Advances in Insect Physiology, Volume 12

Advances in Insect Physiology

Volume 12

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Advances in Insect Physiology edited by

J. E. TREHERNE M. J. BERRIDGE and V. B. WIGGLESWORTH Department of Zoology, The University Cambridge, England

Volume 12

1976 ACADEMIC PRESS LONDON NEW Y O R K S A N FRANCISCO A Subsidiary of Harcourt Brace Jovanovich, Publishers

ACADEMIC PRESS INC. (LONDON) LTD 24-28 Oval Road London NW1 US edition published by ACADEMIC PRESS INC. 111 Fifth Avenue, New York, New York 10003

Copyright @ 1976 by Academic Press I n c (London) Ltd All Rights Reserved

No part of this book may be reproduced in any form, by photostat, microfilm or any other means, without written permission from the publishers

Library of Congress Catalog Card Number: 63-14059 ISBN: 0-12-0242 12-5

Printed in Great Britain at The Spottiswoode Ballantyne Press by William Clowes & Sons Limited London, Colchester and Beccles

Contributors Fotis C. Kafatos

The Biological Laboratories Harvard University, 16 Divinity Avenue, Cambridge, Massachusetts 02138, USA, and Department o f Biology, University of Athens, Panepistemiopolis, Kouponia, Athens (621), Greece

E. David Morgan Department of Chemistry, University of Keele, Keele, Staffordshire ST5 5BG, England Colin

F. Poole’

Department of Chemistry, University of Keele, Keele, Staffordshire ST5 5BG, England Hugh Fraser Rowell

Department of Zoology, University of California, Berkeley, California 94720, USA Klaus Sander

Biologisches Institu t I ( Z ool ogie), D er Albert-Ludw igs- Universitat, Katharinenstrasse 20, 7800 Freiburg im Breisgau, Federal Republic of Germany J. E. Steele

Department of Zoology, University of Western Ontario, London 72, Ontario, Canada

Present Address: Department of Pharmacy, 7 h e University of Aston in Birmingham, Gosta Green, Birmingham 8 4 7 E T.

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Contents Contributors

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Sequential Cell Polymorphism: A Fundamental Concept in Developmental Biology FOTlS C. K A F A T O S . . . . . . . . . . . . . . .

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The Extraction and Determination of Ecdysones in Arthropods E. DAVID M O R G A N AND C O L I N F. POOLE . . . . . .

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17

The Cells of the Insect Neurosecretory System: Constancy, Variability, and the Concept of the Unique Identifiable Neuron HUGH FRASER R O W E L L . . . . . . . . . . . . . .

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Specification of the Basic Body Pattern in Insect Embryogenesis K L A U S SANDER . . . . . . . . . . . . . . . . . . 125 Hormonal Control of Metabolism in Insects J. E.STEELE . . . . . . . . . . . . .

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Subject Index

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Cumulative List of Authors

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345

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Cumulative List of Chapter Titles

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Sequential Cell Polymorphism: A Fundamental Concept in D eve lo p m ent a I B io lo g y Fotis C. Kafatos The Bioiogicai Laboratories, Harvard University, Cambridge, Massachusetts, USA, and Department of Biology, University of Athens, Athens, Greece 1 Introduction 2 Cellular metamorphosis in the saturniid labial gland . 3 The sphingid labial gland: a more complex case of cellular metamorphosis 4 The cocoonase organules of the silkmoth galea: multistage sequential polymorphism in epidermal derivatives . 5 The follicular epithelial cells of silkmoths: biochemically defined sequential polymorphism 6 General discussion Acknowledgements References

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1 Introduction Many years ago, Wigglesworth (e.g. 1954) introduced the concept of sequential cell polymorphism. The outstanding example was the production of distinct types of cuticle and of cuticular derivatives by insect epidermal cells undergoing metamorphosis in a succession of moults. For historical reasons, the concept has been primarily associated with developmental changes occurring during discrete moulting cycles, under the impetus of changing hormonal states. I believe that this formulation of the concept is only a special one, and that the concept is fundamentally applicable t o a very wide variety of developmental phenomena. I believe that developmental regulation centrally involves the programmed expression of specific gene sets in an orderly and often overlapping sequence. The programming of this sequential expression is what makes cell types fundamentally different from each other, and must ultimately be understood if we are to understand development. The actual sequential expression can be 1

2

FOTIS C. KAFATOS

described as a succession of visibly differentiated states. According to the physiological exigencies of the system, this succession may or may not be entrained by hormonal changes; sequential polymorphism may be relatively ,gradual, as well as saltatory. In this formulation, sequential polymorphism refers to a succession of distinguishable but possibly overlapping states of determination and differentiation-regardless of the physiological mechanisms which may have evolved t o control the cell's progress through the succession. The value of this concept to the developmental biologist is that it gives proper attention to the dynamic, temporal aspects of regulation, instead of placing the commonly excessive emphasis on a static end state of differentiation. In this paper, I will discuss polymorphism while reviewing the work that my colleagues and I have performed over approximately the last ten years. Many additional examples (including some that are more apt) could be adduced t o illustrate the concept of sequential polymorphism in this extended formulation.

2 Cellular metamorphosis in the saturniid labial gland The paired labial glands of larval silkworms are known as silk glands. In saturniids, as in B o m b y x (Yamanouchi, 1921), they are long tubes, ending blindly at their distal end and opening together in the spinneret on the labium. Two main parts can be distinguished. The posterior division is secretory, producing and storing the silk proteins, fibroin and sericin. The anterior division (Kafatos, 1971) is a narrower duct, which serves to conduct the liquid silk from the secretory division to the spinneret during spinning. This duct consists of approximately 1000 highly polyploid cells. Their main function appears t o be the production and maintenance of a thick (up to 20 pm) cuticle, which forms a rigid tubular channel. Shearing forces developing during the extrusion of silk through this narrow channel may serve to orient the metastable fibroin molecules, facilitating their subsequent crystallization into an insoluble thread (Lucas et al., 1958). It is also possible that the duct serves of modify the silk solution in subtle ways, by addition or removal of components. However, comparison of the abundance (relative to dry mass) of radioisotopes of CaZ+,K? and C1- in the cocoon and in stored liquid silk gives no evidence that the duct adds ions to silk. If anything, slight reabsorption of I C and C1- from liquid silk may occur during spinning. At pupation, the secretory division completely degenerates (as does the entire silk gland in B o m b y x ) . The duct, however, is preserved. The cells shed their characteristic larval cuticle and their cytoplasm regresses. The

CELLULAR POLYMORPHISM

3

nuclear branches, highly extended in the larval cell, are pulled together into a compact mass, surrounded by a thin layer of cytoplasm (Fig. 1 in Selman and Kafatos, 1974). Evidently, the pupal cells are at a developmental standstill. However, during the subsequent adult development they grow again and undergo a dramatic metamorphosis. First they secrete a thin (iao, T. H., Hsiao, C. and deWilde, J. (1975). Moulting hormone production in the isolated larval abdomen of the Colorado beetle. Nature, 255, 727-728. lIktber, R. and Hoppe, W. (1965). Zur Chemie des Ecdysons VII. Die Kristall-u. Molekiilstrukturanalyse des insektenverpuppungs-hormons Ecdyson mit der Automatisierten Faltmolekulmethode. Chem. Ber. 98, 2403-2424. ILrkawa, N., Hattori, F., Rubio-Lightbourn, J., Miyazaki, H., Ishibashi, M. and Mori, C. (1972).Gas chromatographic separation of phytoecdysones. J. Chronat. Sci. 10,

233-242.

