydrocarbons, and chlorinated hydrocarbons) feature. Reduced length of newly hatched larvae larvae is is frequently correl correlfeature. Reated with larger yolk-sac yolk-sac sizes, sizes, suggesting suggesting impaired development. development. Re duced length in itself is is not considered to lower larval larval fitness. fitness. One characteristic characteristic of of several several abiotic abiotic factors factors influencing fi fish sh larvae incubation is is the altered altered size size and shape shape of yolk sac sac and the length of

4. 4.

EFFECTS OF POLLUTANTS ON FISH EGGS EGGS AND AND LARVAE LARVAE

297 297

the newly hatched larvae. Basicly, yolk-sac size and shape as well as larval length change in relation to various abiotic factors, such as incu incubation salinity and temperature or oxygen. Examples for a decrease in S . gairdneri) gairdneri) at reduced oxy oxylength of newly hatched rainbow trout ((S. gen tension are given by Hamdorf 1961). Salinity and temperature Hamdorf ((1961). effects on length of newly hatched larvae are known to occur in the veincubation of several fish fish species such as English sole Parophrys ve 1968), herring C. C. harengus and C. tulus (Alderdice and Forrester, 1968), C. pallasi (von (von Westernhagen Westernhagen et al., 1974; 1974; Alderdice Alderdice and and Velsen, Velsen, 1971), 1971), and B. belone (Fonds (Fonds et aI., al., 1974). 1974). Frequently Frequently they they occur occur in in and garpike garpike B. conjunction conjunction with with aa prolonged prolonged (larger (larger larvae) larvae) or or shortened shortened (short (short lar larvae) vae) incubation incubation period period until until hatching. hatching. Variations Variations in in larval larval size size are are known known from from rearing rearing experiments experiments with with herring C.. pallasi) ( C . harengus, C pallasi) eggs eggs in in cadmium, cadmium, zinc, zinc, and and copper. copper. herring (C. In all effective treatments, larvae hatch early and total lengths are smaller smaller than than in in controls. controls. Effective Effective concentrations concentrations are are given given by by Rosen Rosenthal 1974) to 1.0 mg/l mg/l for for cadmium cadmium and and 0.1 0.1 mg/l mg/l in in aa thal and and Sperling Sperling ((1974) to be 1.0 pulse pulse exposure exposure of of copper copper (Rice (Rice and and Harrison, Harrison, 1978), 1978), while while Ojaveer Ojaveer et 1 980) report that cadmium concentrations as low as 3.0 al. 3.0 ILg/I pg/l reduce al. ((1980) larval larval length. length. Zinc Zinc increases increases larval larval total total length length in in concentrations concentrations up u p to to 2.0 2.0 mg/l; mg/l; starting starting at at 6.0 6.0 mg/I, mg/l, the the length length of larvae larvae hatched hatched from from zinc zinc incubated incubated eggs eggs decreases decreases (Somasundaram (Somasundaram et al., al., 1984b). 1984b). Yet Y e t under under chronic (Poecilia reticu reticuchronic exposure exposure to to sublethal sublethal levels levels of of zinc, zinc, guppies guppies (Poecilia .7 ILg lata) 0.88 and 11.7 p g zinc/l zincll (Uviovo (Uviovo Zata)are very sensitive to levels of only 0.88 and and Beatty, Beatty, 1979). 1979). Offspring Offspring produced produced under under these these conditions conditions are are smaller than controls smaller than controls and and have have not not absorbed absorbed the the yolk yolk completely, completely, indicating that zinc reduces energy utilization. The authors suggest that that zinc zinc has has an an "uncoupling" “uncoupling” effect effect in in the the mitochondria, mitochondria, similar similar to to the effect that Hiltibran ((1971) 1971) has demonstrated in the mitochondria of the bluegill Lepomus macrochirus liver. liver. Effects of cadmium on herring larvae are depicted in Table II, 11, show showlength of newly hatched herring decreasing length with increasing cadmium at different salinities salinities.. ing decreasing Eaton (1974) L. macrochirus (1974) also found that incubation ooff bluegill L. shorteggs in cadmium concentrations higher than 0.08 0.08 mg/l leads to a short ening of the total length of the hatching larvae. larvae. ening Reduced length of newly hatched larvae is frequently correlated Reduced as noted for yolk sacs sacs of herring larvae with larger yolk-sac sizes, as incubated at different different cadmium and salinity conditions. Besides the infl uence of salinity salinity on on yolk-sac yolk-sac size size (May, (May, 1974a; 1974a; Alderdice Alderdice and and influence Velsen, ndings indicate Velsen, 1971), 1971), these these fi findings indicate low low yolk yolk utilization utilization under under cad cadS . salar). salar). Reduced (Rombough and Garside, 1982; 1982; S. mium exposure (Rombough length of newly newly hatched larvae larvae is also also caused by the exposure of the

Table II I1 Table Clupea Clupea harengus horengus Larvae: Total Length, Diameter of of Eye, and Otic Capsule at Hatching" ~~~~~~~

~~

~

~

~

S (%0) (%)

Cd. conc.

n n

5 5 5 5 5

Control 0.1 ppm 0.1 0.5 0.5 ppm 1.0 ppm 5.0 ppm

80 91 9 1

16 16 16 16 16 16 16 16 16 16

Control 0.1 0.1 ppm 0.5 ppm 1.0 ppm 5.0 ppm

100 100 62 93 105 105

25 25 25 25 25 25

25

Control 0.1 0.1 ppm 0.5 ppm 1.0 ppm 1.0 5.0 ppm

32 32 32 32 32

Control 0 . 1 ppm 0.1 0.5 ppm 1.0 ppm 1.0 5.0 ppm

50 50

63 72 91 9 1 50 54 61 61 74

x r

ss

Eye diameter (mm) (mm)

Otic capsule (mm) (mm)

Total length (mm) (mm)

Experimental design design

si Sf

n

n

x f

SS

si. Sf

si. Sf

125 125 137 137 39

0.002 0.003 0.002 0.002 0.003

76 4 1 41 43 129 129 100 100

0.287±0.0l4 0.28720.014 0.284±0.017 0.28420.017 0.285-tO.009 0.285±0.009 0.283±0.014 0.283+0.014 0.258 2 0.020 0.258±0.020

0.002 0.003 0.001 0.001 0.001 0.001 0.002

0.311 1120.015 0.3 ± 0.015 0.312C0.021 0.312±0.021 0.315±0.017 0.315-tO.017 0.31120.034 0.31 1 ±0.034 0.24420.062 0.244±0.062

0.002 0.003 0.002 0.004 0.006

63 46 91 9 1 82

0.288 0.288 0.27720.018 0.277±0.018 0.289-tO.010 0.289±0.010 0.293±0.0 1l 0.293+0.011 0.263±0.024 0.26320.024

0.002 0.001 0.001 0.001 0.003

0.279±0.027 0.27920.027 0.286-tO.020 0.286±0.020 0.285±0.027 0.28520.027 0.28820.022 0.288±0.022 0.178±0.035 0.17820.035

0.004 0.003 0.003 0.003 0.002 0.006

0.274?0.008 0.274±0.008 0.26520.020 0.265±0.020 0.268 -t0.0 14 0.268±0.014 0.272+0.018 0.272±0.018 0.234-tO.019 0.234±0.019

0.002 0.001 0.001 0.002 0.002 0.002 0.003

0.002 0.002 0.002 0.002 0.007

0.06 0.08 0.05 0.04

100 100 65 93 129 129 141 141

0.3 1420.023 0.314±0.023 0.311 120.025 0.3 1±0.025 0.310±0.021 0.31020.021 0.301?0.027 0.30l±0.027 0.244±0.033 0.244 L0.033

7.9820.17 7.98±0. 17 7.90-tO.46 7.90±0.46 7.8820.41 7.88±0.41 7.48±0.60 7.4820.60 Not measurable

0.02 0.06 0.05 0.06

49 62 71 7 1 9 1 91 102 102

7.0550.28 7.05±0.28 7.0020.34 7.00±0.34 6.94±0.42 6.9420.42 6.8220.41 6.82±0.41 Not measurable

0.04 0.05 0.05 0.05

53 56 63 73 35

7.7820.58 7.78±0.58 7.8820.62 7.88±0.62 7.7020.51 7.70±0.51 7.13-tO.38 7. 13±0.38 Not measurable

S S

0.002 0.001 0.001 0.001 0.002 0.003 0.003

0.32720.020 0.327±0.020 0.3 16?0.024 0.316±0.024 0.313±0.026 0.31320.026 0.31420.025 0.314±0.025 0.2 12-t0.039 0.212±0.039

0.04 0.06

x f

0.288 20.0 18 0.288±0.018 0.280±0.01 l 0.28020.011 0.251 ±0.0l6 095120.016 0.25220.018 0.252±0.018 0.22020.016 0.220±0.016

80 89 171 171 179 179 33

8.2720.34 8.27±0.34 7.77?0.54 7.77±0.54 Not measurable Not measurable Not measurable

n n 55

64 64

25

50 50

38 74 45

a n, n, Number of of larvae measured; x, f , mean; s, s, standard deviation, s., si, error of of the mean. Larvae derived from incubation incubation trials in 32%0 32% salinity originated 1974). Westernhagen et et al. al. ((1974). originated from a second female. After von Westemhagen a

4. FISH 4. EFFECTS EFFECTS OF OF POLLUTANTS POLLUTANTS ON ON FISH

EGGS AND EGGS AND LARVAE LARVAE

299 299

eggs to petroleum hydrocarbons, usually applied as the water-soluble (WSF) of crude oil or its derivates derivates.. Thus Pacific herring (C. (C. fraction (WSF) pallasi) eggs exposed in a 48-h 48-h pulse exposure of of 12. 12.1 pallasi) 1 mg benzene/l 10.3 yield larvae with a mean standard length of 9.2 mm, compared to 10.3 controls.. The same is true for anchovy (E ( E.. mordax) mordax) eggs ex exmm in controls 40-55 ppm (Struhsaker et al., 1974). 1974). Also, Also, Baltic herring (C. (C. posed to 40-55 rnembras) eggs exposed to 5.4-5.8 5.4-5.8 mg/l total oil hydrocar hydrocarharengus membras) (Linden, bons yield significantly shorter hatching larvae than controls (Linden, 1978). The same effects are described by Carls and Rice ((1984) 1978). 1984) after of the walleye pollock T. T . chalcogramma chalcogrumma and embryos exposing eggs of of oil (Leung (Leung and O. latipes to the WSF of of the Japanese medaka 0. Bulkley, 1979). 1979). In the walleye pollock, reduction in length amounts to 0.5 mm at a total length of of only 4.5 mm. In the killifish F. F . heteroclitus, exposure of eggs to the WSF of number 2 fuel oil leads to a shortening of hatching larvae with increasing strength of of the applied WSF (Sharp (Sharp et al., 1979; 1979; Linden et al., 1980). 1980). In this species the latter authors note a simultaneous decrease in the number of vertebrae. vertebrae. Reduced lengths of newly hatched larvae are also known to occur after treatment of cod (G. (G. marhua) morhua) eggs with DDT and DDE. Within the range of DDT applied (0, 0.0095, 0.0413, 0.09, 0. 15, 0.39, 0.69 mg/l), (0,0.0095,0.0413,0.09,0.15,0.39,0.69 mg/l), emerging larvae are progressively smaller with increasing insecticide concentrations; the mean total length is only 4 mm at the highest DDT concentration, while control larvae measure 4.75 mm (Dethlefsen, (Dethlefsen, 1977). 1977).A reduction concentrain length of yolk-sac fry of pike Esox lucius incubated in concentra tions as low as 0.1 0.1 ng 2,3,7,8-tetratchlorodibenzo-p-dioxin (TCDD)/l (TCDD)/I is also known through the experiments of Helder ((1980). 1980). The significance of of the hatching size for the fitness of of fish larvae is not clear, although it is generally accepted that it is of of disadvantage for the larvae to hatch small. This assumption depends on an "uneasy “uneasy feeling" feeling” rather than on facts. facts. Swimming velocity, for instance, does not seem to be altered significantly in different-size different-size herring larvae 1.0 mm total length (von (von Westernhagen and Rosenthal, within 7.0 to 111.0 1979), 1979), and thus prey catching behavior will not be impeded. Of course, smaller larvae have aa smaller range of of food availability, since organs such as eyes and otic capsules (von Westernhagen et al., 1974) 1974) as well as the head and jaw apparatus are smaller, thus limiting the choice of food particles. However, the limitation to smaller food parti particles does not reduce survival if enough food is available, as shown by my unpublished lennius pavao unpublished data on larvae of B Blennius pauo. It seems reasonable to argue that it is not the absolute size of of a larva but its size in relation to its ontogenetic stage of development and the remaining yolk volume that is is important for survival. If If the

300


embryo encounters encounters unfavorable conditions at the end of its intracho intrachorion development and it "decides" “decides” to hatch prematurely, as reported reported for the effects of several abiotic factors including low oxygen, the prematurely liberated larva, unless otherwise damaged, is generally only shorter than normal, but is holding a larger yolk sac. sac. If If the caus causative agent of premature hatch does not have any lasting effects, de development will proceed normally. B Yolk-Sac Size and Yolk Metabolism B.. Yolk-Sac A large or deformed yolk sac is taken as an indicator for metabolic or osmotic disturbances that may be caused by mitochondrial mal malfunction, induced by b y heavy metals or petroleum hydrocarbons. It is not always true that a large yolk sac at hatching is is due to premature hatching. There are indications that a large yolk sac occurs because of metabolic or osmotic disturbances in the embryo/larva embryoAarva that prevent proper use of the energy stored in the yolk. Lanning 1977), Lonning ((1977), from observations of cod, plaice, and fl ounder eggs (G. flounder (G. morhua, Pleuronectes platessa, Platichthys Jesus) fiesus) exposed to Ekofisk oil, thinks that the use of the energy-rich substances in the yolk becomes delayed by an inhibition of the mitochondrial system. For instance, newly hatched larvae from Pacifi C. pallasi eggs exposed to Pacificc herring C. Prudhoe Bay crude oil for 4-144 h and then returned to uncontami uncontaminated seawater show no gross abnormalities micros abnormalities.. Yet transmission microscopy of exposed organisms reveals inter- and intracellular spaces in brain and muscle tissue that are not found in controls (Cameron and 1980). Many mitochondria ((13%) exSmith, 1980). 13%) in the body muscle of ex posed animals are swollen, some with deteriorating cristae. Changes in mitochondrial functions would affect the total respiration and me metabolism of the larvae and thus explain the previously previously mentioned gen general suppression of of embryo activity and metabolism after prolonged exposure to petroleum 1980) inferred petroleum hydrocarbons. Linden et al. ((1980) that at low hydrocarbon hydrocarbon levels, when the homeostatic mechanisms are ect increased costs of homeo not overwhelmed, overwhelmed, respiration rates refl reflect homeostasis. When the stress is more severe, but still sublethal, the response would be mediated by lack of metabolic integration because of of poorly functioning homeostatic mechanisms. They believe that exposure to oil predominately predominately impedes mobilization of of nutrient uptake from the yolk through the breakdown of mitochondria in in the cells, leading to glycogen and lipid depletion such as demonstrated by Sabo and SteSte geman ((1977). 1977). This would ultimately lead to reduced tissue growth, as

4. 4.


301

found in embryonic embryonic herring C. C. pallasi exposed to sublethal sublethal concentra concentra1977). Yolk utilization is likewise (Eldrige et al., 1977). tions of benzene (Eldrige pentachlorophenate (Na (Na PCP), reduced by exposure to sodium pentachlorophenate PCP), as shown by Chapman and Shumway ((1978), for example, in the alevins 1978), of steelhead trout S. S. gairdneri. The bioenergetic data obtained in their consistent with the concept that PCP disrupts energy metab metabstudy are consistent deleterious to the early larva, since energy olism. This is particularly deleterious requirements increase rapidly by approximately tenfold shortly after 1977; C Clupea pallasi). Struhsaker et al. (Eldridge et al., 1977; hatching (Eldridge lupea pallasi). of yolk at high concentrations of of ((1974) 1974) relate the impaired utilization of of the animal; this aspect will benzene to an increasing narcotization of be treated later in a different context. Inhibited yolk utilization is also E . lucius larvae incubated at known under conditions of low pH. Pike E. p H 4.2 are smaller than controls but have larger yolk sacs, which pH 14 days, indicating poor utilization of yolk reserves. Yolk persist for 14 frequently appears appears coagulated, ultimately leading to death of the af affrequently 1975). Retarded yolk absorption fected fry (Johansson and Kihlstrom, 1975). at pH 55 is also known in brook trout (S. fontinalis) alevins (Menendez, (S.fontinaZis) (Menendez, 1976). 1976). outer appearance appearance of the yolk shows signs of abnor abnorFrequently, the outer appearance of an empty space between yolk mality or one notices the appearance flesus (Mazmanidi and Bazhashvili, 1975; 1975; Platichthys ./lesus sac and yolk (Mazmanidi luscus) or anteriorly adjacent to the pericardium as demonstrated by luscus) (1978)for newly newly hatched herring herring (C. ( C . harengus membras) membras) lar larLinden (1978) Linden These features are similar to those observed by von Westernha Westernhavae. These fish gen ((1970) 1970) and Alderdice and Velsen ((1971) 1971) when marine fi sh larvae outside their optimum temperature temperature and salinity re reare incubated outside indicating additional stress stress on the larvae, larvae, resulting resulting in gimes, thus indicating functions.. failure of osmoregulatory functions Cadmium and zinc are also known to interfere with osmoregula osmoregula(C. pallasi) pallasi) eggs (Alderdice et al., 1979c). 1 9 7 9 ~ )Cad Cad. Pacific tion in Pacifi c herring (C. reduces osmolality of perivitelline fl fluid, probmium exposure of eggs reduces uid, prob ably due to the marked tendency of cadmium to form complexes (Remy, (Remy, 1956), 1956), particularly with iodide, bromide, and chloride ions. ions. The effect of the the formation of these complexes is is to to marshal other ions The into complex formation [e.g. [e.g.,, Cd(I3h]' Cd(I&], reducing the number of active solution, thus reducing reducing osmotic pressure. Rosenthal and particles in solution, Sperling (1974) (1974) report a disproportionate shortening of the yolk sacs sacs of cadmium C. harengus larvae larvae at at cadmium cadmium levels levels of5.0 of 5.0 cadmium exposed exposed herring herring C. and 10.0 10.0 mg/l, mg/l, which may be caused by reduced perivitelline fluid turgor. turgor. al. effects on fish larvae. larvae. Somasundaram et al. Zinc has similar effects

302 302


((1984d), 1984d), working working with with C. C. harengus, harengus, speculate, speculate, on on the the basis basis of of their their histological histological investigations, investigations, that that the the swelling swelling of of mitochondria mitochondria and and sar sarcoplasmic coplasmic reticulum reticulum caused caused by by zinc zinc treatment treatment suggests suggests the the creation creation of of an an osmotic osmotic imbalance imbalance through through zinc. zinc. In In mammals mammals zinc zinc causes causes swelling swelling of of mitochondria mitochondria and and appears appears to to alter alter potassium potassium permeability, permeability, uncou uncouples ples oxydative oxydative phosphorylation, phosphorylation, and and inhibits inhibits the the electron electron transport transport chain chain (Cash (Cash et aZ., al., 1968; 1968; Kleiner, Kleiner, 1974; 1974; Bettger Bettger and and O'Dell, O’Dell, 1981}. 1981). Uncoupling ultimately lead Uncoupling of of oxydative oxydative phosphorylation phosphorylation will will ultimately lead to to an an energy deficit, even though the in energy deficit, even though the embryo embryo may may compensate compensate with with increased decomposition creased decomposition of of carbohydrates carbohydrates (Stelzer (Stelzer et aZ., al., 1971); 1971); this this would would have have its its bearing bearing on on the the osmoregulatory osmoregulatory capacities capacities of of the the em embryo. bryo. C. Morphological Aberrations C. Aberrations:: Eye Deformities, Skeletal Abnormalities Gross malformations such as eye Gross eye deformation and reduction, as well as skeletal deformities, are caused by all types of pollutants and are not pollutant-specific. Typical anomalies, which may also be caused by extreme temperatures and salinities, are spirality and cur curvature of the notochord and abnormal development of of the jaw. The severity of the effects can be generally related to the doses applied and diminishes with exposure during later stages of development. Since cadmium interferes with calcium metabolism, it is suspected to impair the calcification process directly. Petroleum hydrocarbons probably act as general stressors and do not have a specifi c effect on specific any any enzyme enzyme or or physiological physiological process. process. Besides aberrations of of length and yolk usage, newly hatched larlar vae display a vast array of gross deformities, such as lack of of organs, of gross extremities, etc. and/or abnormal behavior due to the action of pollu of pollutheir embryonic stage. behavtants during their stage. These malformations and behav ioral aberrations may play major or minor roles in their survival. In general, aberrations that stem from tissue injury or enzyme inhibition follows : various types of of during earlier stages can be categorized as follows: eye deformation or reduction, jaw anomalies, malformations of of the vertebral column, minor morphological aberrations (i.e., fin defects, otic capsule defects, change in color pattern), impairment of of swimswim of these ming and prey catching behavior, and reduced growth. Some of have obvious effects on survival. The bearing of of others may be insiginsig nificant or difficult to recognize. Eye deformations are common in fish larvae exposed to sublethal

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of stressors such as heavy metals. Microphthalmia (Ojaveer et levels of 1980) in Baltic herring incubated in copper and cadmium solusolu al., 1980) al., tions of of BO.01 >0.01 and 0.05 0.05 mg/l have been noted, as well as cyclopia in morhua) reared in sublethal (0.01-0.5 mg Cu/l; Cull; 0.5-10.0 0.5-10.0 mg cod (G. morhua) Zn/l) concentrations of of copper and zinc (Swedmark and Granmo, 1981). de198 1 ) . These gross abnormalities are similar to those already de scribed for the effects of of mercury on killifish ((F. F . heteroclitus) em embryos, by P. Weis and Weis (1977). (1977). The spectrum of of eye and head F . heteroclitus by exposure to inorganic mercury defects produced in F. reflects interference with inductive processes at a relatively early stage. stage. The severity of of the response diminishes with exposure during later stages of 0. S. S. Weis and Weis, 1977). of development (J. 1977). Fundulus of malforma malformaheteroclitus appears to have a propensity for this type of tion; further, Stockard ((1907) 1907) produced cyclopia in this species by treatment with magnesium chloride. chloride. It is is interesting to note that optic abnormalities are not produced in F. F . heteroclitus by exposure to to in insecticides (Weis (Weis and Weis, Weis, 1974). 1974). Other subtle deviations from the normal are displayed by herring (C. harengus) (C. harengus) embryos exposed to cadmium (Rosenthal (Rosenthal and Sperling, al., 1974). 1974). Exposure to cadmium concen 1974; 1974; von Westernhagen et al., concentrations higher than 1.0 1.0 mg/l leads to a reduction in eye diameter (Table 1984a) showed a significant (Table II). 11). Also, Somasundaram Somasundaram et al. al. ((1984a) reduction in eye size size at a concentration of 6 and 12 12 mg/l of zinc in herring (C. (C. harengus), harengus), even considering reduced length at hatching. The same is true for the the reduced otic capsule capsule diameter. diameter. Gross eye eye deformations deformations are are one of the typical effects occurring occurring after sublethal exposure exposure to metals and other teratogenic compounds compounds such such as ben benzo[a]pyrene (BAP). (BAP).When exposing rainbow trout (S. ( S . gairdneri) gairdneri) to the mutagen BAP, BAP, Kocan and Landolt (1984) (1984) and Hannah et al. al. (1982) (1982) always nd gross always fi find gross physical defects in the ocular and cephalic region of larvae similar to those resulting from exposure exposure to heavy metals metals.. Skele Skeletal tal and cephalic abnormalities of newly hatched fish, fish, encountered most frequently upon exposure to BAP, BAP, are believed to be caused by the mutagenic action of the BAP. BAP. Implied possible mutagenic action of copper and cadmium is is not supported by experimental evidence, and thus the the question remains open. open. Another sublethal stressor affecting the the eyes eyes of young fish fish is is low pH. 1980), when sectioning alevins pH. Daye and Garside ((1980), alevins from from exposed eggs eggs of Atlantic salmon (S. ( S . salar), salar), found that incubation at at pH 4.0 4.0 yields alevins alevins with with eye lens lens fibers fibers less less differentiated than that of con controls. trols. The The lenses also also suffer severe sloughing of epithelium, epithelium, a common common pathologic pathologic change change due due to to acid acid environment. environment. Anatomically, Anatomically, the the prime prime

304 30 4


sites cial tissues. sites of of injury injury are are the the superfi superficial tissues. Internal Internal structures structures are are af affected fected secondarily, secondarily, both both in in time time and and degree. degree. A A wide wide array array of of skeletal skeletal malformations malformations (Le., (i.e., jaw, jaw, head, head, pelvic pelvic and and pectoral pectoral girdle, girdle, vertebral, vertebral, and and opercular opercular anomalies) anomalies) occurs occurs commonly commonly in in freshwater freshwater and and marine marine fish fish species species (Wunder, (Wunder, 1971 1971;; Kroger Kroger and and Guthrie, 1973; 1973; Dethlefsen, Dethlefsen, 1980, 1980, 1984). 1984). Accordingly, one would also expect expect these these anomalies anomalies to to occur occur in in fish fish larvae, larvae, and and this this is is the the case case under laboratory conditions. For example, anomalous formation of the jaw, mentioned during earlier investigations, is caused by extreme temperatures and salinities in several fish species [von [von Westernhagen 1974), Platichthys fiesus, jlesus, B ((1970, 1970, 1974), B.. belone; belone; Alderdice and Velsen C. pallasi] pallasi].. These anomalies are also caused by sublethal sublethal ef ef((1971), 1971), C. cranio-facial fects of metal pollutants and are likely to be expressed as cranio-facial as a reaction toward mer merand mandibular malformations; particularly as (Weis, 1984 1984;; F. F. heteroclitus) heteroctitus) and zinc (Somasundaram et al., cury (Weis, 1984a; C. C . harengus). harengus). Different types of of malformations of the head 1984a; (C. harengus) harengus) larvae (Fig. 7). Due to the region are found in herring (C. (Fig. 7). incomplete development of the feeding apparatus of many fish larvae (mouth opening still closed), closed), symptoms of jaw at the time of hatching (mouth immediately detectable, in particular when defects are not always immediately hatched larvae are not given additional time to de deexperimentally hatched 7 shows sev sevvelop their mouth apparatus before assessment. Figure 7 eral types of jaw deformations in herring. These differ depending on development. Jaw deformations are also known to occur the stage of development. spontaneously” in hatchery enterprises in North America. The open open"spontaneously" of salmonids is of of particular particular concern in hatchery-reared jaw syndrome of al., 1973). salmon (Crouch et al., 1973). Jaw deformities may also result from the treatment of treatment of eggs with crude oil (Tilseth et al., al., 1984; 1984; Solberg et al., 1984). At concentrations of of about 150-1245 1984). 150- 1245 kg/l J,tg/l (WSF), (WSF), cod (G. (G. of the upper jaw, which may morhua) larvae suffer from deformation of have a later bearing on feeding. Also, short-term exposure of of newly 24-96 h at concentrations spawned Pacific Pacific herring (C. (C. pallasi) pallasi) eggs for 24-96 of 4800-45,000 p g benzene/l causes severe anomalies in the head of 4800-45,000 J,tg region of of hatching larvae, including including jaw deformations (Struhsaker et al., 1974). of the head region of 1974). Similar pictures of of Baltic herring larvae after treatment with the WSF of crude oil (up g hydrocarafter treatment with of (up to 59,000 p J,tg hydrocar bondl) are given by Linden (1978). Also, when exposing 6-day-old bons/I) ( 1 978). Pacific herring embryos to the WSF of Prudhoe Bay oil at concentraof concentra tions of g total hydrocarbons/l for only 48 h, of around 1000 1000 p J,tg h ' advanced larvae display a high incidence of of gross morphological abnormalities, such as improperly formed mouth, misfit of of the lower jaw into the upper, missing of of the premaxillary bone, and failure of of the jaw to fully “

4. 4. EFFECTS

OF POLLUTANTS ON FISH EGGS AND LARVAE

305

Fig. Fig. 7. 7. Head of of herring (Clupea (Clupea harengus) harengus) larva showing different types of jaw malformations after incubation in Zn-contaminated 2, 55 Zn-contaminated water. Upper row, normal 11,, 2, days (arrow); third row, secondary days old; second row, rudimentary lower and/or upper jaw (arrow); pugheadedness, branchial arches (arrows); (arrows); fourth row, row, pugheadedness, cross bite, bite, and and protrusion protrusion of branchial normal lateral and dorsal view, and same view of of larva with exophthalmus. [From Somasundaram et al. ((1984a).] 1 984a).]

differentiate (Smith (Smith and Cameron, 1979). 1979). Another abnormality no noticed i ticed only only under the the electron electron microscope microscope was was the the absence of of branch branchiostegal membranes, a phenomenon observed also by von Westernha Westernhagen al. ((1987) 1987) after gen et al. after treatment treatment of of herring herring embryos embryos with with surface surface microlayer hexane extracts. extracts. Abnormal development development of the jaw is seen in larvae exposed as eggs to pesticides like the moluscicides Bayluscid and Lebaycid (Paflits (Paflitschek, 1979; Tilapia leucosticta, leucosticta, Heterotilapia multispinosa) chek, 1979; multispinosa) or or as as aa result result of of high PCB PCB content content (2.8 (2.8 JLg/g pg/g wet wet wt) wt) in in eggs eggs of of rainbow trout trout (S. (S. gairdneri) (Hogan (Hogan and and Brauhn, Brauhn, 1975). 1975). In In the the natural natural environment environment these anomalies are these jaw jaw anomalies are likely likely to to interfere interfere severely severely with with feeding feeding and and

306 306

H. VON WESTERNHAGEN VON WESTERNHAGEN

thus reduce survival. In the most severe cases, starvation will follow the inability to feed. In my observations, I frequently noticed that swimming is greatly impaired in gaping-mouth larvae of of marine fish, fish, probably probably due to increased water resistance. Mouth and jaw anomalies constitute a relatively small part of the gross abnormalities occurring after treatment of eggs with pollutants. The bulk of the symptoms observed are related to axis formation. Injury to the vertebral column or its anlage in response to pollutants is commonly seen by most investigators working with fish eggs and lar larvae. The range of damage is extensive: from very slight flexures to bends or spiral distortions, shortening of the body axis, axis, or reductions of the brain. Within the range of possible damage, none of the sub substances seem to cause substance-specific damage, which can undoubt undoubtedly be attributed to a particular pollutant. Responses seem to be general and ubiquitous without regard to the stressor. Exposure time and substance concentration influence the severity of the symptoms. The physical appearance of affected larvae resembles that of individ individuals incubated under natural stress of of extremes of temperature and 1970) and described by sev salinity, as shown by von Westernhagen ((1970) several other authors. Damage of the vertebral column expressed as curvature of the larval body axis axis is caused by all metals currently termed "heavy “heavy metals" metals” when present in the incubating medium. The most common metal pollutants are cadmium, copper, mercury, lead, and zinc, em emof a few micro microployed singly or in combinations at concentrations of grams per liter in the case of the acutely toxic metals such as mercury, and up to several thousand micrograms micrograms per liter with metals such as lead or zinc. facThe toxic levels of the different metals differ and depend on fac tors such as susceptibility of fish species, temperature and salinity, or chemical chemical speciation of the metal. Thus cadmium causes vertebral damage in developing fish eggs at concentrations between 80 I-tg/l pg/l 1974; Lepomis macrochirus) and 300 I-tg/l pgll (Rombough (Rombough and (Eaton, 1974; 1982; S. S. salar) salar) in fresh or brackish water (Voyer (Voyer et ai., al., 1977; 1977; Garside, 1982; americanus), but at higher concentrations of be bePseudopleuronectes americanus), 1000 and 2000 I-tg/l pgIl in seawater (von Westernhagen et aZ., 1974, al., 1974, tween 1000 belone). Since cadmium interferes with calcium 1975; C. 1975; C. harengus, B B.. belone). metabolism-cadmium replacing calcium-the calcium-the effect of cadmium on metabolism-cadmium investivertebrae formation might be a direct one, as suggested by the investi gations of Rombough and Garside (1984) (1984) observing the impairment of of the calcification process in Atlantic salmon alevins. Earlier effects of the metal, prior to ossification, ossification, must be considered general effects on

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dismetabolism. Copper is also known to be effective in producing dis torted larvae at fairly low concentrations. At 30 IL p copper/I, copper/l, 30% 30% of of the hatching herring larvae are deformed (Blaxter, (Blaxter, 1977). 1977). At the same level zebrafish (B. ( B . rerio) rerio) develops scoliosis (Ozoh, (Ozoh, 1979a), 1979a), and for bass) this level is is only slightly higher ((100 Dicentrarchus labrax (sea bass) 100 pg/l) (Cos (Cosson 1981). The initial concentrations for mer merILg/I) son and Martin, 1981). cury causing sublethal effects on axis formation are even lower. J. J. S S.. Weis and Weis ((1977), 1977), in experiments with the killifish (F. ( F . heterocli heteroclitus), prove that concentrations of only 10 pg Hg/I Hg/l cause lordosis and tus), 10 ILg larvae. Although affected larvae are still scoliosis in newly hatched larvae. able to swim, movements are impaired. Experiments of Sharp and Neff 1 980, 1982) 1977) with F. Neff ((1980, 1982) and P. Weis and Weis ((1977) F . heteroclitus confirmed the low effective concentrations for mercury that cause sim similar effects to those described above. Still lower detrimental concen concentrations of 1978) with of mercury were reported by Servizi and Martens ((1978) sockeye (0. (0. gorbuscha) (0.nerka) nerka) and pink salmon (0. gorbuscha) eggs. Apparently, mercury concentrations above 2.5 ILg/1 pgIl increase vertebral deformities; at 4.3 ILg p g mercury/I, mercury/l, 46% of the alevins are crippled with impaired swimming. swimming. Exposure of brook trout (Salvelinus (Salvelinusfontinalis) over three genera generations shows that lead is also an effective teratogen at low concentra concentrations. Alevins of the third generation from eggs exposed to 1119 19 ILg pg lead/l display 2 1 % scoliosis, scoliosis, compared to only 2% 21% 2% in the controls al., 1976). 1976). In short exposures during embryonic devel devel(Holcombe et al., opment, lead causes sublethal effects on axis formation, but concen concentrations must be around 1000 1977; F. 1000ILg/1 pg/l (J.S. (J.S. Weis and Weis, 1977; F . hetero heteroclitus). clitus). Small aberrations from normal axis formation caused by zinc are first detectable at the micrograms per liter level. Slight bends of of the tail tip of herring larvae at hatching can be observed after incuba incubation at 50 ILg/1 pg/l (Somasundaram (Somasundaram et al., 1984a). 1984a).Animals with this type of deformity still swim like normal larvae. With increasing zinc concen concentrations, damage to the vertebral column becomes more severe. Fur Further information on damage of cod (G. (G. morhua) and herring (C. (C. harengus) harengus) larvae hatched from zinc-exposed eggs are given in reports by Swedmark and Granmo ((1981) 1981) and Ojaveer et al. ((1980); 1 980); effective concentrations for herring are in the same range, although higher for cod. A series of photographs of cod. of various degrees of of spinal malforma malformations caused by metal is given in Fig. 8 from our own experiments with herring and fl ounder. In the herring larva (Figs. flounder. (Figs. 8b,8c), 8b78c),the dam damage done to the vertebrae is is visible. Disturbed axis formation is also common in larvae incubated in hydrocarbons-although not a typical phenomsolutions of petroleum hydrocarbons-although

308

d


e Fig. 8. Newly hatched larvae of herring (Clupea (Clupea harengus) harengus) and flounder (Platichy (Platichythysflesus) thysjesus) incubated in water contaminated with 55 mg cadmium/I. cadmium/l. (a) (a)Crippled herring larva with exophthalmus; (b, c) exophthalmus; (b, c) herring larvae with damaged or almost disintegrated notochord (arrows); (arrows); (d-f) (d-f) bent and severely crippled flounder larvae; y, yolk. Horizontal bars indicate pm. indicate 200 /Lm.

enon. According to the Sharp et al. 1979) interpretation of their exper al. ((1979) experF . heteroclitus) heteroclitus) embryos: embryos: imental results with oil-exposed killifish ((F. Hydrocarbon pollutants act iin n fish embryos embryos as general stressors and do not have a hydrocarbon pollu polluspecific effect on any single enzyme or physiological process. Thus hydrocarbon mortants may shunt limited metabolic energy away from critical differentiation and mor phogenetic processes to maintenance functions.

The depressant or retarding effects of hydrocarbons during early de development may be reflected in the effects effects on gross morphology of emergent fry, where the degree of the effects depends on the strength of the hydrocarbon applied. A variety of of species has been used in exposure studies with hydrocarbons (mostly (mostly WSF of crude oil), oil), one of the most common being herring (C. (C. harengus). harengus). Effects of of 680 ILg/l pgll WSF for 48 h on developing herring (C. (C. harengus pallasi) eggs leads abnormalito a significantly higher incidence of gross morphological abnormali (Smith and Cameron, Cameron, 1979). 1979). Most of the abnor abnorties than in controls (Smith malities are bent vertebral columns leading to larvae with L, S, or helical configurations. Affected larvae are usually unable to swim in aa straight line or not able to swim at all. The same aberrations have been provoked by Linden ((1976, 1 976, 1978) 1978) using somewhat higher concentra-

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30 309 9

bentions, 3100 to 111,900 1,900 pgll, JLg/I, and by Struhsaker et al. al. ((1974) 1974) using ben zene. The eggs of several other species have been subjected to the same or similar treatment using crude oil or other petroleum hydrocar same hydrocarbons. Thus Thus Mironov (1969) (1969) used anchovy Engraulis encrasicolus pon ponticus; Hakkila and Niemi (1973) (1973) used northern pike pike Esox lucius; Kiihnhold 1 974) used cod C. Kuhnhold ((1974) G. morhua; Mazmanidi and Bazhashvili ((1975) 1975) used flounder Platichthys flesus luscus; Stoss and Haines (1979) (1979) Japanese medaka Oryzias latipes; and Kiihnhold et al. (1978) (1978) used the winter fl ounder Pseudopleuronectes americanus. americanus. All authors flounder report deformation of axis axis to a greater or lesser extent. Effective con concentrations of of the WSF of petroleum hydrocarbons are in the range of 100 100 JLg/1 pgll (Mazmanidi and Bazhashvili, 1975) 1975) to 4000 JLg/1 pg/l (Stoss and Haines, 1979). 1979). When combining oil oil with oil-spill dispersants (Wilson, (Wilson, 1972; 1972; Linden, 1974, 1974, 1976), 1976), deleterious effects on axis formation are usually aggravated, reaching levels known from the teratogenic ef effects of benzo[a]pyrene 1983) on notochord benzo[a]pyrene (24 (24 JLg/I; pg/l; Winkler et al., al., 1983) abnormalities in the grunion Leuresthes tenuis. tenuis. Body flexure occurs also also in newly hatched larvae from eggs con containing chlorinated chlorinated hydrocarbons such as DDT [Dacre [Dacre and Scott ((1971); 1971); S. 1973), Pseudopleuronectes S. gairdneri; gairdneri; Smith and Cole ((1973), americanus] americanus] in in the range of 2.4-4.6 2.4-4.6 mg/kg mglkg wet weight. Hogan and 60-70% deformed rain rainBrauhn ((1975) 1975) assume that the occurrence of 60-70% bow trout in aa trout hatchery was caused by the high PCB (Aroclor (Aroclor 1242) 1242) content (2.7 (2.7 JLg/g) pg/g) in the eggs. eggs. When exposing eggs to these incubating water at concentrations of about 100 100 JLg pg substances in incubating DDTIl Dethlefsen, 1977; DDTl1 ((Dethlefsen, 1977; C. G. morhua) morhua) or only 13 13 JLg p g PCB/I PCBll (Mauk et al., 1978; Salvelinus fontinalis), fontinalis), a considerable percentage of the al., 1978; hatching larvae displays curvature of the body in different degrees. degrees. The severity of these effects increases with the concentration of of the organochlorine employed. From the data on viable hatch of cod under influence 10 JLg pg DDT/l DDTll or higher already the infl uence of DDT it appears that 10 reduces viable hatch. Similar effects can be caused by other pesti pestireduces cides, such as malathion, but at considerably higher concentrations of 10,000 JLg/l pgll for sheepshead sheepshead minnow (Cyprinodon (Cyprinodon variegatus) variegatus) eggs 10,000 (Weis and Weis, 1976) 1976) or carp carp (C. (C. carpio) carpio) eggs treated with various (Weis Yadav, 1982). 1982). herbicides (Kapur and Yadav, crippled and and distorted larvae is is also ob obOccasional hatching of crippled served when eggs eggs are incubated incubated at at low pH. Thus at pH p H 4.0 4.0 to 4.5, served perch Perca fluviatilis hatching is delayed and the few few (3%) (3%) larvae hatched show vertebral deformations (Runn (Runn et al., al., 1977). 1977). This effect is is known to occur at pH 5.0 5.0 in white sucker Catostomus commersoni

310 310


(Trojnar, (Clupea pallasi) eggs (Trojnar, 1977a). 1977a). In Pacific Pacific herring (Clupea eggs subjected subjected to to (pH 6.7), low pH during development development (pH 6.7), hatching hatching is is reduced reduced to to almost almost zero although a few bent individuals emerge (Kelley, (Kelley, 1946). 1946). Runn et 1977) judged these deformities to be secondary and not caused by al. al. ((1977) a disturbance of the early organogenesis but developing during the prolonged nonhatch period at low pH-possibly pH-possibly aggrevated by the smaller inner volume of the egg and the reduction of of the diffusion of metabolites uid. The metabolites through through the the perivitelline perivitelline fl fluid. The same same phenomenon phenomenon is is known for cod (G. (G. morhua) morhua) embryos that fail to hatch. When the fully developed larva (with jaws and no more yolk) yolk) is liberated by dissec dissection, it remains curled in an embryonic posture (von (von Westemhagen, Westernhagen, 1970), indicating that the growth of the larva inside the egg was re 1970), responsible for the malformation. D. Minor Morphological Aberrations

Aside from the above-mentioned obvious gross gross malformations, an array of minor deformities and deficiencies are known to be caused by pollutants. These cannot be dealt with in detail, detail, although they may represent the real sublethal effects at the individual level. In other words, effects of the pollutants may be expressed as minor changes not large enough to cause immediate or ultimate death, but large enough to reduce overall fitness. Typical effects may follow different kinds of treatment. Thus, fi n erosion or sloughing of epithelial tissue fin occurs after exposing embryos to cadmium, copper, zinc, or lead (von Westernhagen et al., 1975; 1975; Ozoh, 1979a; 1979a; Somasundaram, 1985), 1985), as Westemhagen well as after incubation in water contaminated with petroleum hydro hydro(Kuhnhold, 1972; 1979; carbons (Kiihnhold, 1972; Linden, 1975; 1975; Smith and Cameron, 1979; Vuorinen and Axell, 1980) 1980) or rearing in organochlorines or or organophosphates (Pafl itschek, 1979; (Paflitschek, 1979; Helder, 1980). 1980). Other symptoms are impaired blood circulation or blockage of blood vessels leading to thrombosis, as seen in ?n larvae hatching from eggs incubated in cad cadmium solutions (Pickering and Cast, Gast, 1972; 1972; Eaton, 1974; 1974; Beattie and 1978), toluene (Stoss (Stoss and Haines, 1979), 1979), or tetrachlorodi tetrachlorodiPascoe, 1978), benzo-p-dioxin (TCDD) 1980). The same effects are known (TCDD) (Helder, (Helder, 1980). (Winkler to be caused in fish larvae by the carcinogen benzo[a]pyrene (Winkler et al., 1983). 1983). Very common is the poor development of pigmentation 1980) or exposure to petroleum hydrocar caused by cadmium (Ozoh, (Ozoh, 1980) hydrocarbons (Mazmanidi (Mazmanidi and Bazhashvili, 1975; 1975; Anderson et al., 1977; 1977; John John1979; Falk-Petersen Falk-Petersen et al., 1985), 1985),malathion (Weis (Weis and Weis, son et al., 1979; 1976), 1976), or low pH (Johansson (Johansson and Kihlstrom, 1975; 1975; Nelson, 1982). 1982).

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EFFECTS EFFECTS OF OF POLLUTANTS POLLUTANTS ON FISH FISH EGGS EGGS AND AND LARVAE LARVAE

3 11 311

E. E. Metabolic Metabolic Alterations Alterations One metals and and oil oil is is an an altera alteraOne major major subcellular subcellular effect effect caused caused by metals tion leads to tion of of the the internal internal structure structure of of mitochondria, mitochondria, which which leads to an an im impairment of the inhi pairment of the intracellular intracellular energy energy transfer transfer system. system. Blockage Blockage or or inhibition bition of of this this system system may may be be the the cause cause for for inadequate inadequate use of of yolk reserves and reserves and retarded retarded development. development. More More subtle subtle impact impact of of pollutants pollutants (stressors) (stressors) may may easily easily escape escape at attention is still or resist resist interpretation. interpretation. Recent Recent studies studies show show that that there there is still aa tention or large large number number of of phenomena phenomena that that are are now being being looked looked at at more more closely closely but but are are not not yet yet fully fully understood. understood. This This is is particularly particularly true true for for events events at at the subcellular levels, which are known to be affected and in turn to affect affect the whole whole organism. organism. Thus Thus herring herring embryos, embryos, incubated incubated in in zinc zincpolluted signs of polluted water, water, show, show, in in addition addition to to signs of epithelial epithelial necrosis, necrosis, changes changes in in mitochondria mitochondria structure, structure, absence absence of of the the Golgi Golgi apparatus, apparatus, and and reduction reduction in in smooth smooth endoplasmic endoplasmic reticulum reticulum (Somasundaram, (Somasundaram, 1985). internal active 1985). Because Because of of reduced reduced mitochondria mitochondria internal active surface, surface, cell cell metabolism might be impaired and total energy budget of of the animal affected al., 1984c); 1984~); zinc is is known known to to interfere interfere with with affected (Somasundaram (Somasundaram et al., oxygen uptake of 1971) and might cause un unof mitochondria (Hiltibran, (Hiltibran, 1971) coupling of oxidative phosphorylation and inhibition of the electron transport chain (Kleiner, 1974; Bettger and O'Dell, O’Dell, 1981), 1981), a theory (Kleiner, 1974; 1979). Uviovo and and Beatty Beatty ((1979). also forwarded forwarded by Uviovo Similar Similar effects effects on on mitochondria mitochondria are are caused caused by by xylene xylene on on the the earli earliest cleavage 1986) and (G. morhua) morhua) eggs eggs (Kjorsvik, (Kjorsvik, 1986) and the the est cleavage stages stages of of cod cod (G. oil on WSF of crude crude oil on herring herring larvae larvae hatching hatching from from oil-treated oil-treated eggs eggs (Cameron and Smith, 1980). Enzyme activity 1980). Enzyme activity in in brook brook trout trout S. fonti fonti(Cameron and Smith, naZis, as as shown by by Christensen Christensen (1975), (1975), is is also also greatly greatly affected affected by naZis, metals such as metals such as cadmium, cadmium, mercury, mercury, and and lead. lead. Activity Activity of of glutamic glutamicoxaloacetic (ALP), acetyl acetyloxaloacetic transaminase transaminase (GOT), (GOT), alkaline phosphatase phosphatase (ALP), choline esterase choline esterase (ACH), (ACH), and and adenosine adenosine triphosphate triphosphate (ATP) (ATP) have have been been either either significantly significantly decreased decreased in in late late embryos embryos or or increased increased in in alevins. alevins. Probably Probably several several malformations malformations and and developmental developmental aberrations aberrations are are ultimately ultimately caused caused by by aa blockage blockage of of the the energy-transfer energy-transfer system, system, leading leading to an arrest of respiration and differentiation, or to dedifferentiation. Inhibition Inhibition of of acetylcholine acetylcholine esterase esterase in in neuromuscular neuromuscular and and brain brain tis tissue, (S. sue, for for instance, instance, as as demonstrated demonstrated to to occur occur in in rainbow rainbow trout trout (S. gairdneri) gairdneri) exposed exposed to to organophosphate organophosphate pesticides pesticides (Matton (Matton and and Lat Lat1969) or in Cyprinodon Cyprinodon variegatus treated with malathion (Weis (Weis tam, 1969) and Weis, 1976), 1976), will will severely severely impair impair locomotion locomotion and/or and/or cause cause death death and Weis, of asphyxia. of the the organism organism by asphyxia. Blockage inhibition of systems or Blockage or or inhibition of intracellular intracellular energy-transfer energy-transfer systems or

312 312


shunting energy from differentiation metabolism to detoxification pro processes may also be the cause for the commonly observed retardation in growth and the inability of of the yolk sac larvae to use yolk reserves adequately. It has already been noted that herring (C. (C. harengus) harengus) lar larvae hatching in cadmium-contaminated water had larger yolk sacs than those from controls (von Westernhagen 1974). Obviously, Westernhagen et al., 1974). resorption of yolk under the influence of of metal stress is impaired. This utilization continues in the larval stage. Thus impairment of yolk utilization brook trout S. S. fontinalis alevins incubated in copper-contaminated copper-contaminated water (32.5 (32.5 JLg/I) pgll) take 4 weeks longer to complete yolk resorption and remain smaller than controls (McKim and Benoit, 1971). 1971). Impairment of yolk utilization on incubation in cadmium-contaminated water also occurs in Atlantic salmon alevins when reared through yolk absorp absorption (Rombough and Garside, 1982). 1982). At 9.6°C 9.6"C concentrations of of 0.47 JLg p g Cd/I Cd/l impaired yolk utilization to the extent that final weight of of alevins was significantly reduced when compared to controls. The same is is known for salmon fry reared at 40-55 40-55 JLg p g copper/l copped1 (Hazel and Meith, 1970; 1970; Servizi and Martens, 1978), 1978), and for the young of the zebrafish B. B.rerio under the influence of lead (Ozoh, (Ozoh, 1979a). 1979a). The last lasting effect of cadmium on yolk utilization is shown in rearing experi experiments with Atlantic salmon (S. ( S . salar). salar).When reared in concentrations of 2 JLg/l, pgll, the fish display reduced growth, growth, which continues even after initiation of feeding (Peterson et al., 1983). 1983). Impaired yolk utilization has also been reported as an effect of petroleum hydrocarbons on embryos of winter flounder Pseudo Pseudo1978) and the killifish F. pleuronectes americanus (Kiihnhold et al., 1978) F. heteroclitus (Sharp (Sharp et al., al., 1979). 1979). The effect of these pollutants is not entirely a direct one; these substances express their activity through storage in the lipid reserves of the yolk and are later mobilized during This system is is particularly active with lipophilic sub subyolk absorption. This stances such as the chlorinated hydrocarbons. Thus larvae of fathead 15JLg pg PCB/l minnows Pimephales promelas hatched after exposure to 15 controls (Nebeker et al., are severely retarded in growth compared to controls 1974), 1974), and this was also reported by Halter and Johnson ((1974) 1974) work work0. nerka eggs and ArocIor Aroclor 1254. 1254. Hogan and ing with Pacific salmon O. S. Brauhn ((1975) 1 975) observed that hatchery-reared rainbow trout S. gairdneri fry display a high percentage ooff deformed animals with a yolk utilization. On variety of skeletal abnormalities and impaired yolk analysis, the eggs eggs of parental fish fish showed 2.7 JLg p g PCB/g egg chemical analysis, 0.09 JLg pg DDT/g. Also, Also, S. S . salar incubated in DDT show wet weight and 0.09 alevin development, particularly in their their behavior (Dill (Dill and retarded alevin Saunders, 1974). 1974). Another substance, substance, sodium pentachlorophenate Saunders,

4. 4.


313 313

(Chapman and Shumway, 1978), 1978), when applied at 40 JLg/I, pgll, decreased (Chapman S. steelhead trout S. yolk utilization, growth, and development in steelhead

gairdneri. The significance of impaired yolk utilization is obvious. With a

given yolk reserve, larvae must develop to a certain ontogenetic stage span. If this is not attained, the larva is likely to find in a given time span. itself in an environment for which it is not yet prepared (swimming itself speed, orientation); in the case of salmonids, larvae may emerge and speed, fall prey to larger predators. In the limited environment of a body of fresh water, the proper timing might be crucial for survival. In the sea this factor might not be of paramount importance, but its bearing should not be underestimated. underestimated. F. F. Behavioral Behavioral Abnormalities Abnormalities E ggs incubated Eggs incubated under under the the influence influence of of metals metals or or petroleum petroleum hydro hydrocarbons carbons may may release release larvae larvae with with reduced reduced activity. activity. While While effects effects of of metals metals are are long-lasting, long-lasting, petroleum petroleum hydrocarbons act act twofold; twofold; tran tranhistopathsiently, with subsequent recovery, and permanently, when histopath ological damage took place. Also, the high chlorinated hydrocarbon contents yolk may responsible for for reduced reduced activity. activity. Low Lowcontents of of larval Iawal yolk may be responsible ered is an an indication indication of of reduced reduced fitness. fitness. ered larval larval activity activity is Several Several authors authors assume assume that that an an additional additional reason reason for for slow slow yolk yolk utilization utilization is is the the reduced reduced activity activity of of larvae. larvae. Frequently, Frequently, larvae larvae hatch hatching eggs lie lie motionless motionless on on the the bottom bottom of the the experimen experimening from from exposed exposed eggs tal sluggish movements tal containers containers or or perform perform only only sluggish movements not not equal equal to to nor normal immobile often mal swimming swimming activity. activity. Hatched Hatched larvae larvae that that remain remain immobile often come from eggs incubated in high concentrations of copper, zinc, or (Eaton, 1974; 1974; Swedmark and Granmo, 1981); 1981); the reason for cadmium (Eaton, a2. (1982) (1982) describe their immobilization is not quite clear. Voyer et al. winter flounder larvae Pseudopleuronectes Pseudopleuronectes americanus incubated in cadmium concentrations of up to 100 100 JLg/I pg/l that show reduced swim swim(10% salinity). salinity). This response sug sugming activity only in low salinity (10%0 susgests a potential long-term effect on larval feeding, growth, and sus gests ceptibility to predation. The reasons reasons for for the the reduced reduced activity activity of of metal-treated metal-treated larvae larvae are are definitely hydrodefi nitely different from those that occur after treatment with hydro carbons. The The effects of hydrocarbons on embryo activity activity can be b e ob obalso in newly hatched larvae. Crude oil fractions (WSF) (WSF) are served also activity. Hatched embryos and larvae suffer very effective in reducing activity. narcotic effects when swimming in water admixed with petroleum

314


hydrocarbons (Sharp (Sharp et al., 1979). 1979). From several experiments by Kiihnhold 1 969, 1972; Kuhnhold ((1969, 1972; Kiihnhold Kuhnhold et al., 1978), 1978), we know that larvae may become completely stunned when swimming into clouds of of pe petroleum hydrocarbons or oil dispersants (Wilson, (Wilson, 1974) 1974) and sink to the processs is bottom. When they reach lower WSF concentrations the proces reversed (Hakkila and Niemi, 1973). 1973). Sometimes Sometimes larvae are not fully immbolized but show only reduced swimming activity (Mazmanidi (Mazmanidi and Bazhasvili, 1975). 1975). Partly anesthetized larvae, although still swim swimming, may lose equilibrium (Stene (Stene and Lanning, Lonning, 1984) 1984) and be unable to catch prey. When kept for prolonged periods at high concentrations of the WSF of petroleum hydrocarbons, growth is negatively affected due to nonfeeding [Struhsaker et al. al. ((1974), 1974), 4000 ILg/I pgll WSF benzene; Solberg et al., (1984), ] . The narcotic effects (1984),30-200 30-200 ILg/I pgll WSF crude oil oil]. of crude-oil extracts (8000 WSF) have a stronger impact on (8000 ILg/l pg/l WSF) starved larvae than on individuals with fully functional yolk sacs (Davenport et al., 1979). 1979). Effects are twofold, depending on WSF concentration and dura duration of exposure, and may be transient with subsequent recovery or long-lasting if there is is histopathological damage to the retina or fore forebrain, as shown for larvae of of the surf surf smelt Hypomesus pretiosus by Hawkes and Stehr ((1982). 1982). A larva with a damaged brain or eye is unable to survive. ed by other organic survive. Acute effects may be intensifi intensified al. (1984) compounds accumulated from the water. Solbakken et al. (1984) (G. morhua) morhua) eggs and larvae exposed to several naph naphfound that cod (G. PCB for 24 h accumu thalenes, phenanthrene, benzo[a]pyrene, and PCB accumulated these lipophilic xenobiotics in the yolk and stored them until the yolk was used, thus causing a delayed effect on later development, resulting in reduced activity, morphological aberrations, and the like. Extensive changes in in behavioral behavioral patterns, such as delay in the occur occurrence of certain swim positions in alevins from DDT-treated Atlantic salmon (Dill (Dill and Saunders, 1974), 1974), or reduced swimming activity, may be observed in fish larvae that contain chlorinated hydrocarbons. Ex Experiments with the cyprinodont Adinia xenica confirm that eggs from DDT- and mirex-contaminated loss in mirex-contaminated parents yield larvae that show loss equilibrium and effects of narcotization (Koenig, 1977). Since the yolk (Koenig, 1977). (Atchison, 1976; 1976; Guiney et is the main storage site for DDT and PCBs (Atchison, al., 1980),the larva, while consuming yolk, takes up more and more of al., 1980), the stored chlorinated hydrocarbons, producing the damaging effects. The consequences of the behavioral abnormalities abnormalities depend on If swimming and prey catching behavior their severity and duration. If anticare impaired, increasing risk of starvation or predation would be antic ipated, leading to reduced survival (see (see Rosenthal and Alderdice,

4. 4.

EFFECTS EFFECTS OF OF POLLUTANTS POLLUTANTS ON ON FISH FISH EGGS AND AND LARVAE LARVAE

315

1976). 1976).Larvae with considerable amounts of of chlorinated hydrocarbons in their yolk (see (see Hogan and Brauhn, 1975) 1975) are usually not viable and are characterized by a high incidence of malformations, failure to com completely absorb their yolk sac, or death during the larval stage (Macek, (Macek, 1968). al. ((1974) 1 974) observed reduced survival of 1968). Schimmel et al. of sheeps sheepshead minnow Pimephales >0.32 p,g Pirnephales promelas fry after incubation in >0.32 pg Aroclor 1254/1. 125411. Reduced survival is common in larvae hatched from eggs treated with PCB or DDT during incubation (Hansen et al., al., 1974; 1974; Freeman and Idler, 1975; 1975; Dethlefsen, 1977). 1977). Proof Proof of of the detri detrimental effects of DDT in yolk does not stem only from laboratory experiments but is also available in fi eld data. 1971) field data. Dacre and Scott ((1971) and Hogan and Brauhn ((1975) 1975) report substantial loss of of trout fry due to hydro high DDT in larval tissues. Effects of high levels of of chlorinated hydrocarbons in eggs on percent viable hatch are also known from the investigations of von Westernhagen et al. 1981) with Baltic flounder al. ((1981) flews, Hansen et al. Platichthys flesus, al. ((1985) 1985) with herring C. harengus, Westin et al. 1985) with striped bass Morone saxatilis and Cameron al. ((1985) et al. al. ((1986) 1 986) with whiting, Merlangius merlangus. Effective chlorin chlorinated hydrocarbons are PCB at a gonad concentration of 120-180 120-180 p,g/kg pglkg wet weight and DDE at 18 18 p,g/kg. pglkg. IV. SUBLETHAL SUBLETHAL EFFECTS ON LARVAE NOT EXPOSED AS EGGS

Outstanding sublethal effects on larvae are impairment of of yolk utilization and ensuing depression in growth, which may be caused either by low levels of metals, petroleum hydrocarbons, or chlorinated hydrocarbons, or by low pH. In addition, petroleum hydrocarbons are particularly effective in reducing larval activity. In the previous section I have discussed deleterious effects of pollutants on larvae resulting from exposed eggs. All of the effects lower the individual's individual’s survival chances even if if the larva is released into an uncontaminated uncontaminated environment after hatching. However, pollutants may also exert their influence on normal lar larvae with (sublethal) (sublethal) reactions that result in reduced survival of of the young fish. fish. Larvae hatched in uncontamined water may come under the influence of of contaminants during larval drift by encountering an oil spill or plumes of of heavily polluted river water. Such experimen experimentally mimicked posthatching encounters with pollutant stressors will now be addressed. Larvae and alevins are generally considered more susceptible to

316


abiotic factors factors than is the egg (Hiikkilii (Hakkila and Niemi, 1973; 1973; Linden, 1974; 1974; Rice et al., 1975). 1975). Effective concentrations concentrations for for the production of of suble sublethal effects should be low. For example, sublethal effects exhibited by larval salmon exposed to heavy metals are skeletal deformities after exposure to low mercury (9.3 (9.3 �g/l; pg/l; Servizi and Martens, Martens, 1978) 1978) or low cadmium (>0.78 (B0.78 �g/l; pgll; Rombough and Garside, 1984) 1984) concentrations. concentrations. Other responses may be inhibition of of enzymatic processes by cad cadmium, mium, lead, and mercury exposure, as seen in brook trout, S. S. fonti fontinalis; 1976; McKim et al., 1976); nalis; alevins (Holcombe et al., 1976; 1976); this may lead to reduced survival due to uncoupling or inhibition of of unknown metabolic functions [Benoit [Benoit ( 1975), 1975), Lepomis macrochirus; macrochirus; Spehar ((1976),JordanellafEoridae], 1976),jordanellajloridae] . Thus, one sublethal reaction of of larval spot Leiostomus xanthurus to cadmium is is a lowering of of its thermal maxi maxi1975). Most of of the information on sublethal mum (Middaugh et al., 1975). effects effects of metals is related to metal-induced depression of growth due to insufficient yolk utilization or decreased larval activity and reduced feeding. Blaxter ((1977), 1 977), using larval herring and plaice, found that in feeding plaice larvae there is a marked reduction in growth in length and development at 90 90 �g pg copper/l or above, above, while in herring, doses of 300 300 �g/l pg/l tend to inhibit activity during their dark phase of migration. The growth inhibiting effects of copper on alevins are documented also for king salmon O. 0. tshawytscha (21 (21 �g/I; pg/l; Hazel and Meith, 1970) 1970) as well as brook trout (McKim (McKim and Benoit, 1971) 1971)starting at 3.4 �g/I. pg/l. In the latter species the influence of copper on metabolism retards yolk absorption for about 4 weeks. Lowered rate of yolk utilization is is also considered to be the reason for the slow growth of alevins of Atlantic salmon salmon kept in water with 2 �g pg cadmium/I. cadmium/l. The growth-depressing effect of cadmium continues after the initiation of feeding (Peterson (Peterson et al., 1983). 1983). The possible role of metal-damaged mitochondria in the (Somasunimpairment of energy transfer and metabolism of protein (Somasun 1984b,c) has already been mentioned and might be re redaram et al., 1984b,c) sponsible for the inability of metal exposed larvae to make adequate effects on metabolism, the latter use of their yolk reserves. Besides the effects C. harengus lar larauthors report considerable brain damage in herring C. from zinc exposure; exposure; this will have a bearing on swim swimvae resulting from prey catching behavior. ming activity and prey effects of low low levels levels of heavy metals on fish fish larvae In general, the effects effects described for petroleum hydrocar hydrocarare less spectacular than effects bons. Due to the increasing numbers of oil spills, spills, many reports have bons. effects of petroleum hydrocarbons on recently been prepared on the effects fish larvae to oil oil has aroused particular marine life; the reaction of fish fish larvae exposed to petroleum interest. Generally, the reaction of fish

4.

O F POLLUTANTS ON FISH EGGS AND LARVAE EFFECTS OF

31 317 7

hydrocarbons resembles that of larvae hatched from eggs incubated in oil-polluted waters. Petroleum hydrocarbons and dispersants clearly yolkdecrease larval activity, as noted by a reduction of heartbeat of yolk 1973).At 18 18 mgn mgil of of sac larvae of pike Esox lucius (Hakkila and Niemi, 1973). (Neste A), A), a reduction in heartbeat from 90 beats min-I min-’ an emulsifier (Neste min-l is observed after 2 days, followed by a period of of to 30 beats min-I swimming activity and narcosis. Also, under the influence of of reduced swimming C. harengus and plaice an oil dispersant, larvae of herring C. Pleuronectes platessa show narcosis within 20 min of exposure to 8 abovemg/l; affected larvae may recover if exposure time is within the above mentioned range (Wilson, (Wilson, 1972, 1974). Linden ((1975) 1972, 1974). 1 975) found narcotic ml l-I) l-l) using her hereffects caused by oil/dispersant mixtures (0.01/0.005 mI ring larvae; Rosenthal and Gunkel ((1967) 1967) noted similar effects. These (>0.5 mg/l) induce impaired swimming and prey catching mixtures (>0.5 hydrobehavior, as do dispersants (Wilson, (Wilson, 1972) 1972) or other petroleum hydro carbons alone (Kuhnhold, (Kiihnhold, 1969; Lanning, 1984). 1969; Stene and Lonning, 1984). Concen Concentrations of oil/dispersants of 100/50 n pri 100/50 p,lpl-ll already destroy larval fi fin primordia (Linden, (Linden, 1975) 1975)or other tissue (Kiihnhold, (Kuhnhold, 1972), 1972), which might later kill the individual. Thus, Thus, one of the major effects of oil and dispersants is reducing larval activity. This may influence larval survival directly, since it has been demonstrated demonstrated experimentally that anesthetized anesthetized larvae are more susceptible to predation than others (see susceptible (see Rosenthal and Alderdice, 1976). uence of a narcotic such 1976). One reason may be that under the infl influence larva’s "critical “critical distance," distance,” that is, the as the WSF of crude oil, the larva's greatest distance between a larva and aa small object (hypothetical predator) to induce a flight reaction, is shortened (Johnson et al., 1979), and flight reactions may then be initiated too late to be success success1979), anesthetized larvae is their re reful. Yet the more obvious effect on anesthetized duced prey catching ability due to slower movements and uncoordin uncoordinated swimming swimming and prey catching maneuvers (Rosenthal, 1969). 1969). determines the volume of a water body a larva is Swimming capacity determines able to search for food per unit time (Rosenthal and Hempel, 1970); 1970); thus, reduced swimming swimming speed decreases the number of of encounters encounters detrimental effects of petro petrowith food particles. Starvation enhances detrimental leum hydrocarbons (Davenport (Davenport et al., 1979). 1979). While 35-day-old starved cod G. C. morhua m o r h a larvae exposed to 88 mg/l WSF (crude (crude oil) get narco narcotized, larvae with functional yolk sacs remain unaffected. An additional factor contributing to the deleterious effects of of petro petroleum hydrocarbons is the apparent reduction in larval growth in oil oilpolluted waters. Since petroleum petroleum hydrocarbons are readily accumu accumulated (Roubal et al., 1977), 1977), even fairly small quantities of of oil (0.075

318


mIll) O. gorbuscha ml/l) in the water inhibit growth in Pacific Pacific salmon 0. alevins exposed for 10 10 days (Rice (Rice et ai., al., 1975). 1975). Length and weight were equally affected. 1980) speculated that the affected. Vuorinen and Axell Axel1 ((1980) poor growth of pike E. 1 mg! E . lucius larvae in oil-contamined water (>0. (>0.1 mg/ 1) 1) may be related to gill damage, which decreases oxygen supply and food utilization. A concentration-dependent concentration-dependent reduction in growth is known for cod C. G. morhua larvae continuously exposed to 50-200 50-200 JLg/l pgll of a WSF of crude oil. Besides a direct impairment of of yolk utilization in the presence of oil (Lanning, (Lonning, 1977) 1977) or benzene (Struhsaker (Struhsaker et al., al., 1974), ect alterations in the 1974), the suppressed larval growth may refl reflect metabolic rate with energy diverted from assimilation to detoxifica detoxification (Eldridge (Eldridge et ai., al., 1977). 1977). Poor yolk utilization in larvae and alevins exposed to chlorinated hydrocarbons, in particular PCBs, is the major feature observed with these pollutants. O. kisutch exposed to poIlutants. Thus, alevins of of coho salmon 0. 15 JLg 15 pg Aroclor 1254/1 1254/1 react with reduced growth and poor yolk absorp absorption (Halter and Johnson, 1974), min 1974), as do the young of of the fathead minnow P. P . promelas (Nebeker et al., al., 1974). 1974). The inhibition of the mito mitochondrial NADH oxidase system by PCB PCB as shown by Pardini ((1971) 1971) may very well be responsible for this phenomenon. Inhibited yolk consumption is also present in fry exposed after hatching to low pH. In pike E. E . lucius, pH >

m

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Fig. Fig. 44.. (A) Temporal changes in hepatic estrogen receptor after estrogen administra administra0 , e) O )or isolated isolated hepatocytes in culture ((*) of * ) were treated with a single dose of tion. Fish ((0, estradiol [5 [5mg (kg (kg live weight)-I weight)-l and I1 IJ-M gM for cultured cells, respectively]. respectively]. Estrogen (0,**)) and cytosolic binding sites were determined after the indicated time in nuclear (e, (0)fractions. Modified from Lazier et al. ((1985) (1986). (0) 1985) and Mommsen and Lazier (1986). temporarily depressed, Numbers of assayable binding sites in the cytosolic fraction are temporarily while numbers of of nuclear binding sites continue to increase until the end of of the experi experiof response is reached about mental period ((120 120 h). h). In cultured liver cells, a maximum of 24 h after the addition of of estradiol. Vitellogenin can first be detected by immunoprecipi immunoprecipitation (B) Estrogen-binding characteristics of 24 h. (B) of tation in the hepatocyte medium after 24 salmon liver nuclei. Cultured hepatocytes were treated with estradiol as indicated in (A). (A).At 12 12 h after hormone exposure, cells were harvested and nuclear nuclear extracts were analyzed for receptor activity and characteristics (Scatchard analysis). The dissociation I-I T. T. constant (�) 3.4 nM 1-I (&) for estradiol is computed by linear regression regression and found to be 3.4 P. Mommsen and C. B. B. Lazier (unpublished). (unpublished). The dissociation constant for highly 5.4and specificc estrogen binders from liver nuclei in estrogen primed fish is between 5.4 specifi al., 1985). 5.9 5.9 nM (Lazier et et al., 1985).

5.

357

VITELLOGENESIS VITELLOGENESIS AND OOCYTE ASSEMBLY

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little little exposure exposure is is needed to to initiate initiate the the transcription transcription of of estradiol-de estradiol-dependent genes, genes, in estrogen-receptor gene, gene, thus setting pendent in this this case case the the estrogen-receptor thus setting up an interesting positive feedback system. Compared vitellogenic vertebrates, vertebrates, the teleost liver liver apap Compared with with other other vitellogenic the teleost pears to to be the the richest richest source source of of highly highly specific specific estrogen estrogen receptors, receptors, pears making the fish fish liver liver an an ideal ideal model model system system to to study study the of making the induction induction of receptor and and analyze in detail the mechanism mechanism of of hormone-receptor hormone-receptor receptor analyze in detail the and receptor-chromatin receptor-chromatin interactions interactions in in lower lower vertebrates. vertebrates . The The values values and listed in in Table Table I compare compare the the magnitude magnitude of of the receptor induction induction in in the receptor listed S. salar with other oviparous vertebrates utilized in the analysis of S. of the biochemistry biochemistry of of the the estrogen estrogen receptor. receptor. Recently, Recently, it it was was found found that that the the salmon is not unique in this respect, nor is such largesse of of response restricted to to salmonid salmonid fish. fish. Liver nuclei isolated isolated from the sea-raven sea-raven restricted Liver nuclei americanus), the the longhorn longhorn sculpin sculpin ((M. octodecimspinosus), winter winter ((H. H . americanus), M . octodecimspinosus), flounder ((P. americanus), and the rainbow trout (S. gairdneri) all accuaccu P . americanus), flounder mulate similarly similarly high high concentrations concentrations of of estrogen estrogen binding binding proteins proteins as as mulate

358 358

THOMAS THOMAS P. MOMMSEN MOMMSEN AND AND PATRICK PATRICK JJ.. WALSH WALSH

Table Table I Magnitude Magnitude of of Estrogen-Receptor Estrogen-Receptor Response Response in in Vitellogenic Vitellogenic Vertebratesa VertebratesD Naive animals animals Salmo saiar salar Saimo laevis Xenopus iaevis Gallus domesticus domesticus Gallus

0.15 0.15 0.2 0.2 0.1 0.1

After After induction induction with with estradiol estradiol

> > 12 12

2.5 2.5 0.4 0.4

a0 Values Values are are given given as as 10-1 10-122 moles moles of of nuclear nuclear binding binding sites sites for for estradiol estradiol per per gram gram of of liver. liver. Concentration Concentration of of cytosolic cytosolic binding sites 0.6 x 10-12 binding sites in in the the untreated untreated salmon salmon was was about about 0.6 mol courses of see mol (g (g liver)-I. liver)-'. For For time time courses of receptor receptor abundance abundance see Fig. maximum number number of nuclear binding binding sites sites in Fig. 4. 4. The The maximum of nuclear in vivo vivo and computes to nuclear estrogen and in in vitro vitro computes to around around 25,000 25,000 nuclear estrogen re receptor molecules liver cell. Hayward et ceptor molecules per per liver cell. Sources: Sources: Hayward et ai. al. ((1980), 1980), Lazier 1975), and Lazier ((1975), and Lazier Lazier et et ai. al. (1985). (1985).

the Atlantic salmon (Mann et al., al., 1988). 1988). In a goby (Gobius (Gobius niger), niger), an elasmobranch (Potamotrygon, (Potamotrygon, ssp.), ssp.),and a hagfish (Eptatretus (Eptatretus stouti), stouti), on on the the other other hand, hand, numbers numbers of of estrogen-binding estrogen-binding sites sites in in the the liver liver are are (Le Menn more than an order of magnitude lower than in the salmon (Le et al., 1980; 1980; Callard and Mak, 1985; 1985; Turner et al., al., 1981). 1981). The The magnitude magnitude of of the the receptor receptor response, response, which which resembles resembles that that in in some mammalian systems (Walter et al., al., 1985), 1985), and the reported sta stability of the nuclear binding protein in many teleost fishes fishes (Lazier et al., 1985) 1985) should should make make it it possible possible to to attain attain receptor receptor preparations preparations of highest purity. To date, this goal has been hampered by the poor response in all other oviparous vertebrates (see (see Table I). I). The anticiantici pated availability of of receptor preparation of of extreme purity may help to shed light on the ongoing controversy over the cellular distribution of hepatic receptor (King, (King, 1984; of 1984; Szego and Pietras, 1985) 1985) and the comcom of occupied estrogen receptors in the cell nucleus (Shapiro, (Shapiro, plex fate of 1982). 1982). Furthermore, comparisons with estrogen receptors from mammam malian tissues will furnish insights into the evolutionary trends of of the receptor gene (Greene (Greene et al., 1986). 1986). While the actual source of of these comparatively large amounts of of receptor in the piscine the gene-regulatory estrogen receptor piscine system has not been elucidated yet, some indirect evidence in other vitellogenic verver tebrates indicates that de novo synthesis of of receptor protein is inin 1975; Perlman et al., 1984). Again, the highly sensitive volved (Lazier, (Lazier, 1975; al., 1984). piscine system seems ideally suited to supply mechanistic insight into

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the source of "induced" “induced” receptor molecules by molecular biology techniques, ssimilar imilar to the ones used to assess the transcriptional activ activity mRNA longevity albumin gene (Wolffe et ity and and mRNA longevity of of the the albumin gene in in Xenopus (Wolffe al., 1985), 1985), for instance. instance. al., In receptor proteins proteins resemble the the In all all parameters parameters analyzed, analyzed, the the fish receptor receptors from other vertebrates vertebrates.. The salmon receptors are character characterprogesterized by a high specificity for extradiol and they do not bind progester one, one, hydrocortisone, hydrocortisone, or or dihydrotestosterone. dihydrotestosterone. In In agreement agreement with with stud studies of receptors from many other vertebrate sources, the fish receptors display high high affinity affinity for for the the nonsteroidal nonsteroidal estrogen estrogen diethylstilbestrol diethylstilbestrol as as 4-hydroxytamoxifen. The es eswell as for the nonsteroidal antiestrogen 4-hydroxytamoxifen. E . stouti, on the of the hagfish E. trogen binding proteins in the liver of other hand, display global verte other hand, display unique unique features, features, different different from from the the global vertebrate brate picture. picture. This species species possesses possesses nuclear nuclear estrogen estrogen receptors with with a lower affinity for extradiol than other vertebrate counterparts (disso (disso&=38 3-6 nM in the salmon; salmon; see Fig. ciation constant Ki = 38 nM versus &= Ki= 3-6 4B). However, the hagfish system is unusual in that estrone or estriol 4B). effidisplaced estradiol from the nuclear binding components as effi ciently ciently as as estradiol estradiol or or diethylstilbestrol diethylstilbestrol (Turner (Turner et al., aE., 1981). 1981). In other other vertebrates, vertebrates, binding binding affinities affinities for for estriol estriol or or estrone estrone are are usually usually more more than an order of magnitude lower than for estradiol. estradiol. In the rainbow of vitellogenin trout, estrone administration leads to the induction of synthesis in the liver and its release into the bloodstream, but estrone displays 12% of the 5%to to 12% the potency potency of of estradiol estradiol (van (van Bohemen et displays only only 5% al., 1982a,b). 1982a,b). It seems that one of the functions of estrone in vivo may al., be to prime hepatic tissue for subsequent exposure to estradiol and the hepatocytes to to es esthus to to potentiate potentiate the the vitellogenic vitellogenic response response of the tradiol. The interesting characteristics of the hagfish receptor, to tocharacteristics of gether with the positioning of the cyclostomes within the vertebrate line, of steroid steroid sex sex line, might might in future future shed shed some some light light on on the the evolution evolution of hormones, their interactions, and receptor specificity. specificity. In In the the annual annual cycle cycle of of the the rainbow rainbow trout, trout, the the liver liver is is exposed exposed to to of estradiol and estrone. differing concentrations and ratios of estrone. While blood concentrations of both estrogens increase during early vitello vitellogenesis, the first phase of vitellogenesis is dominated by estrone, which increases by a factor of 10 10 altogether (van (van Bohemen and Lam Lambert, 981). During bert, 11981). During the the later later stages stages of of exogenous exogenous vitellogenesis, vitellogenesis, estra estradiol reaches blood concentrations of60 of 60 ng/ml, reflecting an increase of 60-fold. It would be interesting to analyze whether similar changes 60-fold. are specific estrogen abundance or or preference preference of of specific estrogen re reare reflected reflected in in the abundance ceptors in the nuclei of the liver, liver, the main target organ for ovarian estrogens.

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THOMAS THOMAS P. P. MOMMSEN MOMMSEN AND AND PATRICK PATRICK JJ.. WALSH WALSH

In In addition addition to to the the estrogens, estrogens, androgens androgens are are able able to to elicit elicit aa vitello vitellogenic genic response response in in teleost teleost fish, fish, albeit albeit only only when when administered administered in in phar pharmacological macological doses, doses, (Le (Le Menn, Menn, 1979; 1979; Hori Hori et al., 1979). 1979).Interestingly, Interestingly, at at least in C. G. niger this response appears to be mediated by androgen binding binding to to the the estrogen estrogen receptor receptor rather rather than than through through the the nuclear nuclear an androgen drogen receptor itself itself (Le (Le Menn Menn et al., 1980). 1980). Similarly, Similarly, high high doses doses of of androgen androgen fed fed to to juvenile juvenile salmon salmon may may lead lead to to aa pronounced pronounced feminiza feminization tion of of some some fish fish (Solar (Solar et al., 1984), 19841, although although the the molecular molecular mecha mechanisms for these phenomena remain to be analyzed. The The sedimentation sedimentation coefficient coefficient of of the the salmon salmon receptor receptor protein protein of of 3.6 3.6 SS indicates indicates that that it it may may be be aa little little smaller smaller than than the the nuclear nuclear estrogen estrogen receptors receptors of of birds birds or or mammals, mammals, but but it it falls falls into into the the same same range range as as the the receptor (Lazier, 1978; receptor isolated isolated from from Xenopus laevis (Lazier, 1978; Wright Wright et al., al., 1983). 1983).

C. Plasma Binding Proteins C. The steroid hormones produced and released by the ovarian cells are transported to their target tissue in the systemic circulation. Al Although probably not a target issue in itself, fish plasma displays a certain degree of steroid-binding steroid-binding capacity. For a variety of of fishes as well as for other vertebrate groups, such "sex-steroid “sex-steroid binding pro proteins" teins” have been characterized numerous times (Wingfield, (Wingfield, 1980). 1980). Their specificities and properties clearly distinguish plasma binders from the cellular steroid receptors, while their exact physiological function, over and above the suggested role in steroid transport, is still under debate. Since steroid hormones exert their biological functions only in the free and not in the bound form, such plasma steroidsteroid binding proteins may serve to buffer free steroid concentrations in of high steroid turnover, thus obviating time consuming de conditions of novo synthesis. compo In the plasma of of SS.. salar, two differing estradiol binding components are abundant, one with high affinity and one with low affinity for al., 1985). 1985). In contrast to the highly estradiol-speestradiol-spe estradiol (Lazier et aZ., cific nuclear of the nuclear receptors inducible in the salmon liver, neither neither of plasma-binding components are competed for by the nonsteroidal eses diethyl s tilbestrol. Furthermore, again differing from the situatrogen diethylstilbestrol. situa 4-hydroxytamoxifen does not comcom tion in the liver, the antiestrogen 4-hydroxytamoxifen pete with estradiol for binding to the high-affinity (&=13 (KI= 13 nM) estrogen binder in plasma. Experiments also indicate that the androandro gen dihydrotestosterone as well as progesterone and estrone reveal

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affinity for the plasma binder and are likely to compete considerable affinity 1985). with extradiol in vivo as they do in vitro (Lazier et al., 1985). D. Hepatic Hepatic Events Events With a time delay of a few hours following the binding of the estrogenlreceptor complex to the nuclear DNA, DNA, a variety of changes in estrogen/receptor liver cells are initiated that are consistent with a substantial increase in the capacity for protein synthesis and export-plasma export-plasma concentra concentration tion of vitellogenin vitellogenin may may reach reach 50 50 mg/ml mg/ml (Ng (Ng and and Idler, Idler, 1983). 1983). Indeed, Indeed, naturally sh reveal fish reveal much much higher higher rates rates of of hepatic hepatic protein protein naturally vitellogenic vitellogenic fi synthesis than nonvitellogenic fi sh (e.g., fish (e.g., Haschemeyer and Mathews, 1983; 1983;Yu et al., 1980; 1980; Emmersen and Korsgaard, 1983), 1983),a phenomenon that can be provoked by estrogen administration in vivo as well as in vitro. vitro. Several Several ultrastructural differences are observed between liver cells from immature and vitellogenic fish. (Epifish. In the red grouper (Epi nephelus akaara), akaara), vitellogenic livers are characterized by expanded nuclear envelope envelope cisternae, swollen swollen mitochondria, and much en enhanced rough endoplasmic reticulum, Golgi apparatus apparatus and secretory vesicles (Ng sh treated with estra (Ng et al., al., 1984). 1984). Hepatocytes of naive fi fish estradiol imilar, but diol showed showed ssimilar, but not not entirely entirely identical identical ultrastructural ultrastructural changes changes (Ng (Ng et al., al., 1984). 1984). Several Several studies studies indicate indicate an an increase increase in in hepatosomatic hepatosomatic index (van Bohemen et al., al., 1982a,b; 1982a,b; Dasmahapatra et al., 1981) 1981)and, at least in the red grouper, this appears to be due to a rise in cell lipid and water content rather than proliferation of cell numbers (Ng al., (Ng et al., 1984). 1984). In the Atlantic salmon, as in the flounder, flounder, estradiol administra administration leads to increases in liver protein, total RNA, and total nuclear count (Korsgaard 1976). Since at (Korsgaard et al., al., 1986; 1986; Korsgaard and Petersen, 1976). the same time the liver volume and weight increase, calculated on a is augmented unit weight basis, only the amount of cellular RNA is signifi cantly. It can be concluded that in these two species of fish, significantly. fish, apdiffering from the grouper, hyperplasia rather than hypertrophy ap pears to be responsible for the enhanced liver weight (Korsgaard (Korsgaard et al., 1986). 1986).The more than 30% 30% increase in cellular RNA content (on (on a al., basis) is is yet another indication of the increased biosyn biosynunit weight basis) thetic activity of the liver (Korsgaard al., 1986), 1986),where where the de novo (Korsgaard et al., some of synthesis of messenger RNA for vitellogenin may account for some the observed increase in total RNA. RNA. A larger proportion of the newly synthesized RNA in hepatic tissue the exposure to estradiol is is due to apparent increases in the following the amounts amounts of ribosomal RNA, RNA, which can be explained by the massive

362

THOMAS P. MOMMSEN MOMMSEN AND PATRICK J. WALSH

increases in rough endoplasmatic reticulum observable in liver micro micrographs of fish and other oviparous vertebrates (Bast (Bast et al., al., 1977; 1977; Selman and Wallace, 1983a). 1983a). Since estrogen administration is respon responsible for the proliferation of of cell structures, such as endoplasmic retic reticulum (ER), (ER), Golgi vesicles (and turnover), turnover), and mitochondria, genes coding for any of these structures must have been activated or estro estrogen administration must have at least led to increased translational activity involving existing mRNAs. mRNAs. Obviously estradiol is able to or orchestrate cell metabolism and biosynthetic activities at a number of different levels. An ancillary question concerns the actual localization of hepatocy hepatocytes active in the synthesis and export of vitellogenin. Contrary to the identical, it widespread belief belief that all hepatocytes are metabolically identical, has been shown rather conclusively that rat hepatocytes in the perive perivenous and periportal regions of the liver possess differiong metabolic functions, with anabolic pathways such as gluconeogenesis, fat syn synthesis, and proteins synthesis being favored in the better oxygenated periportal cells (Jungermann (Jungennann and Katz, Katz, 1982). 1982). It would be interesting to know whether a similar hepatic zonation exists in the lower verte vertebrates in general and extends to estrogen receptors and the vitello vitellogenic response. E strogen treatment of fish also appears to result in a general gear Estrogen gearing up of of metabolism to provide the large amounts of of energy and reducing power (NADPH) (NADPH) necessary for protein and lipid synthesis (Ng 1984) fur (Ng et al., 1984; 1984; Petersen and Korsgaard, Korsgaard, 1977). 1977). Ng et al. al. ((1984) further report significant and substantial increases in transaminases and enzymes of the Krebs cycle and glycolysis. glycolysis. On the other hand, natu naturally vitellogenic vitello genic female sockeye salmon (Oncorhynchus (Oncorhynchus nerka) nerka) on their spawning migration do not increase any specific metabolic ma machinery in liver (on (on a weight basis, Mommsen et al., ul., 1980), 1980),apart from the general augmentation due to an-probably an-probably estradiol-dependent estradiol-dependentincrease in liver weight (Idler and Clemens, 1959). 1959). In vivo treatment of male flounder (Platichthys flesus) results in an (Platichthysjesus) increase of protein synthetic activity when assessed in an in vitro system (Korsgaard (Korsgaard et al., 1983). 1983).That such stimulation may be a direct effect of estrogen on liver cells was recently demonstrated in hepato hepatocytes isolated from juvenile coho salmon (0. (0.kisutch; Bhattacharya et al., al., 1985). 1985). Hepatocytes treated with 17f3-estradiol 17p-estradiol and exposed to 4C]serine or [['*C]glycine either [ l14C]serine 14C]glycine exhibit an increase in radioactivity precipitable by trichloroacetic acid (TCA), (TCA), a decrease in TCA-soluble radioactivity, and enhanced release of TCA-precipitable radioactivity into the medium compared with untreated untreated controls (Bhattacharya et

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Table Table II I1 Cellular Events Associated with the Estrogen-Dependent Induction Vitellogenesis in Teleost Hepatic Tissue of Vitellogenesis Transient decrease in cytosolic estrogen receptor protein Induction of nuclear estrogen receptor protein Increase in hepatosomatic hepatosomatic index due to hyperplasia or hypertrophy Golgi apparatus Proliferation of Colgi Increase in cisternae of the nuclear envelope Synthesis Synthesis of ribosomes Polysome assembly assembly Increase in rough rough endoplasmatic endoplasmatic reticulum reticulum of mitochondria mitochondria Swelling of Appearance species of mRNA (vitellogenin) Appearance of of a new species Increase in protein synthetic activity Synthesis Synthesis of vitellogenin Increase in cellular RNA (?)n Increase in lipid metabolism (?)" Increase lipoproteins (VLDL) (VLDL) Augmented output of very low density lipoproteins Decrease in glycogen content per cell Increase in metabolic enzymes enzymes Increase Higher amount of hepatic DNA Increase Increase in in hepatic hepatic water water content content "0 For the the fishes, fishes, to to date date only only circumstantial circumstantial evidence evidence suggests suggests this this particular particular alteration. alteration, See See text text for for relevant relevant references. references.

al., 1985). 1985). Originally Originally it it was was observed observed that that liver liver slices slices from from cod cod (G. (G. 1 4C]leucine into morhua) rnorhua)treated treated with with estradiol estradiol incorporated incorporated labeled labeled [[‘4C]leucine into "egg evidence confi rmed 1971).All this this evidence confirmed “egg proteins" proteins” (Plack (Plack and and Fraser, Fraser, 1971).

that teleost fishes, just as that in in the the teleost fishes, just as in in other other oviparous oviparous vertebrates, vertebrates, estra estradiol rapid and hepatic synthesis and specific specific hepatic synthesis of of the the egg-yolk egg-yolk diol leads leads to to the rapid precursor vitellogenin. The most apparent ultrastructural and bio bioexperience during exogenous vichemical changes that hepatocytes experience exogenous vi 11. tellogenesis are summarized in Table II.