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Imai, S., Fujioka, S., Nakanishi, K., Koreeda, M. and Kurokawa, T. (1967). Extraction of ponasterone A and ecdysone from podocarpaceae and related plants. Steroids, 10, 557-565. Imai, S., Fujioka, S., Murata, E., Sasakawa, Y. and Nakanishi, K. (1968a). The structures of three additional phytoecdysones from Podocarpus macrophyllus, makisterone B, C and D. Tetrahedron Lett. No. 36, 3887-3890. Imai, S., Hori, M., Fujioka, S., Murata, E., Goto, M. and Nakanishi, K. (1968b). Isolation of four new phytoecdysones, makisterone A, B, C, D and the structure of makisterone A, a Czs steroid. Tetrahedron Lett. No. 36, 3883-3886. Jizba, J. and Herout, V. (1967). Plant Substances XXVI. Isolation of constituents of common polypody rhizomes. Colln Czech. Chem. Commun. 32,2867-2874. Kaplanis, J. N., Robbins, W. E., Thompson, M. J. and Dutky, S. R. (1973). 26-Hydroxyecdysone: new insect moulting hormone from the egg of the tobacco hornworm. Science, 180, 307-308. Kaplanis, J. N., Tabor, L. A., Thompson, M. J., Robbins, W. E. and Shortino, T. J. (1966a). Assay for ecdysone (moulting hormone) activity using the house Ely, Musca domestica. Steroids, 8, 625-631. Kaplanis, J. N., Thompson, M. J., Yamamoto, R. T., Robbins, W. E. and Louloudes, S. J. (1966b). Ecdysones from the pupa of the tobacco hornworm Manducn sexta. Steroids, 8 , 605-623, Karlson, P. (1956a). Chemical investigation of the metamorphosis hormone of insects. Ann. Sci Nut. Zoot. Biol. Animale, 18, 125-137. Karlson, P. (1956b). Biochemical studies on insect hormones. Vitamins and Hormones, 14, 227-266. Karlson, P. (1963). Chemistry and biochemistry of insect hormones. Angew, Chem. (Int. Edn.) 2, 175-183. Karlson, P. (1974). Mode of action of ecdysones. In “Invertebrate Endocrinology and Hormonal Heterophylly” (Ed. W. J. Burdette), pp. 43-54. Springer, New York. Karlson, P., and Schmialek, P. (1959). Isolation of N-phenethylmalonamide from shrimp. Hoppe-Seyler’s Z. physiol. Chem. 316, 83-88. Karlson, P. and Shaaya, E. (1964). Der Ecdysontitre wahrend der Insektentwicklung I. Eine Methode zur Bestimmung des Ecdysongehalts. J. Insect Physiol. 10, 79 7-804. Karlson, P., Hoffmeister, H., Hoppe, W. and Huber, R. (1963). Zur Chemie des Ecdysons. Liebigs Ann. Chem. 662, 1-20. Karlson, P., Hoffmeister, H., Hummel, H., Hocks, P. and Spiteller, G. (1965). Zur Chemie des Ecdysons VI. Reaktionen des Ecdysonmolekuls. Chem. Ber. 98, 2394-2402. Katz, M. and Lensky, Y. (1970). Gas chromatographic analysis of ecdysone. Experientia, 26, 1043. King, D. S. (1972). Metabolism of a-ecdysone and possible immediate precursors by insects. In uivo and in vitro. Gen. Comp. Endocr. Suppl. 3, 221-227. King, D. S. and Marks, E. P. (1974). The secretion and metabolism of a-ecdysone by cockroach (Leucophaea maderae) tissues in vitro. Life Sciences, 15, 147-154. King, D. S. and Siddall, J. B. (1969). Conversion of a-ecdysone t o b-ecdysone by crustaceans and insects. Nature, 221, 955-956. King, I). S., Bollenbacher, W. E., Borst, D. W., Vedeckis, W. V., O’Connor, J. D., Ittycheriah, P. I. and Gilbert, L. I. (1974). The secretion of a-ecdysone by the

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prothoracic glands of Manduca sexta in vitro. Proc. natn. Acad. Sci. U.S.A. 71,

793-796. Kirkland, J. J. and Dilks, C. H. (1973). In situ coating of supports with stationary liquids for high-performance liquid-liquid column chromatography. Analyt. Chem.

45, 1778-1781. Koolman, J., Hoffmann, J. A. and Dreyer, M. (1975). Moulting hormone in Locusta migratoria: rate of excretion during the last larval instar. Experientia, 31, 247-249. KopCc, S. (1922). Studies on the necessity of the brain for the inception of insect metamorphosis. Biol. Bull. mar. biol. Lab., Woods Hole, 42, 323-342. Koreeda, M., Nakanishi, K., Imai, S., Tsuchiya, T. and Wasada, N. (1969). Mass spectrometric studies of ecdysone derivatives. Mass Spectroscopy, 17, 669-680. Kruppa, R. F., Henly, R. S. and Smead, D. L. (1967).Improved gas chromatography packings with fluidized drying. Analyt. Chem. 39, 851-853. Lafont, R., Delbecque, J. P., De Hys, L., Mauchamp, B. and Pennetier, J. L. (1974). Proportion of B-ecdysone in the hemolymph of Pieris brassicae (Lepidoptera) during the nymphal stage. C.r. hebd. S 'eanc. Acad. Sci. Ser. D . 279,1911-1814. Lauer, R. C., Solomon, P. H., Nakanishi, K. and Erlanger, B. F. (1974).Antibodies to the insect moulting hormone 0-ecdysone. Experientia, 30, 560-562. Levinson, H. Z. and Shaaya, E. (1966). Occurrence of a metabolite related to pupation of the blowfly Calliphora erythrocephala. Riv. Parassitologica, 27,203-209. Lowe, M. E. and Horn, D. H. S. (1967). Bioassay of the red chromatophore concentrating hormone of the crayfish. Nature, 213,408-410. Mathews, R. G., Schwartz, R. D., Stouffer, J. E. and Pettitt, B. C. (1970). New Polyamide liquid phases for gas chromatography. J.. Chromat. Sci. 8 , 508-512. Mathews, R. G., Schwartz, R. D., Pfaffenberger, C. D., Hen-Nan-Lin, S. and Homing, E. C. (1974). Polyphenyl ether sulphones. Thermally stable polar phases for gas chromatography. J. Chromat. 99, 51-61. Miyazaki, H., Ishibashi, M., Mori, C. and Ikekawa, N. (1973). Gas phase microanalysis of zooecdysones. Analyt. Chem. 45, 1164-1168. Morgan, E. D. and Poole, C. F. (1975). Formation of pentafluorophenyl-dimethylsilyl ethers and their use in the gas chromatography of sterols. J. Chromat. 104,351-358. Morgan, E. D. and Poole, C. F. (1976). The formation of trimethylsilyl ethers of ecdysones: a reappraisal. J. Chromat. 116,333-341. Morgan, E. D. and Woodbridge, A. P. (1971).Insect moulting hormones (ecdysones). Identification as derivatives by gas chromatography. Chem. Commun. 475-476. Morgan, E. D. and Woodbridge, A. P. (1974). Mass spectrometry of insect moulting hormones: trimethylsilyl-methoxime derivatives of ecdysone and 20-hydroxyecdysone. Org. Mass Spectrom. 9, 102-110. Morgan, E. D., Woodbridge, A. P. and Ellis, P. E. (1975a). Studies on the moulting hormones of the desert locust, Schistocerca gregaria. J. Insect Physiol. 21, 979-993. Morgan, E. D., Woodbridge, A. P. and Ellis, P. E. (1975b). Isolation of moulting hormone from the desert locust, Schistocerca gregaria (Forskal). Acrida, 4,69-81. Moriyama, H., Nakanishi, K., King, D. S., Okauchi, T., Siddall, J. B. and Hafferl, W. (1970). On the origin and metabolic fate of a-ecdysone in insects. Gen. comp. Endocr. 15, 80-87. Nakanishi, K. (1971).The ecdysones. Pure and Applied Chem. 25, 167-195. Nalaanishi, K., Moriyama, H., Okauchi, T., Fujioka, S. and Koreeda, M. (1972).

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Biosynthesis of (Y- and P-ecdysones from cholesterol outside the prothoracic gland in Bombyx mori. Science, 176, 51-52. Nakanishi, K., Erlanger, B. F. and Lauer, R. C. (1973). Control of insect behaviour by natural products. In “New Methods Environ. Chem. Toxicol” (Ed. F. Coulston), pp. 149-52. Int. Acad. Print. Co., Tokyo. (Chem. Abs. 1975, 83, 1 0 9 6 2 3 ~ ) . Nigg, H. N., Thompson, M. J., Kaplanis, J. N., Svoboda, J. A. and Robbins, W. E. (1974). High-pressure liquid-solid chromatography of the ecdysones-insect moulting hormones. Steroids, 23, 507-516. Ohtaki, T., Milkman, R. D. and Williams, C. M. (1967). Ecdysone and ecdysone analogues: their assay on the fleshfly Sarcophaga peregrina. Proc. natn. Acad. Sci. U.S.A. 58,981-984. Ohtaki, T., Milkman, R. D. and Williams, C. M. (1968). Dynamics of ecdysone secretion and action in the fleshfly Sarcophaga peregrina. Biol. Bull. mar. biol. Lab., Woods Hole, 135,322-334. Poole, C. F. (1975). An investigation into the determination of ecdysones and other steroid hormones. Ph.D. Thesis, Keele University. Poole, C. F. and Morgan, E. D. (1975a). Structural requirements for the electron capturing properties of ecdysones. J. Chromatop. 115, 587-590. Poole, C. F. and Morgan, E. D. (1975b). Electron impact fragmentation of pentafluorophenyldimethylsilyl ethers of some sterols of biological importance. Org. Mass Spectrom. 10, 537-549. Poole, C. F., Morgan, E. D. and Bebbington, P. M. (1975). Analysis of ecdysones by gas chromatography using electron capture detection. J. Chromatop. 104, 172-175. Rees, H. H. (1971). Ecdysones. In “Aspects of Terpenoid Chemistry and Biochemistry” (Ed. T. W. Goodwin), pp. 181-222. Academic Press, New York and London. Rogers, W. P. (1973). Juvenile and moulting hormones from nematodes, Parasitology, 67, 105-113. Romer, F., Emmerich, H. and Nowock, J. (1974). Biosynthesis of ecdysones in isolated prothoracic glands and oenocytes of Tenebrio molitor in vitro. J. Insect Physiol. 20, 1975-1 987. Sannasi, A. and Karlson, P. (1974). Metabolism of ecdysone: phosphate and sulphate esters as conjugates of ecdysone in Calliphora vicina. 2001. Jb. Physiol. 78, 378-386. Sardini, D. and Krepinsky, J. (1974). Densitometric determination of ecdysones. Farmaco Ed. Prat. 29, 723-731. Sato, Y., Sakai, M., Imai, S. and Fujioka, S. (1968). Ecdysone activity of plantoriginated moulting hormones applied to the body surface of lepidopterous larvae. Appl, Entomol. 2001. 3, 49-51. Schooley, D. A. and Nakanishi, K. (1973). Application of high pressure liquid chromatography to the separation of insect moulting hormones. In “Modern Methods of Steroid Analysis” (Ed. E. Heftmann), pp. 37-54. Academic Press, New York and London. Schooley, D. A., Weiss, G. and Nakanishi, K. (1972). A simple and general extraction procedure for phytoecdysones based on reversed-phase adsorption chromatography. Steroids, 19, 377-383. Schmit, J. A. (1971). Applications of high-speed liquid chromatography using controlled surface porosity support. In “Modern Practice of Liquid Chromatography” (Ed. J. J. Kirkland), pp. 375-415. Wiley-Interscience, New York.