E.. Vitellogenin E 10years vitellogenin molecules from a number of differ differIn the last lO biochemicent fishes have been isolated and partially characterized biochemic ally. Interestingly, Interestingly, the fishes fishes display a much higher variability in the ally. phosphorylsuch as as molecular weight, weight, degree of phosphoryl different parameters, such ation, degree of lipidation, or subunit subunit composition than their amphib amphibation, 111).As the example of the tetrameric counterparts (Table (Table III). ian or avian counterparts shows (Hara (Hara et al., al., 1980), 1980),not all fish vitellogenin in the Japanese eel shows are dimers-for dimers-for instance, and in the case of the brown vitellogenins are

364 364

THOMAS THOMAS P. MOMMSEN MOMMSEN AND AND PATRICK J. WALSH

Table Table III 111 Molecular Weights of of Native Vitellogenin and Subunits from Fish Other Than Rainbow Trout Native molecular mass (kD4 (kDa)

Subunit molecular mass (kDa) (kD4

Carassius Carassius auratus auratus (goldfi sh) (goldfish)

326 380

140-156 140-156 140-147 140-147

Gadus Gadus morhua morhua (cod) (cod) Anguilla japonica (Japanese eel) eel) Fundulus heteroclitus (killifish) (killifish) Ameiurus Ameiurus nebulosus nebulosus (brown bullhead) Platichthys Plotichthys flesus Jesus (flounder) (flounder) Oncorhynchus Oncorhynchus kisutch kisutch (coho (coho salmon) salmon) Salmo Salmo salar salar (Atlantic salmon) (Atlantic salmon) Salrno Salmo trutta trutta (brown trout) Heteropneustes Heteropneustes fossilis fossilis (catfish) (catfish)

400

-

350

85 85

-

200

-

145 145

550

-

Gel fi ltration filtration

390” 390c

-

Gel filtration

495 and 520 495and520

-


440

-

Gel filtration

550

-


Species

Method" Methodu

Reference

Native/SDS-PAGE 1979) Native/SDS-PAGE Hori et et al. al. ((1979) Native/SDS-PAGE Native/SDS-PAGE de Vlaming - et al. (1980) (1980) Plack et Gel filtration et al. (1971) (1971)

Gel filtration and SDS-PAGE SDS-PAGE SDS-PAGE SDS-PAGE b

Hara et al. ((1980) 1980) et al. Selman and Wallace ((1983a) 1983a) Roach and Davies (1980) (1980) Korsgaard and Petersen ((1976) 1976) Markert and Vanstone ((1971) 1971) (1985) So et al. al. (1985) Norberg and Haux ((1985) 1985) Nath and Sundararaj (198 1) (1981)

SDS-PAGE: : sodium dodecyl sulfate polyacrylamide polyacrylamide gel electrophoresis. " SDS-PAGE prevailing mRNA induced by estradiol treatment treatment of of bullhead b Translation product of the prevailing catfish. catfish. c Lipovitellin from coho salmon eggs. eggs. C

bullhead, is substantially bullhead, the the messenger messenger RNA RNA for for vigellogenin vigellogenin is substantially smaller smaller than that for any other vertebrate (Table III). 111). Even within the same species, S . gairdneri), gairdneri),aa large large variation variation in species, in in this this case case the the rainbow rainbow trout trout ((8. the the apparent apparent molecular molecular weight weight of of the the vitellogenin vitellogenin is is noticed, noticed, which which may be due to different methodologies used, different degrees of pro proteolytic breakdown, or teolytic breakdown, or dephosphorylation dephosphorylation occurring occurring during during the the isola isolation IV). Some Some degree degree of of heterogeneity heterogeneity in in vitellogenin may may be tion (Table (Table IV). due due to to the the fact fact that that in in the the fishes, fishes, as as in in other other vertebrates vertebrates,, vitellogenin vitellogenin is is not of slightly slightly differ differnot encoded encoded by by aa single single gene, gene, but but rather rather by by aa family family of ent ent genes. genes. As long as the translation of of one isolated vitellogenin messenger (mRNA) in this salmonid fish leads to different molecular-weight RNA (mRNA) estimates estimates for for the the vitellogenin monomer, monomer, more more attention attention will have have to to

5. VITELLOGENESIS AND 5.

365 365

OOCYTE ASSEMBLY

Table IV IV Molecular Weight of Native Vitellogenin and Subunits from gairdneri S. gairdneri Rainbow Trout S. Native molecular mass (kDa) Wa)

342 342 440 440 440 440 470 470

500 500 500 500 535 600

Subunit molecular molecular mass (kDa) (kDa)

170" 170" 200 200 250 250 16-103" 16103c 220

Method

Reference

Ultracentrifugation Ultracentrifugation Gel filtration Gel filtration Gradient PAGE Gradient PAGE poly(A)+ SSDS-PAGE DS-PAGE of poly(A)+ translation product filtration/SDS-PAGE Gel filtration/S DS-PAGE Not stated SDS and gradient PAGE SDS Gel fi ltration/SDS-PAGE filtration/SDS-PAGE

Campbell and Idler (1980)

(1985) Norberg and Haux (1985) Campbell and Idler ((1980) 1980) 1983) Chen ((1983) Valotaire et et al. (1984) (1984)

1981 ) Sumpter ((1981) (1984)b Y. Valotaire (1984)b Maitre et al. (1985a) (1985a) Hara and Hirai (1978)

modifications, polypeptide alone = = 160 160 kDa. "a Polypeptide plus posttranslational modifi cations, polypeptide al. (1984). (1984). b Unpublished observation, cited in Valotaire et al. prec Eleven polypeptides that represent breakdown products from handling or pre cursors.

be devoted to multiple vitellogenin genes, to strain differences within one species, or to possible partial degradation of of this large molecule or its mRNA-which mRNA-which do not critically affect the immunological reactiv reactivity-before ity-before a definitive answer with respect to molecular weight and phosphorylation sites can be given. Estradiol treatment leads to the appearance of a specific high-mo high-molecular-weight species of of messenger RNA (6300 (6300 or 7200 nucleotides) in the rainbow trout ((Chen, Chen, 1983; 1983; Valotaire et al., aI., 1984; 1984; see Table IV). Using cytoplasmic polyadenylated RNA isolated from thes thesee estradiol estradiolexposed trout in a cell-free translation system, Chen (1983) (1983)was able to synthesize a 160,000-Da 160,000-Da polypeptide poIypeptide that was chemically, immuno immunologically, and electrophoretically identical to the authentic vitello vitellogenin monomer. Similar results were obtained for the same species by Valotaire and co-workers ((1984), 1984), although their larger mRNA (7200 (7200 nucleotides) upon translation yielded a considerably larger (200,000 (200,000 Da) polypeptide, which was immunoprecipitable with antibodies Da) against trout serum vitellogenin. The same authors also synthesized DNA complimentary to the estrogen-stimulated mRNA and back-hy back-hybridized with liver RNA to determine the increase in RNA due to estrogen treatment; the treatment increased it by 9%.

366

THOMAS P. MOMMSEN AND PATRICK JJ.. WALSH

F. F. Posttranslational Modifications Modifications The biochemical information concerning vitellogenin clearly indiindi cates that a great deal of posttranslational modification must occur in the liver cell to reach the finished product seen in the serum. serum. First the protein backbone of the vitellogenin is membrane is synthesized on membranebound ribosomes, a feature that it shares with other proteins destined to be secreted from the hepatocyte (Lewis al., 1976). 1976). In subsequent (Lewis et al., steps, the molecule must be lipidated, glycosylated, and phosphoryla phosphorylated. It has been suggested that all these processes occur on the mem membranes of the endoplasmatic reticulum and that they are already initi initiated while the polypeptide chain is being translated (Tata (Tata and Smith, 1979), although this view has been debated by Gottlieb and Wallace 1979), (1982). (1982). Finally, existing "pro" “pro” sequences or signal peptides have to be removed before vitellogenin is packaged into Golgi vesicles and se secreted into the bloodstream. While some information exists concerning the nature and extent of modifications on the vitellogenin molecule, rather limited information is is available for fish with respect to the mechanism, sequential events, or locale of these transformations. Therefore, the following discussion has to be confined to a description of of nonprotein components found on the circulating vitellogenin molecule. Just as do the vitellogenins from other oviparous vertebrates, fish vitellogenins carry a certain number of of phosphate groups, some of of it as protein phosphorus, in a region that in the mature oocytes becomes deposited as phosvitin. Generally, the molecule is phosphorylated on serine moieties, and since the degree of of phosphorylation of delipida delipidated piscine vitellogenins ranges around 0.6-0.7% 0.6-0.7% (by weight) weight) (Le., (i.e., only about 50% of the protein phosphate content in other vertebrates), vertebrates), the serine content must be comparatively lower. lower. Experimentally, this alkaline-labile protein phosphorus, which is specific to naturally or induced vitellogenic animals, has been utilized utilized repeatedly for the determination of the degree of the vitellogenic response in fish. fish. Vitel Vitellogenic female fish contain between 20 and 100 100 JLg pg of of protein phos phosphorus per milliliter of plasma, while untreated males contain less than 5JLg/mi 5pg/ml (Craik and Harvey, 1984). 1984). In spite of the large amounts of protein phosphate moved through the plasma compartment during vitellogenesis, in the unfertilized egg inorganic phosphate, and phos phospholipid, and not protein-bound phosphate, make up the bulk of of the 3 IP]phosphate (Grasdalen nuclear magnetic resonance (NMR) (NMR) visible [[31P]phosphate and JJgrgensen, flSrgensen, 1985). 1985). This observation indicates that additional ma maternal sources must supply phosphate and phospholipids to the 00oo-

5. 5. VITELLOGENESIS VITELLOGENESIS AND AND

OOCYTE OOCYTE ASSEMBLY ASSEMBLY

367

vitellogenin-derived cyte. Also substantial dephosphorylation of vitellogenin-derived phosphoproteins during transmit through the oocyte or following dep depof osition in the yolk may explain the low protein phosphate content of compo(Craik, 1982). mature oocytes (Craik, 1982). The highly charged phosphate compo nent also gives the vitellogenin molecule its high ion-binding capac capacity. Teleost vitellogenins are known to bind ions such as calcium, magnesium, or iron efficiently (Hara, (Hara, 1976; 1976; Hara and Hirai, 1978; 1978; Hara et al., 1980) thus may 1980) and and thus may designate designate an an important important vehicle vehicle for for mineral mineral supply In fact, supply to to the the growing growing oocyte. oocyte. In fact, the the competition competition of of vitellogenin vitellogenin with chelating substances has been used successfully to isolate fish vitellogenins from other plasma proteins (Ng (Ng and Idler, 1983). vitellogenins 1983). In In contrast contrast to to the the phosphate phosphate content content of of fish fish vitellogenins, vitellogenins, which which is is lower lower than than that that of of other other oviparous oviparous vertebrates, vertebrates, the the amounts amounts of of lipid lipid material material carried carried on on the the vitellogenin vitellogenin molecule molecule are are generally generally about about groups. The lipid content of twice as high as for other vertebrate groups. vitellogenin ranges around 20% fishes as different in 20% by weight in fishes lifestyle (21%; Hori et al., lifestyle and feeding preferences as the goldfish (21%; 1979), 1979), rainbow trout (21%; (21%;Wiegand and Idler, 1982; 1982; Fremont Frbmont et al., 1984; 19%; Norberg 1984; 18%, 18%, Norberg Norberg and and Haux, Haux, 1985), 1985), sea-trout sea-trout ((19%; Norberg and and Haux, 1985), 1985), or the elasmobranch dogfish ((18%; 18%; Craik, 1978a). 1978a). The bulk of this lipid material, moiety of bulk of this lipid material, which which later later forms forms the the lipovitellin lipovitellin moiety of the yolk, yolk, can can be classified classified as as polar polar lipid lipid (Hori (Hori et al., 1979). 1979). In In rainbow rainbow trout 82%of of the the trout vitellogenin, vitellogenin, for for instance, instance, polar polar lipids lipids make make up up some some 82% total oocyte, how 1982). Generally Generally the the mature mature oocyte, howtotal (Wiegand (Wiegand and and Idler, Idler, 1982). ever, ever, contains contains much much larger larger percentages percentages of of triglyceride, triglyceride, and and it it is is there therefore fore reasonable to to assume assume that that sources sources other other than than vitellogenin vitellogenin must must supply teryl supply the the oocyte oocyte with with nonpolar nonpolar lipids, lipids, such such as as triglycerides, triglycerides, ssteryl esters, is interesting interesting to esters, sterols, sterols, and and wax wax esters. esters. In In this this context context it it is to note note that that dietary manipulation of free fatty acids in trout is reflected in altered lipids, but altered fatty fatty acid acid composition composition of of serum serum lipids, but not not of of the the lipopro lipoproteins, which are most important during vitellogenesis (Fremont et al., 1984). 1984). While fish vitellogenin vitellogenin is known to contain carbohydrate groups, little of the the little concrete concrete information information on on the the amount, amount, nature, nature, and and linkages linkages of carbohydrates is available. However, it is known that for many pro proteins, of the the teins, successful successful glycosylation glycosylation is is aa prerequisite prerequisite for for excretion excretion of export protein. In other cases, such as the chicken ovalbumin, ovalbumin, which usually usually occurs occurs in in glycosylated glycosylated form, form, no no glycosylation glycosylation is is required required for for excretion. excretion. Experiments Experiments utilizing utilizing tunicamycin, tunicamycin, aa specific specific inhibitor inhibitor of of N-glycosylation, side N-glycosylation, revealed revealed that that the the absence absence of of the the oligosaccharide oligosaccharide side chains chains from from the the ovalbumin ovalbumin molecule had had no no effect effect on on its its secretion secretion (Colman 1981). For comparative comparative purposes purposes and and from from an an evoluevolu(Colman et al., 1981).

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THOMAS P. MOMMSEN AND J. WALSH AND PATRICK J. WALSH

tionary perspective, it would be a rewarding task to determine the group of glycoproteins to which the fish vitellogenins belong and whether successful glycosylation is a prerequisite for excretion from Obviously, there is a large information gap between the hepatocyte. Obviously, the process of glycosylation of the vitellogenin molecule in the liver and the presence of large amounts of of sialoglycoproteins in fish eggs (Inoue (Inoue and Iwasaki, Iwasaki, 1980a,b). 1980a,b). Vitellogenin could be detected in the blood but not in the livers of of estradiol-treated rainbow trout, a result that was first interpreted to indicate that vitellogenin is rapidly secreted following synthesis (van (van Bohemen et al., 1982b). al. (1983), 1982b). However, Nunomora et al. (1983), using the peroxidase-anti peroxidase complex method (immunologically spe peroxidase-antiperoxidase specifi c for vitellogenin), were able to localize Significant cific significant amounts of vitellogenin in livers of of estradiol treated rainbow trout (Salmo (Salmo gairdneri), (0. keta), keta), or charr (Salvelinus leucomaenis). leucomaenis). gairdneri), chum salmon (0. Similarly, Similarly, So and co-workers (1985) (1985) detected cross-reactivity of anti antibodies against salmon (Salmo (Salmo salar) salar) vitellogenin with liver extracts of vitellogenic fish. fish. These results can be reconciled by the fact that van Bohemen et al. al. (1982b) (1982b) assayed for vitellogenin by molecular weight determination on sodium dodecyl sulfate (SDS) (SDS) polyacrylamide gels and thus screened for mature vitellogenin rather than immunoreactive components (see (see below). below). availMore information regarding posttranslational modification is avail able from other vertebrate systems. Recently, Recently, rooster hepatocytes were used to determine the probable sequence of events in hepatic vitellogenesis (Wang (Wang and Williams, 1982). 1982). Precursors (pVTG (pVTG I and pVTG II) 11)for each of of the two types of avian vitellogenin (VTG (VTG I and 11)were found in hepatocytes of of roosters treated with estrogen VTG II) 3H ]serine and pulse-chase experiments. The by pulse-labeling with [[3H]serine molecular weights of the precursors were lower than those of the SDS gel electrophoresis. How Howmature vitellogenins as determined by SDS of these polypeptides by immunological meth methever, further analysis of ods, peptide mapping, and molecular-weight determinations by gel chromatography revealed that the precursors are similar to mature vitellogenin in size and degree of glycosylation, but are not phos phosphorylated. Wang and Williams (1982) (1982) could also show that highly phosphorylated proteins, such as mature avian vitellogenin, will yield erroneously high molecular weights on SDS SDS gels. The very small quantities of phosphorylated vitellogenin inside of these hepatocytes (1982) to suggest that phosphorylation is is rap rapled Wang and Williams Williams (1982) idly followed by secretion. Their determination of vitellogenin molec molec(Wang ular weight by gel chromatography caused the same authors (Wang

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369

and Williams, 1982) “accepted” molecular weight for the 1982) to revise the "accepted" avian vitellogenin monomer from above 235,000 to 180,000. 180,000. In light of of the obvious controversies about the actual molecular weights of of pispis cine vitellogenins, even within the same species (see Table 111), III), this of multifaceted multifaceted approach represents a fertile area for research on type of fish. In this field, the isolated hepatocyte systems vitellogenesis in fish. would appear to be an excellent, as yet underutilized, experimental tool (Moon (Moon et al., aZ., 1985). 1985).Recently, several laboratories have been able to prove that fish hepatocytes in suspension or in primary culture are highly 1985; Mommsen Mommsen highly responsive responsive to to estrogen estrogen (Bhattacharya (Bhattacharya et al., 1985; 1986), and that hepatocytes isolated from primed fish will and Lazier, 1986), synthesize and excrete large amounts of of vitellogenin in vitro (Hasche (Haschemeyer and Mathews, 1983). 1983). From From the the reviewed reviewed studies studies on on fishes fishes and and other other egg-laying egg-laying verte vertebrates, brates, aa preliminary preliminary picture picture of of the the sequence sequence of of events events implicated implicated in in exogenous synthesized (Table (Table V). V). Unfortu Unfortuexogenous vitellogenesis vitellogenesis can can be synthesized nately, many parts fishes, many parts of of the the scheme scheme require require aa nately, especially especially for for the fishes, major major concerted concerted research research effort effort from from biochemists, biochemists, physiologists, physiologists, and and molecular replace speculation speculation and and add add information information molecular biologists biologists alike to replace on on actual actual mechanisms. mechanisms. The The major major task task would would be be to to successfully successfully utilize utilize the piscine system the vast vast potential potential of of piscine system to to elucidate elucidate and and understand understand the the estrogen with the estrogen receptor receptor mechanism, mechanism, its its interaction interaction with the nuclear nuclear DNA, DNA, and and not not least least the the subsequent subsequent gene gene activation. activation. Further Further challenging challenging topics topics include include the the diverse diverse posttranslational posttranslational modifications modifications of of the the vitel vitellogenin logenin molecule molecule occurring occurring in in the the liver liver cell. cell. Furthermore, Furthermore, the the particu particular lar intracellular intracellular structures structures where where the the individual individual steps steps occur occur have have to to date date eluded eluded identification. identification. Recent of similar similar vitellogenin vitellogenin molecules molecules of of Recent work work on on the the number of Xenopus has has revealed revealed that that the the situation situation is is not not quite quite as as clear-cut clear-cut or or simple simple as as it it first first appeared. appeared. Rather Rather than than being being just just one one protein, protein, coded coded for mRNA, aa whole whole family family of of vitellogenin vitellogenin genes genes is is in in for by by one one type type of mRNA, existence aZ., 1981), 1981), all all of of which which give give rise rise to to slightly slightly differ differexistence (Wahli (Wahli et al., ent ent vitellogenin vitellogenin molecules. molecules. These These in in turn turn supply supply the the growing growing oocyte oocyte with with the the different different building building blocks blocks for for at at least least five five different different types types of of yolk and 2, 2, phosvitin, phosvitin, and and phos phosyolk polypeptides, polypeptides, namely namely lipovitellins lipovitellins 11and vettes 198 1)], which and 22 [nomenclature [nomenclature of of Wiley Wiley and and Wallace Wallace ((198l)l, which in in vettes 11and themselves and 22 can can themselves are are somewhat somewhat heterogeneous. heterogeneous. Lipovitellins Lipovitellins 11 and each each be be resolved resolved into into three three differing differing polypeptide polypeptide components, components, while while dephosphorylated two polypeptide polypeptide bands bands of of different different dephosphorylated phosvitin phosvitin yields yields two molecular SDS electrophoresis. electrophoresis. Phosvettes Phosvettes are are relatively relatively molecular weights weights on on SDS small small phosphorylated components components with single single polypeptide chains. From on the the vitellogenin vitellogenin of of Xenopus, Wiley Wiley and and Wallace Wallace From their their study study on

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THOMAS P. P. MOMMSEN MOMMSEN AND AND PATRICK PATRICK J. WALSH THOMAS J . WALSH

Table V V Table of Events during Hepatic Synthesis Synthesis of of Vitellogenin" Vitellogenina Suggested Sequence of Nuclear compartment compartment Nuclear of vitellogenin viteIlogenin gene through through binding binding of of receptor-homone receptor-honnone complex complex to to Activation of regions of of the the nuclear nuclear DNA specific regions Transcription and presence presence of of primary primary transcript transcript in the nuclear compartment compartment Transcription Processing of of primary primary transcript transcript Processing Translocation to cytoplasm cytoplasm Translocation reticulum Rough endoplasmatic reticulum Polysome assembly Translation of of vitellogenin viteIlogenin mRNA Translation Processing of of previtellogenin previteIlogenin subunits Processing Phosphorylation at serine residuesb residuesb Phosphorylation Lipidationc Lipidation' reticulum Translocation to smooth endoplasmatic reticulum Smooth endoplasmatic reticulum phosphorylation at serine residuesb residuesb Further phosphorylation Translocation Translocation to Golgi apparatus Golgi apparatus Glycosylation Glycos ylation Mannose N-Acetylglucosamine N-Acetylneuraminic acid, etc. Lipidationc LipidationC Removal of existing signal peptides Dimerization Phosphorylation Phosphorylation at serine residuesd Excretion into systemic circulation circulation Adapted from Tata and Smith (1979), (1979), Wang and Williams (1982), (1982), and Gottlieb and Wallace Wallace (1982). (1982). b b In Xenopus, Xenopus, phosphorylation phosphorylation occurs occurs in the rough rough endoplasmatic endoplasmatic reticulum reticulum and the the smooth smooth endoplasmatic endoplasmatic reticulum only. The exact cellular site covalent attachment of lipid to vitellogenin is still site of the non noncovalent under under debate. debate. d In the the chicken, chicken, vitellogenin is phosphorylated phosphorylated during its time in the the Golgi appa apparatus, followed by rapid excretion from the hepatocyte. •

C

((1981) 1981) came to the conclusion that the whole gamut of yolk yolk proteins is is also that the phos phosderived from multiple vitellogenin molecules and also vettes are are alternate cleavage products products from from homologous homologous regions of vettes different parent vitellogenins copies of vitellogenins.. Using complimentary DNA copies mRNA, Wahli Wahli et al. al. ((1981) that vitello vitelloXenopus vitellogenin mRNA, 1 98 1 ) deduced that genin is is encoded in a family family of at least four expressed genes. genes. A similar similar situation situation appears appears to to exist in the the chicken, chicken, where where three different genes genes of of the the vitellogenin vitellogenin family family are are expressed, expressed, producing different three polypeptide polypeptide chains chains with with molecular molecular weights weights ranging ranging from from three

5. 5.

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Table VI Estradiol-Dependent Lower Vertebrates· Vertebratesa Selected E stradiol-Dependent Genes in Lower Proteins Proteins

Organ

Organism(s) Organism(s)

Effect

Ovalbumin Lysozyme Conalbumin Ovomucoid Avidin

Oviduct

Birds

Induced Induced Induced Induced Induced

Vitellogenin Vitellogenin Albumin

Liver

Oviparous vertebrates Xenopus, Oncorhy Oncorhynchus ?) nchus nerka nerka ((?) (?) Chicken, Chicken, teleosts (?) Birds

Induced

ApoB, Apoll ApoII (VLDL) (VLDL) Vitamin-binding Vitamin-binding proteins Biotin Thiamin Cobalamin Ribofl avin Riboflavin Transferrin Transferrin receptor Estrogen receptor

Teleosts, chicken

Depressed Induced Induced Induced Induced Induced Induced Induced

For structural structural and further biochemical biochemical changes initiated in the hepatocyte under the influence of estradiol, II. References: Muniyappa estradiol, see Table 11. Muniyappa and Adiga (1980), (1980), Leger et 1981), Lazier et ai. (1985), et ai. al. ((1981), et al. (1985), White (1985), (1985), Wolffe et et ai. al. (1985). (1985). •

170,000 190,000. Moreover, 170,000 to to 190,000. Moreover, the the three three subunits subunits possess possess different different degrees degrees of of phosphorylation, phosphorylation, and and subsequently subsequently make make up up the the native native vitellogenin By analogy, vitellogenin dimer dimer (Wang (Wang et al., 1983). 1983). By analogy, aa similar similar multi multigene gene family family can can be expected expected to to code code for for vitellogenin vitellogenin in in the the fishes. fishes. The The observed observed multitude multitude of differing differing egg egg phosphoproteins phosphoproteins further further suggests suggests widespread widespread heterogeneity heterogeneity within within the the vitellogenin vitellogenin molecule. molecule. C.. Other Actions of of Estradiol G

The specific specific action of estradiol estradiol at nuclear level level in in hepatic hepatic tistis The action of at the the nuclear sue is is by by no no means means restricted restricted to to the the activation activation of of the vitellogenin gene, gene, sue the vitellogenin although, at least least in in the fishes, vitellogenin single most most although, at the fishes, vitellogenin constitutes constitutes the the single important de novo of protein. In the chicken, which which has has important de novo synthesis synthesis of protein. In the chicken, attracted most attention this respect, number of other genes genes are are attracted most attention in in this respect, aa number of other activated concomitantly, apoVLDLII-the activated concomitantly, including including those those coding coding for for apoVLDLII-the major lipoproteins) in in laying laying chickens chickens (Deel(Deel major VLDL (very-low-density (very-low-density lipoproteins) ey, 1985)-as well well as as various proteins. A A ey, et al., 1985)-as various vitamin-binding vitamin-binding proteins. preliminary (i.e., constantly constantly growing) growing) list list of genes that that are are directly directly preliminary (i,e., of genes affected by estradiol in various various vertebrates vertebrates is is given given in in Table Table VI. VI . In In aa affected by estradiol in

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mechanistically unknown fashion, estradiol also induces the multi multitude of ultrastructural changes occuring in the liver of all vertebrates actively undergoing vitellogenesis (cf. (cf. Table II). 11). As As pointed out above, the teleost fishes add another estrogen-induced gene to this growing list, namely, the gene for the nuclear estrogen receptor pro protein. Estradiol also exerts negative effects effects on the synthesis of other ex export proteins, among them the ubiquitous serum albumins. In many vertebrates, estradiol administration leads to a pronounced reduction in the concentration of albumin circulating in blood, an effect that is especially especially apparent apparent in in chronically chronically estradiol-exposed estradiol-exposed male male Xenopus. Xenopus. Experiments conducted by Tata and co-workers co-workers (Wolffe (Wolffe et al., 1985) 1985) led to the conclusion that in this amphibian, two levels of estrogen action action on on albumin synthesis synthesis can can clearly clearly be be distinguished. distinguished. First, First, estra estradiol diol administration administration leads leads to to aa deinduction deinduction of of transcription transcription of of the the two two genes kDa) and more abundant albumin. genes coding coding for for the the larger larger (74 (74 kDa) albumin. Second, Second, it also causes a substantial destabilization of of the messenger RNA for albumin, which is reflected in a decrease in the actual half halflife of the messenger RNA by two-thirds (Wolffe (Wolffe et al., 1985). 1985). The same deinduction of albumin synthesis in the presence of of estradiol can also be observed in vitro using isolated hepatocytes (Wangh, (Wangh, 1982). 1982). In fishes, a similar reduction in the amount of of circulating albumin is apparent in naturally vitello genic sockeye salmon (0. (0. nerka) vitellogenic nerka) during their spawning migration (T. S. MookeIjea, C. French, (T. Mommsen, S. Mookerjea, and C. results). Conversely, in the rooster, estradiol withdrawal unpublished results). results in the destabilization of vitellogenin and apoVLDLII mRNAs, while the stability of of the serum albumin mRNA is not affected (Wisko (Wiskocil et al., 1980). 1980). In addition to inducing the de novo synthesis of of vitellogenin mRNA, estradiol has been shown to accelerate the rate of transcription of of other genes, while not necessarily necessariIy altering the amounts of mRNAs coding for different genes. of mRNAs genes. In all lower vertebrates, estradiol exerts a pronounced lipogenic action on peripheral tissues, while in Xenopus it also enhances the activities of enzymes involved in the hepatic synthesis of lipids (Phil (Phillips and Shapiro, Shapiro, 1981). 1981). It can be speculated that the de novo synthe synthesized lipid will be partly destined for the lipidation of vitellogenin and partly for inclusion in the increased output of VLDL by the liver. To date, only one study has addressed this topic in fishes: female capelin (Mallotus (Mallotus villosus) uillosus) displayed considerably higher total activi activities of of fatty acid-catabolizing acid-catabolizing enzymes than did their male counter counterparts (Henderson et al., 1984). 1984). However, as long as only one part of

5.

VITELLOGENESIS AND QOCYTE OOCYTE ASSEMBLY ASSEMBLY

3 73 373

fatty acid metabolism (either anabolic or catabolic direction) is anaana lyzed, no conclusive statement can be made about the lipid turnover turnover in the respective respective tissues. Ultimately, the ratio of of fluxes in the two net flux, and. and hence determine net directions will influence the actual net import or export. The increased potential in vitellogenic grouper to sup generate cytosolic NADPH furnishes circumstantial evidence supporting of increased hepatic fat synthesis during this period porting the notion of al., 1984). 1984). In light of of the general observation of of increased lipid (Ng et aZ., content in the blood of of vitellogenic fishes (Plack and Pritchard, 1968; 1968; 1 976; Sand et al., 1980), 1980), it can be speculated Petersen and Korsgaard, 1976; that while Iipid lipid turnover is stepped up, net flux is increased in the direction of lipid export from hepatic tissue. Strong lipogenic action of oflipid of Heteropneustes fossifossi estradiol has been reported for SS.. gairdneri and Heteropneustes lis (Haux and Norberg, 1985; 1985; Dasmahapatra and Medda, 1982). 1982). MicroMicro Zis graphs of of vitellogenic livers of heteroclitus contain less of Fundulus heteroclitus lipid depositions than livers from male fish (Selman and Wallace, 1983b), while the livers of cryso 1983b), of two other teleosts (Notemigonus crysoZeucas leu cas and Brachydanio rerio) redo) increased the amounts of of lipid under the influence of of estradiol in a dose-dependent fashion (de (de Vlaming et al., 1977; 1977; Peute et al., 1978). aI., 1978). In the blenny (Zoarces (Zoarces viviparus), uiuiparus),lipid is accumulated in the liver before vitellogenesis is hormonally induced, and subsequently, the vitello lipid is mobilized and can be found in the bloodstream during vitelloof pregnancy-a pregnancy-a genesis. Also, estradiol treatment during the course of nonvitellogenic period in the blenny-leads blenny-Ieads to a a dose-dependent ac accumulation in vitellogenin and a concomitant increase of lipids in the 979). blood (Korsgaard (Korsgaard and Petersen, 11979). In conclusion, two different strategies can be envisaged with re respect to lipid mobilization and estradiol action in different species of fish. The simpler situation exists in fishes that accumulate lipids fish. within the liver, such as the cod or the blenny. Here, estradiol is likely into first cause a mobilization of intrahepatic lipid stores and later in crease the output of VLDL from the liver. In fishes that use extrahe extrahe(Lepto patic sites for lipid deposition, such as salmonids or a sculpin (Leptococcus armatus; armatus; de Vlaming et aI., al., 1984), 1984), estradiol first induces the mobilization of extrahepatic extrahepatic lipids, and perhaps subsequently paces their uptake into the liver leading to increased hepatic output of VLDLs. The treatment of goldfish with salmon gonadotropin leads to an augmentation of plasma triglycerides and cholesterol (Wiegand (Wiegand and 1980)in goldfish with undeveloped ovaries, a phenomenon that Peter, 1980) is most likely mediated by gonadotropin-dependent gonadotropin-dependent estradiol producis

374


tion by the ovary. ovary. In animals undergoing the final stages of of ovarian development, the same treatment decreases plasma lipid concentra concentration, which is possibly due to a gonadotropin-enhanced (progester (progesterone-dependent?) lipid uptake into the ovary. one-dependent?) ovary. Varied results are reported for the changes changes in intracellular glyco glycogen content following estradiol treatment, although the generally ob observed trend seems to support the notion that hepatic glycogen is is decreased in vitellogenic females. However, variable results for the contents of hepatic glycogen can be expected, expected, since of of all storage materials they are the most likely to be dependent on the preexperi preexperimental state of the experimental organism with respect to variables such as diet, photoperiod, and temperature. Vitellogenic females of the killifish F. F . heteroclitus or estrogen estrogeninjected males contained less glycogen in their livers than uninjected uninjected male fish (Selman and Wallace, 1983a). 1983a).A similar picture can be found in many other teleost fishes [H. [ H . fossilis, Dasmahapatra and Medda ((1982); 1982); Z. viviparus, 1979); S. uiuiparus, Korsgaard and Petersen ((1979); S . gairdneri, Haux and Norberg ((1985); 1985); Anguilla anguilla, Olivereau and Olivereau ((1979)], 1979)], where estradiol-primed vitellogenic fish generally contain less glycogen in their livers than do vehicle-injected controls. In the grouper, in contrast, induction of exogenous vitellogenesis leads to a marked increase in hepatic glycogen (Ng (Ng et at., al., 1984). 1984). Sockeye salmon (0 (0nerka) build up maximum liver glycogen levels at the end of the spawning migration, when exogenous vitellogenesis is approaching completion, and the fish subsequently call upon liver glycogen to fuel the exhausting spawning process (French (French et at., al., 1983). 1983). An integral part of of estradiol action is the observed hypercalcemia in vitellogenic fish, fish, which can largely be ascribed to the calcium calciumbinding properties of phosphorylated, and hence highly charged, components of the native vitellogenin molecule. Furthermore, this hypercalcemia has been employed to confirm the vitellogenic state of of experimental animals. animals. Fish scales have been singled out as the sug suggested source of the bound calcium (Mugiya and Watabe, 1977), 1977), while, for once, estradiol does not seem to be implicated in the uptake of environmental calcium, neither through the gills nor through the intestine (Mugiya (Mugiya and IchU, Ichii, 1981). 1981). As in the case of carotenoid bind binding, the actual site for the attachment of calcium to the vitellogenin molecule has not been identified, although liver seems the most likely If in the future it can be confirmed that other metals, such candidate. If as copper or cadmium, travel from their hepatic deposition site to the ovary bound to vitellogenin (Shackley et al., at., 1981), 1981), it will be appreci appreciated how easily heavy metals will be able to impair the fi ne-tuned ion fine-tuned balance of the growing oocyte. oocyte.

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It is interesting to note that that cortisol, a steroid hormone, which is known to exert direct metabolic effects by way of of enzyme induction and permissive effects on peptide hormones such as glucagon, also possesses a pronounced enhancing influence on the estrogen-induced H.. fossilis (Sundararaj (Sundararaj et al., synthesis of vitellogenin in the catfish H 1982a). This situation is somewhat reminiscent reminiscent of of the estrone-depen estrone-depen1982a). of vitellogenesis through estradiol. Glucocorticoid ad addent priming of ministration to cultured hepatocytes curtails the vitellogenic response to estradiol, while at the same time enhancing the production of of al albumin, a protein whose synthesis may be suppressed in the presence sugof estrogen (cf. (cf. Table VI). of VI). Furthermore, recent experiments also sug gest an important role for thyroxine, which is tightly bound by isolated fish liver nuclei (Bres and Eales, 1986), 1986), as an accelerating factor in exogenous vitellogenesis in the guppy (Poecilia reticulata; reticulata; Lam and Loy, Loy, 1985). 1985). Evidently, a number of other hormones interact in an as yet undetermined manner with estradiol during exogenous vitello vitellogenesis (cf., (cf., Leatherland, 1985); 1985); these interactions should provide a endocrinologists and mo momultitude of challenging topics of study for endocrinologists biologists. lecular biologists. H. H. Male Male Fish Fish It vitellogenesis that that the the It is is an an interesting interesting facet facet of of the the induction induction of vitellogenesis estrogenic elicited in in males males of of oviparous oviparous verte verteestrogenic response response can can also also be elicited brates 1979; Korsgaard Korsgaard et al., 1983; 1983; brates,, including including fish fish (Emmersen (Emmersen et al., 1979; Maitre Maitre et al., al., 1985a). 1985a). It It clearly clearly indicates indicates that that the the administration administration of of estradiol estradiol can can activate activate normally normally silent silent genes. genes. The The complete complete absence absence of of products products of of these these unexpressed unexpressed genes genes has has made made male male animals animals aa prime prime model model for for the the analysis analysis of of gene gene regulation regulation and and activation. activation. Basically, Basically, the the male “reprogrammed” to to synthesize synthesize and and export export large large male liver liver can can be "reprogrammed" amounts amounts of of vitellogenin vitellogenin and and other other proteins, proteins, aa process process that that appears appears to to occur occur without without involving involving DNA DNA replication. replication. Since Since an an appropriate appropriate depo deposition vitellogenin in in the sition site site is is lacking lacking in in the the male, male, the the fate fate of of the vitellogenin bloodstream bloodstream differs differs:: it it builds builds up up to to rather rather high high concentrations concentrations and and eventually eventually is is taken taken up up by by the the liver liver and and degraded degraded along along with with other other blood blood proteins. proteins. The The actual actual process process of of vitellogenesis vitellogenesis is is accompanied accompanied by by identical identical patterns patterns of of hepatocyte hepatocyte differentiation differentiation in in both both sexes, sexes, including including the the pro proliferation RNA liferation in in Golgi Golgi vesicles, vesicles, rough rough endoplasmatic endoplasmatic reticulum, reticulum, and and RNA mentioned mentioned (cf. (cf. Table Table II). 11).In In the the male male Atlantic Atlantic salmon salmon (S. ( S . salar), salar), the the estrogenic estrogenic response response also also includes includes an an increase increase in in the the amount amount of of assaya assayable ble nuclear nuclear estrogen estrogen receptor receptor to to levels levels characteristic characteristic of of induced induced fefe-

376


male fish, fish, which is probably due to de novo synthesis of of the receptor al., 1985). 1985). The identical situation in hepatocytes protein (Lazier et al., from male Xenopus has made it possible to unequivocally identify tranreceptor synthesis as the rate-limiting step in vitellogenin gene tran scription (Perlman (Perlman et ai., al., 1984). 1984). In male fish, fish, vitellogenin synthesis synthesis cannot be stimulated by the administration of pituitary extracts, indicating two specifi c properties specific of the vitellogenic response in fishes: ( 1 ) with regard to exogenous fishes: (1) exogenous horvitellogenesis, the liver is not a direct target organ for pituitary hor mones, and (2) (2) in males, vitellogenesis is specifically dependent on estrogen administration, because of the inability of the gonad to pro produce estrogen (Idler and Campbell, Campbell, 1980). 1980). 1. I. Elasmobranch Fishes

In general, vitellogenesis and its hormonal control in the elasmo elasmobranch fishes have received much less attention than in teleost fishes. The few studies on elasmobranchs suggest that, even in species from temperate zones, vitellogenesis and oviposition appear to occur throughout the year, with a maximum during winter (Sumpter and Dodd, 1979). 1979). As a consequence, vitellogenin is detectable in dogfish 1978b) and skate (Raja (Raja erinacea; erinacea; T. T. P. (Scyliorhinus canicula; Craik, 1978b) Mommsen, unpublished) blood throughout the year, albeit in a low concentration compared with vitellogenic teleosts. The biochemical properties of the elasmobranch vitellogenins and their relationship to vitellogenins from other vertebrates remain to be analyzed. analyzed. Injection of estradiol results in a much smaller vitellogenic re re1978a). While the synthesis of vitello sponse than in teleosts (Craik, (Craik, 1978a). vitellogenin in the female dogfi sh is a slow process compared with teleosts, dogfish its uptake into the ovary is fine-tuned to the rate of its synthesis. synthesis. This results in an unusually long half-life for vitellogenin (9 (9 days; days; Craik, sh plasma, and a similar result can be expected for 197813) in dogfi dogfish 1978b) other elasmobranchs that are vitellogenic throughout the year. The systems where such such long half-lives half-lives for vitellogenin repre repreonly other systems sent the rule rather than the exception are the estrogen-primed males of other vertebrates that possess no tissue that would recognize vitel vitellogenin for uptake. In male, estrogen-injected Xenopus, for instance, vitellogenin is removed from the bloodstream at a rate of less than 11% % per day-which day-which resembles plasma protein turnover-compared turnover-compared to more than 12% 12% per day in the vitello genic female (Wallace vitellogenic (Wallace and Jared, 1968). 1968).

5. 5.


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111. III. OOCYTE ASSEMBLY ASSEMBLY A. Transport of Vitellogenin maAfter the Golgi vesicles of the hepatocyte have unloaded the ma ture vitellogenin into the plasma, the circulatory system delivers it to the ovary. ovary. It appears that vitellogenin is dissolved freely in the plasma, plasma, since since no no special special carrier carrier molecule molecule for for it it could could be be identified identified in in teleosts different situation situation is is found found in in the the blood blood of of teleosts or or amphibians. amphibians. A different birds. birds. In In these these vertebrates, vertebrates, vitellogenin vitellogenin is is carried carried from from its its hepatic hepatic site site of of synthesis to the gonad as part of of the high-density lipoproteins (HDL), (HDL), which are regularly synthesized and excreted by the avian liver.

B. Uptake of Vitellogenin In the females of oviparous vertebrates, with the possible excep excepfishes, tion of elasmobranch fi s hes, circulating vitellogenin is rapidly and specifi cally cleared from specifically from the the bloodstream bloodstream by by the the growing growing oocyte. oocyte. In In vitellogenic Xenopus, some 12% 12% of the vitellogenin circulating in the 1968). In blood is taken up by the gonad per day (Wallace (Wallace and Jared, 1968). specific the absence of a specifi c target tissue in estrogen-primed males, the vitellogenin continues to exist in the circulatory system until it is fi nally removed by the liver and degraded along with other plasma finally proteins. The mechanism of vitellogenin recognition and the selectivity of of its uptake into the oocyte remain open questions, especially for the fishes. fi shes. Here only a single study on the rainbow trout has critically 1979), with looked into into these mechanisms mechanisms (Campbell (Campbell and and Jalabert, Jalabert, 1979), with conclusions that do not support the picture that has emerged from a multitude of studies on Xenopus and the chicken. animals, it appears that vitellogenin In the latter two experimental animals, is bound on the oocyte membrane by specifi c, high-molecular-weight specific, (molecular weight -500,000), receptors (molecular -500,000), which are taken up into the oocyte independent of vitellogenin binding. binding. The oocyte and and turn turn over over independent of vitellogenin The recep recepnonspecificc binding, are saturable, and appar appartor proteins display low nonspecifi ently specifi cally recognize vitelently specifically recognize and and bind bind the the phosvitin phosvitin region region of of the vitel logenin molecule; logenin molecule; again, again, phosphorylation phosphorylation is is crucial crucial to to the the process process of of receptor 1; 1981; receptor recognition recognition and and vitellogenin vitellogenin uptake uptake (Opresko (Opresko et al., 198 Yusko eett al., 1981). 1981). Other studies, in addition, have implicated the

378


importance importance of of N-glycosylation N-glycosylation of of the the vitellogenin vitellogenin molecule molecule on on its its up uptake Similar receptors take by by the the oocyte oocyte (Lane (Lane et al., aZ., 1983). 1983). Similar receptors are are presumed presumed to to exist exist for for VLDL in in avian avian oocytes, oocytes, and and it it has has been been pointed pointed out out else elsewhere where that that the the vitamin-binding vitamin-binding proteins proteins are are only only recognized recognized and and taken taken up up into into the the oocyte oocyte if if adequately adequately phosphorylated phosphorylated (Miller (Miller et al., al., 1982). 1982).After After its its binding binding to to the the oocyte oocyte surface surface receptor, receptor, the the vitellogenin vitellogenin molecule, possibly in conjunction with its receptor, is is taken up into the oocyte by micropinocytosis. In the fishes, vitellogenin contains only about about half the the protein protein phosphorus phosphorus of of other other vertebrates vertebrates,, and and the phosvitins comprise a more heterogeneous group altogether. There Therefore, fore, a critical analysis of the involvement of of phosphate groups in these lower vertebrates may lead to interesting insights into receptor recognition and receptor mechanism in general. In subsequent steps, the vitellogenin is is directed toward different yolk sites sites within the oocyte, depending on the stage during vitello vitellogenesis. While the receptor molecule appears to be recycled, the vi vitellogenin molecule is cleaved proteolytically into the main yolk com components in the course of its translocation from the oocyte surface to the yolk deposition sites. From an enzymatic point of of view, the system responsible for the cleavage of of the vitellogenin molecule is poorly characterized, but the lysosomal system seems to be implicated (see (see below). below). Finally, the components such as the lipovitellins, phosvitins, and phosvettes are deposited within membrane-bound spherical yolk bodies, in many marine teleosts constituting fluid yolk globules rather than the well-known insoluble platelets. Such yolk bodies form the so-called "extravesicular “extravesicular yolk," yolk,” which may fuse at some point during oocyte development (Wallace Selman, 1981). (Wallace and Selman, 1981). The "intravesicular “intravesicular yolks" yolks” that have been described for growing teleost oocytes are supposedly precursors of the cortical alveoli, which shed their endogenously synthesized "yolk" “yolk” at fertilization (te 1977; Wallace and Selman, Heesen, 1977; Selman, 1981). 1981). The discussion in this chapter will be restricted to the egg components derived from exoge exogeautonous vitellogenesis and will therefore not be concerned with the auto synthetic intravesicular yolk as defined above. Considering the rapidity and specificity of of vitellogenin deposition in teleost oocytes in the course of of exogenous vitellogenesis, Campbell and Jalabert (1979) (1979) obtained surprising results results:: developing trout 00oocytes in vitro did not take up vitellogenin selectively over serum al albumin and at a rate that amounted to less than 10% 10% of that observed in (Campbell and Xenopus under comparable experimental conditions (Campbell 1979). Obviously, more research is needed before any gen genJalabert, 1979). eral statements about diversity or conservation in the mechanism of

5. 5.


37 379 9

of the vertebrate line can be vitellogenin uptake in the evolution of made. Interestingly, Xenopus oocytes oocytes are selective for vitellogenin vitelloover albumin or ferritin, while the vertebrate source of the vitello genin-which teleost-possessed little influence on the genin-which included a teleost-possessed rate of uptake (Wallace et aZ., 1980). Similarly, al., 1980). Similarly, microinjected vitello vitellogenin mRNAs from different species gave rise to mature vitellogenins and led to subsequent export from the Xenopus oocyte. Amphibian vitellogenin was later taken back up and deposited in the yolk plate platelets. Locust vitellogenin, on the other hand, was synthesized and exex (Lane et aZ., ported but not sequestered from the medium (Lane al., 1983). 1983). With respect to the hormonal regulation of vitellogenin uptake by the oocyte, aa rather scant body of information is available, apart from the fact that estrogen does not seem to be involved. involved. Instead, uptake may be dependent on the presence of progesterone, with its exact mode of action on the surface of of the oocyte and not on the transcrip transcriptional level still being under debate. This steroid may possess some general maturation function or act specifically to induce micropinocy micropinocytosis in vitellogenin (Tata (Tata and Smith, Smith, 1979). 1979). Studies on Xenopus have indicated that once the oocytes have entered into the vitellogenic state, the rate of vitellogenin sequestering is regulated by by the follicle cells and not by the oocyte itself (Wallace, (Wallace, 1983). 1983). There is an ongoing debate on the number of gonadotropins present in fishes, but inde independent of the outcome of this perceived perceived controversy, two statements can be made with respect to exogenous vitellogenesis. One pituitary hormone, rich in carbohydrate, leads estrogen production in the fe female gonad and thus initiates the events outlined in Fig. 11.. Another pituitary hormone, which is characteristically low in carbohydrate content, specifically enhances the uptake of of vitellogenin from the bloodstream into the growing oocyte while at the same time being devoid of vitellogenic action per se (Burzawa-Gerard, (Burzawa-Gerard, 1982). 1982). Only if the oocyte has taken up the vitellogenin by micropinocyto micropinocytosis will the molecule be processed correctly, cleaved at predeter predeteryolk. On the mined sites, and directed toward specific sites in the yolk. other hand, if if microinjected into the oocyte, the vitellogenin molecule is is rapidly degraded in its entirety and degradation products never reach the yolk platelet (in Xenopus; Wallace and Hollinger, 1979). 1979). These findings reconcile the observations made when messenger RNA for vitellogenin vitellogenin is microinjected into growing oocytes during translational or modification studies. In this case, after the mRNA has been translated and the molecule has undergone the required post posttranslational translational modifications, the mature vitellogenin is excreted from the oocyte and subsequently sequestered from the medium by micro-

380 380


pinocytosis and only then directed toward the yolk, where it is is stored as phosvitin and lipovitellin (Lane ai., 1983). (Lane et al., 1983). Campbell and Idler ((1976) 1976) found that some degree of dephosphorylation of cine vitello of pis piscine vitellogenin may occur during incorporation into ovarian yolk. yolk. At the later stage of meiotic maturation, many fi sh eggs take up fish up substantial amounts of water, and this hydration may be accompanied by a marked drop in protein phosphorus assayable in the oocyte (Craik, (Craik, 1982). 1982). During the previtellogenic part of of oocyte development in the trout, microvesicular bodies (MVB) (MVB) accumulate and later occupy the larger part of the cell. These bodies contain acid hydrolase activity and can be classified as a lysosomal-like compartment. compartment. In the course of of exogenous vitellogenesis, large yolk vesicles form, form, which contain yolk as well as the remnants of of the microvesicular bodies. At the comple completion of vitellogenesis, the microvesicular bodies have disappeared (Busson-Mabillot, (Busson-Mabillot, 1984) 1984) and as a general observation, observation, acid phospha phosphatases are absent from fully developed oocytes (Korfsmeier, (Korfsmeier, 1980), 1980), while cathepsin and a-glucosidase activities are present in unfertil unfertilized eggs (Vernier Sire, 1977). (Vernier and Sire, 1977). Although lysosomal lysosomal activities oc occur associated with yolk platelets in most lower vertebrates, this rule is not without exception. exception. For example, example, the oocytes oocytes of two species of (herring and plaice) plaice) are alto altomarine fishes with polylecithal egg cells (herring gether devoid of acid hydrolases (Korfsmeier, (Korfsmeier, 1980). 1980). The exact role of the lysosomes lysosomes in the proteolytic cleavage of vitel vitellogenin that has been sequestered from the bloodstream by micro micropinocytosis remains an enigma to date. date. From the informatiCill information that has been gathered from other vertebrate systems, systems, it is is not obvious what type of enzymes are responsible for the breakdown of vitellogenin originate. The observation of efficient, nonspecifi nonspecificc and where they originate. breakdown and subsequent removal of microinjected vitellogenin in the Xenopus oocyte suggests that micropinocytosed vitellogenin is not available for full lysosomal lysosomal attack and may be only exposed to en enavailable zymes that will specifi specifically zymes cally cleave it into lipovitellins, phosvitins, and phosvettes.. Obviously, the vitellogenin molecule itself is not resistant phosvettes attack. On the other hand, it seems seems that to other types of proteolytic attack. the microvesicular bodies bodies transporting vitellogenin and its its products to yolk are somewhat related to the lysosomal lysosomal system, system, since they the yolk display some some enzyme enzyme activities activities with characteristic characteristic acidic max maxclearly display ima. The microvesicular bodies, bodies, however, however, do not display the full com comima. lysosomal enzymes, which would most likely lead to the plement of lysosomal its receptor. receptor. degradation of vitellogenin and its

5. 5.

VITELLOGENESIS AND OOCYTE ASSEMBLY VITELLOGENESIS

381 38 1

C. C. Phosvitin and Lipovitellin of the vitellogenin mole moleThe general picture of cleavage products of noncule in piscine systems is not quite as clear-cut as in the other non mammalian vertebrates. Although it has long been known that fish eggs contain lipovitellins and phosvitins with by and large similar properties to those from other vertebrates, a much more pronounced apparent. Such variability is interspecific variation on the theme is apparent. of different components as well as some somereflected in high numbers of times unusual chromatographic behavior. The most extreme example to date is found in the eggs of an Antarctic fish (Chaenocephalus (Chaenocephalus aceratus) that possesses no less than nine different phosphorylated proteins 1984). Fish Fish lipovitellins lipovitellins are are proteins (Shigeura (Shigeura and and Haschemeyer, Haschemeyer, 1984). much more those from much more heterogeneous heterogeneous than than those from other other vertebrate vertebrate eggs, eggs, are are smaller, smaller, contain contain higher concentrations concentrations of of lipids, lipids, and and possess possess little little or or no protein phosphorus. Numerous low-molecular-weight low-molecular-weight phosvitins have been found in teleost eggs, characterized by widely varying, albeit generally low, low, amounts of alkali-labile protein phosphorus (Mano and Yoshida, 1969; 1969; Markert and Vanstone, 197 1 ; Inoue et al., 1971; (Mano 1971; 1971; de de Vlaming Vlaming et al., 1980; 1980; Craik, Craik, 1982). 1982). In In the the killifish killifish Fundulus the native native vitellogenin vitellogenin molecule molecule (200 (200 kDa; cf. cf. Table Table III) 111) heteroclitus, the cannot efficannot be be localized localized within within the the oocyte, oocyte, indicative indicative of of aa rapid rapid and and effi cient degradation into smaller components (Wallace (Wallace and Selman, 1985) 122, lO3, 45, 26, and kDa) 1985).. In In fact, fact, five five major major protein protein bands bands ((122, 103,45,26, and 20 kDa) of which are allegedly derived are abundant in growing oocytes, all of from from proteolytic proteolytic breakdown breakdown of of vitellogenin. vitellogenin. During During final final maturation, maturation, this 122- and and 45-kDa 45-kDa proteins proteins are are this pattern pattern is further further changed changed in in that that the the 122degraded It has number oflower-molecular-weight of lower-molecular-weight proteins. proteins. It has degraded to to yield yield aa number been implied that new proteins been implied that these these new proteins are are involved involved in in the the hydration hydration process during final 1985), but but their their process during final maturation maturation (Wallace (Wallace and and Selman, Selman, 1985), physiological the physiological function(s) function(s) and and their their relationship relationship to to phosvitin phosvitin or the nature of the proteolytic machinery responsible await identification. identification. D. Oocyte Lipids In the course of exogenous vitellogenesis, teleost oocytes accumu accumulate amounts of lipids in lipids delivered late large large amounts of lipids in addition addition to to the the polar polar lipids delivered as as part of the vitellogenin molecule. In spawned eggs, which contain between 8 and and 32% 32% lipid lipid (based (based on on dry dry weight), weight), several several classes classes of of between lipids are represented, where the emphasis varies strongly between different species of different species of fish. fish. Depending Depending on on the the preferred preferred type type of of lipid lipid

382 382

THOMAS P. MOMMSEN AND PATRICK JJ.. WALSH WALSH

accumulated in the eggs, three strategies can be distinguished: distinguished: the first group, which includes rainbow trout, sole (SoZea (Solea vuZgaris), vulgaris), and a whitefish (Coregonus aZbula), atbula), is characterized by equally high levels of polar lipids and triglycerides (Kaitaranta 1 ; Devau (Kaitarantaand Ackman, Ackman, 198 1981; Devauchelle et aZ., al., 1982). 1982). Baltic herring, roach, and turbot (ScophthaZmus (Scophthalmus maximus) belong'to a second group, which accumulates mainly polar lipids (75-90%) 1 ; Devauchelle et aZ., (75-90%) (Kaitaranta (Kaitaranta and Ackman, 198 1981; al., 1982). 1982).A third group, encompassing a wide variety of species such as the gourami (Trichogaster (Trichogaster cosby; cosby; Sand et aZ" al., 1971), 1971), sea bass (Dicen (Dicen1982), striped bass (Morone trarchus Zabrax; al., 1982), (Morone sax saxlabrax; Devauchelle et aZ., atilis; Eldridge et aZ., jluviatilis), burbot (Lota al., 1983), 1983), perch (Perea (Perca fluviatilis), (Lota Zota; 1), and many others, accumulates lota; Kaitaranta and Ackman, 198 1981), large amounts (>80%) (>80%) of wax and steryl esters in the so-called egg oil globules. globules. In fact, all fish eggs harboring oil globules, which are dis distinct from the yolk or yolk platelets, have been shown to contain substantial amounts of wax and sterol esters (Kaitaranta (Kaitaranta and Ackman, 1981). consists almost 1981). In species such as M. M . saxatilis, the oil globule consists entirely of steryl esters and wax esters (90%) (90%)as well as some some triglycer triglycerides ((lo%), 10%), whereas the small the yolk lipids is small bulk of ofthe is dominated by phospholipids (79%, (79%, Eldridge et aZ., al., 1983). 1983).With wax esters generally belonging in the domain of the marine environment, environment, the above list shows that the occurrence of these compounds in fish eggs is by no means restricted to marine species. species. The physiological advantages of of accumulating large amounts of wax esters in eggs eggs (71% (71% of the total caloric value of the egg in M. M . saxatiZis; saxatilis; Eldridge et aZ., al., 1983) 1983)have not been analyzed yet, although it can be hypothesized that in addition to serving as an energy supply, supply, they will play an important role in buoy buoyancy control for the embryo and developing developing larva. exists in our knowledge with respect to Unfortunately, a large gap exists the maternal source of these wax and steryl esters. It appears that the glulipid components of vitellogenins from species accumulating oil glu bules in their eggs have not been given any attention to date. Vitello VitellobuIes fishes is known to transport some 20% (by (by weight) weight) of genin from other fishes some 20% (Wiegand, 1982; 1982; lipid, the bulk of which consists of phospholipids (Wiegand, Haux, 1985). 1985). If this is is verified verified for vitellogenins of fishes fishes Norberg and Haux, eggs, vitellogenin can be ruled ruled out that synthesize oil globules in their eggs, as the transport form form for their unique unique lipid complement. complement. Alternatively, as wax esters esters may be synthesized endogenously in the the oocyte from the wax acids delivered as part of lipoproteins or bound to serum al alfatty acids T . cosby, cosby, where wax esters esters constitute the bumins. In the gourami T. lipids of the egg, egg, the ovarian fatty acyl acyl alcohols alcohols can be synthemajor lipids

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sized de de novo novo from from dietary dietary acetate acetate or or longer longer dietary dietary carbon carbon chains, chains, but but 1971). the site of biosynthesis could not be identified (Sand et al., 1971). Since Wiegand and Idler ((1982) 1982) reported that the ovary of the rainbow trout possesses the metabolic machinery to reduce exogenously ad administered fatty acids to the corresponding alcohols, the endogenous excluded. Another interesting facet synthesis of wax esters cannot be excluded. of fish fish accumulating wax esters in their eggs is the fact that none of the adults of these species use wax esters as their lipid storage forms, forms, but generally rely on triglycerides instead. Comparable Comparable to to the the situation situation in in avian avian systems, systems, some some experimental experimental evidence evidence for for rainbow trout trout suggests suggests that that circulating circulating lipoproteins lipoproteins may may enter and serve the major major source source of of polyunsaturated polyunsaturated free free enter the the ovary ovary and serve as the fatty fatty acids, acids, the the bulk bulk of of which which is is transported transported in in lipoproteins lipoproteins and and not not on on the (Fremont et al., al., 1984). 1984). The experimentally experimentally the vitellogenin vitellogenin molecule molecule (Fremont induced fishes is induced or or naturally naturally occurring occurring vitellogenesis vitellogenesis in in fishes is accompanied accompanied by large increases in liver biosynthesis and export of of VLDL (cf. (cf. Ta Tables II and and VI). bles I1 VI). Just Just as as in in the the hepatic hepatic synthesis synthesis of of vitellogenin, vitellogenin, the the synthesis initiated in synthesis of of lipoproteins lipoproteins may may be be initiated in vivo vivo through through the the influ influence ence of of circulating circulating estradiol. estradiol. In In the the annual annual cycle cycle of of fishes, fishes, increases increases in in blood blood VLDL VLDL are are positively positively correlated correlated with with vitellogenesis. vitellogenesis. Comparative data on avian vitellogenesis and VLDL metabolism indicate that the ovary is capable of of the uptake of of lipoproteins directly from independent of of the the from the the bloodstream bloodstream and and that that this this process may may be independent sequestration vitellogenin through through micropinocytosis. micropinocytosis. While this sequestration of vitellogenin lipoproteins, it it has has general scheme scheme may not not be be applicable applicable to to all serum lipoproteins, been proven to hold for VLDL in the hen (Holdsworth at., 1974), 1974), (Holdsworth et al., where the basal lamina appears to be permeable to circulating VLDL al., 1979). 1979). (Evans et at., (Evans While Wiegand and Idler ((1982) 1 982) determined some capacity for endogenous in the endogenous triglyceride triglyceride biosynthesis biosynthesis from from acetate acetate in in the the ovary ovary in the rainbow trout, Leger and 1981) on of Leger and co-workers co-workers ((1981) on the the same same rainbow trout, the the results results of species suggest serum lipoproteins such as VLDL or LDL as the more likely sources for the triglycerides accumulated in the egg. Lipid of phospholipids, phospholipids, is is first first Lipid material, material, composed composed to to aa large large extent extent of accumulated accumulated in in the the perinuclear perinuclear cytoplasm cytoplasm of of the the oocyte. oocyte. However, However, these bodies, the these early early lipid lipid bodies, the source source of of which which still still awaits awaits identification, identification, do yolk since known to do not not constitute constitute true true yolk since they they are are known to disappear disappear before before or or during during exogenous exogenous vitellogenesis. vitellogenesis. The The study study of of Wiegand Wiegand and and Idler Idler ((1982), 1 982), which which showed showed for for an an in in vitro vitro system system that that labeled labeled acetate acetate was was incorporated incorporated into into ovarian ovarian polar polar lipids, lipids, remained remained inconclusive inconclusive with with regard cell fraction lipid was regard to to the the cell fraction with with which which polar polar lipid was associated. associated.

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THOMAS THOMAS P. MOMMSEN AND PATRICK PATRICK JJ.. WALSH WALSH

E. Carotenoids Carotenoids Fish Fish eggs eggs are are known known to to contain contain other other secondary secondary products products such such as as carotenoids, carotenoids, which which sometimes sometimes contribute contribute to to the the colorful colorful appearance appearance of of the (0.keta), keta),almost almost 1% 1%of of the the fresh fresh weight weight the eggs. eggs. In In the the chum chum salmon salmon (0. of of the the spawned spawned egg egg consists of of carotenoids, carotenoids, mainly mainly astaxanthin astaxanthin (Kita (Kitahara, 1984). In this many other the egg egg carot 1984). In this species, species, as as in in many other salmonids, salmonids, the carothara, enoids enoids are are presumably presumably derived derived from from previous previous depositions depositions in in the the mus muscle 1970). In In view view of of the the absence absence of of aa specific specific transporting transporting cle (Crozier, (Crozier, 1970). vehicle, carotenoids are hypothesized that that the the carotenoids are passively passively vehicle, it it can can be hypothesized transported transported out out of of the the tissue tissue together together with with storage storage lipids lipids according according to to their In the the course the their lipid lipid solubility. solubility. In course of of the the spawning spawning migration, migration, the lipid lipid deposits deposits within within the salmon's salmon’s body are are mobilized mobilized in their their en entirety, tirety, partly partly for for energy energy production production during during migration migration and and in in the the female female also Depending on also as as part part of of the the estrogenic estrogenic response. response. Depending on the the composition composition of the individual lipids that the carotenoids are associated with, differ differing envisaged for for their their transport transport into into the the gonad gonad of of the the ing routes can can be envisaged vitellogenic female: female: the carotenoids may form part of the vitellogenin molecule itself or, alternatively, they may travel with the abundant lipoproteins, Rolipoproteins, especially especially VLDL, in in salmon salmon blood (cf., (cf., Skinner and and Ro gie, Sire and puri gie, 1978; 1978; Sire and Vernier, Vernier, 1983). 1983). The light light pink pink hue hue of of highly highly purified sockeye salmon salmon vitellogenin suggests that least some some of fied sockeye vitellogenin suggests that at at least of the the carotenoids travel to the ovary bound to the lipid component of vitel vitellogenin (T. (T. P. Mommsen and C. C. J. French, unpublished). In fact, it has been reported that a crustacean lipovitellin moiety possesses a cova covaaZ., 1983). 1983). Obviously, Obviously, lent binding binding site for for carotenoids carotenoids (Zagalsky (Zagalsky et ai., more cations of more research research on on posttranslational posttranslational modifi modifications of the the vitellogenin vitellogenin polypeptide polypeptide and and on on possible possible association association of of carotenoids carotenoids with with other other li lipophilic components of the fish blood is required before even a pre preliminary picture will emerge. emerge. Another Another interesting facet of the carotenoid deposition deposition in in the the oocyte oocyte is is the the fact that not all of the carotenoids are are localized localized in in the the yolk, yolk, but but some 20% 20%is associated with other structures in in the the oocyte oocyte (Kitahara, (Kitahara, some 1984) 1984) leaving the question of the physiological function function of of such such aa heterologous heterologous group as carotenoids in embryo embryo nutrition nutrition and and survival survival wide wide open. open. F. F. Glycoproteins In addition to their ubiquitous glycogen stores, fish eggs are protein-bound carbohydrate moieties, but their ex exknown to contain protein-bound act localization and their biochemical nature have only been given

5. 5. VITELLOGENESIS AND OOCYTE OOCYTE ASSEMBLY

385 385

attention. Even more surprisingly, surprisingly, the physiological rather cursory attention. function of these carbohydrate-containing proteins, which have lately been been charaterized as sialoglycoproteins, is is entirely unknown, despite abunthe fact that these compounds may surpass the egg phosvitins in abun (Inoue and Iwasaki, 1980a,b). 1980a,b). dance by almost an order of magnitude (Inoue fish Sialoglycoproteins are apparently rather common constituents of fi sh eggs, eggs, since they have been described and partially characterized for a (Ctupea pallasii), pallasii), Alaskan number of species, namely Pacific Pacific herring (Clupea poIlack (Theragra (Theragru chalcogramma), chulcogramma), Japanese common charr (Salve (Sahepollack (Salmo gairdneri), gairdneri), and three spe speleucophaenus), rainbow trout (Salmo linus leucophaenus), 0. masou, and O. 0 . nerka) nerka) cies of Pacific salmon (Oncorhynchus keta, O. (Inoue and Iwasaki 1978, 1978, 1980a,b; 1980a,b; Iwasaki and Inoue, 1985). 1985). (Inoue sialoglycoproteins are associated with the soluble fraction of The sialoglycoproteins the egg, namely, the cortical vesicles, and thus do not form part of the purified demembrane superstructure of the egg. egg. The purifi ed molecules are de void of phosphorus and derive their acidity from the abundant sialic, acids. In fact, fact, these three strongly acidic com comglutamic, and aspartic acids. 20% (by weight) weight) of the sialoglycoproteins ponents make up more than 20% (Inoue and Iwasaki, 1980a). 1980a). In this species, the in the herring egg (Inoue molecular weights of the three main sialoglycoproteins range from 40-50% of 8800 to 13,000. 13,000. Their protein backbone, comprising some 40-50% the molecules, is unique in its amino acid composition and displays contrast, is very little variability. The carbohydrate composition, in contrast, variable, especially in the content of N-acetylglucosamine, constitut constituting 6.2% of the total weight of the sialoglycopro sialoglycoproing between 12.5 12.5 and 6.2% N-acetylteins. Other abundant carbohydrates, in addition to N-acetyl neuraminic acid (sialic (sialic acid) acid) and N-acetylglucosamine, are neurammIC N-acetylgalactosamine, fucose, galactose, and mannose. co-workers (Iwasaki (Iwasaki and Inoue, Inoue, More recent work by Inoue and co-workers 1985) on polysialoglycoproteins isolated from un un1985; Inoue et al., 1985) 1985; summafertilized egg from different species species of salmonid fishes fishes can be summa rized as follows: follows: l1.. The The glycoproteins glycoproteins are are characterized characterized by by

high high molecular molecular kDa. 150 to 300 kDa. weights, ranging from 150 2. 2. More More than than 50% 50% of of their their weight weight is is comprised comprised of sialic sialic acid, acid, and and total carbohydrate content may reach 85%. 85%. 3. 3. They They contain contain poly(oligo)sialyl poly(o1igo)sialyl groups groups linked linked to to O-glycosidic O-glycosidic carbohydrate carbohydrate units. units. 4. The polypeptide polypeptide backbone backbone is is made made up up of of seven seven acidic acidic or or neu neu4. tral tral amino amino acids acids only, only, namely, namely, alanine, alanine, aspartatic aspartatic acid, acid, glu glutamic acid, glycine, proline, threonine, and serine. serine.

386


5. 5. Amino Amino acids acids are are arranged arranged in in two kinds kinds of of repeated repeated polypeptide polypeptide 13 amino sequences ((13 sequences amino acid acid residues). residues). 6. 6. All of of the the threonine threonine and and half half of of the the serine serine residues residues are are glycosy glycosylated. lated.

It It is interesting to to note note that that the the accumulated accumulated polysialoglycopro polysialoglycoproteins teins in in the the rainbow-trout rainbow-trout egg egg undergo undergo aa drastic drastic reduction reduction in in size upon upon fertilization, fertilization, at at which which time time they they decrease decrease from from 260 to to 9 kDa kDa (Inoue (Inoue et ai., 1985). 1985). A similar al., similar reduction reduction in in size size can can be be expected expected for for the the large large glycoproteins prevalent in is glycoproteins prevalent in other other salmonid salmonid fishes, fishes, and and the the situation situation is somewhat reminiscent of the further breakup of vitellogenin break breakdown products into even smaller units during final oocyte maturation in in F. heteroclitus (Wallace (Wallace and and Selman, Selman, 1985). 1985). In both both cases, cases, the the dras drastic decreases in size are due to highly specific proteolytic attack, and the carbohydrate moieties on the polysialoglycoproteins remain unal unaltered. In the case of of the glycoproteins, the drastic reduction in size occurs occurs simultaneously simultaneously with with cortical cortical vesicle vesicle breakdown breakdown and and exocytosis exocytosis (Inoue (Inoue et ai., al., 1985). 1985). Unfortunately, Unfortunately, despite despite the the fact fact that that the the sialoglycoproteins sialoglycoproteins com compounds are are prevalent prevalent in in fish eggs eggs and and that that the the timing of of their their break breakdown implies some involvement upon fertilization (block to poly polyspermy?), spermy?), no no data data exist exist on on such important important aspects aspects as as their their physiological physiological function, function, their their source, transport transport form form or or mechanism, mechanism, and and timing of uptake into the developing developing oocyte. oocyte. If If these multitudes of carbohydrates carbohydrates are synthesized in the maternal liver as part of of the posttranslational cation of posttranslational modifi modification of the the vitellogenin vitellogenin molecule, molecule, the the codes codes for for the the small, small, but but unique, unique, polypeptide polypeptide chains chains should be be identifiable identifiable with with relative relative ease ease within within the the recently recently purified purified vitellogenin vitellogenin messenger messenger RNA (Chen, (Chen, 1983; 1983; Valotaire Valotaire et ai., al., 1984). 1984). Biochemical and histochemical studies have identified lectins as an integral part of the soluble fraction of fish oocytes (Nosek et al., 1983). 1983). However, However, just just as as in in the the case case of of the the sialoglycoproteins sialoglycoproteins found found in in mature eggs, sources or physiological function is a matter of of specula speculation (Nosek (Nosek et ai., al., 1983). 1983). G. Vitamin-Binding Proteins As mentioned before for the chicken, estradiol induces the hepatic synthesis of a number of vitamin-binding proteins destined for uptake into the growing oocyte (Table (Table IV). IV). One of these vitamin-binding proteins is the well-characterized riboflavin-binding protein, which is glycosylated as well as phosphorylated and is responsible for the

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carbohytransport of riboflavin to the oocyte. While in many instances carbohy drate side chains are important in the recognition of glycoproteins by their target cells, it was recently demonstrated that, in the case of the glycosylariboflavin-binding protein, correct phosphorylation and not glycosyla tion is crucial for the uptake of the molecule into the oocyte, as was shown for the uptake of experimentally administered phosvitin (Miller et al., al., 1982). 1982). For the fishes, however, information on occur occur(Miller rence of such vitamin-binding proteins is limited to the observation (0. that the specific riboflavin-binding protein is absent from salmon (0. nerka) oocytes. Riboflavin, Riboflavin, on the other hand, occurs in salmon 00oonerka) (H. B. cytes in similar concentrations as in the chicken egg (H. B. White and M. of its source and its M. A. Letavic, unpublished), leaving the question of possible transport open to speculation. Vitamin-binding proteins can be expected to play an integral role in the survival of embryos or larvae, supplying them with vitamins at poscritical stages of their development. Such proteins may further pos unsess antimicrobial action by rendering vitamins stored in the egg un available to infesting bacteria. H. H. Hormones While it has been known for some time that fish larvae respond to exogenously administered hormones hormones,, the physiological relevance of such observations remained unclear, especially at a time when the intraembryonic existence and availability of such hormones had not been established. In the context of the hormonal status of fish oocytes, an avian concept may deserve attention by researchers interested in embryonic fish metabolism and morphogenesis. In addition to known nutrients and secondary compounds, the chicken egg contains signifi significant amounts of thyroxin, and the embryonic chick liver already dis displays highly specific specific steriod receptor activities activities for hormones such as 17,8-estradiol ) )(Bella 17p-estradiol (Lazier, (Lazier, 1978) 1978) and 3,5,3'-triiodothyronine 3,5,3’-triiodothyronine (T (T33 (Bellabarba and Lehoux, 11981). 981). Recent analyses by 1987) and Kobuke 1987) by Brown et al. al. ((1987) Kobuke et al. al. ((1987) unequivocally unequivocally established the presence of substantial amounts of thy thyroxin and T T33 in unfertilized ova and embryos embryos of salmonids salmonids (Oncorhy (Oncorhynchus sp.) s p . ) and striped bass bass (M. ( M . saxatilis). saxatilis). These hormones hormones,, which are localized preferentially in the embryonic yolk, yolk, are apparently of ma maternal origin. The The suggested route of transfer from from the maternal circu circulatory latory system into the growing oocyte is is through vitellogenin, since since this compound compound displays appreciable binding capacity for thyroid horhor-

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(0. ki mones in in the the plasma collected collected from from vitellogenic vitellogenic coho coho salmon (0. kiC. V. Sullivan, unpublished sutch; A. A. Hara, W. W. Dickhoff, and C. results). results). The absolute amount of hormone transferred into the ova, (in the range of 5 ng/oocyte, Brown et al., 1987; however, is small (in 1987; Kobuke Kobuke et al., 1987), 1987),and it is therefore unlikely that such transfer will be reflected in concentration changes of hormone in the maternal circulatory system. While the presence of a hormone does not necessarily imply its physical availability or functionality, 1987) functionality, the data of Brown et al. ((1987) and Kobuke et al. (1987) (1987) already indicate that yolk thyroxin and T T3 3 undergo turnover during early development. development. Thus both hormones can be assumed to be available to the embryo and to infl uence physiologi influence physiological functions. Considering how many other lipophilic compounds from the maternal system reach the growing oocyte, the deposition of of thyroxin is not surprising, and the same principle is likely to be appli applicable to other steriod hormones. hormones. However, as the present discussion reveals (cf., (cf., III,F, G, and I), I), similar arguments can also also be made for the potential transfer of peptide hormones (insulin, (insulin, glucagon, etc.) etc.) from the maternal system into the oocyte. oocyte. Two important conclusions conclusions can can be drawn from these novel findings findings:: 1. 1.The fact that thyroxin and T3 T3 are present in the growing oocyte oocyte and undergo undergo changes changes during oocyte development long before a functional hypothalamo-adenohy hypothalamo-adenohypophysial-thyroid axis axis is established, established, implies that these hormones hormonesyet unidentified-exert unidentified-exert physiological roles during early larval mor morphogenesis and 2. If, If, as it seems seems possible, hormone stores supplied by the maternal system are a common feature in fi sh eggs, fish eggs, an entirely new window on the endocrinology and physiology of developing fish has been opened. opened.

Yolk-DNA I. Yolk-DNA exclusive, route for the uptake of vitellogenin The main, if not exclusive, oocyte is micropinocytosis (Brummett (Brummett from the blood into the growing oocyte and Dumont, 1977). 1977). As pointed out elsewhere in this review, the and fate of the vitellogenin molecule inside inside of the oocyte oocyte is is subsequent fate yolk. cleavage into different components which are later stored in the yolk. However, it can can be hypothesized that the the uptake of the large molecule However, may not entirely exclude exclude smaller, smaller, vitellogenin by micropinocytosis may blood-borne molecules, such as as sugars, sugars, lipids, lipids, plasma proteins, or blood-borne even DNA. DNA. To To exemplify this phenomenon, phenomenon, a short short comparative comparative ex exeven cursion to amphibian systems systems is required, since to date no data on cursion similar phenomena phenomena have have been analyzed for fishes. fishes. similar

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of the oocyte chromatin, the yolk platelets In addition to the DNA of of the amphibian Xenopus laevis Iaevis contain yolk-bound DNA. DNA. This yolkyolk of DNA is found to be double-stranded, and characterized by a high molecular weight (Hanocq et al., al., 1972), 1972), but its actual concentration is small at about 20 ng per oocyte, compared with some 280 jLg pg vitello vitellogenin derived protein contained in the fully grown oocyte. When experimentally exposed to Xenopus, bovine, or bacterial DNA, iso isolated vitellogenic oocytes of of Xenopus sequestered it from the incuba incubation medium, and the DNA was later found to be associated with the yolk platelets (Opresko (Opresko et al., 1979). 1979). However, there was no discrimi discrimination in uptake rates for DNA from the different sources, and further furthermore yolk-DNA yolk-DNA was determined to undergo relatively rapid turn turnover. This circumstantial evidence suggests that in this particular amphibian system, the DNA associated with the yolk is not involved in information transfer during the embryonic development. On the other hand, the indisciminate uptake of DNA from the maternal bloodstream, which has been shown to contain small small (25 (25 jLg pg ml-1) ml-l) amounts of of DNA (Opresko (Opresko et al., 1979), 1979),presents a good example of of an adventitious uptake of maternal blood components into the growing oocyte, which is is solely a byproduct of the mode of vitellogenin uptake by micropinocytosis. It may also also help to explain the otherwise surpris surprising presence of other components of maternal plasma or their deriva derivatives in growing oocytes. J. M etabolism Metabolism In addition to the uptake and and processing of vitellogenin and other blood-borne proteins such as as VLDL, the growing fish fish oocyte synthe synthesizes sizes and and accumulates accumulates aa number number of of high-molecular-weight high-molecular-weight compo components. First, First, the oocyte displays displays aa whole complement of RNA (some (some 10 lo44 more than in somatic cells), cells), mainly rRNA (95%), (95%),mRNA (2-3%), (values (values for Xenopus) Xenopus) and tRNA, tRNA, including an oocyte-specific oocyte-specific 5-S 5-S RNA (Denis (Denis and Ie le Maire, 1983), 1983),which are likely to be of importance in early embryonic development. development. Second, Second, the oocyte oocyte can can perform pro protein tein biosynthesis as as well well as as aa multitude of of posttranslational modifica modifications, specifi cally glycosylation, specifically glycosylation, phosphorylation, and lipidation. In the the course course of of their their development, development, Xenopus oocytes oocytes increase increase their their bio biosynthetic synthetic activity by more than 100-fold, 100-fold,from from 4.3 4.3ng protein per day in stage stage 11 oocytes oocytes to over 0.5 0.5 jLg p g per day day in stage 66 oocytes oocytes (Taylor (Taylor and Smith, Smith, 1985). 1985). Each of the mentioned activities activities requires specific sub subsets sets of enzymes. enzymes. This high biosynthetic potential made made the Xenopus oocyte oocyte the system system of choice choice to study translation and posttranslational posttranslational

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THOMAS THOMAS P. MOMMSEN AND PATRICK JJ.. WALSH

modification of microinjected messenger RNAs from a variety of of ani animal sources, including insect mRNAs (Lane 1983; Soreq, Soreq, 1985). ul., 1983; 1985). (Lane et al., As the the example example with with microinjected microinjected vitellogenin vitellogenin shows shows (see (see above), above), the oocyte is also capable of totally degrading "foreign" “foreign” proteins. On account of these results and some histochemical studies, the oocyte en usually gets credited with a limited complement of lysosomal enzymes that are supposedly also involved in the breakdown of of vitello vitellogenin into phosvitin, etc. Furthermore, adventitiously sequestered yolk-DNA yolk-DNA has been shown to undergo turnover (Opresko (Opresko et al., 1979), 1979), again a metabolic activity that demands a specific set of en enzymes. zymes. Other metabolic activities of the growing teleost oocyte apparently include the synthesis of urea, which is absent in most adult teleosts, and results in oocyte urea concentrations surpassing those of the ma maternal system by two- to fivefold (Depeche (DBpGche et al., ul., 1979). 1979). All of these actions, as well as acid-base acid-base regulation and the active vesicle transport through the cell, require energy in the form of ATP. ATP. The ATP must somehow be generated inside of the oocyte, oocyte, since it is unlikely that it is furnished by the follicle cells cells.. Unfortunately, the questions concerning the energy supply and preferred substrates for the growing oocyte have yet to be investigated, particualarly for the fishes. fishes. This is a deplorable situation, especially since the answers to these questions may have particular relevance to the early survival of of the fish embryos and larvae. larvae. Even before exogenous vitellogenesis has been initiated, the 00oocytes of Misgurnus fossilis augment their contents of of metabolic en enzymes, specifically those involved in glycolysis, glycolysis, the pentose shunt, and gluconeogenesis. During the entire course of oocyte develop development, glucose sequestered from the maternal circulation serves as an important energy source and also supplies the building blocks for accumulating glycogen. In fact, the activity of one of the key enzymes in this pathway, glycogen synthetase, increases 100-fold 100-fold during vitel vitellogenesis (Yurowitzky 1975). Following maturation, the (Yurowitzky and Milman, 1975). Misgurnus oocyte completely loses hexokinase activity and with it the ability to use exogenously administered glucose. glucose. At the same time, the switch from exogenous to endogenous energy use, at least as far as carbohydrate metabolism is concerned, is reflected in alterations of the enzymes regulating glycogen synthesis and its degradation. The moment that hexokinase is lost from the oocyte, glycogen synthetase activity decreases by half, while glycogen phosphorylase activity in increases by an order of magnitude (Yurowitzky (Yurowitzky and Milman, 1972). 1972). It can can be be concluded concluded from from the the presence presence and and high high activities activities of of enzymes enzymes

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mainvolved in glucose catabolism that during oocyte development ma ternal glucose may constitute one of the major energy sources for the different ATP-demanding reactions. It would also be interesting to confirm whether vitellogenesis might be correlated with increased glucose output from the maternal liver, as the decrease in hepatic glycogen exact extent extent of of its its importance importance is is not not clear clear yet, yet, glycogen suggests. suggests. The exact mainly because data for enzymes involved in other pathways, such as fatty acid utilization, are lacking. It should be recalled that during exogenous vitellogenesis, the availability of of lipid material through increased VLDL output by the liver is stepped up, as is lipid turnover in general. Once the oocyte has been matabolically "cut “cut off' off’ from the hexmaternal continuum of energy supplies, as the disappearance of hex metabolically distinct, okinase from the oocyte suggests, suggests, it exists as a metabolically and closed, unit, which from this point on has to rely on accumulated substances glycogen will will serve substances for for survival. survival. It It can can be speculated speculated that that glycogen serve of the relative ease as the first supplier of metabolic energy, because of with which it can be mobilized. Considering their overall bulk and their caloric contents, yolk lipids will be of overwhelming importance during ensueing parts of of embryonic and larval development, while the accumulated amounts of protein and amino acids are most likely to be funneled into anabolic and not ATP-delivering pathways. IV. EPILOGUE

The processes of hepatic vitellogenin synthesis and yolk-compo yolk-component deposition in fishes represent nent deposition in the the oocyte oocyte in in the the fishes represent aa wide-open wide-open field for fields. From for researchers researchers in in aa variety variety of of fields. From comparative comparative and and evolution evolutionary pis cine systems ary viewpoints, viewpoints, studies studies on on piscine systems are are likely likely to to supply supply valu valuable able insights insights into into hepatic hepatic steriod steriod receptor receptor mechanisms, mechanisms, estrogen estrogen in interactions genes, and mechanisms of multitude of genes, and mechanisms of teractions with with a multitude posttranslational posttranslational modifications, modifications, as as well well as as into into the the nature nature of of hormone hormone interactions level. On interactions on on the the receptor receptor and and gene gene level. On the the level level of of the the oocyte, oocyte, central topics will include the vitellogenin receptor mechanism, the regulation regulation and and control control of of the the enzymatic enzymatic machinery machinery involved involved in in the limited breakdown of vitellogenin, and the sources-maternal sources-maternal or in internal-of sialoglyco ternal-of such differing compounds as wax esters, lectins, sialoglycoproteins, to proteins, or or hormones, hormones, or or vitamins. vitamins. In In each each case, case, the the fishes seem to present the experimenter with a variety of species ideally suited for ease with with which which massive massive the individual individual topic, topic, not not least least because because of the ease vitellogenesis 7,8-estradiol. vitellogenesis can can be induced induced by by the the administration administration of of 117P-estradiol. The with respect respect The apparent apparent variability variability among among different different species of of fish with

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THOMAS P. MOMMSEN AND PATRICK J. J. WALSH WALSH

to each each theme theme will will help help to to prevent prevent the the emergence emergence of of generalizing generalizing to statements from from the the study study of ofjust just one one species. species. This This approach approach is is unforunfor statements tunately prevalent prevalent in in the the literature literature on on other other vertebrates, vertebrates, where where one one tunately toad (Xenopus (Xenopus laevis) laevis) represents represents all all amphibians, amphibians, or or the the chicken chicken porpor toad trays the birds. birds. trays the Finally, it is imperative imperative to described biochemical biochemical events events into into Finally, it is to put put described context of of the the life life history history of of fishes. fishes. In In contrast contrast to to most most other other eggegg the context laying vertebrates, vertebrates, the the fishes are are known known to to invest invest large large amounts amounts of of laying their body body reserves reserves into into the the production production of of gonadal gonadal products. products. The The most most their extreme examples examples of of the striking metabolic metabolic effort effort exerted exerted by fishes fishes extreme the striking during the the time leading up up to to the the spawning spawning period period are are some some anadroanadro during time leading Pacific salmons (Oncorhynchus spp.) or the mous fishes such as the Pacific American shad shad ((Alosa (Idler and and Clemens, Clemens, 1959; 1959; Glebe Glebe A h a sapidissima) (Idler American and Leggett, Leggett, 1981). 1981). and It can be appreciated appreciated that that only only an an unperturbed unperturbed sequence sequence of the It can of the outlined events in the maternal system will lead to mature oocytes development. The with an optimized starting point for embryonic development. fine-tuning of of the the orchestrated orchestrated maternal events leading leading to to mature mature oo00fine-tuning maternal events cytes makes makes it necessary to to consider consider potential potential points points of of environmental environmental cytes it necessary interference. While potentially potentially interfering interfering infl uences range range from from interference. While influences acid-base disturbances disturbances (Tam (Tam et al., al., 1987) 1987) and and thermal thermal pollution pollution to acid-base anything that will invoke stress reactions reactions in in fish, fish, the the following following will will anything that will invoke stress focus with potentially potentially detrimental effects on the focus on two specific cases with survival of of the the young young of of the the ensuing ensuing generation, generation, namely, namely, lipophilic lipophilic survival toxicants and and heavy heavy metals. metals. toxicants Although Although carotenoids carotenoids are are possibly possibly rather rather ancillary ancillary compounds compounds in the the egg egg per per se, se, the the case case of of the the accumulated accumulated carotenoids carotenoids shall shall serve serve to emphasize emphasize the the point point of of the the potential potential importance importance that that the the maternal maternal history events may egg components. history and and events may bear bear to to the the formation formation of of egg components. Ca Carotenoids fish in usually deposited in their their food food and and usually deposited due due rotenoids are are taken taken up up by fish to to their their chemical chemical properties properties together together with with functional functional lipids-in lipids-in the the case As part part of of case of of salmonid salmonid fishes, fishes, usually usually in in the the white white muscle muscle tissue. tissue. As the the general general lipogenic lipogenic action action of of estradiol estradiol and and thus thus during during the the course course of of vitellogenesis, vitellogenesis, extrahepatic extrahepatic lipid lipid stores stores are are mobilized mobilized and and transported transported to to the the liver; liver; due due to to their their hydrophobicity, hydrophobicity, carotenoids carotenoids are are translocated translocated to liver together to the the liver together with with mobilized mobilized lipids. lipids. At At this this point point it it should should be recalled recalled that that during during exogenous exogenous vitellogenesis, vitellogenesis, hepatic hepatic tissue tissue consti constitutes central organ organ with with respect respect to to lipid lipid metabolism, metabolism, in in that that it it takes takes tutes the central up up triglycerides triglycerides and and phospholipids phospholipids to to utilize utilize them them for for different different meta meta(1) fatty acids serve as major oxidative substrates to fuel bolic tasks tasks:: (1) metabolic (2) as as part part of of the the posttranslational posttranslational modifications modifications metabolic processes; processes; (2) performed lipids are the liver, liver, lipids are attached attached to to that that particular particular part part of of the the performed by the

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forms the lipovitellin of the oocyte; vitellogenin molecule that later forms (3)the rate of hepatic lipoprotein synthesis and export is sharply and (3) increased during vitellogenesis. Carotenoids may associate passively of the lipid utilized in these processes, or it may actively be with any of bound to a covalent binding site on the vitellogenin molecule. The fi rst route will lead to carotenoid deposition in the liver. The second first option will result in carotenoid-colored vitellogenin, as the example of of the pink hue of sockeye-salmon vitellogenin shows. shows. The third alter alternative will also deliver carotenoids from the liver to the gonad, which during vitellogenesis displays possibly the highest rates of uptake for lipoproteins, especially VLDL, from the bloodstream. All lipophilic substances accumulated in the maternal system are likely to behave like the carotenoids. carotenoids. It is is known that chlorinated hydrocarbons and many other lipophi lipophilic pesticides are transported in the bloodstream by lipoproteins lipoproteinsDDT, for instance, has been found associated with serum lipoproteins (Salmo gairdneri; Plack et e t al., 1979). simiin exposed rainbow trout (Salrno 1979). A simi lar behavior can be anticipated for other lipophilic environmental toxicants, such as aliphatic or polycyclic hydrocarbons and many of of toxicants, their derivatives derivatives.. As a consequence, it is is reasonable to assume that sh such lipophilic compounds that have found their way into adult fi fish will eventually be translocated-just translocated-just as the carotenoids are-to are-to the exogenous vitellogenesis. Considering the facts facts that un ungonad during exogenous der the influence of estradiol, hepatic lipoprotein synthesis is in in(cf. Table II) 11) and that vitellogenin itself contains a highly creased (cf. lipophilic region, it does not come as a surprise that DDT and other fish (Plack et al., hydrophobic pesticides are accumulated in fi sh eggs (Plack 1979). Subsequently they will severely impair egg survival and hatch hatch1979). ability (Johnson and Pecor, 1969). 1969). The massive oil globules, com composed of wax esters and steryl esters, prevalent in the eggs of a large fish, number of fi s h, designate a potentially detrimental sink for pesticides, petrochemicals, or other lipophilic environmental toxicants. toxicants. Further Furthermore, at the level of the gonad, exposure of vitellogenic fish sublefish to suble thal concentration of pesticides led to a signifi cant decrease in the significant 32P]phosphate by the growing oocytes, uptake of [[32P]phosphate oocytes, thus probably com compromising their normal composition ((Singh Singh and Singh, Singh, 1981). 1981). hydrocarbons, it To compound the problems posed by halogenated hydrocarbons, has been reported that such compounds not only bind to the vitello vitellogenin molecule, but also decrease the estradiol-dependent vitello vitellogenic response in the rainbow-trout liver (Chen (Chen and Sonstegard, Sonstegard, 1984). 1984).Inducers of the hepatic mixed-function oxidase oxidase system, system, such as beta-naphthofl avone, exert an inhibitory influence on the production beta-naphthoflavone,

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of (Chen and Sonstegard, of vitellogenin mRNA in the rainbow trout (Chen Sonstegard, 1984). It should be recalled that during exogenous vitellogenesis, the 1984). matabolic demand put on the liver is enormous (cf. (cf. Table II). 11).Conse Consequently, it can be expected that any additional additiona1 metabolic require requirements placed on the liver, such as the synthesis of of elements involved in detoxification, are likely to reduce the effort expended on vitello vitellogenesis and thus may imbalance the maturing of of the oocytes. oocytes. A further example for the costly metabolic expenditure incurred is the occur occurrence of a novel vitellogenin-like protein in the blood of of pesticide pesticideexposed fish (Denison et al., 1981). 1981). Also on the level of the liver, vitellogenesis may be impaired or its timing imbalanced by the known estrogenic action of some insecticides insecticides.. Examples in mammals and birds show that the chlorinated insecticide chlordecone interacts directly and rather persistently with the uterine estrogen receptor (Hammond et al., 1979). 1979).As As pointed out, mammalian and piscine estro estrogen receptors reveal numerous similarities, similarities, making the exertion of biological effects highly likely in fish systems. A similar line of reasoning applies to the exposure of of fish to envi environmental heavy metals. In Blennius pholis, cadmium and copper are known to accumulate in hepatic tissue, and in the course of the final oocyte oocyte maturation and massive yolk deposition, these heavy metals are transferred from the liver to the gonad and accumulate in the egg (Shackley et al., 1981). (Shackley 1981). Whereas this designates one passive way for the female fish to decrease its own hepatic concentration of these trace metals, it may develop into an important, potentially lethal, strategy for the oocyte. It is not too far-fetched to suggest that in situations where the environmental load of these or other heavy metals to the adult is increased from trace amounts to sublethal levels, transfer to the gonad in the course of oocyte maturation may result in the accu accuHow of mulation of highly toxic levels in the oocyte. oocyte. While such flow of poten potential toxicants may presently not affect marine fish, it is already fright frightfully relevant for freshwater and brackish-water fishes in many parts of the world. The vitellogenin molecule itself may be implicated in the transport of hepatic heavy metals to the gonad due to its protein phos phosphorus-dependent phorus-dependent charge and ion-binding capacity (Hara et al., 1980; 1980; Hara and Hirai, 1978; 1978; Lange, 1981). 1981). An additional problem may be introduced through the potential competition of hepatically accumu accumulated heavy metals for those metal ions that are transported to the gonad during undisturbed vitellogenesis, namely magnesium, cal calcium, and iron. Although adult fish are able to bind and detoxify heavy metals quite efficiently through the specific hepatic synthesis of metallothio-

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(Roch and McCarter, 1984), 1984), the process does not rid the parent nein (Roch body of the heavy metal load rapidly and thus sets the stage for the oocyte. Also, since metallothionein is inpotential poisoning of the oocyte. in duced duced in in the the liver, liver, its its synthesis synthesis effectively effectively competes competes with with vitellogenin vitellogenin (cf. aZ., 1984) 1984) and and therefore therefore can can be be expected expected to to impair impair the the (cf. Seguin Seguin et al., balanced flow How of vitellogenin to the gonad. of Xenopus and some other amphibian eggs is a The green tinge of reflection of the maternal biliverdin deposited adventitiously. colorful reHection It also presents an additional example of how the maternal system may may dispose of of an an excretory excretory product product via via the the eggs. eggs. However, However, as as the the above compounds deposited deposited in in the the maternal maternal above examples examples show, show, not not all compounds liver and and eventually eventually accumulating in in the the eggs eggs are are as as inocuous inocuous as as bili biliverdin in Xenopus.