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Siddall, J. B., Cross, A. D. and Fried, J. H. (1966).Steroids CCXCII. Synthetic studies o n insect hormones 11. The synthesis of ecdysone. A m . chem. SOC. 88, 862-877. Simpson, R. M. (1972). “The Separation of Organic Chemicals from Water”, pp. 1-26. Rohm and Haas, Philadelphia. Slama, K., Romanuk, M. and Sonn. F. (1974).Chemistry and physiology of ecdysoids. I n “Insect Hormones and Bioanalogues”, pp. 303-387. Springer, Vienna and New York. Stahl, E. (1962).“Dunnschicht-Chromatographie”, p. 51 5. Springer, Berlin. Stamm, M. D. (1958). Isolement d’hormones de mPtamorphose dans I’orthopt6re Dociostaurus maroccanus. Rev. Esp. Fisiol. 14,263-268. Takemoto, T,, Ogawa, S. and Nishimoto, N. (1967a).Constituents of Achyranthis radix 111. Structure of inokosterone. Yakugaku Zasshi, 87, 1474-1475. Takemoto, T., Ogawa, S., Nishimoto, N. and Hoffmeister, H. (1967b).Steroide mit Hautungshormon-Aktivitat aus Tieren und Pflanzen. 2. Naturf. 22b, 681-682. Takemoto, T., Ogawa, S., Nishimoto, N. and Taniguchi, S. (1967~).Constituents of Achyranthis radix IV. Isolation of the insect moulting hormones from Formosan achyranthis. Yakugaku Zasshi, 87, 1478-1480. Takemoto, T., Ogawa, S., Morita, M., Nishimoto, N., Dome, K. and Morishima, K. (1968). Studies on the constituents of Achyranthis radix VI. Determination of insect moulting hormones. Yakugaku Zasshi, 88, 39-43. Thompson, M. J., Kaplanis, J. N., Robbins, W. E., Dutky, S. R. andNigg, H. N. (1974). 3-Epi-20-hydroxyecdysone from meconium of the tobacco hornworm. Steroids, 24,

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3 59-366. Thompson, M. J., Kaplanis, J. N., Robbins, W. E. and Yamamoto, R. T. (1967). 20,26-Dihydroxyecdysone, a new steroid with moulting hormone activity from the tobacco hornworm, Manduca sexta. Chem. Commun. 650-653. Thompson, M. J., Robbins, W. E., Kaplanis, J. N., Cohen, C. F. and Lancaster, S. M. (1970). Synthesis of analogues of a-ecdysone. A simplified synthesis of 2/3,3/3,1&trihydroxy-7-en-6-one-5/3-steroids. Steroids, 16,85-104. Thomson, J. A. (1974). Standardization of dipteran bioassay for moulting hormones. In “Invertebrate Endocrinology and Hormonal Heterophylly” (Ed. W. J. Burdette), pp. 121-129.Springer, New York. Thomson, J. A,, Imray, F. P. and Horn, D. H. S. (1970). An improved calliphora bioassay for insect moulting hormones. Aust. J. exp. Biol. med. Sci. 48,321-328. Vedeckis, W. V., Bollenbacher, W. E. and Gilbert, L. I. (1974).Cyclic AMP as a possible mediator of prothoracic gland activation. 2001.Jb. Physiol. 78, 440-448. Wigglesworth, V. B. (1934). Factors controlling moulting and “metamorphosis” in an insect. Nature, 133, 725-726. Willig, A. and Keller, R. (1973). Moulting hormone content, cuticle growth and gastrolith growth in the moult cycle of the crayfish Orconectes Eimosus. J. Comp. Physiol, 86, 377-388. Woodbridge, A. P. (1971). Studies of the moulting hormones of the desert locust, Schistocerca gregaria. Ph.D. Thesis, Keele University. Zatsny, I. L., Gorovits, M. B., Rashkes, Ya. V. and Abubakirov, N. K. (1975).Phytoecdysonesof Serratula. 111. Mass spectrometric study of ecdysterone and viticosterone E acetates and acetonides. Khirn. Prir. Soedin. 11, 155-158.

T h e Cells of t h e Insect Neurosecretory System: Constancy, Variability, and t h e Concept o f t h e Unique Identifiable Neuron Hugh Fraser Rowell University of California, Berkeley, California, USA

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1 Introduction 63 65 2 The anatomy of the insect neurosecretory system 65 2.1 Recognition of neurosecretory cells . 2.2 Differences between “specific” staining techniques for neurosecretory 67 . cells with light microscopy . 2.3 General results of morphological studies of neurosecretory cells 70 71 2.4 The distribution of neurosecretory ceIIs in insects 75 2.5 Diversity of neurosecretory cell complement in insects . 3 Implications of the identifiable cell concept for the insect neurosecretory 99 system. . 3.1 Anatomically significant differences between neurosecretory cells and 99 other neurons 3.2 Constancy, uniqueness, and reduplication in neurosecretory cells 100 106 4 Available techniques, research strategy, and some examples . 106 4.1 Filling of neurons with dye from severed nerve stumps . 4.2 Filling of neurons with dye through an intracellular micropipette electrode 107 107 4.3 Intensification of cobalt staining by silver precipitation . 110 4.4 Electrophysiological recording and intracellular current injection 111 Acknowledgements Abbreviations . 112 References 112

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7 Introduction Invertebrate neurophysiology has been revolutionized in recent years by the general application of the concept of the unique identifiable neuron. Broadly stated, this concept runs as follows. In many invertebrate taxa, 63

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most of the nervous system is comprised of neurons each of which has a i:li,iracteristic morphology, connectivity and function. Both the individual ni’urones and the neuronal circuits they form are effectively identical in di 1 ferent conspecific individuals. The number of neurones is fixed, within narrow limits of variation. The system is “hard-wired”, there appears t o t:sist no large pool of neurons whose connectivity is essentially plastic and which potentially show large differences between individuals as a consec pence of environmental experience. Within the central nervous system as a whole, there is very little “redundancy”, in the sense of duplication of u i i its with effectively identical function, other than that inherent in the s q n e n t a l organization of the body or in spatially organized sensory arrays such as the retina. Nervous organization of this sort is characteristic of taxa with determined growth, little plasticity of adult form, short lives, and a high degree of neural complexity combined with relatively small numbers of neurons in t h c CNS. It is most obvious in the Annelida, Arthropoda, the gastropod Mollusca, and in the Nematoda and other askhelminth groups. It is not yet dcar whether the same principles apply t o taxa with nervous systems diaracterized by very numerous cells and plastic behaviour (v-rtebrates and ce1)halopod molluscs) or with less complex nervous systems or great mor111 lological plasticity (such as echinoderms, or coelenterates). It is, however, n o t excluded that the concept of the unique identifiable neuron is applicable in these cases too; it is merely the technical difficG!tv of the proof which has discouraged investigation. ‘The foundations of the concept were laid by the early invertebrate ~ic.uroanatomistssuch as Retzius on the basis of methylene blue staining. Its rec.ent wide acceptance has come from the development of newer techiiiques: a. 1)evelopment of markers suitable for intracellular labelling of cells in both light microscopy (LM) and electron microscopy (EM). h. Axonal iontophoresis or diffusion of these markers into cells via the cut axon. c. Anatomical identification of recorded cells by injection of marker through the intracellular recording electrode. d. The ability to return repeatedly t o homologous neurons in different individual animals, and t o check the identity both by physiological characterization and by injected marker. Neuronal circuit analysis, cellular neuronal function, and genetic studies of neuronal morphology and function are now dominated by those invertebrate preparations t o which the concept of the unique identifiable neuron is currently most applicable.

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This paper seeks t o examine the applicability of the concept t o the insect neurosecretory system, and t o define areas and preparations which seem suitable for analysis by the new techniques. The physiology of the insect neurosecretory system is a very active field, and one frequently reviewed (most recently by Goldsworthy and Mordue, 1974, and by Maddrell, 1975). I will not attempt t o deal with this aspect, and have confined myself to anatomical data. I deal with anatomical identification of neurosecretory cells (NSCs), and then with their distribution in the insects. I have attempted to ascertain whether their numbers are relatively constant between individuals, in which taxa the numbers of NSCs are large or small, and where cells suitable for electrophysiological techniques are likely to be found. In the second part of the paper, I present the biological implications of the concept of the unique identifiable neuron for insect neurosecretion, and give some indication of the sorts of experiments potentially feasible, with a few recent examples.

2 The anatomy of the insect neurosecretory system

2.1

RECOGNITION OF NEUROSECRETORY CELLS

Neurosecretory areas are often initially localized by physiological work involving lesion or ligature, and in some cases the cells themselves are visible upon inspection of the living tissue, a blue coloration deriving from their vesicular contents by the Tyndall effect. In general, however, NSCs are recognized from histological preparations. There are obvious dangers in attributing neurosecretory function t o a neuron on purely morphological grounds, a point stressed for many years by Scharrer and by Bern (e.g. 1966), but experience t o date suggests that the correlation is in fact a good one. Most neurones with NSC morphology do indeed turn out t o differ from regular neurons in their functioning. As the boundary between the two types is arbitrary, there are always borderline cases, and these are especially obvious in the case of direct neurosecretory innervation, as discussed on p. 74, or in the development of neurosecretory facies in injured and regenerating regular neurons (Boulton and Rowell, 1969; Milburn, personal communication). I will in general take the position that the morphological identification of NSCs has some validity, and discuss the available data accordingly. Three distinct types o f histological display of NSCs can be recognized, using respectively (a) general purpose LM staining, (b) “specific” LM staining for NS material and (c) EM techniques. a. General purpose LM stains The original descriptions of NSCs in insects were made using orthodox general stains. Some of these fortuitously

66

HUGH FRASER ROWELL

inc luded dyes with a special affinity for neurosecretory material, such as tlic. fuchsin component of both Masson’s and Mallory’s triple stains, and tliiis presage the use of “specific” stains. The majority of accounts were, hoivever, based not on specific dye affinity but on the structural correlates of ,ecrction, and especially on the presence of numerous “granules” visible in i.he cytoplasm of soma and sometimes axon. These granules have sullsequently proven to represent aggregates of submicroscopic membraneboiind vesicles or other organelles such as lysosomes, and the aggregation is oft(m a fixation artifact (Bloch et al., 1966; Bern, 1966). b. *‘Specific” LM staining f o r NSCs Modern NSC cytology dates from the ap1)lication by Bargmann (1949) of two of Gomori’s staining procedureschi ome alum-haematoxylin phloxine and aldehyde fuchsin. These were the fir\t of many which rely on a special affinity of a dye for some compon,ent o f the NSC. The component in question is almost certainly not the h o i mone itself, but is possibly the carrier protein associated with it (see e.p. Schreiner, 1966). They are here referred t o as “specific” stains, the q u ( Itation marks stressing that their specificity is actually for certain chl.mica1 groups which have only a statistical probability of association wiih NSCs. They were first applied t o insects in the early fifties by rl’hl)msen, Scharrer, Nayar, Gabe and their co-workers. A section on the ch.rracteristics of the various techniques is given below, but basically the abicling dilemma is that no dye stains all NSCs as opposed to all other netirons. As the functional distinction between the two is of degree rather tli;in of kind, this is t o be expected. Consequently, the more chemically pr‘.cise the staining reaction the smaller the number of NSCs it reveals, and coiiversely the number of NSCs discovered is usually directly related t o the nuiiiber of staining methods utilized in the study. With any one dye, there a x usually differences between NSCs in the distribution of dye within the cc:ll, and the apparent granule size is sometimes the main distinguishing ch.iracteristic (e.g. in Lampyris, Coeloptera: Naisse, 1966a). Modern LM claysifications typically use both stain affinity and intracellular distribut ion of stain as criteria.

c. f:M techniques The most gratifying aspect of the EM examination of NS(k was to find that virtually wherever neurosecretory function had been postulated on the basis of LM staining, the cells did in fact contain a plaiisible facies of endoplasmic reticulum, ribosomes, vesicles, etc., when vielved by EM. Additionally, some cells have been discovered which have thc appearance of NSCs by EM, but which do not stain with any of the uxu.il LM procedures (Sandifer and Tombes, 1972; Chalaye, 1974a); it is no1 known how common such cells are. The morphological classification oI)i,iined from EM profiles, based on size, shape, electron density and

N EUROSECR ETO RY CELLS

67

number of vesicles, is as diverse as that obtained from LM procedures (up to 1 0 categories can be recognized by either means) and in at least some cases the two classifications conflict. That is, for example, a population of NSCs which appears homogenous after staining for LM may show a variety of vesicle types under EM (e.g. Maddrell, 1967; Unnithan et aE., 1971; Geldiay and Edwards, 1973; Musko and Novak, 1973) or vice versa (Schooneveld, 1974a). Many attempts have been made to correlate LM and EM pictures of the same populations (Ramade, 1966; Block et al., 1966; Bassurmanova and Panov, 1967; Brady and Maddrell, 1967; Cassier and Fain-Maurel, 1970; Smalley, 1970; Geldiay and Edwards, 1973; Chalaye, 1974a; Schoonveld, 1974b), but no study has been made with truly identified cells or in which the extent of correlation was the main object of the investigation. (See Note added in proof, p. 123.)

2.2

DIFFERENCES BETWEEN “SPECIFIC” STAINING TECHNIQUES FOR NEUROSECRETORY CELLS WITH LIGHT MICROSCOPY

As mentioned in the preceding section, the LM staining techniques used to demonstrate NSCs vary between two extremes. At one extreme, there are techniques which apparently stain all NSCs, but tend to stain regular neurons as well; the distinction between the two becomes subjective and tenuous, and requires much experience for consistency. The best example of such a technique is azan. At the other extreme are histochemical stains which react with a precisely defined chemical substrate, and the techniques employ chemical pretreatment to turn neurosecretory products or carrier proteins into these reactive groups. An example is the use of Red Sulfhydryl Reagent which stains sulfhydryl or disulphide groups. Type A NSCs (as defined below) are rich in cystine and cysteine; after oxidation sulphydryl and disulphide groups are formed, which appear t o be the basis for the selectivity of several basic dyes for these cells (see Schreiner, 1966, and Table 1).There are many undoubted neurosecretory cells which do not stain at all with these stains, and the same pretreatment applied t o type C and some type B cells, for instance, results in predominantly pyrrhol and indol radicals (Baudry and Baehr, 1970; Raabe and Monjo, 1970). The classification of insect NSCs by their staining properties has been the subject of much controversy. Most authors have found it convenient t o recognize, for descriptive purposes at least, four broad classes, as follows.

1. Cells staining red with the azocarmine component of azan; staining dark blue or black with chrome alum haematoxylin (CH), sometimes referred to as “Gomori positive” in the older papers; staining heavily in shades of purple with paraldehyde fuchsin (PF). These cells correspond to the type A cells of Nayar (1955).

68

HUGH FRASER ROWELL

2. Cells staining blue with the aniline blue component of azan; not stained by CH but stained shades of red with phloxin’e (P); stained weakly or not at all by PF, but often staining with one of the counterstains used with this technique, such as picroindigocarmine (PIC) or Halmi’s mixture (HMChromotrope 2R, orange G, light green, phosphotungstic acid). These cells are always more capricious and difficult t o demonstrate than A cells, and correspond t o Nayar’s (1955) type B cells. 3. Cells stained red in azan, but not stained by either CHP nor b y PF or its counterstains. These cells are called type C b y Raabe (1965, 1967, and subsequent works) and the numerous subsequent French workers who have made a study of their occurrence. 4. Finally, by exclusion, one can define a heterogeneous category comprising cells which stain differently from any of the above. They are less often described, or investigated, than the others. Classifications based on staining affinities have been endlessly proposed, subdivided, criticized, modified and acclaimed. Much of the discussion appears t o be generated by: a. A natural tendency to subdivide. b. An apparent belief that classifications should be both all embracing and also correspond t o real biological distinctions between NSCs or their products. This belief is surely unjustifiable; it is no criticism of an arbitrary morphological classification t o prove that there are intermediates (e.g. cells which stain. heavily with PF but also stain with phloxine, as described by Fletcher (1969)). All that matters is whether the diagnostic characters are stable, repeatable, and precisely definable. c. Unfamiliarity with the material used by other workers, leading t o a reluctance to use the same terminology as was applied originally t o a different species, in case it proves t o be different.

d. A simple failure t o follow precedent in nomenclature; thus the appellation “C cell” has been applied by successive workers t o distinct populations of cells t o the point where, in the absence of further description, it now means only “not A or B”. The result of all this is semantic chaos. It is impossible to compare the results of two workers without knowing not only the details of their histological procedure, but also which school (if any) of nomenclature they follow. Occasional noble attempts to rationalize the literature (e.g. Delphin, 1965) have not led to subsequent improvement. For the purposes of this section, I will use the terms A, B, and C cells in the senses indicated above, and make no value judgement. There is some histochemical evidence that the proteins elaborated by these 3 types of cells show real differences (Raabe and Monjo, 1970; Prentb, 1972).