ACKNOWLEDGMENTS We would like to thank Dr. Catherine B. Lazier (Dalhousie (Dalhousie University) and Dr. Harold B. White III I11 (University of Delaware) Delaware) for helpful discussions. We are grateful to Dr. Bodil Korsgaard (Odense University) for critically reading the manuscript manuscript,:

REFERENCES

of estradiol on serum calcium, phosphorus and protein of of Bailey, R. E. ((1957). 1957). The effect of J . Exp. Erp. Zool. 136, 136,455-469. 455-469. goldfish. ]. Bast, eld, S. S. A., Gehrke, L., and Ilan, J. (1977). Garfield, (1977). Coordination Coordination of of ribosome Bast, R. E., Garfi content and polysome formation during estradiol stimulation of vitellogenin syn synthesis in immature male chick liver. Proc. U.S.A. 74, Proc. Natl. Natl. Acad. Acad. Sci. Sci. U.S.A. 74, 3133-3137. 3133-3137. Bellabarba, D., and Lehoux, J.-G. (1981). (1981). Triiodothyronine Triiodothyronine nuclear receptor in chick 1017-1025. embryo: of hepatic receptor. receptor. Endocrinology Endocrinology 109, 109,1017-1025. embryo: Nature and properties of Bhattacharya, SS.,. , Plisetskaya, E., Dickhoff, W. W., and Gorbman, A. ((1985). 1985). The effects of estradiol and triiodothyronine triiodothyronine on protein synthesis synthesis by hepatocytes of of juvenile coho salmon (Oncorhynchus (Oncorhynchw kisutch). kisutch). Gen. Gen. Camp. Comp. Endocrinol. Endocrinol. 57, 103-109. 103-109. Bres, O., 0., and Eales, J. G. (1986). (1986). Thyroid hormone binding to isolated trout trout (Salmo (Salmo gairdneri) liver nuclei in vitro: oitro: Binding Binding affinity, capacity, and chemical specificity. Gen. 61, 29-39. Gen. Camp. Comp. Endocrinol. Endocrinol. 61,29-39.

1983). Gonadotropine Breton, B., Fostier, A., Zohar, Y., Le Bail, P. Y., and Billard, R. ((1983). glycoprotc�ique glycoprotkique maturante et oestradiol-17{3 oestradiol-17/3 pendant Ie le cycle reproducteur chez la 220-231. truite Fario (Salmo (Salmo trutta) trutta) femelle. Gen. Gen. Compo Comp. Endocrinol. 49, 49,220-231. N. R., Whitehead, C., and Breton, B B.. (1982). (1982). Relationships between behveen serum Bromage, N. levels of of ovarian of gonadotropin, gonadotropin, oestradiol-17{3, oestradiol-l7P, and vitellogenin vitellogenin in the control of development in the rainbow trout. 11. Effects of alterations in environmental photo phototrout. II. period. Gen. Compo Endocrinol. 366-376. Gen. Comp. Endocrinol. 47, 47,366-376.

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Browder, L., ed. (1985). 1. Pergamon, New York. (1985). "Developmental “Developmental Biology," Biology,” Vol. 1. York. Brown, C. L., L., Sullivan, C. V., V., Bern, H. A., and Dickhoff, W. W. ((1987). 1987). Occurrence of of thyroid hormones in early developmental stages of teleost fish. Trans. Trans. Am. Am. Fish. Fish. Soc. S O C . (in press). press). Brummett, A. R., and Dumont, J. N. ((1977). 1977). Intracellular Intracellular transport transport of vitellogenin in Xenopus Xenopus oocytes: An autoradiographic autoradiographic study. Dev. Deu. Bioi. Biol. 60, 482-486. 482-486. Burzawa-Cerard, 1982). Existe-t'i1 Burzawa-Gerard, E. ((1982). Existe-t’il plusieurs gonadotropines gonadotropines (CTH) (GTH) chez les pois poissons? Donnees In "Reproductive “Reproductive Physiol PhysiolDonnbes biochimiques et vitellogenese vitellogenbse exogfme. exogbne. In ogy of Fish" J. J. J. Richter and H. J. T. Coos, Goos, compilers), compilers), pp. 19-22. 19-22, Pudoc, Fish” (C. J. Wageningen. Busson-MabilIot, S. (1984). (1984). Endosomes transfer yolk proteins to Iysosomes lysosomes in the vitel vitelBusson-Mabillot, S. Bioi. Cell. Cell. 51, 53-66. logenic oocyte of the trout. Biol. 53-66. Callard, G C.. V. (1982). (1982). Aromatase in the teleost brain and pituitary: Role in hormone action. In In "Reproductive “Reproductive Physiology of of Fish" Fish” (C. (C. J. J. Richter and H. J. T. Coos, Goos, compilers), pp. 40-43. 40-43. Pudoc, Wageningen. Callard, C. 1985). Exclusive nuclear nuclear localization of of estrogen receptors G. V., and Mak, P. ((1985). Squalus testis. Proc. Sci. U.S.A. in Squalus Proc. Natl. Natl. Acad. Acad. Sci. U.S.A.82, 82, 1336-1340. Callard, C. V., Petro, Z., Ryan, K. J., and Claiborne, J. B. ((1981). 1981). Estrogen synthesis G. V., synthesis in in in vivo viva in the brain of of a marine teleost (Myaxacephalus). (Myorocephalus).Gen. Gen. Camp. Comp. vitro and in vitro 243-255. Endacrinal. 43,243-255. Endocrinol. 43, Campbell, C. M., and Idler, D. R. ((1976). 1976). Hornlonal Campbell, Hormonal control of vitellogenesis vitellogenesis in hypoph hypophysectomized winter flounder (Pseudapleuronectes (Pseudopleuronectes americanus americonus Walbaum). WaIbaum). Cen. Gen. Comp. Endocrinol. 28, 143-150. 143-150. Camp. Campbell, 1 980). Characterization Campbell, C. M., and Idler, D. R. ((1980). Characterization of of an estradiol-induced protein protein from rainbow trout trout serum as vitellogenin vitellogenin by the composition and radioim radioimmunological cross reactivity to ovarian yolk fractions. Biol. Bioi. Reprod. Reprod. 22, 22, 605-617. 605-617. Jalabert, B. (1979). (1979). Selective protein incorporation by vitellogenic Campbell, C. M., and Jalabert, Salma Salmo gairdneri gairdneri oocytes in in vitro. uitro. Ann. Ann. Bioi. Biol. Anim., Anim., Biochim., Biochim., Biophys. Biophys. 19, 429429437. 437. Chen, T. T. 1983). Identifi cation and characterization of T. ((1983). Identification of estrogen-responsive estrogen-responsive gene Cell B Bioi. Can.}. J . Biachem. Biochem. Cell i d . 61, 802-810. 802-810. products in the liver of rainbow trout. Can. Chen, T. T., and Sonstegard, R. A. (1984). (1984). Development of of a rapid, sensitive and quanti quantiof xenobiotics on reproduction reproduction in fish. tative test for the assessment of the effects of Mar. Environ. Enuiron. Res. Res. 14, 14, 429-430. 429-430. Mar. A., Lane, C., Craig, R., Boulton, A., Mohun, T., and Morser, J. J. (1981). Colman, A,, (1981). The influence of pro of topology and glycosylation on the fate of heterologous secretory pro]. Biochem. 113,339-348. teins made in Xenopus oocytes. Eur. }. Biochem. 113, 339-348. Craik, J. 1978a). The effects of J. C. A. ((1978a). of oestrogen treatment on certain plasma constituents Scyliorhinus canicula L. Gen. Gen. elasmobranch Scyliarhinus associated with vitellogenesis in the elasmobranch 35,455-464. Comp. Endacrinal. Endocrinol. 35, 455-464. Compo of vitellogenin vitellogenin metabolism in the elasmobranch Craik, J. C. C. A. ((197813). 1978b). Kinetic studies of Scyliorhinus Compo Biochem. Scyliorhinus canicula canicula L. L. Comp. Biochem. Physiol. Physiol. A A 61A, 355-361 355-361.. Craik, J. C. A. (1982). (1982). Levels ooff phosphoprotein iinn the eggs and ovaries of of some fish species. Camp. Comp. Biochem. Biochem. Physial. Physiol. B 72B, 72B, 507-510. 507-510. A., and Harvey, S. (1984). The magnitudes of three phosphorus-contain phosphorus-containCraik, J. C. A., S. M. (1984). ing fractions in the blood plasma and mature eggs of Conp. Biochem. Biochem. Phy Phyof fishes. Camp. 78B,539-543. 539-543. siol. B 78B, sial. G. F. (1970). (1970). Tissue carotenoids Crozier, C. carotenoids in prespawning and spawning sockeye salmon (Oncorhynchus 973-975. (Oncorhynchus nerka). nerka).). J . Fish. Fish. Res. Res. Board Board Can. Can. 27, 27,973-975.

5. 5.


397

of estradiol dipropionate on the Dasmahapatra, A. K., and Medda, A. K. ((1982). 1982). Effect of glycogen, lipid and water contents of liver, muscle, and gonad of male and female (Heteropneustesfossilis Bloch). Bloch). Gen. Gen. (vitellogenic and nonvitellogenic) nonvitellogenic) Singi (vitellogenic Singi fish (Heteropneustes 48,476-484. Comp. Endocrinol. Endocrinol. 48, Camp. 476-484. (1981). Temperature dependent Dasmahapatra, A. K., Ray, A. K., and Medda, A. K. (1981). (Heteropneustesfossilis Bloch). fish Bloch). Endokrinologie 78, action of estrogen in Singi Singi fi sh (Heteropneustes 107-1 10. 10. 107-1 Deeley, R. G., Tam, S.-P., and Archer, T. K. (1985). (1985). The effects of of estrogen on apopro apoproCan.]. J . Biochem. 882-889. tein synthesis. synthesis. Can. Biochem. Cell Biol. 63, 882-889. M. (1983). Denis, H., and Ie le Maire, M. (1983). Thesaurisomes, a novel kind of of nucleoprotein 263-297. particle. Subcellular Biochem. 9, 263-297. Denison, M. S., S., Chambers, J. E., and Yarbrough, J. D. D. (1981). (1981). Persistent vitellogenin vitellogeninlike like protein and binding of of DDT in the serum of of insecticide-resistant insecticide-resistant mosquitofish (Gambusia finis). Camp. 12. (Gambusia af affinis). Comp. Biochem. Biochem. Physiol. Physiol. C C 69C, 69C, 109-1 109-112. Depeche, Gilles, R., Daufresne, S., S., and Chiapello, H. (1979). DBpSche, J., Gilles, (1979). Urea content and urea production via the ornithine-urea omithine-urea cycle pathway during the ontogenic development of two teleost fishes. Camp. Comp. Biochem. Physiol. A A 63A, 51-56. 51-56. Devauchelle, N., Brichon, Brichon, G., G., Lamour, F., and Stephan, G. (1982). (1982). Biochemical compo composition of ovules and fecund eggs of sea bass (Dicentrarchus labrax), labrax), sole (Solea (Solea vulgaris) maximus). In "Reproductive (Scophthalmus maximus). “Reproductive PhYSiology Physiology of of uulgaris) and turbot (Scophthalmus Fish" T. Goos, compilers), pp. 155-157. Pudoc, Wa WaFish” (C. (C. J. J. Richter and H. J. T. geningen. de Vlaming, D. L., Shing, J., Paquette, G., 1977). In vivo G,, and Vuchs, R. ((1977). oivo and in vitro uitro of oestradiol-17-{3 oestradiol-17-P on lipid metabolism in Notemigonus crysoleucas. ]. J. Fish effects of Biol. 10, 273-285. 273-285. Biol. 10, de Vlaming, V., FitzGerald, R., Delahunty, G., Cech, J. J., Selman, Selman, K., and Barkley, M. (1984). (1984). Dynamics of of oocyte development and related changes in serum estradiol17{3, 17P, yolk precursor, and lipid levels in the teleostean fish, Leptocottus armatus. armatus. Comp. Biochem. Physiol. A 77 A, 599-610. Comp. 77A, 599-610. de L., Wiley, H. S., d e Vlaming, V. L., S., Delahunty, G., and Wallace, R. A. (1980). (1980). Goldfish (Carassius (Carassius auratus) auratus) vitellogenin: vitellogenin: Induction, isolation, properties and relationship to yolk proteins. Comp. Comp. Biochem. Biochem. Physiol. Physiol. B B 67B, 613-623. 613-623. Dougherty, J. J., Puri, R. K., and Toft, D. (1982). (1982). Phosphorylation Phosphorylation in vivo of of chicken oviduct progesterone receptor. ]. J. Biol. Biol. Chem. Chem. 257, 257, 14226-14230. Eldridge, M. B., Joseph, J. J. D., Taberski, Taberski, K. M., and Seaborn, Seaborn, G. T. (1983). (1983). Lipid and fatty acid composition of the endogenous energy sources of (Morone of striped bass (Morone saxatilis) Lipids 18, 510-513. 510-513. saxatilis) eggs. Lipids mmersen, J., E Emmersen, J., and Korsgaard, B. (1983). (1983). Measurements Measurements of of amino acid incorporation capacity of liver cell-free systems through the breeding season and following estra estra(Platichthys flesus L.). Physiol. 2, 192. L.). Mol. Mol. Physiol. 2, 181181-192. diol treatment of flounder (Platichthys$esus Emmersen, J., Korsgaard, B., and Petersen, 1. I. M. (1979). (1979). Dose response kinetics of of serum serum vitellogenin, vitellogenin, liver liver DNA, DNA, RNA, RNA, protein protein and and lipid lipid after after induction induction by by estradiolestradiolComp. Biochem. 17{3 flounders (Platichthysflesus 17p in in male male flounders (Platichthyspesus L.). L.). Comp. Biochem. Physiol. Physiol. B B 63B, 63B, 1-6. 1-6. Evans, A. J., Perry, M. N., and Gilbert, A. B. ((1979). 1979). Demonstration Demonstration of of very low density lipoprotein in the basal lamina of the granulosa layer in the hen’s hen's ovarian follicle. Biochim. Biochim. Biophys. Biophys. Acta Acta 573, 184-195. Fostier, A., Jalabert, B.., B., Billard, R., Breton, B., B., and Zohar, Y. (1983). (1983). The gonadal steroids. Fish Physiology" In "“Fish Physiology” (W. (W. SS.. Hoar, D. J. Randall, and E. Donaldson, Donaldson, eds.), eds.), steroids. In Vol. 9, 9, pp. pp. 277-372. 277-372. Academic Press, New York.

398


Fremont, 1984). Effects of FrBmont, L., Leger, C., Petridou, B., and Gozzelino, M. T. ((1984). of a (n-3) (n-3) polyunsaturated fatty acid deficient diet on profiles of of serum vitellogenin vitellogenin and lipo lipoprotein in vitellogenic trout (Salmo (Salmo gairdneri). 19, 522-528. gairdneri).Lipids Lipids 19,522-528. French, C. J., Hochachka, P. 1 983). Metabolic organization of C . J,, P. W., and Mommsen, T. T. P. ((1983). liver during spawning migration of Am. J. J. Physiol. Physiol. 245, R827R827of sockeye salmon. Am. R830. R830. 1981). Latitudinal Glebe, B. D., and Leggett, W. C. ((1981). Latitudinal differences differences in energy allocation and sapidissima) and use during the freshwater migrations of (Alosa sapidissima) of American shad (Alosa their life history consequences. Can. 806-820. Can.J. I . Fish. Fish. Aquat. Aquat. Sci. Scf.38, 38,806-820. Smith, D., and Notides, A. ((1968). 1968). Hormone receptors: Gorski, J., Toft, D., Shyamala, G., Smith, receptors: Studies on the interaction Recent Prog. Prog. Horm. Horm. Res. Res. 24, interaction of estrogen with the uterus. Recent 45-80. 45-80. Gottlieb, T. A., and Wallace, R. A. ((1982). 1982). Intracellular vitellogenin in Intracellular glycosylation glycosylation of of vitellogenin the liver estrogen-stimulated Xenopus laevis. laeuis. ]. J. Bioi. Biol. Chem. Chem. 257, 95-103. 95-103. liver of estrogen-stimulated Grasdalen, 1985). 31P-NMR Grasdalen, H., and J�rgensen, Jgkgensen, L. ((1985). 31P-NMR studies on developing eggs and larvae of Comp. Btochem. Biochem. Physiol. Physiol. B B 81B, 291-294. 291-294. of plaice. Compo Greene, G. L., Gilna, P., Waterfield, M., M., Baker, A., Hort, Y., Y., and Shine, J. J. (1986). (1986). Sequence and expression expression of human estrogen receptor complementary complementary DNA. Sci Science 150-1 154. ence 231, 11150-1154. Hammond, 1979). Hammond, B., Katzenellenbogen, Katzenellenbogen, B. S., S., Krauthammer, N., and McConnell, J. ((1979). Estrogenic activity of the insecticide chlordecone (Kepone) and interaction interaction with Sci. U.S.A. 6641-6645. uterine estrogen receptors. Proc. Natl. Natl. Acad. Sci. U.S.A.76, 76,6641-6645. receptors. Proc. Hanocq, F. E., and Steinert, G. F. M., Kirsch-Voiders, J., Hanocq-Quertier, Hanocq-Quertier, J., Baltus, E., (1972). (1972). Characterization Characterization of yolk-DNA from Xenopus laevis laewis oocytes ovulated in vitro. U.S.A.69, 1322-1326. uitro. Proc. Proc. Natl. Natl. Acad. Sci. Sci. U.S.A. Hara, A. ((1976). 1 976). Iron-binding activity of female-specific serum proteins of of rainbow trout (Salmo Biochim. Biophys. (Salmo gairdneri) gairdneri) and chum salmon (Oncorhynchus ketal. keta).Biochim. Biophys. Acta 437, 549-557. 437,549-557. Hara, H. ((1978). 1978). Comparative studies on immunochemical Ham, A., and Hirai, H. immunochemical properties of omp. Biochem. female speCific (Salmogairdneri). gairdneri).C Comp. specific serum proteins in rainbow trout (Salmo Physiol. 339-343. Physiol. B 59B, 339-343. Hara, A., Yamauchi, K., and Hirai, H. ((1980). 1980). Studies Studies on female-specifi female-specificc serum protein (vitellogenin) (Anguilla japonica). japonica). Compo Comp. (vitellogenin) and egg yolk protein in Japanese eel (Anguilla Biochem. Physiol. B 65B, 315-320. 315-320. Haschemeyer, Haschemeyer, A. E. V., and Mathews, R. W. (1983). (1983). Temperature dependency of pro protein synthesis synthesis in isolated hepatocytes of Antarctic fish. fish. Physiol. I. 56, Physiol. Zoo 2001. 56, 78-87. 78-87. of estradiol-17f3 estradiol-17P on the liver content of of Haux, C., and Norberg, B. ((1985). 1985). The influence of protein, protein, lipids, glycogen and nucleic acids in juvenile rainbow trout, Salmo gairdnerii. B 81B, 275-279. 275-279. gairdnerii. Compo Comp. Biochem. Biochem. Physiol. Physiol. B Hayward, M. M. A., A., Mitchell, T. A., and Shapiro, D. J. 1980). Induction of J. ((1980). of estrogen recep receptor and reversal of of the nuclear/cytoplasmic receptor ratio during vitellogenin syn synBioI. Chem. 1312. thesis and withdrawal Xenopus laevis. laewis.]. J. Biol. Chem. 255, 11308-1 11308-11312. withdrawal in Xenopus Henderson, R. J., Sargent, J. R., and Pirie, B B.. J. S. S. (1984). (1984). Fatty acid catabolism in the capelin, Mallotus villosus willosus (Muller), (Muller), during sexual maturation. Mar. Mar. BioI. Biol. Lett. Lett. 5, capelin, Mallotus 1115-126. 15-126. Holdsworth, G., Michell, R. H., and Finean, J. B. ((1974). 1974). Transfer of of very low density lipoprotein from hen plasma 275-277. F E E S Lett. 39, 39,275-277. plasma into egg yolk. FEBS S. H., Kodama, T., and Tanahashi, Tanahashi, K. (1979). Hori, S. (1979). Induction of of vitellogenin synthesis in Compo Endocrinol. 37, 306-320. goldfish by massive doses of androgen. Gen. Gen. Comp. 306-320. of androgen.

5. 5.


399

Housley, demonstration of of glucocorticoid receptor Housley, P. R., R, and Pratt, W. B. ((1983). 1983). Direct demonstration J . Biol. Chem. 258, 258,4630-4635. phosphorylation by intact L-cells. ]. Bioi. Chem. 4630-4635. Idler, D. R., and Campbell, C. M. ((1980). of estrogen and yolk 1980). Gonadotropin stimulation of Gen. Compo Comp. EndocrinlJl. Endocrinql. 41, 384384precursor synthesis in juvenile rainbow trout. Gen. 391. D. R R.,, and Clemens, W. A. ((1959). of Fraser River Idler, D. 1959). The energy expenditures of sockeye salmon during spawning spawning migration to Chilko Chilko and Stuart Lakes. Int. Pac. Salmon Fish. Fish. Comm., Comm., Prog. Rep. Rep. 6, 1-80. Inoue, S., (1978). Isolation of a novel glycoprotein glycoprotein from the eggs of of S., and Iwasaki, M. (1978). rainbow trout-Occurrence Biotrout-Occurrence of disialosyl groups on all carbohydrate carbohydrate chains. Bio 83,1018-1023. chem. Biophys. Biophys. Res. Res. Commun. Commun. 83, 1018-1023. chem. Inoue, S., S., and Iwasaki, M. (1980a). (1980a). Sialoglycoproteins from the eggs of of Pacific herring. 1 1, 131-135. Isolation and characterization. /. Biochem. 1111, characterization. Eur. ]. Characterization of of a new type of of glycoprotein glycoprotein Inoue, S., and Iwasaki, M. ((1980b). 1980b). Characterization Biophys. Res. Res. Commun. saccharide containing polysialosyl sequence. Biochem. Biophys. saccharide Commun. 93, 162-165. 162-165. Inoue, S., S., Kaneda-Hayashi, T., Sugiyama, H., and Ando, T. 1971). Studies on phospho T. ((1971). phosphoI. Isolation and characterization of of a phosphoprotein from proteins from fish eggs. 1. the eggs of Pacific herring. ]. J . Biochem. (Tokyo) (Tokyo)69, 1003-1011. 1985). Localization and Inoue, S., Iwasaki, M., Kitajima, K K.,, Inoue, Y., Y., and Kudo, SS.. ((1985). Proc. polysialoglycoprotein in rainbow rainbow trout eggs. Proc. activation-induced proteolysis of polysialoglycoprotein 1985. Glycocoajugates, 8th, 8th, 1985. Int. Symp. Glycoconjugates, Iwasaki, M., and Inoue, S. ((1985). 1985). Structures of of the carbohydrate carbohydrate units ofpolysialoglyco of polysialoglycoproteins isolated from the eggs of GlycoconjugateJ. of four species of of salmonid fishes. Glycoconjugate]. 2, 209-228. 2,209-228. Jensen, E. E. V., Suzuki, T., Kawashima, T., Stumpf, W. E., Jungblut, P. W., and De Suzuki, T., Sombre, E. E. R 1968). A two-step mechanism for interaction R. ((1968). interaction of of estradiol with rat Sombre, Sci. U.S.A. 632-638. Proc. Natl. Natl. Acad. Acad. Sci. U.S.A.59, 59,632-638. uterus. Proc. Johnson, H., and Pecor, C. (1969). cohos. In "Sport (1969). DDT and Great Lakes cohos. “Sport Fishing 4-5. Sport Fish. Inst., Washington, D.C. Bulletin,” No. 203, pp. 4-5. Institute Bulletin," heterogeneity. Hepato HepatoK.,, and Katz, N. (1982). (1982). Functional hepatocellular heterogeneity. Jungermann, K logy 385-395. logy 2, 2,385-395. Kaitaranta, J. K K.,, and Ackman, R R. G. (1981). (1981). Total lipids and lipid classes of of fish roe. C omp. Biochem. Physiol. 69B, 725-729. Comp. 725-729. 1 984). Enlightenment and confusion over steroid hormone receptors. King, King, R R. J. B. ((1984). receptors. Nature 701-702. Nature (London) (London)312, 312,701-702. King, W. J., J., and Greene, G. L. (1984). (1984). Monoclonal antibodies localize oestrogen recep recepof target cells. (London)307, 745-747. 745-747. of the nuclei of tor of cells. Nature (London) Kitahara, T. T. (1984). (1984).Behaviour of of carotenoids carotenoids in the chum salmon Oncorhynchus keta during development. Bull. Jpn. ]pn. SSoc. Sci. Fish. 50, O C . Sci. 50, 531-536. 531-536. Kobuke, L., Specker, Specker, J. L., and Bern, H. A. (1987). (1987).Thyroxine content of of eggs and larvae of coho salmon, Exp. 89-94. kisutch.]. J. E r p . Zool. Zool. 242, 242,89-94. of coho salmon, Oncorhynchus kisutch. Korfsmeier, K-H. K.-H. (1980). (1980). Lysosomal enzymes and yolk platelets. platelets. Eur. ]. /. Cell Bioi. Biol. 22, 208. 208. Korsgaard, B. ((1983). 1 983). The chemical composition of of follicular and ovarian fluids of of the pregnant blenny (Zoarces 101-1 108. (Zoarces viviparus uiuiparus (L.» (L.)).. Can. Can.]. J . Zool. Zool. 61, 11101-1108. 1976). Natural occurrence and experimental Korsgaard, B., and Petersen, 1. I. M. ((1976). experimental induc induction by estradiol-1713 (Platichestradiol-17p of of a lipophosphoprotein (vitellogenin) (vitellogenin) in flounder (Platich thys ./lesus (L). (L). Compo Comp. Biochem. Physiol. B 54B, 443-446. 443-446. thysjesus

400

THOMAS MOMMSEN AND THOMAS P. P. MOMMSEN AND PATRICK PATRICK J. WALSH WALSH

Korsgaard, Korsgaard, B., and and Petersen, Petersen, I. I. M. (1979). (1979).Viteliogenin, Vitellogenin, lipid lipid and and carbohydrate carbohydrate metabo metabolism during vitellogenesis and pregnancy, and after hormonal induction in the Comp. Biochem. Zoarces viviparus oiuiparus (L.). (L.). Comp. Biochem. Physiol. Physiol. B B 63B, 63B, 245-251 245-251.. blenny Zoarces Korsgaard,.B., 1983). Estradiol-induced M.. ((1983). Estradiol-induced hepatic hepatic Korsgaard,,B., Emmersen, Emmersen, JJ.,. , and and Petersen, Petersen, I. M fiesus protein ounder, Platichthys Platichthysflesus protein synthesis and transaminase activity in the male fl flounder, (L). Gen. Comp. Endocrinol. 50, 1- 17. (L). Gen. Comp. Endocrinol. 50, 111-17. Korsgaard, 1986). The T. P., P., and and Saunders, Saunders, R. R. L. L. ((1986). The effect effect of of temperature temperature Korsgaard, B., B., Mommsen, Mommsen, T. on the vitellogenic salar). Gen. (Salmo salar). Gen. vitellogenic response in Atlantic salmon post-smolts post-smoks (Salmo Comp. 193-201. 62,193-201. Comp. Endocrinol. Endocrinol. 62, Krebs, E. G. (1985). (1985).The phosphorylation of of proteins: A major mechanism for biological 813-820. regulation. Biochem. Biochem. Soc. SOC. Trans. Trans. 13, 13,813-820. Lam, T. J,, J., and Loy, G. G. L. (1985). (1985). Effect of of L-thyroxine L-thyroxine on ovarian development and gestation in the viviparous guppy, Poecilia Poecilia reticulata. reticulata. Gen. Gen. Compo Comp. Endocrinol. Endocrinol. 60, 324-330. 324-330. Lambert, J. G. D., and van Oordt, P. G. W. J. (1982). (1982).Catecholestrogens in the brain of the female rainbow trout, Salmo Salmo gairdneri. gairdneri. Gen. Gen. Comp. Comp. Endocrinol. Endocrinol. 46, 401. Lane, C. D., Champion, J., J,, Colman, A., A,, James, T. T. C., and Applebaum, S. S. W. (1983). (1983).The fate of Xenopus Xenopus and and locust vitellogenins made made in Xenopus Xenopus oocytes. An export export529-535. import processing model. Eur. Eur.J. ]. Biochem. Biochem. 130, 130,529-535. Lange, R. H. 1981). Are yolk phosvitins carriers for H. ((1981). for specific cations? Comparative microanalysis in vertebrate yolk platelets. Z. forsch., C: Z . Natur Naturforsch., C: Biosci. Biosci. 36C, 36C, 686686687. 687. Lazier, (1975). (3Hl-Estradiol [3H]-Estradiol binding by chicken liver nuclear extracts extractsLazier, C. C. B. (1975). Mechanism of increase in binding following estradiol injection. Steroids 281Steroids 26, 26,281298. 298. Lazier, 1978). Ontogeny Lazier, C. B. B. ((1978). Ontogeny of the vitellogenic response to oestradiol and of the soluble nuclear oestrogen receptor in embryonic chick liver. 143Biochem.}. J. 174, 174,143liver. Biochem. 152. 152. Lazier, C. C. B., Lonergan, K., and Mommsen, T. P. (1985). (1985).Hepatic estrogen receptors and plasma estrogen-binding the Atlantic salmon. salmon. Gen. Gen. Comp. Comp. Endocrinol. Endocrinol. estrogen-binding activity in the 57, 234-245. 57,234-245. Leatherland, 1985). Effects of 17{3-estradiol Leatherland, J. F. F. ((1985). 17p-estradiol and methyl testosterone testosterone on the activ activGen. Comp. ity of the thyroid gland in rainbow trout, Salmo Salmo gairdneri gairdneri Richardson. Gen. Comp. 343-352. Endocrinol. 60,343-352. Endocrinol. 60, I., and Desfarges, M.-F. ((1981). FrBmont, L., Leger, C., Fremont, L., Marion, D., Nassour, I., 1981). Essential 16,593593fatty acids in trout serum. Lipoproteins, vitellogenin and egg lipids. Lipids 16, 600. 600. (1979).Induction de vitellogenine vitelloghine par l'oestradiol l’oestradiol et par des androgfmes androgknes Menn, F. (1979). Le Menn, L. C. C . R. R. Hebd. Hebd. Seances Acad. Acad. Sci. Sci. 289, 289,413tkl6ost6en marin: Gobius niger L. chez un te\eosteen 413416. 416. (1980). Effect of androgen mediated Le Menn, Menn, F., F., Rochefort, H., and Garcia, M. (1980). Le Steroids 35, 35, 315� 315fish liver: ViteIIogenin Vitellogenin accumulation. Steroids by estrogen receptor of fish 328. 328. Lewis, J. A., A,, Clemens, Clemens, M. J., J., and Tata, Tata, J. J. R. R. (1976). (1976). Morphological and biochemical changes in the hepatic Xenopus laevis laeois hepatic endoplasmatic and golgi golgi apparatus of male Xenopus after after induction induction of egg-yolk egg-yolk protein synthesis by estradiol-17{3. estradiol-17p. Mol. Mol. Cell. Cell. Endo Endo1-329. crinol. 4, 31 311-329. crinol. 4, Liley, N. N. R., R., and Stacey, Stacey, N. N. E. E. (1983). (1983).Hormones, Hormones, pheromones, pheromones, and reproductive behav behavior in fish. In "Fish “Fish Physiology" Physiology” (W. (W. S. S . Hoar, Hoar, D. D. J. J. Randall, and E. E. M. M. Donaldson, fish. In eds.), eds.), Vol. Vol. 9B, 9B, pp. pp. 1-63. Academic Press, New York. York.

5. 5.


40 1 401

Y. ((1985a). Maitre, J. L., Le Guellec, C., Derrien, S., S., Tenniswood, M., M., and Valotaire, Y. l 985a). of vitellogenin from rainbow trout by rocket rocket immunoelectrophoresis: immunoelectrophoresis: Measurement of Application Can. ]. J. Application to the kinetic analysis of of estrogen stimulation in the male. male, Can. Biol. 63, 982-987. 982-987. Biochem. Cell Bioi. Maitre, J. L., M ercier, L., Dolo, Dolo, L., and Valotaire, Y. (1985b). (1985b). Caracterisation de d e recep r6cepMercier, oestradiol, induction de la vitellogenine et de son mRNA dans la teur specifique I’oestradiol, spkcifique aii l' (Salmo gairdneri). gairdneri). Biochimie 67, 215-225. 215-225. d e truite arc-en-ciel arc-en-cieI (Salmo foie de Mann, M., Lazier, C., C., and Mommsen, T. (1988). (1988). In preparation. f A novel composition of (1969F of phosvitins from salmon and Mano, Y., and Yoshida, M. (1969 (Tokyo) 66, 105-108. trout roe. ]. J. Biochem. Biochem. (Tokyo) 105-108. Markert, J. R., and Vanstone, W. E. ((1971). 1971). Eggproteins of of coho salmon (Oncorhynchus (Oncorhynchus kisutch): h u t c h ) :Chromatographic Chromatographic separation and molecular weights of of the major proteins Res. Board in the high density fraction and their presence in salmon plasma.]. Fish. Res. Board plasma. j . Fish. Can. 1853-1856. 28,1853-1856. Can. 28, Miesfeld, R., Okret, S., S., Wikstrom, A.-C., Wrange, 0., O., Gustafsson, J.-A., and Yamamoto, Yamamoto, K. R. (1984). (1984). Characterization Characterization of of a steroid hormone receptor gene and mRNA in wild-type and mutant cells. Nature 312, 779-781. Nature (London) (London)312, 779-781. Migliaccio, A., Rotondi, A., F. (1984). (1984). Calmodulin-stimulated Calmodulin-stimulated phospho phosphoA,, and and Auricchio, F. rylation of 17,B-estradiol Proc. Natl. Natl. Acad. Acad. Sci. Sci. U.S.A. U.S.A. 81, 81, 17p-estradiol receptor on tyrosine. Proc. 5921-5925. 5921-5925. Miller, M. 1982). Dephosphorylation M. S., S . , Benore-Parsons, M., and White, H. B. ((1982). Dephosphorylation of chicken riboflavin-binding ooriboflavin-binding protein and phosvitin decreases decreases their uptake by 00cytes. cytes. ]. J. Bioi. Biol. Chem. Chem. 257, 6818-6824. 6818-6824. Mommsen, T. T. P., P., and Lazier, Lazier, C. C. B. B. (1986). (1986). Stimulation of estrogen estrogen receptor accumula accumula269tion by estradiol in primary cultures of salmon hepatocytes. FEBS FEBS Lett. Lett. 195, 195,269271. T. P., French, C. J., and and Hochachka, P. P. W. W. (1980). (1980). Sites and and pattern of Mommsen, T. protein and amino acid Can.J. acid utilization during the spawning spawning migration of salmon. Can.]. Zool. 58, 58, 1785-1799. 1785-1799. Moon, T. W., T. P. P. (1985). (1985). Fish hepatocytes: A model W., Walsh, P. P. J., and Mommsen, Mommsen, T. metabolic system. Can.]. f. Fish. Fish.Aquat. Aquat. Sci. Sci. 42, 42, 1772-1782. system. Can. Mugiya, Y., and Ichii, T. 1981). Effects of estradiol-17,B T. ((1981). estradiol-17p on branchial and intestinal calcium uptake in the rainbow trout, Salmo Salmo gairdneri. gairdneri. Comp: Comp. Biochem. Biochem. Physiol. Physiol. A A 70A, 97-101. 97-101. Mugiya, Y., 1977). Studies on fi sh scale formation and resorption. II. Y., and Watabe, Watabe. N. ((1977). fish 11. Effect of estradiol on calcium homeostasiS homeostasis and skeletal tissue resorption resorption in in the Carassius auratus and the killifish, Fundulus heteroclitus. heteroclitus. Compo Comp. Bio Biogoldfish, Carassius chem. Physiol. A 57A, 197-202. chem. Muniyappa, R. (1980). (1980). Estrogen-induced synthesis synthesis of thiamin-binding Muniyappa, K., and Adiga, P. R. protein in immature immature chicks-Kinetics chicks-Kinetics of induction, induction, hormonal specificity specificity and modu moduJ. 186, 186,201-210. 201-210. lation. Biochem. ]. Nagahama, Y. ((1983). 1983). The The functional morphology of teleost gonads. In In "Fish “Fish Physiol Physiology" ogy” (W. (W. S. S. Hoar, D. D. J. Randall, and E. E. M. M. Donaldson, Donaldson, eds.), eds.), Vol. Vol. 9A, 9A, pp. 223-275. 223-275. Press, New York. York. Academic Press, Nath, P., 1981). Isolation and identification of female-specific P., and Sundararaj, B. B. I.I. ((1981). serum Heteropneustes fossilis. fossilis. serum phospholipoproteins (vitellogenin) (vitellogenin) in the catfish Heteropneustes Gen. Gen. Comp. Comp. Endocrinol. Endocrinol. 43, 43, 184-190. Ng, 1983). Yolk formation and differentiation in teleost T. B., B., and Idler, D. R. ((1983). teleost fishes. In In Ng, T. "Fish “Fish Physiology" Physiology” (W. (W. S. S. Hoar, D. D. J. J. Randall, and E. E. M. M. Donaldson, Donaldson, eds.). eds.). Vol. Vol. 9, 9, pp. 373-404. 373-404. Academic Press, Press, New York. York.

402

THOMAS WALSH THOMAS P. P. MOMMSEN MOMMSEN AND AND PATRICK PATRICK J. WALSH

Ng, Ng, T. T. B., Woo, Woo, N. Y. A., Tam, P. P. L., L., and Au, C. Y. Y.W. (1984). (1984).Changes in metabolism and hepatic ultrastructure ultrastructure induced by estradiol and testosterone in immature Epinepholus akaara Serranidae). Cell female Epinepholus akaara (Teleostei, (Teleostei, Serranidae). Cell Tissue Tissue Res. Res. 236, 651651659. 659. Norberg, Haux, C. 1985). Induction, isolation C. ((1985). isolation and a characterization of of the lipid Norberg, B., and Haux, content of Salmo species two Salmo species:: Rainbow Rainbow trout (Salmo (Salmo of plasma vitellogenin from two gairdneri) (Salmo trutta). trutta). Compo Comp. Biochem. Biochem. Physiol. Physiol. B B 81B, 869-876. 869-876. gairdneri) and sea trout (Salmo Nosek, J., 1983). Studies Studies on lectins. LVII. LVII. Immuno ImmunoNosek, J,, Krajhanzl, Krajhanzl, A., and Kocourek, Kocourek, J. ((1983). 131fluorescence localization Histochemistry 79, 79,131localization of of lectins present in fish ovaries. ovaries. Histochemistry 139. 139. Nunomura, Takano, K., and Hirai, H. (1983). (1983).Immunohistochemical locali IocaliNunomura, W., Hara, A., Takano, zation of vitellogenin in hepatic cells of Fac. Fish., Fish., of some some salmonid fishes. fishes. Bull. Fac. Hokkaido 79-87. 34,79447. Hokkaido Univ. Univ. 34, Olivereau, M., and Olivereau, J. ((1979). 1979). Effect of of estradiol-17f:l estradiol-17P on the cytology of of the liver, gonad and pituitary, and on the plasma electrolytes in the female freshwater eel. Cell 431-454. Cell Tissue Tissue Res. Res. 199, 199,431-454. Opresko, H. S., and Wallace, Wallace, R. A. (1979). (1979).The origin of of yolk-DNA yolk-DNA in Xenopus Xenopus Opresko, L., Wiley, Wiley, H. laevis. J. Exp. E x p . Zool. 2001.209, 367-376. 367-376. laeuis. ]. Opresko, H. S., and Wallace, Wallace, R. A. (1981). (1981). Receptor-mediated binding and Opresko, L., Wiley, H. 91, 218a. internalization of oocytes. ]. J. Cell Biol. Biol. 91,218a. of vitellogenin by Xenopus oocytes. Pan, M. L., Bell, W. J., J,,and Telfer, W. H. (1969). (1969).Vitellogenic blood protein synthesis by insect fat body. Science Science 165, 165, 393-394. 393-394. Perlman, A. J., Wolffe, Champion, J., and Tata, J. R. ((1984). 1984). Regulation of Wolffe, A. P., Champion, of estrogen receptor of vitellogenin gene transcription in Xenopus Xenopus hepatocyte cultures. Mol. Mol. Cell. Cell. Endocrinol. Endocrinol. 38, 151-161. Peter, R. E. (1983). (1983). The brain and neurohormones neurohonnones in teleost reproduction. In In "Fish “Fish Physiology" S. Hoar, Hoar, D. J. J. Randall, and E. M. Donaldson, eds.), eds.), Vol. 9, pp. 9797Physiology” (W. (W. S. 137. 137. Academic Press, New York. Petersen, I. M., and Korsgaard, 1977). Changes in serum glucose and lipids in liver Korsgaard, B. ((1977). glycogen and phosphorylase during vitellogenesis in nature in the flounder (Platichthysflesus, Comp. Biochem. (Platichthys Jesus, L.). L.).Comp. Biochem. Physiol. Physiol. B B 58B, 167-171. 167-171. Peute, J., van der Gaag, Gaag, M. A., and Lambert, Lambed, J. J. G. D. (1978). (1978). Ultrastructure and lipid content of Brachydanio rerio, rerio, related related to vitellogenin of the liver of the zebrafish, zebrafish, Brachydanio synthesis. Cell Cell Tissue Tissue Res. Res. 186, 186, 297-308. 297-308. B. W., and Shapiro, J. (1981). Shapiro, D. J. (1981). Estrogen regulation of of hepatic 3-hydroxy-33-hydroxy-3Phillips, B. methylglutaryl coenzyme A reductase and acetyl-CoA acetyl-CoA carboxylase carboxylase in Xenopus laevis. Chem. 256, 2922-2927. 2922-2927. 1.Bioi. Biol. Chem. laeois.]. Plack, A., and Fraser, N. W. (1971). into egg proteins (1971).Incorporation of of L-(l4C)-leucine ~-(‘~C)-leucine Plack, P. A., 857-862. by liver slices from cod. Biochem. ]. J. 121, 121,857-862. cod. Biochem. Plack, J. (1968). (1968).Effects of of oestradiol 3-benzoate on concentra concentraPlack, P. A., and Pritchard, D. J. 257-262. tions of retinal and lipids in cod plasma. Biochem. ]. J. 106, 106,257-262. plasma. Biochem. Plack, (1971). Egg proteins in cod serum. serum. Plack, P. A., Pritchard, D. J., and Fraser, N. W. (1971). Natural occurrence and induction by injections of of oestradiol-3-benzoate. oestradiol-3-benzoate. Biochem. 847-856. ]. J. 121, 121,847-856. Plack, P. A., Rogie, A., and Mitchell, A. I. I. (1979). A., Skinner, E E.. R., Rogie, (1979). Distribution of of DDT between the lipoproteins of 19of trout serum. Comp. Comp. Biochem. Biochem. Physiol. Physiol. C. C . 62C, 1119125. 125. Pottinger, T. 1986). Estrogen-binding sites on the liver of T. G. ((1986). of sexually sexually mature male and female brown trout, Salmo trutta L. Gen. Comp. Endocrinol. 61, 120-126. Gen. Comp. 120-126.

5. VITELLOGENESIS VITELLOGENESIS AND 5.

OOCYTE ASSEMBLY

403 403

L. (1980). (1980).Catfish Catfish vitellogenin vitellogenin and its messenger RNA are Roach, A. H., H., and Davies, P. L. counterparts. Biochim. Biochim. Biophys. Biophys. Acta 610, smaller than their chicken and Xenopus counterparts. 400-412. 400-412. Roch, M., and McCarter, J. A. ((1984). metallothionein production and and resis resisRoch, 1 984). Hepatic metallothionein tance to heavy metals by rainbow (Salmogairdneri). gairdneri). I. I. Exposed to an artifical rainbow trout (Salmo Biochem. Physiol. 77C, 71-75. 71-75. cadmium. Comp. mixture of zinc, copper and cadmium. Comp. Biochem. Physiol. C 77C, Sand, D. D. M., Schlenk, H. H. (1971). (1971). Biosynthesis Biosynthesis of wax esters in fish. fish. Sand, M., Hehl, J. H., H., and Schlenk, 10, 2536-2541 2536-2541.. Metabolism of dietary alcohols. Biochemistry 10, O..,, Petersen, I. M M.,. , and Korsgaard, B. B. (1980). (1980). Changes iin carbohydrate Sand, 0 n some carbohydrate metabolizing enzymes and glycogen in liver, glucose and lipid in serum during (Platichthys vitellogenesis and after after induction induction by estradiol-17-,8 estradiol-17-p in the flounder (Platichthys vitellogenesis flesus p e w s L.). L.). Comp. Comp. Biochem. Biochem. Physiol. Physiol. B. B . 65B, 65B, 327-332. 327-332. Seguin, Carter, A. D., D., and Hamer, D. D. H. (1984). Competition for Seguin, C., Felber, B. K., Carter, H. (1984). (London), metallothionein gene transcription. Nature (London), cellular factors that activate metallothionein 312,781-785. 312, 781-785. Selman, K., K., and Wallace, Wallace, R. A. (1983a). (1983a).Oogenesis in Fundulus heteroclitus. heteroclitus. 11. II. The Selman, Gen. Comp. Comp. Endocrinol. Endocrinol. 42, 42, 345345vitellogenesis into maturation. Gen. transition from vitellogenesis 354. 354. Selman, K., and Wallace, R. A. (1983b). (1983b).Oogenesis Oogenesis in Fundulus heteroclitus. III. 111. Vitel VitelSelman, J. Exp. E x p . Zool. 2001.226, 226,441-457. logenesis. J. logenesis. 441-457. S. E., E., King, P. E., E., and Gordon, Gordon, S. S. M. (1981). (1981).Vitellogenesis and trace metals in Shackley, S. J. Fish Bioi. Biol. 18, 18, 349-352. 349-352. a marine teleost. j. Shapiro, D. 1 982). Steroid hormone regulation D. ((1982). regulation of vitellogenin vitellogenin gene expression. expression. CRC CRC 8,187-203. Crit. Rev. Rev. Biochem. 8, Crit. 187-203. Shigeura, H. H. T., E. V. V. (1984). (1984). Purification of egg-yolk proteins Shigeura, T., and Haschemeyer, A. E. aceratus. Comp. Comp. Biochem. Biochem. Physiol. B 80B, fish, Chaenocephalus aceratus. from the Anarctic fish, 935-939. 935-939. H., and Singh, Singh, T. T. P. P. (1981). (1981). Effect of parathion and aldrin on survival, ovarian Singh, H., 32P uptake and gonadotrophic gonadotrophic potency in a freshwater catfish, caffish, Heteropneustes 32P (Bloch). Endokrinologie 77, 77, 173-178. fossilis (Bloch). Sire, M M.-F., J.-M. (1983). (1983).Enterocytes Enterocytes et synthese de lipoproteines lipoprotkines chez les Sire, .-F., and Vernier, J.-M. VLDL. E h tude d e ultrastructurale ultrastructurale et biochimique. Bioi. Biol. Chylomicrons ou VLDL. poissons. Chylomicrons

Cell. 49, A24. A24. Cell.

Skinner, E. R., and Rogie, A. (1978). (1978). The isolation and partial characterization of the Skinner, serum lipoproteins 173, 507Biochem.J. J. 173,507lipoproteins and apolipoproteins apolipoproteins of the rainbow rainbow trout. Biochem. 520. 520. So, Y. P., Idler, D. D. R., and Hwang, S. S. J. ((1985). 1985). Plasma vitellogenin in landlocked So, (Salmo salar Ouananiche): Ouananiche): Isolation, homologous radioimmunoas radioimmunoasAtlantic salmon (Salmo vitellogenin from other other teleosts. teleosts. say and immunological cross-reactivity with vitellogenin Comp. Biochem. Biochem. Physiol. B 81B, 81B, 63-71 63-71.. Compo Solar, II.. G G., E.. M M.,. , and Hunter, G G.. A. (1984). (1984). Optimization ooff treatment Solar, . , Donaldson, E sex differentiation differentiation and sterilization in wild rainbow rainbow trout for controlled sex regimes for (Salmo gairdneri Richardson) Richardson) by oral administration administration of o f 17a-methyltestosterone. 17a-methyltestosterone. (Salmo Aquaculture 42, 129-139. mRNA-microinSoreq, H. ((1985). 1985). The biosynthesis of biologically active proteins in mRNA-microin Crit. Rev. Reu. Biochem. Biochem. 18, 18, 199-238. 199-238. jected Xenopus oocytes. CRC Crit. Sumpter, J. J. P. P. ((1981). purification and radioimmunoassay of vitellogenin vitellogenin from the Sumpter, 1981). The purification rainbow trout, Salmo Salmo gairdneri. gairdneri.Abstr., Abstr., Int. Int. Symp. Symp. Comp. Comp. Endocrinol., Endocrinol., 9th, 9th, 1981, 1981, p. 243. 243.

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THOMAS THOMAS P. P, MOMMSEN MOMMSEN AND AND PATRICK PATRICK JJ.. WALSH WALSH

Sumpter, J. P., and Dodd, J. M. ((1979). 1979). The annual reproductive cycle cycle of of the female sh, Scyliorhinus Scyliorhinus canicula canicula L., and its endocrine control. }. J , Fish Fish lesser spotted dogfi dogfish, 687-695. Bioi. 15, Biol. 15,687-695. Sundararaj, B. I., Nath, P., Synthesis of P., and Burzawa-Gerard, Burzawa-Gerard, E. (1982a). (1982a). Synthesis of vitellogenin Sundararaj, B. and its uptake by the ovary in the catfish, (Bloch) in re Heteropneustes fossilis (Bloch) recatfish, Heteropneustes sponse to carp gonadotropin and its subunits. subunits. Gen. 93-98. Gen. Camp. Comp. Endocrinol. Endocrinol. 46, 46,93-98. Sundararaj, S. V., and Lamba, V. J. (1982b). Goswami, S. (1982b). Role of of testosterone, estra estraSundararaj, B. I., Goswami, Heteropneustes fossilis diol-17/3, and cortisol catfish, Heteropneustes diol-1713, cortisol during during vitellogenesis in the catfish, (Bloch). Compo Endocrinol. 390-397. (Bloch). Gen. Gen. Comp. Endocrinol. 48, 48,390-397. Szego, J. (1985). (1985). Subcellular distribution of of oestrogen receptors. Szego, C. C. M., and Pietras, R. J. Nature (London) 88-89. (London)317, 317,88-89. Tam, W. Payson, P. D., Whitney, D. K., and Yu, C. K.-C. K.4. W. H., Birkett, L., Makaran, Makaran, R., Payson, (1987). cation of carbohydrate metabolism and liver vitellogenic function in (1987).Modifi Modification brook trout (Salvelinus ]. Fish. Aquat. (Salvelinusfontinalis) by exposure to low pH. Can. Can.]. Aquat. Sci. 44,630-635. 44, 630-635. Tata, J. Smith, D. F. ((1979). 1979). Vitellogenesis: J. R., and Smith, Vitellogenesis: A versatile model for hormonal 47-95. regulation of Recent Prog. Prog. Harm. Horm. Res. Res. 35, 3547-95. of gene expression. expression. Recent Taylor, durin� (1985).Quantitative changes in protein synthesis during Taylor, M. A., and Smith, L. D. (1985). Bioi. 110,230-237. 110, 230-237. Xenopus laevis. laevis. Dev. Dev. Biol. oogenesis in Xenopus te Heesen, D. D. (1977). (1977). Immunologische Untersuchungen an exoexo- und endogenen Dot Dotterproteinen von Brachydanio Brachydanio rerio rerio (Teleostei, Cyprinidae) Cyprinidae) und verwandten venvandten Ar Arten. Zool. Jahrb., Abt Anat. Anat. Ontog. 566-582. 2001.Jahrb., Ontog. Tiere, Tiere, 97, 97,566-582. M.. P. R., R., Searle, P. F., Wolffe, Wolffe, A. P., and Tata, J. R. (1983). (1983).Rapid estrogen Tenniswood, M metabolism and vitellogenin gene expression in Xenopus hepatocyte cultures. Mol. Cell 329-345. Cell Endocrinol. Endocrinol. 30, 30,329-345. Dickhoff, W. W., Gorbman, A. ((1981). Turner, R. T., Dickhoff, W., and Gorbman, 1981). Estrogen binding to hepatic stouti. Gen. Camp. Endocrinol. pacific hagfish hagfish Eptatretus stouti. Gen. Comp. Endocn'nol. 45, 26-29. 26-29. nuclei of of pacific 1984). Changes in serum Ueda, O., Hara, A., Yamauchi, Yamauchi, K., and Nagahama, Nagahama, Y. Y.((1984). Ueda, H., Hiroi, 0., concentrations of of steroid hormones, thyroxine, thyroxine, and vitellogenin during spawning migration of keta. Gen. Gen. Comp. Comp. Endocrinol. 53, of the chum salmon, salmon, Oncorhynchus keta. 203-211.1 . 203-21 Valotaire, Y.,Tenniswood, Tenniswood, M M.,. , L Lee Cuellec, Guellec, C., and Tata, J. J. R R.. (1984). (1984).The preparation Valotaire, Y., and characterization of (Salmo of vitellogenin vitelIogenin messenger RNA from rainbow trout (Salmo gairdneri). 217, 73-77. gairdneri). Biochem. Biochem. }. J . 217,73-77. van Bohemen, C. G., C., and Lambert, G. D. (1981). (1981). Estrogen synthesis in relation Lambert, J. C. to estrone, estradiol and vitellogenin plasma levels during during the reproductive cycle Camp. Endocrinol. 45, 105of Gen. Comp. 105of the female rainbow trout, Salmo gairdneri. Gen. 114. 114. van Bohemen, C. C., G. D., Coos, Coos, H. J. T., and van Oordt, P. G. G. W. J. G., Lambert, J. C. (1982a). (1982a).Estrone and estradiol participation during exogenous exogenous vitellogenesis in the Compo Endocrinol. female rainbow trout, Salmo gairdneri. gairdneri. Gen. Gen. Comp. Endocrinol. 46, 81-92. 81-92. van Bohemen, C. C. C., G., Lambert, J. J. C. G. D., D., and van Oordt, P. G. G. W. J. (1982b). (1982b).Vitellogenin ViteIIogenin induction by estradiol in estrone-primed rainbow trout, Salmo Salmo gairdneri. gairdneri. Gen. Gen. 136-139. Camp. Comp. Endocrinol. 46, 46,136-139. J.-M., and Sire, Sire, M.-F. ((1977). activit6 hydrolasique Vernier, J.-M., 1977). Plaquettes vitellines et activite acide au cours du developpement dheloppement embryonnaire de Ie le truite arc-en-ciel. arc-en-ciel. Etude ultrastructurale et biochimique. Biol. Bioi. Cell. Cell. 29, 99-112. 99-112. Wahli, Ryffel, C. G. U., and Weber, Weber, R. (1981). (1981). Vitellogenesis and the Wahli, W., Dawid, II.. B., Ryffel, 298-304. vitellogenin gene family. family. Science 212, 298-304.

5. 5.


405

Wallace, R. A. (1983). (1983). Interactions between between somatic cells and the growing oocyte of of WaUace, Xenopus cLaren Xenopus laevis. laeuis. In In "Current “Current Problems in Germ Cell Differentiation" Differentiation” (A. (A. M McLaren eds.), pp. 285-306. and C. L. Wylie, eds.), 285-306. Cambridge Univ. Press, London and New York. York. Vitellogenesis and oocyte growth in non-mammalian vertebrates. Wallace, R. A. ((1985). 1 985). Vitellogenesis In In "Developmental “Developmental Biology" Biology” (L. (L. Browder, ed.), ed.), Vol. Vol. 1, pp. 127-177. Pergamon, Pergamon, New York. York. Wallace, R. A., and Hollinger, T. G. (1979). T. G. (1979). Turnover of of endogenous, microinjected, and oocytes. Exp. 19, 277-287. sequestered protein in Xenopus Xenopus oocytes. E x p . Cell Cell Res. Res. 1119,277-287. Wallace, R. A., A., and Jared, D. W. (1968). (1968). Studies on on amphibian yolk. VII. Serum-phos Serum-phosphoprotein synthesis by vitellogenic Xenovitellogenic females and estrogen-treated estrogen-treated males of Xeno pus J. Biochem. Biochem. 46, 953-959. 953-959. pus laevis. laeuis. Can. Can.J. Wallace, R. A., and Selman, K. ((1981). 1981). Cellular and dynamic aspects of of oocyte growth in 325-343. teleosts. Am. Am. Zool. Zool. 21, 21,325-343. Wallace, R. A., A., and Selman, K. (1985). (1985). Major changes during vitellogenesis vitellogenesis and matura maturation of Fundulus Dev. Bioi. 10, 492-498. Fundulus oocytes. Deu. Biol. 1110,492-498. Wallace, R. A., A., Deufel, R. A., and Misulovin, Z. (1980). (1980).Protein incorporation by isolated amphibian oocytes. VI. Comparison of autologous and xenogeneic vitellogenins. vitellogenins. Comp. Biochem. Physiol. B 65B, 151151-155. Compo 155. Walter, P., Green, S., Greene, G., Krust, A., A., Bornert, J.-M., Jeltsch, J.-M., Staub, Staub, A., A,, Jensen, E., Scrace, G., Waterfield, M., M., and Chambon, P. (1985). (1985). Cloning of the Natl. Acad. Acad. Sci. Sci. V.SA. Proc. Natl. U S A . 82, 82, 7889-7893. 7889-7893. human estrogen receptor cDNA. Proc. Wang, S.-Y., S.-Y., and Williams, D. L. (1982). (1982). Biosynthesis of of the vitellogenins. vitellogenins. Identification Identification and characterization characterization of of nonphosphorylated precursors to avian vitellogenin I and Bioi. Chem. vitellogenin II. 11. J. J. Biol. Chem. 257, 3837-3846. 3837-3846. Wang, S.-Y., D. E., D. L. ((1983). 1983). Purification of S.-Y., Smith, Smith, D. E., and Williams, D. of avian vitellogenin III: II. Biochemistry 6206-6212. 111: comparisons with vitellogenins I and 11. Biochemistry 22, 22,6206-6212. Wangh, L. J. ((1982). 1 982). Glucocorticoids act together with estrogens estrogens and thyroid hormones in regulating the synthesis and secretion of Xenopus Xenopus vitellogenin, vitellogenin, serum albumin and fibrinogen. Dev. 294-298. D e n Bioi. Biol. 89, 89,294-298. Welshons, W. V., V., Lieberman, M. E Liebennan, M. E.,. , and Gorski, J. (1984). (1984).Nuclear Nuclear localization of of unoc unoc747-749. Nature (London) (London)307, 307,747-749. cupied oestrogen receptors. Nature White, H. H. B. ((1985). 1985). Biotin-binding oocytes. Ann. Biotin-binding proteins and biotin transport transport to oocytes. Ann. N. N.Y. Y. Acad. Sci. Sci. 447, 202-21 1. 447,202-211. Acad. Wiegand, M. D. ((1982). 1 982). Vitellogenesis Vitellogenesis in fishes. In In "Reproductive “Reproductive Physiology of Fish" Fish” (C. J. J. Richter and H. H. J. J. T. Goos, compilers), 146. Pudoc, Wageningen. compilers), pp. 136136-146. Wageningen. (1982).Synthesis of lipids by the rainbow trout (Salmo (Salmo Wiegand, M. D., and Idler, D. D. R. (1982). J. Zool. 60, 2683-2693. gairdneri) Can.J. 60,2683-2693. gafrdneri) ovary in vitro. Can. 1980). Effects of the salmon gonadotropin (SG-GlOO) Wiegand, M M.. D., and Peter, R. E. ((1980). (SG-G100) on plasma lipids in the goldfish, Carassius 967-972. Carussius auratus. aurutus. Can. Can.J. J. Zool. 58, 58,967-972. Wiley, H. S., ultiple vitello S . , and Wallace, R. A. (1981). (1981). The structure of of vitellogenin. M Multiple vitellogenins in Xenopus Xenopus laevis laeuis give rise to multiple forms forms of of the yolk proteins. J. J . Bioi. Biol. 8626-8634. Chem. 256,8626-8634. Chem. 256, Wingfield, J. J. C. ((1980). 1980). Sex-steroid binding proteins in vertebrate blood. In In Hormones Hormones:: Adaptation and Evolution" (S. Ishii, T. Hirono, and M. Wada, eds.), eds.), pp. 135-144. 135-144. Evolution” (S. Jpn. Soc. Press, Tokyo. Jpn. Sci. SOC. Wiskocil, R., Bensky, P., Dower, W., W., Goldberger, F., Gordon, J. I., and Deeley, R. G. Goldberger, R. F., (1980). (1980).Coordinate regulation regulation of of two two estrogen-dependent genes in avian liver. liver. Proc. Proc. Natl. Acad. Sci. A. 77, 4474-4478. Sci. U.S USA. 77,4474-4478. Natl. Acad.

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Wolffe, A. P., Glover, J. F., F., Martin, S. S. C., C., Tenniswood, Tenniswood, M. P. R., Williams, J. L., and Tata, J. J. R. ((1985). 1985). Deinduction of transcription transcription of of Xenopus Xenopus 74-kDa albumin genes ]' and destabilization ofmRNA mRNA by estrogen in in vivo uiuo and in hepatocyte cultures. Eur. Eur.J. destabilization of Biochem. 489-496. Biochem. 146, 146,489-496. Wright, C. V. E., E., Wright, S. S. C., C., and Knowland, J. (1983). (1983).Partial purification purification of of estradiol receptor from Xenopus Xenopus laevis laeuis liver and levels of of receptor in relation to estradiol 973-977. concentration. EMBO], EMBO], 2, 2,973-977. 1980). Sexual patterns of Yu, J. Y.-L., Dickhoff, W. W., and Gorbman, Gorbman, A. ((1980). of protein metab metabolism in liver and plasma of of hagfish, Eptatretus stouti, with special reference to Compo Biochem. Biochem. Physiol. 1 1- 1 17. vitellogenesis. Physiol. B B 65B, 65B, 1111-117. vitellogenesis. Comp. Yurowitzky, Y. G., and Milman, L. S. ((1972). 1972). Changes in enzyme activity of of glycogen fassilis L. and hexose metabolism Misqurnusfossilis metabolism during oocyte maturation in a teleost, Misqurnus Wilhelm 171, 48-54. Wilhelm Roux' Roux’ Arch. Entwicklungsmech. Org. Org. 171, 48-54. Yurowitzky, Y. G., and Milman, L. S. S. ((1975). 1975). Changes Changes in activity of of enzymes enzymes of of glycogen metabolism in loach oocytes and embryos. Biochemistry (Engl. 821(Engl. Transl.) Transl.) 40, 40,821825. 825. Yusko, S., 1981). Receptor-mediated Yusko, S., Roth, T. F., and Smith, T. T. ((1981). Receptor-mediated vitellogenin vitellogenin binding to 43-50. Biochem. ]. J . 200, 200,43-50. chicken oocyte. Biachem. Zagalsky, P. P. F., F., Gilchrist, Gilchrist, B. M., M., Clark, R. J. J. H., and Fairclough, D. D. P. P. (1983). (1983). The canthaxanthin-lipovitellin canthaxanthin-lipovitellinof Branchipus Branchipus stagnalis stagnalis (L). (L). (Crustacea: (Crustacea: Anostraca) Anostraca):: A A resonance resonance Raman and circular dichroism study. study. Compo Comp. Biochem. Biochem. Physial. Physiol. B 73B, 73B, 163-167. 163- 167.

6 YOLK ABSORPTION ABSORPTION IN EMBRYONIC EMBRYONIC YOLK LARVAL FISHES AND LARVAL

A . HEMING HEMING THOMAS A Pulmonary Division Division Pulmonary Department of of Internal Internal Medicine Medicine Department University of of Texas Texas Medical M edical Branch Branch University Galveston, Texas Texas 77550-2780 77550-2780 Galveston,

BUDDINGTON RANDAL KK.. BUDDINGTON Department of of Physiology Physiology Department University of of California California University Los Angeles, Angeles, California California 90024 90024 Los

I. Introduction I. Introduction II. 11. Structural Structural Aspects Aspects of of Yolk Yolk Absorption Absorption A. A. Yolk Yolk Morphology Morphology B. B. Meroblastic Meroblastic Fishes Fishes C. C . Holoblastic Holoblastic Fishes Fishes Ill. 111. Yolk Yolk Composition Composition during during Development Development A. A. Dry Dry Matter Matter and and Water Water Content Content B. B. Protein Protein C. C . Lipid Lipid D. D. Carbohydrates Carbohydrates E. E. Caloric Caloric Content Content IV. IV. Rate Rate of of Yolk Yolk Absorption Absorption V. V. Efficiency Efficiency of of Yolk Yolk Utilization Utilization A. A. Biotic Biotic Factors Factors B. B. Abiotic Abiotic Factors Factors VI. VI. Nonyolk Nonyolk Nutrient Nutrient Sources Sources during during Early Early Development Development A. A. Piitter's Putter’s Theory Theory B. embranes and B. Egg Egg M Membranes and Perivitelline Perivitelline Fluid Fluid C. C. Viviparity Viviparity D. D. Mixed Mixed Feeding Feeding VII. VII. Nutrition Nutrition of of Embryos Embryos and and Larvae Larvae References References 407 407 FISH FISHPHYSIOLOGY, PHYSIOLOGY, VOL. VOL.XIA XIA

Copyright©0 1988 1988by byAcademic AcademicPress, Press, Inc. Inc. Copyright All rights rightsof ofreproduction reproduction in inany anyform formreserved. reserved. All

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THOMAS A. A. HEMING HEMING AND AND RANDAL RANDAL K. K. BUDDINGTON BUDDING TON THOMAS

I. INTRODUCTION INTRODUCTION I.

of our knowledge regarding yolk absorption is A major portion of based on on species species possessing possessing large, large, demersal demersal eggs, eggs, which which are are adapted adapted based pre for colder waters and long incubation periods. This is evident in previous reviews reviews of of yolk yolk utilization by Hayes Hayes (1949), ( 1949), Smith Smith (1957, ( 1957, 1958), 1958), vious utilization by ( 1967), Blaxter (1969), ( 1969), Terner (1979), (1979), and Boulekbache Williams (1967), ( 1981). Development Development of of culture culture techniques techniques for for other other species species and and inin (1981). of addiaddi creasing ecological concerns, however, have elicited research of particularly marine fishes. This information has been tional groups, particularly incorporated and contrasted in in the present review. review. incorporated and contrasted the present A fish egg can be considered a semiclosed system. Once the egg membrane(s) has been hardened mem membrane(s) hardened by exposure to water, the membrane(s) permits permits gas gas exchange but is is relatively impervious to to most most brane(s) exchange but relatively impervious of fish embryos are dependent solutes. As a consequence, the majority of on endogenous yolk reserves to supply the substrates for energy pro pro(see Section duction and growth. Viviparous fishes are are an an exception (see VI,C). Both the rate of of yolk utiliutili VI,C). of yolk absorption and the efficiency of zation of early zation are are important determinants of early development, growth, and ultimately dependent on the availability survival. Larval Larval survival is ultimately of food food in in sufficient sufficient quantity and of of adequate quality after after yolk yolk rere of quantity and adequate quality pres serves are exhausted. It follows that there are strong selective presof yolk absorption, development sures synchronizing completion of of the capability of of feeding, and the availability of of suitable food of (Barns, 1969; 1969; Rosenthal and Alderdice, 1976). 1976). As well, since large (Barns, size size confers confers certain certain advantages advantages on on larvae, larvae, there there are are strong selective selective pressures pressures to to maximize the the efficiency with with which which yolk yolk is is converted converted into into tissues. tissues. Larger Larger larvae larvae of of aa given given species species can can be be expected to to be stronger swimmers (Hunter, 1972), 1972), less affected by competition (Hulata (Hulata et al., al., 1976), 1976), more more resistant resistant to to starvation starvation (Blaxter (Blaxter and and Hempel, Hempel, 1963), 1963), less susceptible to predation (Ware, (Ware, 1975), 1975), able to com commence mence feeding feeding earlier earlier (Wallace (Wallace and and Aasjord, Aasjord, 1984a), 1984a),and and able able to to have have increased success at first feeding (Braum, (Braum, 1967; 1967; Ellertsen et al., al., 1980). 1980). The The rate rate and and efficiency efficiency of of yolk yolk absorption absorption are are influenced influenced by by a number number of of environmental environmental factors, factors, including including temperature, temperature, light, light, oxy oxygen gen concentration, concentration, and and salinity. salinity. Fish Fish eggs eggs are are not not motile, motile, however, however, and and thus thus developing developing embryos embryos are are unable unable to to actively actively exploit exploit the the most most favorable favorable environments environments available, available, at at least least until until after after hatching. hatching. Only Only species species that that utilize utilize reproductive reproductive strategies strategies such such as as viviparity viviparity or or mouth mouth brooding may, through parental behaviors, be able to manipulate egg may,

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YOLK ABSORPTION IN IN EMBRYONIC EMBRYONIC AND AND LARVAL FISHES YOLK LARVAL FISHES

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incubation conditions. It is selectively advantageous, therefore, for a species to produce eggs that can develop successfully within a range of “expected” incubation conditions. The scope of “expected” of "expected" of these "expected" conditions will depend on those conditions experienced during evo evoof the species. For some fishes, the fl fluctuation environmenlution of uctuation in environmen tal parameters may be relatively slight (e.g., abyssal marine habitats), while for others it may be large (e.g., some temperature freshwater habitats). habitats). From an applied standpoint, there has been an interest in of environmental factors factors on yolk absorp absorpdetermining the influence of tion-particularly the effects of temperature, since it is generally the tion-particularly most variable parameter and the most easily controlled in culture settings. settings, A review review of of present present knowledge knowledge regarding regarding yolk yolk absorption absorption in in fish is is hindered somewhat by the use of of many different and often impre imprestaging. For the pur purcisely defined terminologies for developmental staging. pose of our review, we have adopted the generalized terms defined below. below. 1. Embryo-the 1. Embryo-the developing developing fish prior prior to to hatch. hatch. 2. sh after 2. Eleutheroembryo-the Eleutheroembryo-the developing developing fi fish after hatch hatch until until the the fish, parturi parturior, in the case case of viviparous fish, initiation of feeding or, tion 1975). For For our our purposes, purposes, feeding feeding refers refers to to the the in intion (Balon, (Balon, 1975). gestion gestion of of exogenous exogenous matter matter into into the the stomach stomach or or the the capability capability to so, rather rather than than behavioral behavioral responses responses to to potential potential food food to do do so, items. comitems. Defined Defined in in this this way, way, feeding feeding is is independent independent of com plete independent of of the the capabil capabilplete yolk yolk absorption absorption and and may may be independent ity ity to to digest digest and and utilize utilize ingested ingested material. material. 3. 3. Larva-the Larva-the developing developing fish fish after after initiation initiation of of feeding feeding or or partu parturition juvenile, characterized characterized by by aa full full complement complement of of rition until until a juvenile, minute minute adult adult features, features, is is attained. attained. 4. 4. Yolk-the Yolk-the nutritional nutritional reserves reserves provided provided in in the ovum, ovum, includ including ing those those associated associated with with the the yolk yolk platelets platelets and and oil oil globules. globules. A number number of of authors authors categorize categorize the the yolk yolk sac sac contents contents into into "yolk" “yolk” and basis of of visual visual appearance. appearance. For For simplicity, simplicity, we we and "oil" “oil” on on the basis have have regarded regarded these these categories categories as as equivalent equivalent to to yolk yolk platelets platelets and and oil oil globules, globules, respectively. respectively. 5. 5. Tissues-the Tissues-the body body of of the the developing developing fish fish including including the the yolk yolk sac sac but but without without the the yolk. yolk. Throughout Throughout this this review, review, we we use use the the common common and and scientific scientific names names of of fishes 1980). by Robins Robins et ai. al. ((1980). fishes listed listed by

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THOMAS A. HEMING AND RANDAL K. BUDDINGTON

II. STRUCTURAL 11. STRUCTURAL ASPECTS OF YOLK YOLK ABSORPTION

A. Yolk Morphology The structural components of fish yolk include yolk platelets and oil globules. The majority of yolk platelets are round or oval in shape, flattened in one plane, and 4-15 4-15 1Lm pm in length. Larger platelets appear to be characteristic of species possessing larger eggs (Grodzinski, (Grodzinski, 1973). 1973).Platelet size also varies within each egg, with the deeper, more centrally located platelets tending to be larger and more homogenous than the superficial peripheral ones (Vernier (Vernier and Sire, 1977; 1977; Hamlett and Wourms, 1984). 1984). Each platelet consists of an outer sheath and a central core (Fig. (Fig. 1). 1). The sheath forms forms a semipermeable bilayer around the core (Grodzinski, (Grodzinski, 1973) 1973)and contains mucopolysaccharides (Ohno al., 1964). (Ohno et al., 1964). The core is is composed of lipovitellin and phos phosvitin, or analogous lipoproteins and phosphoproteins (Fujii, (Fujii, 1960; 1960; Wallace et al., al., 1966; 1966; Jared and Wallace, 1968). 1968). These core proteins may or may not be arranged in 1 , 1982; in a crystalline lattice (Lange, (Lange, 198 1981,1982;

'-l----- sm

��\--- a l

�m1.mlt=fC �

cc

}

8

Fig. 1. structure of ofAmia Fig. 1. Model of a yolk platelet based mainly mainly on the structure Amia platelets. The (A) outer sheath; sheath; (B) (B) main body; body; sm, superficial membrane; aI, al, interior: (A) cuts reveal its interior: sm, superfi cial membrane; amorphous superficial superficial layer; layer; fc, fibrillar fibrillar cortex; cortex; cc, crystalline crystalline core. [From [From Grodzinski Grodzihski amorphous (1973).] (1973).]

6.

FISHES YOLK ABSORPTION ABSORPTION IN EMBRYONIC EMBRYONIC AND AND LARVAL LARVAL FISHES YOLK

411 411

Lange et et al., al., 1982). Moreover, M oreover, the crystalline structure may be lost as 1970). the ova mature (Balinsky, 1970). Oil globules are located among the yolk platelets. Globule number vary greatly among species, from innumerable small globules and size vary in the micrometer micrometer diameter range to singular large globules in the millimeter millimeter diameter range. The globules contain primarily triglycertriglycer ides, although proteins (Grodzinski, 1973), 1973), wax esters (Vetter et al., al., 1983), and carotenoid pigments (Nakagawa and Tsuchiya, 1971) 1983), 1971) are also present in some species. M eroblastic Fishes B. Meroblastic of most fishes fishes (elasmobranchs and Meroblastic cleavage, typical of teleosts), results in the formation of of an extraembryonic yolk sac. A characteristic feature of of this extraembryonic sac is the yolk syncytium, a specialized tissue responsible for absorption of yolk. The presumppresump tive yolk syncytium, the periblast, is recognizable in the fertilized 1982). As cleavage pro teleost egg at the one-cell stage (Yamamoto, (Yamamoto, 1982). proceeds, numerous free nuclei appear in the periblast, thus transforming transforming the layer into a true syncytium. meso In teleost eggs, the yolk syncytium together together with overlaying mesoEndo derm and ectoderm spreads to enclose the entire yolk mass. Endoderm does not follow the movement of of the teleost blastodisc rim and, consequently, Ab consequently, the yolk is not enclosed by an endodermal layer. Absorption of yolk nutrients in teleosts, therefore, occurs without any involvement of endodermal cells or the gut (Bachop (Bachop and Schwartz, 1974). 1974). A system of blood vessels, the vitelline circulation, develops within the walls of the yolk sac. In some areas, the endothelial wall of vitelline capillaries is incomplete incomplete and embryonic blood is in direct contact with the syncytium (Shimizu and Yamada, 1980) 1980).. Absorption of yolk involves endocytosis by the syncytium, intrasyncytial diges digestion and synthesis, and finally the release of yolk metabolites to the vitelline circulation circulation.. When yolk reserves are exhausted, the syncy syncytium is resorbed; it does not take part in formation of the permanent fish fish body (Yamada, (Yamada, 1959; 1959; Yamamoto, 1982). 1982). Two regions of the yolk syncytium can be distinguished distinguished on the basis of their fine structure (Shimizu and Yamada, 1980). 1980). One region, characterized by smooth endoplasmic endoplasmic reticulum, numerous mitochon mitochondria, and glycogen granules, is proposed to be responsible for carbo carbohydrate and/or lipid metabolism. This This region extends throughout the syncytium. syncytium. The The second second region region is is characterized characterized by by rough rough endoplasmic endoplasmic

412

BUDDINGTON THOMAS A. HEMING AND RANDAL K. BUDDING TON

reticulum reticulum and and Golgi Golgi complexes, complexes, and and extends extends in in portions portions across across the the syncytium region is is thought syncytium forming forming aa stratified stratified structure. structure. This This latter latter region thought to involved in proteinaceous sub to be be involved in the the synthesis synthesis and and transport transport of of proteinaceous subsoluble. protein must be dephosphorylated to become soluble. stances. Yolk protein Amirante 1972) suggested suggested that that fish yolk yolk proteins proteins are are solubilized solubilized by by Amirante ((1972) the Syncytial Golgi Golgi the action action of of calcium calcium and and phosphoprotein phosphoprotein phophatase. phophatase. Syncytial complexes complexes probably probably supply supply acid acid hydrolases hydrolases for for the the degradation degradation of of yolk Sire, 1977; 1977; Hamlett, Hamlett, et al., 1987). 1987). yolk platelets platelets (Vernier (Vernier and and Sire, In In addition addition to to the the syncytial syncytial layer, layer, the the yolk yolk itself itself contains contains enzymes enzymes (Hamor 1973) that that probably probably facilitate facilitate the the breakdown breakdown of of (Hamor and and GarSide, Garside, 1973) yolk (1977) described described yolk into into its its constituent constituent nutrients. nutrients. Vernier Vernier and and Sire Sire (1977) two types types of of yolk yolk platelets platelets with with different different enzyme enzyme contents. contents. One One form, form, the the embryonic embryonic platelet platelet type, type, has has an an enzyme enzyme load load that that allows allows nutrients nutrients to released prior second or of the the syncytium. syncytium. The The second or to be be released prior to to establishment establishment of

Ectoderm

Capill ories

Endoderm

Yol Syncyllum

Fig. 2. 2. Idealized diagram diagram of the cellular organization organization in a preimplantation preimplantation shark Fig. yolk sac. sac. The teleost yolk sac is similar in structure structure except that it it lacks endoderm. endodem. [From [From Woums (1984).] (1984).1 Hamlett and Wourms

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YOLK ABSORPTION ABSORPTION IN IN EMBRYONIC EMBRYONIC AND AND LARVAL FISHES YOLK LARVAL FISHES

413

usual platelet type lacks this enzyme load and is digested by syncytial enzymes. While the extraembryonic yolk sac with its yolk syncytium is the sole site of of yolk absorption in teleosts, this is not the case in chonchon ratfish). In holocephali holocephalidrichthyean fishes (sharks, skates, rays and ratfish). of the yolk mass is enclosed by ans, for instance, only a small portion of the 1906). The The remainder remainder breaks breaks up up into into aa viscous viscous the yolk yolk sac sac (Dean, (Dean, 1906). fluid, fl uid, which is first absorbed via the external gills of the embryo and later ingested through the mouth. This ingestion of yolk nutrients is comparable, in a general sense, to that exhibited by oophagous sharks, (Fuwhose viviparous embryos ingest ova present in the same uteri (Fu 1981; 1983). jita, 198 1 ; Gilmore et al., 1983). In I n elasmobranchs, elasmobranchs, the the formation formation of of an an archenteron archenteron at at the posterior posterior edge blastodisc during during gastrulation gastrulation results results in in aa yolk yolk sac sac that that edge of of the blastodisc possesses 2). This endodermal layer medi medipossesses an an endodermal endodermal layer layer (Fig. (Fig. 2). ates transfer of yolk yolk metabolites metabolites from from the the syncytium syncytium to to the the vitel vitelates the transfer 1987). Moreover, the elasmobranch line circulation (Hamlett et al., 1987). yolk sac sac is continuous with the alimentary tract via a yolk stalk, and thus majority of of yolk yolk is is digested digested within within the the intestine. intestine. Yolk plate platethus the majority lets moved by yolk sac lets are are moved by ciliary ciliary action action from from the the yolk sac through through the yolk yolk stalk (TeWinkel, Winkel, 1943; 1943; Baranes Baranes and and Wen Wenstalk and and into into the the spiral spiral intestine intestine (Te dling, 1981). 1981). An An internal internal yolk yolk storage storage organ organ may may or or may may not not be dling, present. Enzymatic activity in the gut is established reilltively relgtively early in development, development, when when the the embryo embryo is is approximately approximately one-quarter one-quarter its its size size at 943). (Te Winkel, Winkel, 11943). at parturition parturition (Te C. C. Holoblastic Holoblastic Fishes Fishes A A few few fish fish species species develop develop holoblastically holoblastically (e.g., (e.g., lampreys lampreys and and chondrosteans). endodermal and and lateral lateral plates plates chondrosteans). In In these these species, species, the endodermal fuse ventral line fuse along along the the mid midventral line forming forming an an intraembryonic intraembryonic yolk yolk sac. sac. As As aa result, result, all all three three germ germ layers layers enclose enclose the the yolk yolk mass mass (Ballard (Ballard and and Ginzburg, 1980). The The resultant resultant intraembryonic intraembryonic yolk yolk sac sac directly directly par parGinzburg, 1980). ticipates ticipates in in formation formation of of the the alimentary alimentary canal. canal. During During posthatch posthatch devel development yolk sac sac is is separated separated into into two two major major opment of of chondrosteans, chondrosteans, the yolk regions, regions, each each of of which which develops develops separate separate blood blood drainages drainages (Ballard (Ballard and and Needham, 1964).The The distal distal region region comprises comprises the the intestine intestine and and spiral spiral Needham, 1964). valve, corresponding blood blood supply supply proceeds proceeds to to the the liver. liver. Yolk Yolk valve, and and the corresponding within is the the first first to to be utilized. utilized. The The second second region region in inwithin this this region region is cludes cludes the the stomach stomach and and esophagus esophagus and and develops develops aa blood blood supply supply that that proceeds sinus venosus. venosus. This This region region is is the the last last portion portion proceeds directly directly to to the sinus

4 14 414

THOMAS THOMAS A. A. HEMING HEMING AND AND RANDAL RANDAL K. K. BUDDINGTON BUDDINGTON

of of the the alimentary alimentary canal canal to to differentiate, differentiate, and and yolk yolk is is retained retained there there longer (Buddington and Christofferson, 1985). longer (Buddington and Christofferson, 1985). Although Although hydrolytic hydrolytic enzymes enzymes are are present present within within the the developing developing alimentary canal, their activities are low (Korzhuev and alimentary canal, their activities are low (Korzhuev and Sharkova, Sharkova, 1967; 1967; Buddington Buddington and and Doroshov, Doroshov, 1986). 1986). The The existence existence of of yolk yolk mate material yolk sac rial within within endodermal endodermaI cells cells lining lining the the yolk sac implies implies that that endocyto endocytosis and sis and intracellular intracellular digestion digestion may may be be the the primary primary mechanisms mechanisms by which which yolk yolk nutrients nutrients are are made made available available (Krayushkina, (Krayushkina, 1957; 1957; Bud Buddington dington and and Christofferson, Christofferson, 1985). 1985). Thus, Thus, the the intraembryonic intraembryonic yolk yolk sac sac of yolk sac of holoblastic holoblastic fishes fishes and and the the extraembryonic extraembryonic yolk sac of of meroblastic meroblastic fishes exhibit exhibit similar fishes similar mechanisms mechanisms for for mobilization mobilization of of yolk yolk nutrients. nutrients.