N E U ROSECR ETORY CELLS

69

To assess the comprehensiveness of a reported investigation, it is essential to know what degree of specificity each of the “specific” staining techniques possesses. The more important facts are presented in Table 1. Note that the names refer not t o dyes but t o entire histological processes, including fixation. The text hereafter will use the abbreviations given in Table 1. This Table shows that the selection of stains from the different categories is important in making a survey of the NSCs of a nervous system. It is common to find a worker using several different stains but restricting TABLE 1 Summary of the commonest “specific” light microscope techniques for neurosecretory cells Abbreviation

Full name

i. Basic dyes with affinity f o r type A NSCs, arranged in decreasing order of selectivity RSR Red suphydryl reagent PAVB Performic acid/victoria blue AB Alcian blue Paraldehyde fuchsin, aldehyde fuchsin. The former has largely replaced PF, AF the latter. CH Chrome alum haematoxylin Paraldehyde thionin. Similar in effect to paraldehyde fuchsin, but said to PTh respond better to type B material. ii. Acid dyes with affinity f o r type B cells P Phloxine PIC Picroindigocarmine iii. Combination techniques designed to display simultaneously and differentiate between A - and 13-type cells CHP Chrome alum haematoxylin/phloxine ABP Alcian blue/phloxine PF/HM Paraldehyde fuchsin, counterstained with Halmi’s mixture (chromotrope 2R, orange G, light green, phosphotungstic acid) PF/PIC Paraldehyde fuchsin, counterstained with picroindigocarmine FTh/NY Paraldehyde thionin, counterstained with naphthol yellow iv. Wide range techniques which demonstrate a variety of NSCs, but rarely provide critical information differentiating between them. Often used to stain NSCs not stained by any of the above methods Az Azan (note that type C material, as defined in the text, can only be demonstrated by use of both azan and other techniques simultaneously) M Mallory’s triple stain M3C Masson trichrome stain PSI Pseudoisocyanin (this is a fluorescence microscopy technique)

70

HUGH FRASER ROWELL

them in such a way as t o stain oniy one of the major cell types. It is especially common that only stains for A’cells, which are readily demonstrated, are used. For this reason, the subsequent Tables, which present the results of surveys of cell numbers, include information about the staining techniques used, in order that results which are likely t o be deficient in one or more cell types can be identified. For example, an investigation that did not use Azan is not likely t o have considered C cells, which seem t o be the commonest NSCs of the ventral nerve cord (VNC) and the tritocerebrum.

2.3

GENERAL RESULTS OF MORPHOLOGICAL STUDIES OF NEUROSECRETORY CELLS

Apart from a variety of observations of relevance t o the problems of cellular secretion per se, at least three general facts of importance have emerged from the anatomical work on insect NSCs. i. The stainability of particular cells or populations of cells varies with time. To what extent this correlates with any “secretory cycle”, and whether intense staining reflects anything about the hormonal content of the cell, is debatable. It is, however, certain that the same cells stain more readily at some times than others, and may be quite invisible t o a “specific” stain at some times. Variation may be cyclical, with either a diurnal o r longer (e.g. stadial) periodicity, or merely show a long-term change with age; examples can be found in Arvy and Gabe (1952, 1953), Khan and Fraser (1962), Huignard (1964), Panov (1965), Brady (1967), Burgess (1971), Steele and Harmsen (1971), Cymborowski (1973) and Kono

(1975). ii. The stainability of the NSC is not necessarily constant throughout its length. With some cells “granules” (actually clumps of submicroscopic membrane-limited vesicles) can be stained in the somatic cytoplasm and out along the axon t o the release point. This is characteristic of A-type cells of the medial NSCs of the pars intercerebralis. In other cells, undoubtedly neurosecretory, no “ganules” are ever seen in the axon by light microscopy, and it is presumed that the secretion is transported for release in a nonstainable form (e.g. in the lateral NSCs of the pars of Orthoptera). Other cases have been reported where the dye affinity of cytoplasm or of granules changes as one nears the morphological release point, again suggesting a chemical change in hormone, carrier protein, membrane, or some other constituent of the cell (Gabe, 1972). iii. Broadly similar NSCs (as described by EM or LM practices) are found in widely different insects, and there is not an infinite range of response t o the microscopical methods. As insects can be expected t o show at least marked similarity, if not identity, in their hormones and carrier proteins, this result

N EU ROSEC R ETO R Y CELLS

71

makes one hopeful that the morphological classifications of NSCs may in fact have some relevance ‘to their biological diversity-e.g. differently staining cells may indeed secrete hormones, and possibly even vice versa.

2.4

THE DISTRIBUTION OF NEUROSECRETORY CELLS IN INSECTS

The physiological and anatomical techniques described above have led t o the description of NSC somata in the following parts of the body. a.

BRAIN

Pro tocerebrum i. Lateral cells of pars intercerebralis. ii. Median cells of pars intercerebralis. These two groups are the source of the NSC axons of the nervi corporis cardiaci I and 11, where these exist as separate nerves (Hanstrom, 1940; Cazal, 1948; Williams, 1948). In at least 2 insects with separate NCC I and 11, the “medial” cells contribute axons t o both nerves (Perzplaneta,Willey, 1961: Leptinotarsu, Schooneveld, 1974b). The median and lateral groups are well separated in some orders (e.g. the Orthoptera) but much less so in the Oligoneoptera as a whole. Usually the lateral cells are defined by exclusion of the medials, and both are probably heterogeneous categories. Optic lobe

NSCs have been described from the optic lobe of neuropterans (Arvy, 1956), saturniid moths (Mitsuhashi, 1962), calliphorid flies (Thomsen, 1965) and cockroaches (Beatty, 1971), and may be of more general occurrence. Ocellar nerue Median NSCs are often associated with the base of the ocellar nerve (e.g. in grasshoppers-Girardie and Girardie, 1972), but in Surcophugu (Diptera) and Gryllus (Orthoptera) NSC somata are found along the length of the nerve (Schlein, 1972; Loher, personal communication). The ocellar “nerve” is really an extension of the brain, containing both cell bodies and synaptic neuropil (C. Goodman, unpublished observations) so this observation is less remarkable morphologically than it at first appears. D&to cerebrum

At least some of these NCSs are the origin of the axons contained in the NCC IV (Brousse-Gaury, 1967).

HUGH FRASER ROWELL

72

rritocerebrum .Qt least some of these cell bodies are the origin of the axons of the NCC 111 (Pflugfelder, 1936, Dupont-Raabe, 1956; Willey, 1961; Raabe, 1965a, b). b. RETROCEREBRAL COMPLEX

Corpus cardiacum (CC) The essential element of the CC is the intrinsic glandular cells and their innervating neurons, derived at least largely from the brain. In many, though not all, insects the CC additionally contains a more or less complex iieurohaemal organ derived from the cerebral NSCs (above). It is not clear how many of the intrinsic secretory cells of the CC are t o be regarded as neurons, nor indeed what would be the distinguishing criteria for NSC as opposed t o some other forms of parenchymatous secretory cell of ectotlermal origin. Some authors assume without question that CC intrinsic cells Lither are or are not NSCs, and those that debate the question appear t o make the attribution of NSC on the basis of either an axon-like process, or a propagated action potential (e.g. Normann, 1975; Cazal et al., 1971). Neither of these features, however, is a necessary or sufficient definition of 25*

2x7* 2 x 3*

“Identical with Ephemeroptera” “Very numerous” 2 x >30* 2 x >40*

Present

“Numerous”

Present

Az, CHP, PF

Cazal (1948) ANY and Gabe (1953) Cazal (1948)

Present Az, Fuchsin, Methyl Green, CHP PSI

Arvy and Gabe (1952) Sterba and Hoheisel (1964) Gabe (1966)

2 x 66

2x6

CHP, PF

Khattar (1968)

2 x 300+

Not seen 2 x 12-16 Present No information

PAVB PF/PIC CHP, PF CHP, PF, PAVB Methylene blue, EM Az, AB, PF, PAVB,

Dogra (1967a) Gaude and Weber (1966) Huignard (1964) Geldiay and Edwards (1972) Girardie (1973)

Very numerous” 2 x 400 No information

2 x 8-12

per formic-acid-Schiff,

EM

5 < rn r r v)

U

a3

TABLE 2-conlinued Taxonomic division

Median NSCs

Lateral NSCs

Schistocerca

2 x c. 1000 2 x c. 400

2 x 10

2 x 604

Anacridium

“Numerous”

Melano pus

2 x 400+

2 x 12

Technique CHP, PF Cobalt Iontophoresis EM of NCC I

Reference Highnam (1961) Mason (1973)

PF, PAVB

Rowell and Mason (unpublished) Girardie and Granier

PAVB. CHP

Dogra and Ewen (1970)