III. YOLK COMPOSITION DURING DEVELOPMENT 111.

Selective sh exploiting wide diverdiver Selective pressures pressures have have resulted resulted in in fi fish exploiting aa wide sity of reproductive strategies. As a consequence, egg size and fecun sity of reproductive strategies. a consequence, egg size and fecundity fishes from 0.7 mm dity vary vary among among species, species, in in oviparous oviparous fishes from about about 0.7 mm egg egg diameter (e.g., convict surgeon fish Acanthurus triostegus) to diameter (e.g., convict surgeon fish triostegus) to greater greater than 10 mm diameter (e.g., chinook salmon than 10 mm diameter (e.g., chinook salmon Oncorhynchus tsha tshawytscha), wytscha), with spawns varying from less than 100 100 eggs per female (e.g., (e.g., mouth-brooding mouth-brooding cichlid cichlid Labeotropheus fuelleborni) fuelleborni) to to more more than Viviparous fi sh tend than 9,000,000 9,000,000 (e.g., (e.g., Atlantic Atlantic cod cod Gadus morhua). morhua).Viviparous fish tend to produce fewer but proportionally proportionally larger eggs. These differences in egg egg size size and and number number imply imply maternal maternal investment investment per per egg egg differs differs widely widely among among species. species. The The deposition deposition of of nutrients nutrients into into the the egg egg during during oogenesis been reviewed Mommsen and oogenesis has has been reviewed in in this this volume volume by by Mommsen and Walsh Walsh (this volume, Chapter 5). 5). (this The nutrient composition of of fish eggs is species-specific (Table I). Within a given species, as well, welI, egg quality varies as a function of of maternal age, weight, and diet (Kamler, (Kamler, 1976; 1976; Kuznetsov and KhaliKhali tov, 1979). 1979). Despite these differences, the dynamics of of yolk absorption are similar among groups. Following fertilization, the developing emem bryo begins to utilize yolk nutrients. This is accompanied by increas increasing consumption of of oxygen, particularly particularly after the blastula stage is reached. As development proceeds, the absolute and relative compocompo 1972, 1974, 1976). sition of of the yolk changes (Nakagawa and Tsuchiya, 1972,1974,1976). Various approaches (proximal ra (proximal analysis, respiratory quotients, radiolabeled substrates) have been used to investigate the sequence Genwith which yolk nutrients are catabolized for energy production. Gen erally, erally, carbohydrate, lipid, and protein are consumed prior to hatch-

Table Table II Chemical Composition Composition of Fish Eggs Eggs Chemical Percentage Percentage of of dry dry weight weight

Dry weight

Species Species

mg

% %

Protein

Lipid

Carbohydrate

Ash

Acipenser transmontanus (white sturgeon) sturgeon) (white Coregonus albula (vendace) (vendace) Coregonus lavaretus (whitefish) (whitefish) Cyprinus carpio

6.25" 6.25"

23.8 23.8

67 67

30 30

-

33

16.2'ib 16.27b

-

64.4

25.8

-

8.5 8.5

Dabrowski Dabrowski and and Luczynski Luczynski (1984) (1984)

15.6b 15.6b

-

60.3

27.7

-

9.8 9.8

Dabrowski 1984) Dabrowski and and Luczynski Luczynski ((1984)

-

30.4" 30.4" 10.2b 1O.2b

64.3 64.3 58.3-59.2 58.3-59.2

5.9 5.4-29.3 5.4-29.3

3.7 3.7 1.5-6.2 1.5-6.2

6.3 6.3

0.283" 0.283" 0.29Bb 0.298b 0.232" 0.232a

22.1 22.1 10.4 10.4 46.3

66.4 66.4 56.7

-

-

20.5 16.8 16.8 52.0

-

2. 2.11 8.4 3.0 3.0

M oroz and 1976) Moroz and Luzhin Luzhin ((1976) M oroz and 1976); Moroz and Luzhin Luzhin ((1976); Kamler 1976) Kamler ((1976) Lapin and Lapin and Matsuk Matsuk (1979) (1979) atsuk (1979) Lapin Lapin and and M Matsuk (1979) 1981a) Eldridge Eldridge et et al. al. ((1981a)

0.051AF" 0.051AFa 0.049AFb 0.049AFb

-

79.3 77.4

15.4 15.4 19.4

5.3 5.3 3.2

7.2 7.2

42.1" 42.1a

-

41.3 33.Bb 33.8b

56.2 59.8-71.3 59.8-71.3

11.4 1 1.4

0.6

3.8-3.9 3.8-3.9

49.7b 49.7b

36.0

52.2

36.1

1.0

2.8

-

29.3"

71.6

13.0 13.0

a >

2 2

E

QI It:

I I

2

Relative weight Re 1 0 1 ive egg we i g hl

3 or

4 volume

Fig. 5. 5. Relationship between the rate of yolk absorption absorption and initial egg size of fish: fish: , Fig. (1)Oncorhynchus keta (Beacham (Beacham and Murray, Murray, 1985); (2)O. 0.keta (Beacham (Beachamet al., 1985); (1) 1985); (2) al., 1985); (3) O. 0. kisutch (Beacham (Beacham et al., 1985); 1985); (4) (4) Salvelinus Saloelinus alpinus (Wallace (Wallace and Aasjord, Aasjord, (3) 1984a); (5) (5) 0. (Yastrebkov, 1966); 1966); (6) (6) Salmo salar (Kazakov, 1981); (7) 1984a); O. gorbuscha (Yastrebkov, (Kazakov, 1981); (7) S. (Escaffreand Bergot, Bergot, 1984); 1984);(8) (8)O. 0.tshawytscha (Rombough, (Rombough,1985); 1985);(9) (9) Clupea gairdneri (Escaffre harengus harengus (Blaxter (Blaxter and and Hempel, 1963); 1963); (10) (lo) Gadus morhua (Knutsen (Knutsen and and harengus 1985). Tilseth, 1985).

6. 6. YOLK YOLK ABSORPTION ABSORPTION

427

IN EMBRYONIC AND IN EMBRYONIC AND LARVAL LARVAL FISHES FISHES

species prolongs the period of endogenous nutrition (fertilization species (fertilization to complete complete yolk yolk absorption) absorption) by about about 1.3 1.3 times. times. In these these latter latter species, species, the the rate rate of of yolk yolk absorption absorption per per unit unit area area of of syncytium syncytium must must decrease decrease as as egg size increases. In terms terms of of the the rate rate of of consumption, consumption, many many teleosts teleosts exhibit exhibit three three rst or pre hatch phase 6). The fi first prehatch distinct phases of yolk absorption (Fig. (Fig. 6). is characterized by slow but steadily increasing rates of of yolk absorp absorption. globules are tion. Yolk Yolk platelets platelets and and oil oil globules are consumed consumed at at approximately approximately the same relative rate during this phase (Nakagawa (Nakagawa and Tsuchiya, 1972). Shortly 1972). Shortly before before and and at at hatching, hatching, the the rate rate of of yolk yolk absorption absorption in increases creases rapidly, rapidly, probably probably in in response to to both both an an increase increase in in absorptive absorptive surface surface area area due due to to changes changes in in yolk sac sac shape shape and and an an increase increase in the the metabolic activity of the yolk syncytium. This marks the beginning of of the the second second or or posthatch posthatch phase phase of of absorption, absorption, which which is is characterized characterized by by aa relatively relatively high absorption. During high and and constant constant rate rate of of absorption. During the the posthatch posthatch phase, over the oil globule phase, yolk yolk platelets platelets are are preferentially preferentially consumed consumed over the oil globule 180

-.�

�: ·1.

140

1\ \

;.I

�

""

P

'j

I

100

100

a J

\.

60

20

20

i b, I

\

•

I 60 20 60 20 Prehatch phase Pre hatch phase

I

100 lao

'\

."",- 0•

I

140 I40

180 I80

I

Terminal phase I Posthatch Porthatch phase phose ITerminol phased I

Days Days

postfertilization postfer t i l izo tion

Fig. 6. Typical changes in dry yolk weight of of teleost fish. fish. Data from chinook salmon Fig. 8°C (T. (T.A. A, Heming, unpublished data). data). The period of of (Oncorhynchus tshawytscha) at 8°C absorption) has been divided into endogenous nutrition (fertilization to complete yolk absorption) of yolk absorption. The broken line represents three phases based on trends in the rate of 50% hatching. hatching. 50%

428 428

THOMAS THOMAS A. A. HEMING HEMING AND AND RANDAL RANDAL K. K. BUDDINGTON BUDDINGTON

(May, (May, 1974; 1974; Eldridge Eldridge et al., 1982; 1982; Li Li and and Mathias, Mathias, 1982; 1982; Quantz, Quantz, 1985). 1985). As As the the reserve reserve of of yolk yolk platelets platelets nears nears exhaustion, exhaustion, the the rate rate of of yolk slows, probably probably in in response response to to both both aa decrease decrease in in yolk absorption absorption slows, absorptive absorptive surface surface area area as as the the yolk yolk sac sac shrinks shrinks and and the the changing changing com composition position of of yolk. yolk. This This marks marks the the beginning beginning of of the the terminal terminal phase phase of of absorption, absorption, during during which which the the remaining remaining yolk, yolk, predominantly predominantly oil oil glob globules, is consumed. consumed. ules, is Factors Factors that that increase increase or or decrease decrease the the metabolic metabolic activity activity of of the the yolk yolk syncytium expected to to increase increase or or decrease, decrease, respectively, respectively, the the syncytium can can be expected rate rate of of yolk yolk absorption absorption is is reduced, reduced, for for rate of of yolk yolk absorption. absorption. The rate example, oxygen concentrations 1965; Ha Haexample, by by low low dissolved dissolved oxygen concentrations (Brannon, (Brannon, 1965; mor 1977b),subsub- and and supraoptimal supraoptimal salinities salinities (May, (May, 1974; 1974; mor and and Garside, Garside, 1977b), Santerre, Santerre, 1976), 1976), high high ammonia ammonia concentrations concentrations (Fedorov (Fedorov and and Smirnova, 1978), and and sublethal sublethal concentrations concentrations of of toxic toxic xenobiotics xenobiotics Smirnova, 1978), (Crawford 1985). Some Some xenobiotics xenobiotics induce induce deformities deformities (Crawford and and Guarino, Guarino, 1985). in 1972). The The struc strucin the the yolk yolk sac sac (e.g., (e.g., crude crude oil oil fractions fractions;; Kiihnhold, Kuhnhold, 1972). ture yolk itself itself may may be be sensitive sensitive to to some some chemicals; chemicals; fuel-oil fuel-oil frac fracture of yolk tions tions can can cause cause coalescence coalescence of of the the oil oil globules globules in in fish yolk yolk (Ernst (Ernst et

8 7 6

3 2

O +---,---4---�--�--� 2 o

4

5

Fig. 7. Frequency distribution distribution of of the QI Qlo absorptionin fish, Fig. fish, based on O values for yolk absorption observations from 23 species. areas designate marine marine fi fish eggs. (From (From nu nu29 observations species. Shaded areas sh eggs. merous sources.) sources.) merous

6. 6.

429 429

YOLK ABSORPTION IN IN EMBRYONIC EMBRYONIC AND LARVAL LARVAL FISHES YOLK

al., 1977). 1977). The extent to to which yolk absorption is is influenced by such al., structural abnormalities is unclear. with temperature throughout most The rate of absorption increases with Qlo the range of thermal tolerance. Figure 7 summarizes the Q of the lO values 23 species species of fi fish, 1of 23 sh, at temperatures spanning the overall range of 130°C. The overall mean value is 2.916 2.916 (SE 0.166, n= = 29 observa observa30°C, (SE = 0. 166, n tions). As the upper limit of thermal tolerance is approached, the rate tions). Qlo (Fig. 8), probably of yolk absorption and hence the Q I O value decrease (Fig. processes. due to a breakdown of normal metabolic processes. Temperature has a differential effect on the absorption of yolk platelets and oil globules. Oil absorption appears to be affected more b y increases in temperature (Kuo (Kuo et al., al., than platelet consumption by 1973; May, May, 1974; 1974; Ehrlich and Muszynski, Muszynski, 1982). 1982). Thus, Thus, the Q Qlo 1973; O value l (Fig. 8). 8). for oil absorption is greater than that for platelet absorption (Fig. Near the lower limit of the tolerated thermal range, early life stages =

a),

--0.015 0..0. 1 5 r

.�

.

Q. ls 1:

0..67 ....• ..

c

0. .0. 10. JI � --0.010 3�

� - ,0 '"
"'" '::f..:;:�:w.:�:�:::�:: ::::�:::�::::::�:::::��:::::::#::�::::.:.::��:::�::�:�::::::�:;::::::: ::!::::::::::::�:::�

I

:',H:'i:" ::.;"::"H

I

" #*,,,,,,.;,,,,8:*"1;1

I

2 3 4

Fig. Fig. 1. 1. Distribution Distribution of of the the hatching hatching enzyme enzyme (EDTA-sensitive (EDTA-sensitive protease) protease) activity activity among among the the subcellular subcellular fractions fractions obtained obtained from from 0.3 0.3 M M sucrose sucrose homogenate homogenate of of medaka medaka embryos embryos at at some some developmental developmental stages. stages. Fraction Fraction H: H : whole whole homogenate; homogenate; fraction fraction 1: 1: 600g 600g Xx 10 1 0 min min pellet; pellet; fraction fraction 2: 2 : 600-1OOOg 600-1000g Xx 10 1 0 min min pellet; pellet; fraction fraction 3: 3 : lOO0g l OOOg Xx 10 10 min min to 15 min min pellet; pellet; fraction fraction 4: 4: supertant. supertant. Proteolytic Proteolytic activity activity was was assayed assayed in in to 10,000g 1O,000g xx 15 principle principle following following Kunitz Kunitz (1947). (1947). Dotted Dotted column column and and open open column column refer refer to to the the activity activity in in the the original original in the the absence absence and and presence presence of of55 mM mM EDTA, EDTA, respectively. respectively. Stages Stages 3 3 and and 44 in report report (Iuchi (Iuchi and and Yamagami, Yamagami, 1980) 1980) should should read read 22 and and 3, 3, respectively, respectively, as as shown shown in in this this figure. figure.

7. 7.

MECHANISMS OF HATCHING IN FISH

453

ing enzyme synthesis in fish fish embryos embryos is initiated in general just after lens formation but in advance of of eye pigmentation. According 1979), the According to to Yamamoto Yamamoto et al. al. ((1979), the hatching hatching gland gland ofmedaka of medaka continues continues to to produce produce secretory secretory granules granules until nearly the the prehatching prehatching stage. of the Golgi stage. A few secretory granules found in the trans face of apparatus were less electron-dense than most other granules, proba probably representing an immature state. Such immature granules could be found sometimes in day-5 1 , 11day before hatching). IIn n d a y 4 embryos (st. (st. 331, embryos close to the hatching stage, there were two types ooff secretory granules granules in in hatching hatching gland gland cells; cells; one one was was homogeneously homogeneously electron electrondense and the other consisted of an electron-dense electron-dense portion and a less dense dense portion. portion. In In the the latter, latter, the the electron electron dense dense portion portion often often took took aa crescent shape shape in the periphery of the granule, like a shell. shell. Such heterogeneity heterogeneity of of electron electron stainability stainability in in aa hatching-enzyme hatching-enzyme granule granule has been seen also in some cyprinid embryos, Brachydanio redo rerio and and has been seen also in some cyprinid embryos, Danio malabaricus (Willemse Denuce, 1973), (Willemse and and Denuc6, 1973), and and salmonid em embryos, Salmo gairdneri, s. S. trutta, S. pluvius trutta, Salvelinus !ontinalis, fontinalis, and s. (Yokoya 1976). As described above, histochemical stain (Yokoya and Ebina, 1976). stainability ability of of aa granule granule was was reported reported to to change change markedly markedly during during develop development. Although it remains uncertain whether or not such a granule of electron density, it is evident that the change is correlated with that of hatching-enzyme granules undergo some some physicochemical changes during drastic change change in in the electron electron density density of of the the during their their maturation. maturation. A drastic granules in their last maturation phase seems to be closely related to the secretion process. This problem will be discussed again in the next section. B. Ultrastructural B. Ultrastructural Changes Changes in in the the Hatching Hatching Gland Gland Associated with Secretion

1. H ISTOLOGICAL S TUDIES 1. HISTOLOGICAL STUDIES After After being being packaged packaged in in the the secretory secretory granules, granules, the the hatching hatching en enzyme secreted into zyme is is secreted into the the perivitelline perivitelline space, space, where where it it gains gains access access to to the egg envelope. In this the egg envelope. In this section, section, the the cellular cellular and and subcellular subcellular changes changes in in the the hatching hatching gland gland associated associated with with secretion secretion will will be be discussed. discussed. There so far far been been only only aa few few studies studies on on the the cellular cellular changes changes of There have have so the the hatching hatching gland gland during during secretion. secretion. In In their their histological histological studies, studies, Ishida 1944b) and Ishida ((1944b) and Ouji Ouji (1959a,b) (1959a,b)observed observed morphological morphological changes changes of of hatching-gland cells in Oryxias latipes and and Odontobutis obscura, obscuru, re rehatching-gland cells in Oryzias spectively. In the spectively. In the former, former, the the nucleus nucleus of of the the gland gland cell cell was was invisible invisible at at

454

KENJIRO YAMAGAMI YAMAGAMI KENJIRO

of secretion and when secretory granules were released. In the time of the latter, the nucleus nucleus remained remained in in the the gland gland cell, cell, while while the the granules granules the latter, the disappeared during secretion. However, a more detailed description disappeared during secretion. However, a more detailed description of gland-cell gland-cell changes changes was was possible possible only only with with the the electron electron microscope. microscope. of Yamamoto (1963) ( 1 963) reported reported that were three of secretory secretory Yamamoto that there there were three types types of of medaka embryos. Type 1 1 grangran granules in the hatching-gland cells of ules were were homogeneously homogeneously electron-dense electron-dense and and were were predominant predominant at at ules earlier develomental develomental stages. stages. Type Type 2 2 granules granules were were as as electron-dense electron-dense earlier as 1 but of higher electron as type 1 but contained a crescent-shaped shell shell of density. Type 3 granules contained somewhat granular contents with Iow an electron density as the cytoplasmic matrix; they also had an as low of electron-dense shell around the granular contents. The granules of this type type were were predominant in the the embryos embryos at later developmental developmental this predominant in at later of stages. Just before secretion, a small hole appeared at the apical end of the cell cell,, and type 3 granules seemed to be disintegrated within the cell. cell. E LECTRICALLY INDUCED S ECRETION I NDUCED SECRETION 2. 2. ELECTRICALLY

hatching It is is sometimes difficult to predict accurately when the hatchinggland cells of of an embryo initiate secretion under natural conditions. As will be discussed in detail later, several reagents or treatments have been reported that induce hatching-enzyme secretion in fish, of elec causing precocious hatching. Among them, an adequate dose of elec(AC) stimulation is quite effective in causing hatching-enzyme tric (AC) (Iuchi and Yamagami, Yamagami, secretion in medaka as well as in rainbow trout (Iuchi 1976a; 1976a; Yamamoto Yamamoto et al., 1979). 1979). Rainbow trout embryos that would hatch normally about day 19-20 19-20 after fertilization at 15°C 15°C could be induced to hatch precociously on day 16-17 16-17 when they were stimu stimulated with 100 100 V AC for 3 ss 10 10 times with 5-min intermissions. In this case, hatching-gland cells on the surface of of embryos became invisible a few minutes after the stimulation. When the dechorionated embryos were stimulated, hatching enzyme as determined by its caseinolytic activity (see (see later) later) increased in the medium (Iuchi (Iuchi and Yamagami, Yamagami, 1976a). 1976a). Medaka embryos embryos also hatch precociously upon electric stimulation (Fig. (Fig. 2). 2). When cultured normally in a shaking incubator at 30°C, 30"C, they hatch on day 6 if the day of fertilization was regarded as day 11(Yama (Yamagami, gami, 1960). 1960).Natural hatching of control embryos embryos begins early on day 6 and it takes almost one more day until all the control embryos embryos com com100 V AC plete hatching. However, stimulation of the embryos with 100 for 5 5 s early on day 55 (-25 (-25 h earlier than the beginning of natural

7. 7.


455

,...,

� 80 Dl c

:i: u

C eo .c

'0 " u

��

'tJ u C

20

O ��=-��=m��m=-x�m�,�oo=-��=-�I��-=��m=-�'��=-�Il�� t-- S th Day

1- 6 th Day

Devel o pm e n t a l slage

� 1 1h Day

Fig. Fig. 2. 2. Induction of of hatching enzyme secretion secretion by electric stimulation in the me medaka embryos. (A) (A) Scanning electron micrographs (SEMs) (SEMs) of median cuts of the head of the 5 min after stimulation. Many the embryos embryos at the prehatching stage stage (Ar) (A*)before and (A2) (A%) hatching-gland in the buccal wall in in (AI) (Al) but not in hatching-gland cells are are seen as round protrusions in (AZ) (B) Incidence of hatching of the the stimulated (0, (0,O) e) or unstimulated (control, x 200). (B) (A 2) ((x200). 0) embryos. The arrow indicates the time of application application of the electric stimulation stimulation (AC (AC 0) embryos. lOOV, 5 s). s). A bold bar in the figure figure indicates indicates the prehatching stage (Yamamoto (Yamamoto et et al., al., lOOV, 1979). 1979).

hatching) gave rise to precocious hatching of some embryos. embryos. Their hatching) as development proceeded, and most em emresponsiveness increased as bryos at prehatching prehatching stages could be induced to hatch. hatch. The gland gland cells bryos embryos at the prehatching stage are are considered to have been of the embryos mature, in the sense that all cells cells were ready to secrete the hatching

456

KENJIRO YAMAGAMI YAMAGAMI

enzyme upon stimulation. It was found found that almost all all gland gland cells cells enzyme upon stimulation. It was that almost completed their min after after the stimulation at latest. the stimulation at the the latest. completed their secretion secretion 5 min Exploiting this electric stimulation, stimulation, a a sequential sequential ultrastructural ultrastructural Exploiting this electric change of the the hatching-gland hatching-gland cells cells during during the the course course of of secretion secretion change of could be followed followed in medaka (Yamamoto al., 1979). 1979). The The gland gland cells cells in medaka (Yamamoto et al., could are arranged arranged side side by side and are covered covered by by aa sheet sheet of of squamous squamous b y side and are are epithelium on the the inner inner wall wall of of the the pharyngeal pharyngeal cavity. Each epithelial epithelial epithelium on cavity. Each cell has a Three adjoining adjoining epithelial epithelial cells cells meet meet at cell has a hexagonal hexagonal contour. contour. Three at the apical apical center center of of each underlying gland cell. Just Just before before electrical electrical the each underlying gland cell. stimulation the the gland gland cells cells were were full full of of secretory secretory granules granules of of homogehomoge stimulation neous electron electron density, density, with with the nucleus at the base. base. Near Near the the Golgi Golgi neous the nucleus at the apparatus, immature secretory granules with with lower electron density were observed. Soon Soon after after the the electric electric stimulation stimulation (usually (usually -30 -30 s), s), aa were observed. swelling of each gland gland cell cell occurred occurred and and the the secretory secretory granules granules within within swelling of each a more clearly clearly discernible discernible as as round round protrusions. protrusions. Every Every a cell cell became became more junction of epithelial cells cells was separated and and the the apical apical surface surface junction of three three epithelial was separated of underlying gland gland cell was exposed exposed (Fig. (Fig. 3). Inside the the gland gland of the the underlying cell was 3). Inside cell, a a coalescence of electron-dense electron-dense secretory secretory granules granules occurred occurred to cell, coalescence of to form large mass mass of of secretory secretory substance substance surrounded limiting form aa large surrounded by aa limiting membrane. The contents the coalesced mass appeared to be be com membrane. The contents of of the coalesced mass appeared to composed of fine fine granules, its electron electron density was reduced reduced remarkremark posed of granules, and and its density was ably. The uncoalesced gran ably. The electron electron density density of of the the contents contents of of aa few few uncoalesced granules, As aa ules, except except for for their their peripheral peripheral part, part, was was decreased decreased slightly. slightly. As result, appeared to result, these these granules granules appeared to have have aa crescent-shaped crescent-shaped shell of of high sehigh electron electron density. density. The The membrane membrane surrounding surrounding the the coalesced coalesced se-

3. SEMs of the hatching-gland hatching-gland cells ofmedaka embryos (A) (A) before and (B) (B) 30 s Fig. 3. hatching-gland cells were swollen and after electric stimulation. Upon stimulation, the hatching-gland granules became discernible. discernible. Every junction (arrow) (arrow) of adjOining adjoining epithe epithethe secretory granules lial cells covering cells was separated et al., 1979). 2 2 0 0(Yamamoto (Yamamoto ) et al., 1979). covering the gland cells separated ((x~2200)

7.

MECHANISMS OF OF HATCHING HATCHING IN IN FISH MECHANISMS FISH

457

cretory mass became united with the cell membrane membrane at the apex of of the orifice gland cell, forming an orifi ce through which the secretory substance ).It seems that the nucleus and flew Hew out into the buccal cavity (Fig. 4 4). cytoplasm including some endoplasmic reticuli still remained in some secretion. About 24 h after the secretion, the openings gland cells after secretion. at the epithelial junctions were reclosed and the open surface of of the flat, since any swollen gland cells were now absent epithelium was Hat, underneath. However, some gland cells containing an electron-dense irregu1ar-shaped irregular-shaped nucleus and many fragmented cisternae of rough en enreticulum but no secretory granules were found to persist doplasmic reticulum under under the the epithelium. epithelium. N ATURAL S ECRETION 3. NATURAL SECRETION

In cially induced In contrast contrast to to the the situation situation with with artifi artificially induced hatching, hatching, the the hatching-gland cells in the process of natural secretion exhibited hatching-gland somewhat 1979). As shown al., 1979). shown in in Fig. Fig. somewhat different different features features (Yamamoto (Yamamotoet ai.,

Fig. Fig. 4. 4. Diagrammatic Diagrammatic illustration illustration of of the the ultrastructural ultrastructural changes changes in in the the hatching hatchinggland process of induced precocious precocious secre gland cells cells of of medaka medaka embryos embryos in in the the process of electrically electrically induced secretion tion and and natural natural secretion secretion (for (for explanation, explanation, see see text) text) (Yamamoto (Yamamotoet et al., al., 1979). 1979).

458

KENJIRO YAMAGAMI KENJIRO

3, gland cells 3, the gland cells were were swollen swollen and and the the epithelial epithelial junctions junctions were were open open as in the Inside the as in the case case of of electrically electrically induced induced secretion. secretion. Inside the gland gland cell, however, however, aa different different pattern pattern of of secretory-granule secretory-granule change change was was ob observed. served. The The granules granules did did not not coalesce coalesce with with each each other other and and each each gran granule became ule became markedly markedly electron electron lucent, lucent, except except at at its its periphery. periphery. Thus, Thus, aa hatching-gland was, as hatching-gland cell cell just just before before natural natural secretion secretion was, as observed observed in in earlier earlier studies studies (Yamamoto, (Yamamoto, 1963), 1963), full full of of electron-lucent electron-lucent secretory secretory granules became some granules bearing bearing electron-dense electron-dense shells. shells. The The granules granules became somewhat what angular angular in in shape, shape, their their membranes membranes were were dissolved dissolved partly, partly, and and their contents were mixed with their contents were mixed with cytoplasm cytoplasm before before they they were were secreted secreted from This process exocyto cell. This process seemed seemed to to be different different from from that that of of exocytofrom the cell. sis. ne granules sis. The The electron-lucent electron-lucent contents were were composed composed of of fi fine granules in in this this case case also. also. In In summary, summary, aa comparison comparison of of the the ultrastructural ultrastructural changes changes of of hatch hatching-gland during the ing-gland cells cells during the electrically electrically induced induced secretion secretion with with those those during during natural natural secretion secretion shows shows two two kinds kinds of of changes changes:: those those that that are are common to both types of c to each type of of secretion and those specifi specific of secretion. The common changes are swelling of gland cells, cells, exposure of cells following of the the apical apical center center of of gland gland cells following the the separation separation of of the the epithelial reduction of epithelial junction, junction, and and reduction of electron electron density density of of secretory secretory sub substance coales secretion. By By contrast, contrast, in in natural natural secretion, secretion, no no coalesstance prior prior to secretion. cence of of secretory granules was observed, while in the induced secre secretion, many secretory granules of high electron density coalesced into a large mass of secretory substance and their electron density was de decreased. creased. A typical typical exocytosis exocytosis was was observed observed only only in in the the induced induced preco precocious secretion, while the secretory granules were disintegrated and mixed with mixed with the the cytoplasm cytoplasm of of the the gland gland cell cell in in natural natural secretion. secretion. In In salmonid salmonid fishes, fishes, secretory secretory granules granules become become electron-lucent electron-lucent and and fused fused together together just before before secretion. secretion. The The gland gland cells cells discharge discharge the the granules other cytoplasmic structures differently granules together together with with some some other cytoplasmic structures differently from ordinary exocytosis. exocytosis. After exhaustion of the secretory granules, the the gland gland cells cells dissociate dissociate from from the the epithelium epithelium (Yokoya (Yokoya and and Ebina, Ebina, 1976). aZ. (19S3a), (1983a), there are three 1976). However, according to Schoots Schoots et al. types pike embryos (1)exocytotic exocytotic discharge discharge via via aa se setypes of of secretion secretion in in pike embryos:: (1) cretion vacuole, (2) (2) exocytosis exocytosis at protruded cell part, and (3) (3) intercel intercellular is predominant. predominant. lular exocytosis. exocytosis. Among Among them, them, type type 1 is Although Although the the reason why why such different different types types of of secretion secretion occur occur in in the hatching gland gland is the hatching is obscure, obscure, the the fusion fusion of of secretory secretory granules granules has has also also been been reported reported in in the the process process of of secretagogue-induced secretagogue-induced secretion secretion of of various cells other 1 ; Ichi various cells other than than hatching-gland hatching-gland cells cells (Kurosumi, (Kurosumi, 196 1961; Ichikawa, 1969; Kanno, Kanno, 1972; 1972; Kagayama Kagayama and and kawa, 1965; 1965; Amsterdam Amsterdam et al., 1969; Douglas, 1977). d., 1977). For For example, example, in in the rat rat peritoneal peritoneal Douglas, 1974; 1974; Lawson Lawson et al., mast cell stimulated mast cell stimulated by by the the treatment treatment with with ferritin-conjugated ferritin-conjugated sheep sheep

7. 7.

MECHANISMS OF MECHANISMS OF HATCHING HATCHING IN IN FISH FISH

459

(S anti-Rlg-FT), anti-RIg-FT), an active degranula degranulaantibody to rat immunoglobulin (S tion occurs and the secretory granules coalesce into a large mass with density. The membrane interaction in association with low electron density. the degranulation leads to an exocytosis exocytosis of of the coalesced granular material (Lawson et al., 1977). 1977). Thus, it seems that a fusion of of granules occurs when the gland cells are forced to secrete somewhat rapidly by areforced stimulants. Reduction of the electron density of secretory substances, irrespective of whether they are in granules or in vacuoles, may be related partly to hydration of the substances. According to recent work (Yamagami (Yamagami et al., 1983), 1983), the electron density of secretory granules (or overly) overly) matured hatching glands of remained high in the fully (or medaka embryos whose hatching had been retarded by an "air-incu “air-incubation" p. 482). bation” (see (see p. 482). This fact strongly suggests that the electron electrondense granules are already mature in the sense that they are ready to be secreted upon stimulation and that the reduction of den of electron density is not an indication of maturation but an indication of of having entered into the secretion process. From the above results, it seems that some facets in the secretory changes, such as the increased fusi fusibility of secretory granules, were manifested exaggeratedly in the electrically stimulated secretion of of hatching gland compared to natu natural hatching. A stimulus for natural secretion may act somewhat more slowly or moderately, although its nature remains still uncertain (see (see later). later). Even after natural secretion, some hatching-gland hatching-gland cells without any secretory granules but full of of fragmented cisternae of of endoplasmic reticulum persist under the epithelium (Yamamoto, (Yamamoto, 1963; 1963; Yamamoto Yamamoto et al., 1979). de 1979). Similar persisting gland cells in the pike reportedly degenerated sooner or later by programmed death (apoptosis) (apoptosis) (Schoots et al., al., 1983a) 1983a).. III. 1 11. HATCHING ENZYME AND CHORIOLYSIS CHORIOLYSIS

A. Purification and Characterization of of Fish Hatching Enzymes Dissolution of the tough egg envelope by the secreted hatching enzyme is, together with the subsequent breakage of the remnant egg envelope (outer layer of of chorion) by the embryo, aa major feature of of hatching in fishes. Thus, the nature of of the hatching enzyme and enzy enzymatic choriolysis have been foci of interest in the study of of hatching. It is is known that the hatching enzyme of fish has a proteolytic activity in

460


addition to to its its egg egg envelope-dissolving envelope-dissolving activity activity (choriolytic (choriolytic activity) activity) addition (Ishida, 1944c; 1944c; Kaighn, Kaighn, 1964). 1964). Therefore, Therefore, the the hatching hatching enzyme enzyme activactiv (Ishida, ity can be assayed tentatively of the the tentatively for its proteolytic activity. Assay of ity proteolytic (or (or peptidolytic) peptidolytic) activity activity of of the the fish fish hatching hatching enzyme enzyme has has proteolytic been performed performed using using different different substrates substrates such such as as insulin insulin (Kaighn, (Kaighn, been casein or or its its derivatives 1972, 1973; 1973; Hagenmaier, Hagenmaier, 1964), casein derivatives (Yamagami, (Yamagami, 1972, 1964), 1974a; Schoots Schoots and and Denuce, Denuce, 1981), 198 1), and and some some synthetic peptides 1974a; synthetic peptides (Yamagami, 1973; 1973; Yasumasu Yasumasu et al., 1985). 1985). However, However, when when aa crude crude (Yamagami, of only the proteolytic (or peptidolytic) activactiv sample is used, the assay of ity is is not not appropriate appropriate for for discriminating discriminating the the reaI real hatching hatching enzyme enzyme from from ity other proteases, if if any. turbidimetric method method of of semi any. A turbidimetric semiother concomitant concomitant proteases, quantitative determination determination of choriolytic activity of medaka medaka enzyme enzyme quantitative of choriolytic activity of (Fig. 1970) was was devised devised to to overcome such difficulty, difficulty, 5 ) (Yamagami, (Yamagami, 1970) overcome such (Fig. 5) although method seems seems not not be be be applicable to the rainbow applicable to rainbow trout trout although the the method 4C-Iabeled chorion was recently (Ohzu et al., 1983). enzyme 1983).1'*C-labeled recently used as a enzyme (Ohzu substrate for enzyme (DiMichele 1981). (DiMichele et al., 1981). substrate for Fundulus enzyme The purification purification of of hatching hatching enzyme enzyme in has been been carried carried out out The in fish fish has

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7. 7.

MECHANISMS MECHANISMS OF OF HATCHING HATCHING IN IN FISH FISH

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1960s. In most cases, the enzyme has been obtained since the early 1960s. and purified from the hatching liquid, that is, is, the medium in which 1964) tried to purify the the embryos were allowed to hatch. Kaighn ((1964) (chorionase) of of Fundulus heteroclitus by gel filtra filtrahatching enzyme (chorionase) tion and sucrose density-gradient ultracentrifugation. ultracentrifugation. The chorionase was sedimented between two molecular-weight markers, ribonucleribonucle ase and hemoglobin, suggesting that its molecular weight was be be15,000 and 40,000. 40,000. Kaighn reported that the chorionase hydro hydrotween 15,000 lyzed tyrosine-threonine tyrosine-threonine and threonine-proline threonine-proline peptide bonds in the B chain of of insulin. Enzyme activity was inhibited with diisopro diisopropylphosphorofluoridate (DFP), er of a serine residue. (DFP), a specifi specificc modifi modifier Ogawa and Ohi (1968) 1970) fractionated an (1968) and Ohi and Ogawa ((1970) aqueous extract of manually isolated hatching glands of of medaka by agar gel electrophoresis and obtained two fractions fractions bearing chorion digesting activity and another fraction active in causing swelling of the chorion. According to a recent report (Schoots (Schoots et al., al., 1983c), 1983c), the swelling of the chorion seems to be an intermediate phase in the proteolytic digestion of of the chorion. chorion. Assuming that this is true, cho choriolytic enzyme(s) enzyme(s) obtained from hatching-gland cells ofmedaka of medaka sepa separated as as three different fractions moving toward the cathode at pH 8.6 on agar gel electrophoresis. Purification of medaka hatching enzyme used Sephadex column chromatography of of the ammonium sulfate pre precipitates of hatching liquid, followed by CM-cellulose CM-cellulose column chro chro(Yamagami, 1972). 1972). As will be shown in Fig. Fig. 7, 7, Sephadex matography (Yamagami, G-75 G-75 column chromatography of of the ammonium sulfate precipitate of hatching liquid gave two peaks of choriolytic and proteolytic activi activities (or enzyme I) I) and PI1 PII enzyme (or ties,, which were named PI enzyme (or (or 1973). The specific specific activity enzyme II), 11),respectively (Yamagami, (Yamagami, 1972, 1972,1973). of PI1 PII was much higher than that of PI. PI. When the PI1 PII enzyme was fractionated by by CM-cellulose column chromatography, a ssingle ingle peak of choriolytic and proteolytic activities coincident with a peak of of pro protein was eluted by 0.02 M NaCI. Specific M Tris HCI HCl (pH (pH 7.1)-0.3 7.1)-0.3 M M NaCl. activities of this enzyme fraction (named PII-0.3) PII-0.3) with respect to cho choriolytic activity and proteolytic activity were 212 212 and 183 183 times those protein eluted as aa sin sinof hatching liquid, respectively. The enzyme protein gle gle peak on Sephadex column chromatography and gave a single band moving toward the cathode on starch gel electrophoreses at pH 8.6 8.6 and 5.2 and and 5.2 and on on polyacrylamide polyacrylamide gel gel disc disc electrophoresis electrophoresis (PAGE). (PAGE). How However, this enzyme preparation showed some heterogeneity on sodium dodecyl sulfate (SDS) (SDS) PAGE (Iuchi (Iuchi et al., 1982). 1982). Thus, it has yet to be determined whether the additional protein(s) protein(s) in PII-0.3 PII-0.3 are merely contaminants or some fragments of of chorion protein associated with the enzyme. Recently, the enzyme. Recently, the the secretory secretory granules granules of of the the medaka medaka hatching hatching

Fig. Fig. 6. 6. Transmission Transmission electron micrographs micrographs(TEMs) (TEMs)of the hatching hatching enzyme granules granules ofmedaka of medaka (A) fixed in situ and (B) (B) isolated from the homogenate of of whole whole prehatching embryos 7000).Insets are higher magnifications magnifications of of a part of of respective granules. granules. Note embryos ((xx 7000). crystalline (Iuchi et crystalline patterns patterns -70 -70 A A wide in both granules granules ( x 130,000) 130,000) (Iuchi et al., al., 1982). 1982).

7. MECHANISMS OF HATCHING IN FISH 7. MECHANISMS

463 463

0.3 M M sucrose (Fig. (Fig. 6). 6). The aqueous extract of gland were isolated in 0.3 representthe isolated granules exhibited a high choriolytic activity, represent single band of protein on SSDS-PAGE (Iuchi et al., 1982). 1982). The ing a single DS-PAGE (Iuchi PII-0.3 enzyme enzyme as determined by Sephadex molecular weight of PII-0.3 (Yamacolumn chromatography was reported at first to be about 8000 (Yama gami, gami, 1972). 1972). When determined on SDS-PAGE SDS-PAGE following Weber and (1969),however, it was about 221,000. Osborn (1969), 1,000. The molecular weight of the enzyme in the aqueous extract of the isolated granules was also about 2 1 ,000 on SSDS-PAGE DS-PAGE (Iuchi et al., 1982). 21,000 1982).A similar discrepancy in the molecular weight was also reported for the pike hatching en enzyme (Schoots and Denuce, DenucB, 1981, 1981, see later). later). This discrepancy may be attributable partly to a high affinity affinity of the hatching enzyme for the supporting medium of gel fi ltration, and this method seems to filtration, to be inadequate for for the estimation of molecular weight of of this enzyme. It seems highly probable that the hatching enzyme of of medaka is a metal metalloprotease but is is not a serine protease nor sulfhydryl protease, as its activity is inhibited by ethylenediamine tetraacetic acid (EDTA) (EDTA) but neither by DFP nor by iodoacetamide (IAM) (lAM) (Ohi (Ohi and Ogawa, 1970; 1970; Yamagami, Yamagami, 1973). 1973). Low concentrations of of some monovalent and diva divalent cations activate the enzyme slightly, slightly, while high concentrations 1973). inhibit it (Yamagami, (Yamagami, 1973). 7A of medaka embryos contains As Fig. 7 A shows, the hatching liquid of apparently two hatching enzyme fractions, PI enzyme (enzyme (enzyme I) I) and PII (enzyme II). PI1 enzyme (enzyme 11). It was found, however, that a part of of PI enzyme could be converted to PI1 PII enzyme (which (which was named enzyme PI-PII) PI-PII) through re-salting out and rechromatography on Sephadex. Such a conversion from PI to PI1 PII was observed if if rechromatography of of the PI enzyme was repeated. The properties of of enzyme I, enzyme II, 11, and enzyme PI-PII PI-PI1 in terms of of the sensitivity to some inhibitors were (Fig. 7C). 7C). These observations strongly found to be almost identical (Fig. PII enzymes were essentially the same enzyme, suggest that PI and PI1 of their different states of of but that that they behaved behaved differently because of association with some heterologous substances such as hydrolyzed (Yamagami, 1975). 1975). This view has been confirmed by our rere chorion (Yamagami, PII enzyme have recently been highly cent work. PI enzyme and PI1 purified from the hatching liquid by repeating Toyopearl gel filtration 10. These procedures resulted in dissociation chromatography at pH 10. of the bound hatching enzymes. As a result, each PI enzyme and PI1 PII of of two types of enzyme was found to consist of of proteases; one was a (HCE) and the other was a protease with high choriolytic activity (HCE) (LCE) (Yasumasu (Yasumasu et al., 1988). 1988). protease with low choriolytic activity (LCE) Their molecular weights are about 24,000 and 25,500 respectively, on SDS-PAGE after Laemmli (1970). (1970).

KENJIRO YAMACAMI YAMAGAMI KENJIRO

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Fig. 7. Fractionation Fractionation of of medaka medaka hatching hatching enzyme enzyme by by Sephadex Sephadex column column chromatog chromatography. hatching liquid raphy, (A) (A) Elution Elution pattern pattern of of the ammonium ammonium sulfate sulfate precipitate precipitate of of the the hatching liquid on on Sephadex (B) Elution column chromatography. chromatography. (B) Elution pattern pattern of of rechromatography rechromatography of of the the Sephadex G-75 column ammonium through the same same column column as as in in (A). (C) (C) Comparison Comparison ammonium sulfate sulfate precipitate precipitate of PI through of the properties I, enzyme II, and properties of of enzyme enzyme I, enzyme 11, and enzyme enzyme PI-PlIo PI-PII. ST!, STI, Soybean Soybean trypsin trypsin (33 /Lg/ml); pg/ml); Leup, leupeptin (33/Lg/ml); (33pg/ml); Pepst, pepstatin (0.33 (0.33 /Lg/ml) pg/ml) (Yama (Yamainhibitor (33 gami, gami, 1975). 1975).

Appearance of multiple hatching enzyme peaks on gel filtration chromatography has been reported also also in some other fish species such as rainbow trout (Ohzu (Ohzu and Kasuya, 1979) 1979) and pike (Schoots and Denuce, Denuck, 1981). 1981). It seems that such a physical heterogeneity is a charac characteristic of the hatching enzyme not only of fish but also of some some other animal species such as sea urchin (see 477). An analysis of (see p. p. 477). of this problem will wiIl be useful for elucidation of the nature and the mecha mechanism of action of this enzyme.

7. 7.

465

MECHANISMS HATCHING IN MECHANISMS OF HATCHING IN FISH FISH

hatchThe hatching enzyme of rainbow trout was purified from the hatch ing liquid through the fractionation procedure similar to that of of the medaka enzyme (Hagenmaier, (Hagenmaier, 1974a; 1974a; Ohzu and Kasuya, 1979). The Kasuya, 1979). enzyme protein seems to be a basic protein from its behavior on chro chromatography and electrophoresis. The molecular weight as determined by gel fi ltration chromatography was about 10,000. filtration 10,000. However, consid considering the probable inadequacy of the gel filtration method for determi detennination of the molecular weight of hatching enzyme, it would be neces necessary to reexamine the molecular weight weight of of the salmonid hatching enzyme with some other analytical methods. This enzyme also ap apA, ethy pears to be a metalloprotease, as it was inhibited by EDT EDTA, ethyleneglycol bistetraacetate (EGTA), KCN, but (EGTA), O-phenanthroline, or KCN, not by uoride (PMSF), (PM SF), tosyl-L-Iysylchlorome by phenylmethyl sulfonylfl sulfonylfluoride tosyl-L-lysylchloromethane (TLCK), (TLCK), tosyl-L-phenylalanylchloromethane (TPCK), or tosyl-L-phenylalanylchloromethane (TPCK), iodoacetamide (Hagenmaier, (Hagenmaier, 1974a,b). 1974a,b). It was reported that the activ activ2 +) ity of the EGTA-inactivated enzyme was restored only by iron (Fe (Fe2+) (Hagenmaier, (Hagenmaier, 1974b). 1974b). The optimal pH of this enzyme was found to be around 8, 8, resembling the medaka enzyme. enzyme. Recently, the hatching enzymes of Fundulus heteroclitus and of the pike Esox lucius have been well studied from biochemical and l4C]iodoace physiological viewpoints. Using chorion labeled with [[l4C1iodoacetamide as substrate, DiMichele et al. 198 1) examined some character al. ((1981) characteristics of Fundulus chorionase. This enzyme was found to be quite chorionase. This stable below 30°C, 30°C, like like the medaka enzyme (Yamagami, (Yamagami, 1973), 1973), and had a Q of 2.2 between 15 and 30°C. The pH optimum for the activity Qlo 2.2 15 30°C. lO was between 8.0 8.0 and 8.5. 8.5. This This enzyme seems to be halophilic; in solutions trength below 0.05 solutions of ionic sstrength 0.05 M, M , approximately 50% 50% of the activity was lost in 18 18 h, but addition of NaCI NaCl within 48 h restored the activity. ionic strength was between 0. 1 and 0 2 M. activity. The The optimum ionic 0.1 0.2 M.Such a salt requirement is is seen in the enzymes of medaka (Yamagami, (Yamagami, 1973) 1973) and the marine fish Gobius jozo j o z o (Denuce, (Denuc6, 1976). 1976). The Fundulus en enIAM but sensitive to EDT EDTA zyme was found to be insensitive to lAM A as well as to PMSF. These results show that the Fundulus enzyme enzyme is is a serine protease and/or metalloprotease but not sulfhydrylprotease. sulfhydrylprotease. Kaighn Kaighn (1964) (1964) also also reported that Fundulus chorionase chorionase was was inhibited by DFP and was, was, therefore, presumably a serine protease. protease. According to Denuce Denuc6 and Thijssen (1975), (1975), the hatching enzyme of zebrafish, Brachydanio rerio, rerio, also seems seems to to be a serine protease. Schoots 1981) purifi ed the pike hatching enzyme Schoots and and Denuce Denuce ((1981) purified enzyme 1600 times from the original original hatching hatching liquid using affinity affinity chromatog chromatog1600 raphy with carbobenzoxy-n-phenylalanyl-triethylenetetramine (Z-D carbobenzoxy-D-phenylalanyl-triethylenetetramine (Z-DPhe-T) Phe-T) Sepharose. This This enzyme enzyme is is a glycoprotein glycoprotein containing 2% 2%carcar.

466 466

KENJIRO KENJIRO YAMAGAMI YAMAGAMI

bohydrate. The molecular weight of this enzyme was 10,000-15,000 by gel fi ltration but 23,500-25,400 filtration 23,500-25,400 with other methods such as PAGE, SSDS-PAGE, DS-PAGE, and sedimentation analysis. The activity was inhibited by some metal chelators such as EDTA, EDTA, EGTA, EGT A, and O-phenanthroline 0-phenanthroline but not by DFP, PMSF, iodoacetic acid, or N-ethylmaleimide (NEM). (NEM). Furthermore, they concluded that this enzyme is a zinc metallopro metalloprotease based on atomic absorption spectrometry and renaturation ex experiments of the denatured apoenzyme. In summary of the above results (Table I), we notice common (Table I), features in some of the enzymes, although we still lack much informa information for drawing a precise picture of the fi sh hatching enzyme. fish enzyme. The hatching enzyme is a choriolytic protease with a broad pH optimum around 8.0. (probably a divalent cation) 8.0. It requires a metal (probably cation) for full activity, although some enzymes are reported to be inhibited by serine active site reagents. The molecular weight of the enzymes seems to be in the range of 15,000-30,000, most probably somewhat higher than 20,000. 20,000. B. Solubilization gg Envelope (Chorion) B. Solubilization of E Egg (Chorion) Following activation or fertilization, the weak and fragile egg en enfish velope of the unfertilized fi sh egg is transformed into a tough structure (water) hardening. The egg envelope (cho (chothrough a process called (water) rion) of the fertilized egg consists of a thin outer layer and a thick rion) (Yamamoto, 1963; Lonning, 1972; 1972; Yamamoto and inner layer (Fig. (Fig. 8). 8). (Yamamoto, 1963; Lanning, subYamagami, 1975), 1975), the former being divided structurally into two sub layers (Anderson, (Anderson, 1967; 1967; Fliigel, Flugel, 1967; 1967; Wourms and Sheldon, 1976; 1976; Dumont and Brummett, 1980). 1980). The salmon egg chorion is composed (Young and of a scleroprotein, which was classified as pseudokeratin (Young 1938) and was later named ichthulokeratin (Young (Young and Smith, Smith, Inman, 1938) 1956). There is a great similarity in amino acid composition of the 1956). Fundulus,and medaka, chorion proteins among the eggs of salmonids, Fundulus, (and/or characterized by an abundance of proline and glutamic acid (andlor glutamine). The hardening occurs mainly in the thick inner layer. layer. glutamine). chemiThis tough structure protects the embryo against mechanical, chemi cal, and biological harm during development but also seems to be a cal, enbarrier to the embryo in terms of hatching. Usually, the hatching en zyme is secreted shortly before an actual hatching occurs. In medaka, secretion occurs less than 11 h before hatching. Thus, the thick chorion temperais digested by the enzyme within 11 h or so, so, depending on tempera ture. We tried to simulate the process of natural choriolysis in medaka

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Fig. Fig. 8. 8. Electron micrographs of intact egg envelopes of of medaka embryos. (A) (A) TEM of shows a of the egg envelope of of day-1 day-1 embryo (middle blastula) ((x~ 3600). 3 6 0 0 )Inset . a higher magnification (8) TEM of magnification of a part part of of the outer layer. (B) of the egg envelope of of day-6 embryo (-1 h before hatching) ((x3600). x3600). (C) (C) SEM of of the outer surface of of egg envelope of of day-1 day-1 (D) SEM of embryo ((x240). x 240). (D) of the inner surface of the egg envelope of of day-l day-1 embryo. embryo. af, Attaching filaments ( x~800) and Yamagami, 8 0 0 (Yamamoto (Yamamoto ) Yamagami, 1975). 1975).

b y incubating chorion pieces in the concentrated hatching liquid eggs by (PII-0.3 enzyme) enzyme) and to fol folor the purified hatching enzyme solution (PII-O.3 low the sequential ultrastructural changes of the chorion pieces (Yamamoto and Yamagami, 1975). 1975). (Yamamoto

Table II Table Some Characteristics of of Fish Hatching Enzymes" Enzymes·

�

Fish Fish species species

Molecular weight (method of of determination)

Medaka Medaka Oryzias Oryzias latipes latipes

21,000 (SDS-PAGE,W,-0)" (SDS-PAGE,W,-O)c 21,000

8,000 (Gel filtration)b filtration)b

Effect of of some inhibitors Optimum pH pH

Inhibited Inhibited by

8.0-9.0 8.0-9.0 (Chorio1ysis)'j (Choriolysis)'J 7.5-8.3 7.5-8.3 (Proteolysis)' (Proteolysis)k

KCN" HZS" EDTA'a

8.0-8.5 8.0-8.5 (Choriolysis)' (Choriolysis)'

EDTA' PMSF' P M S F' DFP" DFp· EDTArO E DTAf,° .o EGTAf,." EGTAf 0-PHEN" O-PHEN° KCN" KCN° EDTAh, EGTAh E DTAh, EGTAh CDTAh, O-PHENh 0-PHENh CDTAh, DTTh, TGAh TGAh DTTh,

24,000 (SDS-PAGE,L)d (SDS-PAGE,L)d

Ip

0, Of) 0

Mummichog Fundulus heteroclitus heteroclitus Fundulus Rainbow Rainbow trout trout

15,000-40,000 (Sedimenta(Sedimenta 15,000-40,OOO analysis)· tion analysis)" 10,000 ltrationf,g 10,OOO (Gel fi filtrationfa

Salmo Salmo gairdneri gairdnel-i

Pi ke Pike

Esox Esox lucius lucius

10,000-15,000 10,000-15,000 (Gel (Gel filtra filtration)h 24,000 24,000 (SDS-PAGE,L)h (SDS-PAGE,L)* 25,400 25,400(Sedimentation (Sedimentation analysis)h analysis)h

8.5 (Proteolysis)! (Proteo1ysis)f 8.5

7.0-9.0 (Proteolysis)h (Proteolysis)* 7.0-9.0

Not inhibited by DFPk,q DFpk.• IAM IAMk SBTI' SBTl' LEUP' LEUP' IAM' lAM' SBTI, LBTlo LBTE" SBTl, PMSFf." P M S Ff o TPCK", TLCK" TPCKo, TLCKo IAA", OVOMo OVOM" lAAo, DFPh, PMSFh PMSFh DFPh, NEMh, SBTIh NEMh, SBTlh OVOMh, TPCKh TPCKh OVOMh, .

Goby Goby Gobius Gobius jozo jozo Zebrafish Zebrafi sh Bruchydanio reno rerio Brachydanio

-

-

8. 1-8.4 (Choriolysis, 8.1-8.4 (Choriolysis, proteolysis)m proteolysis)m

-

EDTAm, EDTA'", EGTAm EGTA" O-PHENm 0-PHENm

SBTI" OVOM"

DFPP

uoridate; DTT, dithiothreitol; EDTA, ethylenedi a Abbreviations: Abbreviations: CDTA, cyclohexanediaminetetraacetate; cyclohexanediaminetetraacetate; DFP, diisopropylphosphorofl diisopropylphosphorofluoridate; ethylenediamine lAM, iodoacetamide; LBTI, lima ethylene glycol glycol bistetraacetate; bistetraacetate; lAA, IAA, iodoacetate; iodoacetate; IAM, lima bean trypsin inhibitor; inhibitor; LEUP, amine tetraacetate; tetraacetate; EGTA, ethylene leupeptin; O-PHEN, TLCK, 0-PHEN, O-phenanthroline; 0-phenanthroline; OVOM, ovomucoid; ovomucoid; PMSF, phenylmethylsulfonyl fluoride; SBTI, soybean trypsin inhibitor; TLCK, tosyl-L-lysylchloromethane; tosyl-L-lysylchloromethane; TPCK, TPCK, tosyl-L-phenylethyl tosyl-L-phenylethyl chloromethyl ketone; SDS-PAGE,W,O, SDS-PAGE,W,O,SDS-polyacrylamide SDS-polyacrylamide gel electrophoresis follow following the method of (1969); SDS-PAGE,L, of Laemmli (1970). of Weber and Osborn (1969); SDS-PAGE,L, SDS-polyacrylamide SDS-polyacrylamide gel electrophoresis following the method of (1970). b Yamagami ((1972). 1972). Iuchi et et al. ul. (1982). (1982). d Yasumasu Yasumasu et al. (1985). (1985). Kaighn (1964). (1964). f f Hagenmaier (1974a). (1974a). g 1979). g Ohzu Ohm and Kasuya ((1979). h Schoots 1981 ) . Schoots and Denuce DenucC ((1981). ; Ishida (1944b). (194413). j Yamagami (1970). (1970). k Yamagami ((1973). 1973). l DiMichele et al. 1981). al. ((1981). Denuce 1976). DenucC ((1976). (1944~). Ishida (1944c). Hagenmaier ((1974b). 1974b). P p Denuce DenucC and Thijssen (1975). (1975). q Ohi and Ogawa ((1970). 1970). Yamagami ((1975). 1975). a

c

'

e

J

m n o

r

470

KENJIRO YAMACAMI YAMAGAMI

As shown in Fig. 8C, 8C, there are a number number of of honeycomb-like patpat of intact chorion, as was first first documented by terns on the outer surface of (1928). A large number of of villi are present present all over the surface Kamito (1928). of of chorion, and many attaching filaments, much longer than villi, are of vegetal pole area of of the egg chorion. The restricted to the surface of of the intact chorion appears to be smooth, showing a inner surface of of chorion pieces in somewhat parallel wavy pattern. On incubation of buf the hatching hatching liquid or in the purified hatching hatching enzyme solution bufTris-HC) at pH 7.2, the outer surface of of chorion became fered with Tris-HC1 enzy rougher and many irregular dents and grooves appeared as the enzymatic erosion proceeded (Fig. (Fig. 9A). 9A). In contrast to the outer surface, the inner surface of of the chorion showed no irregular erosions during didi gestion; the partially digested inner surface remained smooth and

Fig. 9. SEMs of the outer and inner surfaces of the egg envelope of medaka embryos (A) After 5-min incu incuduring the process of enzymatic digestion by the hatching liquid. during liquid. (A) (B) After I5-min 15-min incubation (from (from inside). The inner layer bation (from (from outside) outside) ((x240), x 240), (B) bation is remaining ((~800) has been digested away and a sheet of outer layer with a villus is x 800) (Yamamoto and Yamagami, Yamagami, 1975). 1975). (Yamamoto

7. 7.

MECHANISMS MECHANISMS OF OF HATCHING HATCHING IN IN FISH FISH

471 471

flat. After complete digestion, a thin outer layer remained, apparently fiat. 9B). When examined successively by only slightly digested (Fig. (Fig. 9B). transmission electron microscopy, the thickness of the inner layer was evenly. It has been reported, however, that in the found to decrease evenly. vivo, instead of in vitro, vitro, the degree of inner enzymatic choriolysis in vivo, layer digestion varied from fish species to species depending on the 1982~).As (Schoots et al., thickness of the inner layer of chorion (Schoots al., 1982c). shown in Fig. 10B, lOB, there were indications of enzymatic solubilization of inner layer at the peripheral (outer) (outer) parts, just beneath the outer layer. These areas seemed to correspond to the dents of the grooves grooves shown in Fig. 9A and to be caused by the enzyme enzyme that had permeated through the outer layer of chorion pieces incubated in the enzyme solution. As shown in Fig. 10, 10, the partially digested inner layer was solution. slightly swollen, swollen, decreased in in its its electron electron density, density, and and loosened loosened into into aa fibrous The solubilized fibrous network. network. The solubilized products products of of the the inner layer could could be be fixed with which suggests with glutaraldehyde glutaraldehyde and and osmic osmic acid, acid, which suggests that that the the solubilized 10D). solubilized products products were were of of high high molecular molecular weight weight (Fig. (Fig. lOD). This This is is also also confirmed confirmed by analyzing analyzing the the enzymatic enzymatic digests digests of of me medaka 1975; Iuchi Iuchi and and daka chorion chorion biochemically biochemically (Yamagami (Yamagami and and Iuchi, Iuchi, 1975; Yamagami, 976b). In a preliminary experiment (Yamagami, Yamagami, 11976b). (Yamagami, 1970), 1970), it was was found found unexpectedly unexpectedly that that the the hatching hatching enzyme enzyme digests of of the the me medaka daka chorion chorion contained contained aa small small amount amount of of free free amino amino acids acids as as detected detected by thin layer chromatography. chromatography. When a large number of chorions chorions iso isolated lated from from blastulae blastulae was was incubated incubated with with the the purified purified hatching hatching enzyme enzyme (PII-0.3), (PII-0.3), most of them were digested digested to a clear clear viscous solution, leav leaving the outer layers with villi and attaching filaments undigested. The G-75 column solubilized material was fractionated using Sephadex G-75 (PI) of high-molecular-weight high-molecular-weight chromatography into a major fraction (PI) glycoproteins and aa.minor one (PH) (PII) of lower-molecular-weight lower-molecular-weight sub subis, small peptides and/or free amino acids. The former stances, that is, was fractionated further into two peaks of glycoproteins on Sephadex G-200 column chromatography: one (named Fr. 1) 1) was eluted at the 2) eluted later. Both peaks are consid consid(Fr. 2) void volume and the other (Fr. ered ered to to be be major major constituents constituents of of the the inner inner layer layer of of the the chorion. chorion. They They were approximately equal in amount and were very similar to each other amino acid well as as in in absorption absorption spectrum. spectrum. other in in amino acid composition composition as well analyses, each of them exhibited symmetrical Upon ultracentrifugal analyses, Schlieren 7.0 S for for Fr. Fr. 11 and and Schlieren profiles profiles with with sedimentation sedimentation constants constants of of 7.0 4.5 4.5 S for for Fr. Fr. 2. 2. However, However, disc disc electrophoretic electrophoretic analyses analyses revealed revealed that that six protein Fr. 11 was highly heterogeneous, being composed of about six bands (C 1-C6), while Fr. 2 (Cl-C6), 2 was homogeneous (Yamagami (Yamagami and Iuchi,

472

KENJIRO YAMAGAMI YAMACAMI KENJIRO

Fig. 10. 10. TEMs of the egg envelope envelope sections during the process of enzymatic diges digesFig. hatching liquid. liquid. (A) (A) After 2-min incubation ((x~4500). 4 5 0 0 )(B) (B) . After 5-min incuba incubation by the hatching beneath the outer layer, layer, is is digested by the tion. Peripheral part of the inner layer, just beneath (C)A higher magnifica.tion magnification enzyme that had permeated through the outer layer ((x3500). x 3500). (C) partially digested part of the inner layer as indicated by a square in (B) (B) ((X24,OOO). of a partially x 24,OOO). (0) (D) After lO-min 10-min incubation. incubation. The sample sample was was carefully fixed fixed to avoid avoid dispersing dispersing the the solubilized material ((x3500). (E) After 15-min 15-min incubation. incubation. Only a sheet of outer outer layer is x3500). (E) solubilized remaining ((x350O) (Yamamoto and Yamagami, Yamagami, 1975). 1975). X 3500) (Yamamoto remaining

7. 7.

473 473

MECHANISMS MECHANISMS OF HATCHING IN FISH

1975; Iuchi and Yamagami, 1976b). 1976b). Thus Thus the major products of enzy enzy1975; matic choriolysis comprise about about seven high-molecular-weight pro promatic (Fig. 111). also found seven proteins including teins (Fig. 1). Denuce ((1975) 1975) also 80,000 and 200,000 200,000 in the those of approximate molecular weight of 80,000 enzymatic hydrolysate of medaka chorion. On further examination of pattern Fr. 1, 1, it was noticed that there seemed to be a regularity the patt, e rn of Fr. Fr. 11 (Iuchi (Iuchi and of chemical characteristics among the components of Fr. 1976b). After determining determining the molecular weights of the Yamagami, 1976b). Fr. 11 components, and of Fr. Fr. 2 following the native forms of the Fr. method of Hedrick and Smith (1968), (1968),it was concluded that the net electric charge of each of the six components of Fr. 11 was approxi-

+

L

Origin Origin

!

to

1.00 0.50

£

'"

,9

C2

0.1 0

C3

C4

005

O

C5 C6

Gel ·1.

D Get

I

10

W

10

15

20

_. 25

Mol.cular Weight X 10

Fig. 111. glycoproteins solubilized from the medaka egg envelope by the Fig. 1 . Major glycoproteins enzyme. (A) Densitometric illustration of of a polyacrylamide polyacrylamide gel action of the hatching enzyme, (Cl-C6) and Fr. Fr. 2. (B) (B) of Fr. 11 (CI-C6) electrophoretic pattern. Major glycoproteins consist of Molecular-weight determination of the six components of Fr. Fr. 11 and Fr. Fr. 2 according to Molecular-weight of Hedrick and Smith ((1968). R , values of the glycoproteins were the method of 1968). The log Rm plotted against acrylamide concentrations concentrations (BI), (B,), and their approximate molecular plotted of the plots (BZ). weights were estimated from the slope of (Bz). M, D, and T refer to monomer 67,000),dimer, and trimer trimer of bovine serum albumin, used as references (Iuchi (MW = 67,000), Yamagami, 1976b). 1976b). and Yamagami, =

474

KENJIRO YAMAGAMI KENJIRO

2. The molecular mately the same, but different from that of Fr. 2. weights of the six components of Fr. 11ranged from 8.6 8.6 x x 104 lo4for C C1l to 4 for C6 with an average molecular weight difference of 2 1.4 x 21.4 x 10 lo4 about 2.6 2.6 x x 104 lo4 between neighboring components, while the molecu molecu4 (Fig. lar weight of Fr. 2 was approximately 7 x X 10 lo4 (Fig. 11). 11). It may be presumed that to the smallest component of 1, C l , is of Fr. 1, C1, is added a kind of repeating unit polypeptide of about 2.6 x X 104 lo4 molecular weight weight to form the second smallest polypeptide, C2, C2, and to C2 is added the repeating unit to form C3, C3, and so on. As described above, the pI PI values of all components of Fr. 11 seem to be identical. From these observations, it seems that these components could be named a Fr. 11 family. family.Moreover, it was found that the molar ratios of of C C1l to C2, C2 to C6, as calculated from their relative molar C3, . . . ,, and of C5 to C6, C3, concentrations in the chorion digests, are all about 3. 3. This might mean that one molecule of C6 is is combined with three molecules of of C5 and one molecule of C5 with three molecules of C4, C4, and so so forth, and that the hatching enzyme could break the connections between the com components (Yamagami, (Yamagami, 1981). 1981). This assumption implicates some cross crosslinking of polypeptide chains in the inner layer of the hardened cho chorion. In this connection, a report of Hagenmaier et al. (1976) (1976) that y-glutamyl-e-Iysine y-glutamyl-e-lysine was present only in the hardened chorion proteins of rainbow trout eggs is of particular interest. An exhaustive chorioly choriolysis with a prolonged enzymatic digestion of chorion resulted in no sis significant change in Sephadex column chromatographic pattern and PAGE pattern of Fr. 11 and Fr. 2 (Iuchi (Iuchi and Yamagami, 1976b; 1976b; S. S. al., unpublished). Yasumasu et al., These results suggest that the hatching enzyme digests the inner layer of chorion by hydrolyzing some restricted peptide bonds of its constituent proteins to give rise to two groups of of soluble glycoprotein compounds, Fr. 11 and Fr. 2. Once these glycoproteins (C I-C6 of compounds, (Cl-C6 of Fr. 1, 1, and Fr. 2) 2) are formed, they seem to be resistant to further enzymatic breakdown. A similar mode of choriolysis in principle may occur in the hatching of other fish species, species, although an accumulation of of free amino acids is reported in the hatching fluid of rainbow trout (Ohzu (Ohzu 1981). (1942,1949) and Kusa, 198 1). Some 40 years ago, Hayes (1942, 1949) suspected that the action of hatching enzyme was not hydrolytic, as the amount of amino-N produced by the enzymatic digestion of a capsule was so small. small. A limited cleavage of of the inner-layer proteins of of chorion by the hatching enzyme would give rise to the result compatible with the Hayes's Hayes’s observations as well as explain the efficient and rapid solubi solubilization of chorion by the hatching enzyme. .

..

7. 7.


475

C. Comparative Studies of Enzymatic Hatching and Related Problems docuAlthough the hatching enzyme or enzymatic hatching was docu rst in fish, fish, hatching has been described in many other animal mented fi first species, and the number of such examples examples is is increasing (Ishida, (Ishida, 1948a; 1981). Whether there is is a phylogenetic correlation 1948a; Davis, 1969, 1969,1981). to the mechanisms of the enzymatic hatching in various animals is still better of fish uncertain, but it would be useful for b etter understanding of hatching to make reference to the enzymatic hatching in other animal groups. In this section a brief brief survey will be made of of hatching in amphibians and sea urchins, Discussion will be extended to the diges digestion of the cocoon by cocoonase in insects and the solubilization of the vitelline envelope by sperm lysins, as as these phenomena are closely related to enzymatic hatching in some respects. MPHIBIAN H ATCHING 1. AMPHIBIAN HATCHING 1. A

Studies on the enzymatic hatching of Amphibia, like those of fish, have a long history. After Bles ((1905) 1905) described the role of of frontal glands in the hatching of Xenopus Zaeuis laevis embryos , and presumed that embryos, a proteolytic enzyme was secreted from it, this gland was studied histologically in urodele as well as in anurans by other workers (Jaensch, 192 1 ; Noble and Brady, 1930; 1930; Holtfreter, 1933; 1933; Yanai, 1950, 1921; 1950, 1953, 1953, 1959). 1959). The hatching gland of Amphibia is is of ectodermal ectodennal origin. Although there was a view that the anuran hatching gland originated from the neural crest (Yanai 1953, 1955, 1955, 1956), 1956), it was found (Yanai et al., ul., 1953, recently that most gland cells were derived from the superfi cial epi superficial epidermal cells situated on the neural crest (Yoshizaki, 1976; Y oshizaki (Yoshizaki, 1976; Yoshizaki and Yamamoto, Yoshizaki ((1979) 1979) succeeded in in Yamamoto, 1979). 1979). Moreover, Yoshizaki inducing the hatching-gland cells from the explanted superficial layers of the presumptive ectoderm in Rana japonica with LiCl. LiC1. Thus, it is believed at present that the anuran hatching glands originate mostly from ectoderm other than the neural crest. In pilocarpine-induced pilocarpine-induced secretion, the electron density of secretory granules of gland cells decreases and a partial coalescence of some granules occurs (Yoshi (Yoshizaki, 1973). 1973).As As to the escaping of anuran embryos from the jelly layers, there have been some reports suggesting a nonenzymatic process (Ko (Kobayashi, 1954a,b). formosus, there are four 1954a,b). In the toad Bufo vulgaris uulgarisformosus, jelly layers, which are named A, B, C, and D, respectively, from the outer to the inner layers. layers. A and B form a jelly string and the innermost

476


D interfaces with the vitelline envelope. When the embryos attain the late late neurula neurula stage, stage, they they escape escape preliminarily preliminarily from from the the jelly jelly string string by perforating layers A and By B, while each embryo remains still covered by layers C and D. Hatching from these layers occurs only when the embryos reach the tail-bud stage. stage. Escape from layers A and B is not due to any proteolytic action but is primarily due to swelling of layer C. Kobayashi 1 954b) argued C. Kobayashi ((1954b) argued that that an an augmented augmented respiratory respiratory activity activity of of the the embryos embryos was was closely related related to to the the swelling of of layer layer C. C. Thus, Thus, enzymatic hatching is preceded by a nonenzymatic process. A similar observation was also made on Xenopus laevis embryos by Carroll and 1974), who reported that the hatching process consisted of Hedrick ((1974), of rally distinct phases, that is, phase 11 and phase 2. two tempo 2. In phase temporally 1, the embryo escapes from the outer jelly layers, J3 1, 53 and J2, without the aid of a hatching enzyme, but probably by a physical process such as water imbibition by the inner jelly layer, JJ1; l ; in phase 2, a hatching protease participates in the dissolution of the vitelline envelope. This This two-step hatching process seems to be of some some interest and suggests that such an analysis should be made also also in fish hatching, although no thick multijelly layers are present. In salmonid embryos, embryos, the hardness of the egg envelope gradually decreases long before actual hatching (Hayes, (Hayes, 1942, 1942, 1949). 1949). It seems improbable that the hatching enzyme had already been secreted and participated in such envelope soften softening. Thus, Thus, there is is a possibility of participation of some factor(s) factor(s) other than the hatching enzyme in a preliminary softening of the egg enve envelope in the hatching of some sh. The amphibian hatching enzyme is some fi fish. also also a protease (Cooper, (Cooper, 1936; 1936; Ishida, Ishida, 1947; 1947; Carroll and Hedrick, Hedrick, 11974; 974; Katagiri, 975; Yoshizaki Katagiri, 11975; Yoshizaki and Katagiri, Katagiri, 1975; 1975; Urch and Hedrick, 1981). 1981). The Rana chensinensis enzyme was purified about lOO-fold 100-fold from its original culture medium. The molecular weight is approxi approxi55,000-60,000 and its optimum pH is 7.4-7.8. 7.4-7.8. This enzyme enzyme is mately 55,000-60,000 not affected by Na+, Na+, K, K,++ or soybean trypsin inhibitor but is strongly 2 + , Mg 2+ , E Ca2+, Mg2+, EDTA, (Katagiri, 1975). 1975).The Xeno Xenoinhibited by Ca DTA, and DFP (Katagiri, pus laevis hatching enzyme was purified 2200-fold over the starting crude hatching media (Urch Hedrick, 1981). (Urch and Hedrick, 1981).This This enzyme has two two enzymatically active charge isomers present with molecular weights enzymatically of 62,500. The activity toward its natural substrate is is optimal optimal at pH 7.7. 7.7. of62,500. 2 + and by E The DTA and seems to The enzyme enzyme is is inhibited by Zn Zn2+ EDTA to be a from inhibition by DFP and PMSF. PMSF. From these char charserine protease from enzyme is different from the enzymes of acteristics, the amphibian enzyme Oryzias O q z i a s and salmonids salmonids but somewhat similar to those of Fundulus and

Brachydanio. Brachydanio.

7. 7.

MECHANISMS MECHANISMS OF OF HATCHING IN I N FISH FISH

477

2. E ECHINOID HATCHING 2. CHINOID H ATCHING The aquatic invertebrate whose hatching enzyme has been best first studied is the sea urchin. The echinoderm hatching enzyme was fi rst documented in Strongylocentrotus (Hemicentrotus) (Hemicentrotus) pulcherrimus, by Ishida ((1936), 1936), and its properties were studied by 1 943). b y Sugawara ((1943). The optimal pH of its proteolytic activity was around 8.5-9.5, and low 2 + seemed to be necessary for its activity. Of concentrations Of all concentrations of of Ca Ca2+ the animal species, purifi cation of of hatching enzyme was tried first in purification echinoderm 1961), who is echinoderm by Yasumasu Yasumasu ((1961), who obtained obtained Anthocidaris Anthociduris crass crussispina enzyme enzyme in in crystalline crystalline form. form. The The optimal optimal pH of of the the crystalline crystalline 8.2-8.4, half of the activity remained enzyme activity was 8.2 -8.4, and nearly a half even even after after heating heating at at 60°C for for 10 10 min. Following Following these these pioneering pioneering studies, cation and studies, there there have have been been many many studies studies of of purifi purification and partial partial char characterization acterization of of hatching hatching enzymes enzymes from from various various species species of of echino echinoderms. characteristics are general in some re rederms. The hatching-enzyme characteristics spects sh hatching spects but but contradictory contradictory in in others others (as (as in in the the case case of of fi fish hatching enzyme). the echinoderm echinoderm hatching hatching enzymes reported reported heretofore heretofore enzyme). All the 2 + for their maxi of Ca Ca2+ maxiare found to require a suitable suitable concentration of 2 + on the activity is not settled Mg2+ mum activity, while the effect of Mg (Barrett 1976; Takeuchi 1979; Nakatsuka, Takeuchi et al., al., 1979; Nakatsuka, 1979). 1979). (Barrett and and Edwards, Edwards, 1976; The enzyme 8.0 or enzyme seems seems to to be retained on on DEAE-cellulose DEAE-cellulose at at pH 8.0 or 8.2. 8.2. There has been considerable variation in the reported molecular purpurutus enzyme was purifi purified weights. On the one hand, the S. S. purpuratus ed and the about 28,500 and and 30,000 30,000 on on the molecular molecular weight weight was was reported reported to to be about SDS-PAGE SDS-PAGE and Sephadex column chromatography, respectively (Bar (Barrett 1976); on the the other hand, hand, the the molecular molecular weight weight of of rett and Edwards, Edwards, 1976); the which was band on on enzyme, which was purified purified to to aa single single band the SS.. intermedius enzyme, SDS-urea-PAGE SDS-urea-PAGE and and separated separated from from concomitant concomitant j3-1,3-glucanase, P-1,3-glucanase7es esterases DS 44,000 on on SSDSterases and and most most proteinases, proteinases, was was found found to to be be about about 44,000 urea-PAGE 45,000 on on Sephadex Sephadex column column chromatography chromatography (Ta (Taurea-PAGE and and 45,000 keuchi et al., d . , 1979). 1979). This variation in molecular weights seems to be attributable in part to a physical heterogeneity of of echinoderm hatch hatchcoming enzymes in the original hatching liquid; the enzyme may be com bined with some heterologous molecules such as various-sized mate mated . , 1971). 1971). Moreover, rials of fertilization envelope (Barrett et al., Nakatsuka ((1985) 1 985) reported recently that the sea urchin hatching en enzyme was present inside the blastula in proenzyme form, form, which had a larger larger molecular molecular weight. weight. Another hatching enzyme enzyme study study in in echinoderms echinoderms is is of of the the Another line line of hatching genetic control of this enzyme protein. Koshihara and Yasumasu

478

KENJIRO YAMACAMI YAMAGAMI KENJIRO

(1966) reported that the Hemicentrotus pulcherrimus enzyme could (1966) of the be synthesized in vitro using chromatin of the embryos embryos about about 3 hr ( 1969) reported, before hatching as a template. template. Barrett and Angelo (1969) however, however, that the echinoid hatching enzyme was entirely maternal based based on on their their studies on on reciprocal reciprocal hybrid hybrid embryos, whose parent parent S. purpuratus echinoid species, species, S. purpuratus and and S.franciscanus, S.franciscanus, had had the the hatching hatching 2 + . Showman and enzymes of of different sensitivities to the added Mn Mn2+. 1980) have recently reported that the messenger RNA of of the Whiteley ((1980) echinoid hatching enzyme is newly transcribed in advance of hatch of hatching, based on their well-devised experiment using the hybrid andro ing, androS. purpuratus Den merogons between the two two echinoid species S. purpuratus and Dendraster excentricus. The developmental stage at hatching is much earlier in echinoids than in fish; fish; the echinoid hatching enzyme seems to to be be one one of of aa few few specific specific proteins proteins that that may may be be synthesized synthesized during cleavage. cleavage. Therefore, Therefore, it it seems highly highly probable probable that that the the fish hatching hatching enzyme is also synthesized under the control of not the "maternal" “maternal” genome but the embryonic genome. It has not yet been observed electron electron microscopically microscopically that that the the echinoid echinoid hatching hatching enzyme enzyme is is pack packaged in any particular structure such as secretory granules, although Nakatsuka ((1985) 1 985) reported that a granular hatching enzyme could be obtained by centrifugation. centrifugation. Thus, the cellular site of synthesis synthesis of the echinoid hatching enzyme has not yet been identified.

3. O OTHER PHENOMENA RELATED TO H HATCHING THER P HENOMENA R ELATED TO ATCHING 3. There are some some enzymes similar to the hatching enzyme in a strict sense, that is, an embryonic enzyme dissolving a fertilization enve envelope. The best studied among them is cocoonase, which is synthe synthesized in and secreted from the maxillary galea of the pupa of certain saturniid moths and participates in the digestion of the cocoon, cocoon, mak makhatching” of the pupa possible (Kafatos (Kafatosand Williams, ing the "“escape escape hatching" 1964; Kafatos, Kafatos, 1972). 1972). This enzyme is an organophosphate-sensitive 1964; specificity, amino amino acid protease, resembling trypsin in its substrate specificity, composition, and molecular weight (�24,000) (-24,000) (Kafatos (Kafatoset al., 1967a,b). 1967a,b). composition, Cocoonase is is synthesized synthesized in zymogen-producing zymogen-producing cells of the the galea galea and Cocoonase transported into 1; into zymogen-storing vacuoles vacuoles (Berger (Berger and and Kafatos, Kafatos, 197 1971; Kafatos, 1975). 1975).A remarkable characteristic characteristic ooff this enzyme enzyme Selman and Kafatos, is that the the active active enzyme is is deposited on the galea as as a semicrystalline semicrystalline is secretion. The enzyme powder is is dissolved in a encrustation after secretion. exudate, which serves serves as as the buffer solvent for the enzyme enzyme galeal exudate,

7. 7.

MECHANISMS HATCHING IN MECHANISMS OF OF HATCHING IN FISH FISH

479

of cocoon. cocoon. Thus, it seems before being applied onto the inner surface of that a natural enzyme solution can be easily obtained from a pupa just before "escape al., 1967a). 1967a).The second feature of “escape hatching" hatching” (Kafatos (Kafatos et al., this this enzyme enzyme is is its its unique unique mechanism mechanism of of action; action; this this enzyme enzyme digests digests the broin, hydrolyzing not not its its main main constituent constituent protein, protein, fi fibroin, the cocoon cocoon by hydrolyzing but but sericin, sericin, which which glues glues the the fibroin fibroin fibers fibers together together (Kafatos (Kafatos and and Wil Williams, 1964). 1964). agents) of of Another group of egg envelope-dissolving enzymes (or (or agents) interest so-called egg interest in in comparison comparison with with fish fish hatching hatching enzymes enzymes is is the the so-called egg membrane sperm lysin, lysin, although although it it is is quite quite different different from from the the membrane lysin lysin or sperm hatching described in so far far been been described in verte vertehatching enzyme. enzyme. Many Many lysins lysins have have so brates brates and and invertebrates invertebrates (Hoshi, (Hoshi, 1985). 1985). The The lysin lysin is is thought thought to to be localized in the acrosome and localized in the sperm sperm acrosome and to to participate participate in in the the dissolution dissolution of of the the egg egg envelope envelope when when the the sperm sperm penetrates penetrates the the envelope. envelope. Mam Mammalian maIian acrosin acrosin is is one one of of the the best-characterized best-characterized vertebrate vertebrate lysins lysins and and is is similar al., 1972, 1972, 1973; 1973; Zaneveld Zaneveld et al., al., 1972; 1972; similar to to trypsin trypsin (Polakoski (Polakoski et al., Polakoski Polakoski and and McRorie, McRorie, 1973; 1973; Parrish Parrish and and Polakoski, Polakoski, 1979). 1979). It It is is as assumed sumed that that mammalian mammalian acrosin acrosin is is functional functional in in aa form form bound bound to to the the acrosomal natural condition acrosomal membrane membrane under under natural condition (Brown (Brown and and Hartree, Hartree, 1976; 1976; Castellani-Ceresa Castellani-Ceresa et al., al., 1983). 1983). There There have have been been many many studies studies on lysins in by sperm sperm lysins in marine marine inverte inverteon the the egg egg envelope envelope dissolution dissolution by brates 1939; Berg, Berg, 1950; 1950; Wada Wada et al., 1956; 1956; Haino, Haino, 1971; 1971; Haino Hainobrates (Tyler, (Tyler, 1939; Fukushima, Heller and 1974; Heller and Raftery, Raftery, 1973; 1973; Levine Levine et al., al., 1978; 1978; Fukushima, 1974; Levine 1 ; Sawada 1984; Levine and and Walsh, Walsh, 1980; 1980; Hoshi Hoshi et al., al., 198 1981; Sawada et al., 1982, 1982,1984; Lewis Lewis et al., al., 1982; 1982; Ogawa Ogawa and and Haino-Fukushima, Haino-Fukushima, 1984). 1984).Among Among them them are some reports which the some gastropod sperm are not are some reports in in which the lysins lysins of of some gastropod sperm are not enzymes enzymes but but rather rather low-molecular-weight low-molecular-weight proteins, proteins, which dissolve dissolve or or loosen vitelline coat coat of of eggs eggs by combining combining with with it it stoi stoiloosen markedly markedly the vitelline to form form aa soluble soluble complex complex (Haino-Fukushima, (Haino-Fukushima, 1974; 1974; chiometrically to Lewis Lewis et al., al., 1982; 1982; Ogawa Ogawa and and Haino-Fukushima, Haino-Fukushima, 1984). 1984). It It seems seems improbable improbable that that such such aa nonenzymatic nonenzymatic action action of of sperm sperm lysins lysins is is preva prevalent it appears appears that this type type are are lent in in marine marine invertebrates; invertebrates; it that lysins lysins of this found in some such as found only only in some restricted restricted animal animal groups groups such as archaeogastro archaeogastropods 1984). However, However, the the mechanism mechanism pods (Ogawa (Ogawa and and Haino-Fukushima, Haino-Fukushima, 1984). of of action action of of this this gastropod gastropod lysin lysin gives gives us us important important information information about about aa facet facet of of the the mechanisms mechanisms of of egg egg envelope envelope dissolution dissolution or or of of the the biologi biological cal breakdown breakdown of of aa noncellular noncellular structure structure composed composed of of scleroprotein. scleroprotein. It It seems high affinity affinity for for its its natural natural seems that that the the hatching hatching enzyme enzyme also also has has aa high substrate. Is it unreasonable to think that the mechanism of of action of the is an example of the archaeogastropod archaeogastropod lysin lysin is an extreme extreme example of the the interaction interaction between the between the egg egg envelope-dissolving envelope-dissolving factor factor and and its its substrate substrate??

480

KENJIRO YAMAGAMI

IV. PHYSIOLOGY OF HATCHING IN IN FISH FISH IV.

A. Factors Controlling Fish Hatching As described described before, before, hatching hatching of of fish fish is is aa developmental developmental stagestage As specific phenomenon. phenomenon. In In fact, fact, the the embryo embryo must must have have attained attained aa particpartic specific ular developmental developmental stage stage and and have have fully fully matured matured hatching-gland hatching-gland cells cells ular before hatching occurs, occurs. However, attainment of develop before of a specific developmental is not not sufficient sufficient to to cause cause actual actual hatching. hatching. Some Some triggering triggering mental stage stage is stimuli, either either extrinsic extrinsic or or intrinsic, intrinsic, have to be be received received by by the the approappro stimuli, have to developed embryo in order to induce hatching enzyme secresecre priately deveIoped ( 1957) pointed out, the onset of of hatching in tete tion. Thus, as Smith (1957) is a a complex phenomena. As shown in Table 11, II, there have been leosts is many factors or treatments that are reported to either stimulate or suppress the the hatching of of fish. They are believed to to influence the se seof the earliest studies of of cretion of of the the fish hatching enzyme. In one of ( 1936) argued that there the factors inducing fish hatching, Armstrong (1936) of the embryonic were two factors involved: the lashing movement of tail and the secreted hatching enzyme. He showed that no hatching of these factors was inhibited. At present, it is is occurred when either of of embryo is is effective only well known that the lashing movement of of after the enzyme has exerted its digesting action on the the inner layer of the chorion. XYGEN A ESPIRATORY M 1. OXYGEN AVAILABILITY RESPIRATORY MOVEMENT 1. O OVEMENT VAILABILITY AND R

embryos, an opercular Ishida ((1944b) 1944b) observed that in medaka embryos, movement took place followed by disintegration of the hatching gland supshortly before hatching. When the opercular movement was sup M KCI, KC1, the gland did not disintegrate. pressed by treatment with 0.25 0.25 M O On of hatching glands occurred when n the other hand, the beakdown of 0.1 afthe embryo was treated with 0. 1 or 0.2% 0.2% Veronal-sodium, which af fected the whole body movement but not the opercular movement. It was further observed that the gland cells could be disintegrated by water flow from a capillary that had been inserted into the pharynx of embryo. Thus, Thus, the enhancement of opercular movement of em emthe embryo. seems to be one of the phenomena most closely correlated with bryos seems the initiation of the hatching enzyme secretion in medaka, although it flow sole cause for the remains obscure whether or not water fl ow is the sole hatching-enzyme secretion. When the shaking of a large number of

Table II I1 Table Factors Influencing the Hatching-Enzyme Secretion Stimulants Stimulants Hypoxia H2 Hz gas gas Respiratory movement of shaking Stoppage of N2 Nz gas gas CN-

MS M S 222 M) ((10-5 10-5 M) Epinephrine Epinephrine Corticosteroid Prolactin Electric current

Rise in temperature temperature Ionophore

Reference Reference

(1937) Trifonova (1937) Ishida ((194413) 1944b) (1970) Yamagami (1970) Hagenmaier ((1972) 1972) Ishida (1944c) (1944~) al. (1985) (1985) Iuchi et ai. (1981) DiMichele and Taylor (1981) DiMichele and Taylor ((1981) 1981)

Cloud (1981) (1981) Schoots et al. (1982a) (1982a) Schoots Schoots et al. ai. (1982a) (1982a) Iuchi and Yamagami (1976a); ( 1976a); Yamamoto Yamamoto et al. ai. (1979); ( 1979); Iuchi et al. ai. (1985); (1985); Luczynski (1984~) (1984c) Luczynski (1984~) (1984c) Schoots Schoots et al. ai. (1981) (1981) Iuchi et al. (1985) (1985)

Suppressants Suppressants Hyperoxia O2 gas

Reference Reference 1954) Milkman ((1954) DiMichele and Taylor ((1980) 1980)

Air incubation

1977) Taylor et et al. al. ((1977) DiMichele and Taylor (1980) (1980) Yamagami et al. al. (1983) (1983) Yamagami et

MS M S 222 M

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