PF, silver impregnation

Willey (1961)

(1973)

Dictyoptera Periplaneta “Numerous” 2 X 45-50 2xc.50

Phasmida Cliturnnus Carausius, Clitu mnus Bacillus, Eurycnema

Not seen 2 x 40, (;;h;r only

Not seen Not seen

CHP, PF CHP, PF

I

C 2 x >40* “Numerous”

Present 2 X 5-6

Az, CHP, M3C

Cazal(l948) Dupont-Raabe (1951 1956,1957)

2 x >40*

CHP

“Un g a n d nombre” Plecoptera ISOPerla

Fiiller (1960) Pipa (1962 and personal communication)

2 x >30*

Present

CHP

Herlant-Meewis and Paquet (1956) Arvy and Gabe (1954)

t)

I n I]

Rrn

R R

0 r r

D errnaptera Forficu la, Lab idu la Anisolabis

3. Paraneoptera Mallophaga Psocoptera Thysanura Anoplura Hemiptera (Gymnocerata) Reduviidae

i

z

“Numerous” 2 x 10-20

CHP, AF, M

Cazal (1948) Ozeki (1958)

m C

a

am 0

a rn

-I

0

< Present

Gabe (1966)

Present

0

rn r r v)

2 x 20* 2x65

----- -- --- 2 Adelphocoris Lygeidae Oncopeltus Stilbocoris Pyrrhocoridae Iphita Dysdercus Pentatomidae Nerara Scutellera

Present Not positively identifiedperhaps 2 x 2

No information 2 x 19-20

CHP, Az 32 total _ _ _ _ _ _ _ _ _ _ _ - - - _ PF/HM

Wigglesworth (1940) Baehr (1968) Steel and Harmsen (1971)

2 x 12

2x3

PF, CHP, PAVB

Ewen (1962)

2x7 2 x 14-16

2x43 2x2

PF, CHP PF, PAVB, CHP, Az

Johansson (1957,1958) Furtado (1971)

2 x 16 2 x 9-10

2 x 3-4 2 X 4-6

CHP, PF PF, PAVB, PATh

Nayar (1955) Dogra (1967)

2 x 5+ 2 x 8-9+

No information PF, PAVB Not seen PF, PAVB, AF

Awasthi (1 969) Srivastava and D o p a (1969)

03 0

TARLE 2-continued Taxonomic division (Cryptocerata) Belostomatidae Belostoma Nepidae Ranatra Homoptera (Cicadoidea) Tettigia

(Aphoidea) Aphis Drepano sip hu m

Median NSCs

Lateral NSCs

2 x 8-10+

Not seen

PF, PAVB

Dogra (1969)

2 x 9-10+

2 x 3-4

PF, PAVB

Dopa (1967)

Present

Present, though not laterally situated

2 x 4-6 2 x 4-6

2x2

No information

Present

Technique

Reference

Cazal (1948)

Az, CHP, Pf

Johnson (1963)

4. Oligoneoptera (Panorpoid complex) Siphonaptera; Trichoptera: Mecoptera; Panorpa Neoptera: Euroleon

Cazal (1948)

I C GI

r n

“Numerous” 2 x >14*

Present

2 x >20*

2 x >5*

Cazal (1948) PF, CHP, F’rennants trichrome

Arvy (1956)

ID

50

Lepidoptera: (Pyraloidea) Ephestia Galleria (Sphingoidea) Herse, Sphinx (Bombicoidea) Bombyx

c

2 x 6-8 2 x 38-47

2 x 2-3 2 x 12

Az, Iron haematoxylin

Rehm (1950,1951)

n

Az, CHP, PF

Delipine (1965)

0, m

2x8

2 x 9-10

CHF, PF, PTh

Panov and Kind (1963)

rn -I 0

2 x 10 (2x8

2x7 2 x 8-10

CHP, PF CHP, PF, PATh

Kobayashi (1 95 7) Panov and Kind (1963)

0

0

n

n


15*

No information

NS

Cazal ( 1948)

2 x>5*

2 x >4*

NS

Cazal (1948)

2x3 2x3 2x3 2 x 5-7 2 x "a few" 2x3

RefIected Iight CHP, PF CHP CHP CHP PF, PAVB

E. Thomsen (1952) M. Thomsen (1965) Langley (1965) Kopf (1957) Nayar (1954) Dogra and Tandan (1966)

2x6

PF

Gawande (1968)

Not seen

CHP

M. Thomsen (1954)

Ca Ilip ho ra Glossina Drosophilu Chaetodacus Sarcop haga 5. Oligoneoptera (nonpanorpoids) S trepsip tera Hymenopt era (Formicoidea) Formica and Camponotus (09) (Vespoidea) Eumenes, Synagris (99) (Apoidea) A p i s (P)

Andrenu ( 0 )

Coleoptera (Caraboidea) Nebrio

(i

;2-13 2 x 8-10 2 X 12-16 2xc.6 2x13

Technique

Reference

N o information

2 x c. 100 2x

>loo

I

2 x c. 60* 2 x c . 65 2 x 20+

N o information Present CHP Not seen Az, CHP Molybdenum haematoxylin

Weyer (1935) Formigoni (1956) Brandenburg (1956)

5 b

I -n II

D

v)

m II SD

0

2x8

2 X 2-4

AF, CHP,'AB/P silver inpregnation

Ganargarajah (1965)

5r... r

(Hydrophiloidea) Hydrous

3

2 x 20

Aulacophora (Curculionoidea) Slaps Hypera Sitophilus

(Cantharoidea) Lampyris

X

4-6

GHP, Qiemsa, Feullgen

De Lerma (1954)

n .. 3

1

(Dermestoidea) Dermestes (Chrysomeliodea) Galeruca Leptino tarsa

2

Ern

2x5

Ladduwahetty (1962) quoted by Siew (1965)

2

0

2 x 54-64 2 x 80

2 x 8-16 2 X 6-27

2 x 8-10

2x2

2

--

X

24-26

2 x 7-12

- - --- - c. 2 x 30 total ------------

No information

2 x 11

2 x 12

Present

PF, ABQ’, CHP, Az CHP, PlF/PIC, PTh, histoochemical stains AF, CHIP

Siew (19 6 5) Schooneveld, (1970)

ABP, P:F EM of INCC PF/HM:I, CHP

Fletcher (1969) Tombes and Smith (1970) Sandifer and Tombes (1972)

AB, C m P , p F / n I C , PTh/NY, Histoochemical stains

Naisse (1966a)

rn r

r cn

Saini (1967)

Figures given without comment are derived directly from the author quoted. Those indicicated with an asterisk are minimum estimates derived by the present author from the quoted work, e.g. from pictures of histological secztions, in cases where the original author gives no numerical information. Unless otherwise stated counts are based on numbers of s o m a t a after staining. The subdass and order are routinely given along with the genus; where several genera of one order have been hvestigisated, they are additionally grouped by family or superfamily as appropriate. W

w

84

HUGH FRASER ROWELL

large numbers of medial NSCs, certainly up t o 2 x 600. In the case of the acridid Orthoptera, comparable estimates have been reached b y three different methods: counting of stained somata from sectioned material; axonal iontophoresis of cobalt up the NCC I and counting somata in whole mount and in section; and counting neurosecretory profiles in an EM transverse section of the NCC I. There is no doubt as t o the large number of cells. Figures are not available for the Phasmida (stick insects, etc.), but the qualitative descriptions suggest that they may have equally large numbers. The number of lateral cells, in contrast, extends only over a narrow range from 2 x 6 to 2 x 16 cells. Three of the orders of the Polyneoptera have not been investigated in any detail, including, surprisingly, the economically important termites. iii. Paraneoptera Only two of the six orders have been investigated, and only the Hemiptera in any detail. At first sight the Hemiptera have remarkably few median NSCs, but they may have suffered from an overly restricted range of staining techniques. It may be significant that all the really low estimates (2 x 5-10) derive from only PF and VB stains (highly specific for A cells) that investigators using CHP in addition have found more (2 x 7-16), and that those using Azan too have found still higher numbers (2 x 16-65). Estimates of lateral cells cluster mostly between 2 x 3-9. With one exception, there appears t o be no marked correlation of variation with taxon, except that the sole reduviid investigated, Rhodnius, has much the most numerous cells in both median and lateral groups and these cells are smaller than the average for the group, making it likely that the high number is indeed not a staining artifact (see p. 104ff). Two independent estimates for this animal differ by a factor of two. One of these investigations did not use azan staining and further employed a quite different method of assessment (see p. 92); the two figures are best regarded as minimum and maximum figures. The suggestion of small numbers of protocerebral cells in the Paraneoptera which is provided b y the Hemiptera is supported by what unfortunately appears to be the only quantitative account from the important order of Homoptera. Here, uniquely low counts were obtained by Johnson (1963), indicating a maximum of 7 pairs of NSCs in the entire brain, median and lateral groups combined. This figure, moreover, was obtained with a wide spectrum of stains. It is not known t o what extent the animals used (Aphidae, which are aberrant in many ways relative t o most insects) are typical of their group. If confirmed, the homopteran situation is a most intriguing one, and one of special significance t o the neurophysiologist (see also p. 103). iv. Oligoneoptera

The arbitrary division between L‘medial’’and “lateral”

NEU ROSEC R ETO R Y CELLS

85

NSCs is least useful in the ,Oligoneoptera (see for example section 4.3) and to make useful comparisons with the other groups it is probably best t o consider the total NSC complement of the brain. First consider the panorpoid complex: figures are not available for the more primitive orders, but published pictures of sections of the brains of scorpion flies and ant-lions make it clear that they have at least moderately large numbers of median NSCs, probably as many as a cockroach. Within the Lepidoptera, numerous investigations have been made, but many confine themselves to larvae or pupae, and use a restricted range of staining techniques. Estimates are available for adult representative of 6 superfamilies, and with one exception all estimates for “median” NSCs lie between 2 x 8-13, and a11 for “lateral” NSCs between 2 x 5-10. Panov and Kind (1963) have claimed a remarkable constancy in number for the A-type cells in the brains of different lepidopteran species. Unfortunately for this tidy state of affairs, the investigation which used the widest range of staining techniques (DelCpine, 1965) estimated 2 x 47 medial NSCs and 2 x 1 2 lateral NSCs in the pyralid Galleria. 2 x 20-25 of the medial cells described were C (azan only) cells. Counts from other species of this superfamily have not given such large numbers, but most did not use azan. A previous worker (Rehm, 1955) with the same animal reported being unable t o assess the number of median NSCs, which might suggest that there are more of them than usual. The remaining panorpoid order, the Diptera or true flies, have also had rather a restricted range of histological techniques applied to them. Such figures as are available, however, suggest that they tend to have rather more “medial” and rather fewer “lateral” NSCs than the Lepidoptera, but that the total number is about the same as in that group. The largest number expressly recorded is from Drosophila, which was estimated to have a total of 2 x 23 NSCs in the brain, a total comparable with many Lepidoptera. No azan preparations were made, however. There do not seem to be large differences between the different suborders. Among the three nonpanorpoid orders of the Oligoneoptera, only the Coleoptera have received much attention. As perhaps befits the taxon with the largest number of species in nature, it shows great variation in its NSC complement. Median cell estimates vary from 8-80 pairs of cells, and lateral cells from 2-27 pairs. Further, this entire range of variation has been found within the superfamily Chrysomeloidea (leaf-beetles) alone. The Hymenoptera have received surprisingly little attention, but it is obvious that the species investigated, all of which are social, have more protocerebral NSCs than any other insects, except the Orthoptera (and possibly the phasmids, dragonflies and mayflies, if hard figures are ever obtained for these groups). These figures, moreover, mostly derive from a single staining technique, either PF or CHP, and so are probably underestimates.

86

HUGH FRASER ROWELL

From these figures the following picture emerges. The lateraI NSCs of in number, though I)( cause they are few, the variation is large if expressed as a percentage. Both minimum (2-3 pairs) and maximum (15-25 pairs) occur in taxonc )mically remote groups, such as Polyneoptera and Oligoneoptera. By contrast, the medial NSCs show great absolute variation in number, but the ;ric,at majority of insect orders have between 20 and 80 pairs of cells. This i n i ludes some of the most primitive and some of the most evolved orders, f o i example the Grylloblattodea and the Diptera. A very few orders are clraracterized by medial NSC numbers well outside this range. The Hemipicra and Homoptera (and conceivably all of the Paraneoptera?) have impressively few medial cells, down t o 7 pairs in some aphids. At the other c.\treme, the Hymenoptera comfortably exceed this norm, and the Orthoptera have uniquely large numbers, up t o at least 600 pairs in some iri\tances. There is as yet no evidence worth considering that a very large number of NSCs is a primitive trait as suggested b y Gabe (1966), though it m ~y eventually turn out t o be so if it can be shown t o be shared by plrasmids and the Palaeoptera; information on the latter would be particula1 ly valuable. The argument that “large numbers” (again without quantit,ii we support) of cerebral NSCs are characteristic of other, allegedly more pi imitive, arthropod groups, such as Thysanura or of polychaete Annelida, does not seem compelling. At the present time, the large numbers of medial cc 11s of the Orthoptera seems t o be a special and isolated feature of that older. It is a tempting speculation t o equate the large number of medial N4Cs of the social Hymenoptera with the diversity of behavioural and plieromonal potential within the individual in those groups, but there is no rc 11 evidence to support this. Studies on termites, and on tenthredenid or oilier nonaculeate Hymenoptera would be an early requisite. It has been rc ported, however, that formicid queens-but not apparently honey bees (kormigoni, 1956)-have more cerebral A cells than do drones (Gawande, 1‘)68). As such a sexual dimorphism has not been reported elsewhere, it tc lids t o suggest that the role of the queen in the colony necessitates a laiger NSC complement. Even in the best investigated orders (Orthoptera, Hemiptera, Lepidoptera atid Coleoptera) the total number of species whose NSCs are known is niinute. It does seem, even so, that the Coleoptera show by far the greatest M. ithin-group diversity. The chysomelid beetles might for example be pc.rfert material t o attempt t o correlate variation in NSC number with di lferent degrees of complexity of environment, behaviour, or life history. ’Iliere is certainly no obvious gross correlation of cell number with “\uccess”, if this can be measured by species number. The most species-rich insect orders, and probably the most diverse, are in order the Coleoptera, t h v protocerebrum show little absolute variation

NEUROSECRETORY CELLS

87

Lepidoptera, Hymenoptera, Diptera, Hemiptera, Homoptera and-a long way behind-the Orthoptera ’ (Freeman, 1970). The remaining 22 orders account for only about 5 per cent of the known insect species. It will be seen that these groups with high diversity include three orders with “typical” numbers of protocerebral NSCs, and also all the extreme deviations towards both greater and smaller numbers of NSCs.

2.5.3 Variation among taxa: NSCs of the ventral nerue cord, and the total complement of NSCs Table 3 compares the numbers of NSCs found in the brain with those found elsewhere in the CNS. The available data are much fewer than for the median and lateral NSCs, and in most cases one is especially handicapped by the lack of information on the numbers of additional brain NSCs, outside of these two groups. In spite of obvious defects, Table 3 shows clearly that in all known cases there are as many, o r more, NSCs in the rest of the CNS as there are in the brain. Frequently the number in the brain is relatively insignificant. The only apparent exception derives from Johansson’s figures for Oncopeltus. His estimates for the VNC are probably much too small, however, as his stains would not be expected t o demonstrate the C cells which comprise roughly 70 per cent of the NSCs in the VNC of other insects. Of these C cells, more than half appear t o be associated with the median NHOs, at least if the phasmids (Raabe, 1965; Maddrell and Brady, 1968) and acridids (Chalaye, 1967) are typical. For the remainder, it is important t o remember the reservations expressed earlier about the identification of NSCs on morphological criteria, and the possibility that a good number of apparent NSCs innervating muscles directly may in fact be motor neurones of one sort or another. If these are discounted, the general case is more nearly one of parity between brain and VNC, in numbers of NSCs. I have attempted, on even less data, to make minimum estimates of the total number of NSCs in some insects. Here the difficulties are much greater, the move obvious being the very few numerical estimates of NSCs in the retrocerebral complex, and the uncertainty as t o how many insects possess the less commonly described NSC populations, such as those of the optic lobes. I know of no single insect in which estimates are available for all the major NSC populations, so extrapolation from the most closely related form, or often just plain guessing, is required t o supplement the data. The results are presented in Table 4. Seven orders are represented. There is enough partial data from other species of these orders t o make it at least plausible that the figures are more or less representative, though the estimates from the Coleoptera are so diverse that the example given here should not be taken as necessarily typical. It is unfortunate that there are

TABLE 3 Comparison of numbers of neurosecretory cells in the brain and in other parts of the CNS Taxonomic division Grylloblattodea Schizodacty lus Orthoptera

Acheta

Schistocerca

Medial and lateral NSCs

144

830

c. 2000 1228

Technique

Author

PF, CHP

Khattar (1968)

PF, CHP, PAVB,EM

Geldiay and Edwards (1972) Gaude and Weber (1966) Highham (1961)

1

PF, CHP EM, PF/ PIC

1

SOG

NSCs

30

VNC NSCs

Technique Reference

no information

PF, CHP

Khattar (1968)

PTh

Panov (1964)

At least A cells closely comparable to figures to Schistocerca below 134-137* 695-1143*

Rowell (unpublished)

Az, PF Delphin (1965) CHP, ABP M3C

Dictyoptera

Periplaneta

c. 120

120 optic

CHP, PF, Fuller (1960) PF, Ag impr. Willey (1961) EM, histochemical Beattie (1971)

20

209

Az, CHP PF, RSR

de Besse (1965,1967) I

C

c, I n

Hemiptera Rhodnius

Oncopeltus

a 170 64 32

Az, CHP PF/HM

Baehr (1968) Steel and

CHP, PF

Harrnsen (1971) Johansson (1957, 1958)

34

None

106

18

A,CHP

CHP, PF

Baehr(1968) Baudry(1968) Johansson (1957, 1958)

9 YJ YJ

0

%

Lepidoptera Galleria Adult

118

“Nymph”

38

“Preiiymph”

32

( Bombyx

Diptera L u c ilia Larva

Culiseta Larva, Pupa Coleoptera Leptino tarsa

36

Az, CHP, PF Az, CHP, PF Az, CHP, PF CHP, PF

[PTh

Delipine (1 965)

8

Del6pine (1965)

None

72

Delkpine (1965)

28

80

Kobayashi (1957)

80-100

129

>500t

Az, CHP, PF Az, CHP, PF Az, CHP, PF CHP, PF

DelCpine (1965) DClepine (1965) Dklepine (1965) Kobayashi (1957)

Panov and Kind (1963)

42

CHP, PF, AB

Fraser (1959a)

None

50

CHP, PF

Fraser (1956b)

28

PF/HM

Burgess (1971, 1973)

None

32-48

PF/HM PAVB

Burgess (1971)

CHP, PF

Schooneveld (1 970)

CHP, P F

Schooneveld (1970)

214

2

No information

dD .(

2r

‘

(D

0

Taxonomic divison

Blaps Adult Larva

Medial and lateral NSCs

76 114

Technique

ABP, PF ABP, PF

Authoi

Fletcher (1969) Fletcher (1969)

SOG NSCs

c. 30 c. 30

VNC NSCs

239 171

Technique

ABP, PF ABP, PF

Reference

Fletcher (1969) Fletcher (1969)

* Schktocerca Two figures are quoted from Delphin. The lower indicates the maximum of cells counted at any one time in any individual. The higher is the sum of the largest number of cells of the different staining characters ever recorded, and gives an estimate perhaps closer to the total number of cells in the ganglia, as opposed t o those active at any one time. t B o m b y x Kobayashi records “between 80 and 120 cells in all ganglia other than the brain and the SOG”. It is not clear to me whether he distinguished between segmentally distinct but morphologically fused ganglia or not, so the figure of 500 is minimal. Delphin quotes 1100 from the same source. With few exceptions the figures reported here for “brain” NSCs include only the cells of the pars intercerebralis, and those for the suboesophageal ganglion and ventral nerve cord include only those NSCs found within the major ganglia. For a discussion of other NSCs, not included in any of these categories, see pp. _ _ 71-75. In most cases the numbers of additional cells are fairly small.

I G) C

-n

30

$ rn sa

m

0

5r

r

91

NEUROSECRETORY CELLS TABLE 4 Estimates of total numbers of NSCs in some selected insects Orthoptera Schisto cerca Medial and lateral NSCs Deuto- and tritocerebral NSCs SOG NSCs VNC NSCs

cc

Dictyoptera Periplaneta Medial and lateral NSCs Optic lobes Deuto- and tritocerebral NSCs SOG NSCs VNC NSCs (Geldiay, 195 9, estimated 300-400 NSCs in SOG + VNC combined)

cc

1232 20 137 695 47% EL -

i

> 37%

+

EL

+, Complete germ band; -, extraembryonic development.

Kobashi (1974)-activation of the nuclei for germ anlage formation by contact with the periplasm of this region-is not altogether convincing because such a contact is possible also after fragmentation within this region during cleavage. However, it appears from these results as well as from formation of multiple germ bands after chilling (section 4.3) that once a group of cells have become capable of turning into germ anlage cells, they also can generate the complete germ band pattern. In Tenebrio the germ anlage cells may not be quite that self-sufficient because after early cautery of the posterior egg region partial patterns are formed. But most of these partial germ bands terminate in a long and nondescript “tail” (see left inset in Fig. 25(d)) which may actually contain the cells programmed to form the missing segments. Formation of this tail is probably due t o aberrant gastrulation; this is indicated by observations of Haget (1953)in Leptinotarsa, and by the fact that partial

INSECT EMBRYOGENESIS

183

germ bands produced after gastrulation do not carry such tails (right inset in Fig. 25(d)). The longer development proceeds, the larger can the posterior burns be made without impairxnent to the final pattern (curve V-v in Fig. 25(d)), until during the germ anlage stage the egg can be cauterized up till 25 per cent EL. The cauterized region then includes nearly the posterior half of the germ anlage, yet a complete pattern will result. Larger bums will result in extraembryonic development so that practically no partial patterns are produced at this stage. These results were taken to mean that the initially rigid dependence of germ anlage formation on a visibly distinguished posterior region preformed in the oocyte gradually diminishes, probably due t o forward spreading of an “activation centre” effect as postulated for Plutycnemis (Ewest, 1937) (see section 4.2.2).

Extensive series of uv irradiations have been carried out by Kiithe (1966) in Dermestes fiischi Kugel, a species which ranks somewhere between the intermediate and short germ-types of development. Applying high doses of approximately 250-320 nm wavelength from both egg sides, Kiithe denaturated the contents of the treated egg regions right through to the centre; the operation may therefore be compared t o heat coagulation rather than to superficial uv irradiation. The results obtained by eliminating anterior or posterior regions prior to 7 h bear some resemblance to those from ligatured Atrachya eggs (see above), in that complete germ bands may be formed by anterior or posterior “fragments” provided a certain intermediate region (43-47 per cent EL) is included. However, somewhat smaller fragments will produce partial germ bands lacking head or abdomen, a type of result not mentioned by Miya and Kobayashi for Atrachya. The results from later stages resemble those obtained in Bruchidius in so far as the more terminal regions (hatched areas) can still compensate for the damage when more equatorial regions have lost this capacity. The territories for different body regions, as indicated by the curves in Fig. 25(c), can be established by both anterior and posterior irradiations (see insets). They show much stronger apparent movements than in Bruchidius (Fig. 25(b)). With Kiithe’s claim that this is not due to cell movements (but see section 4.9.5) the phenomenon still awaits a satisfactory explanation. In this respect, experiments with other techniques such as pinching the egg might be worth while, especially since this species has now been studied t o some degree with biochemical and autoradiographic methods (Kiithe 1972, 1973).

4.5.7 Summary of results obtained with beetle eggs The results described and discussed in this section may be summed up as follows: a. In an extreme short germ beetle (Atrachya), specification of the body pattern must essentially occur after the germ anlage stage. A certain region

184

KLAUS SANDER

of the egg cell initially appears to be. indispensable for germ anlage formation. A large anterior egg region, comprising in Atrachya more than the anterior egg half, is without essential function in pattern specification.

b. In a beetle with intermediate-type germ anlage (Necrobia), the anterior egg region not crucially involved in pattern specification is still rather long. Changes spreading from near the posterior pole during cleavage and blastoderm formation can be interpreted as the forward movement of instructing influences for various body regions. Towards the end of this apparent movement, at the germ anlage stage, a complete pattern can be formed after removal of the posterior 30 per cent of the egg. The instructions provided by the posterior region are by themselves not sufficient to specify the whole pattern; at least for some pattern elements to be formed, an anterior influence probably from the region around or in front of 60 per cent EL is needed. This is evident also in the “gap phenomenon” (see section 4.4.5). c. In a long germ-type beetle (Bruchidius), anterior and posterior terminal egg regions are apparently of equal importance for pattern specification and exert their influences before the germ anlage stage; the mode of action of these regions appears comparable to that inferred for the leaf hopper (section 4.4). This conclusion is completely opposite to that which Haget (1953) has drawn from his numerous experiments on the egg of the Colorado beetle. The resulting discrepancy will again be discussed in section 5.3.

d. The capacity to compensate for regional uv damage persists longer in the more terminal regions. Pattern defects resulting from localized uv irradiation after cleavage in Bruchidius may be due rather to interference with transmission of instructing signals than to destruction of determined nuclei or cells. Some results from regional denaturation of Dermestes eggs by uv are comparable to results from fragmentation of short germ beetle eggs, but some other results are without parallels in other insect species studied so far.

4.6

HYMENOPTERA AND LEPIDOPTERA: POTENCY REGIONS AND EARLY ORGAN DETERMINATION CHALLENGED

Embryonic pattern specification in hymenopterans and lepidopterans has received relatively little attention so far (section 5.1). For each group, an early major paper exists (M. Schnetter, 1934; Luscher, 1944). In both papers influential concepts were developed which may need to be revised in the light of more recent evidence.

INSECT EMBRYOGENESIS

185

4.6.1 Hymenoptera In hymenopterans, even more extreme differences concerning type of early development are found than in beetles; but this wide range is due to complete reduction of the yolk plasmodium in polyembryonic forms (Ivanova-Kasas, 19721, which doubtless requires some mechanism of pattern specification during proliferative growth (short germ-type, probably secondary). The other hymenopterans classified so far range from more or less intermediate ( Vespu) to extreme long germ-types (Apis, Krause, 1939). For the honey bee, M. Schnetter (1934) constructed a fate map of the 24-h stage which was based on partial patterns from anterior as well as posterior fragments obtained by ligaturing the egg. This map (Fig. 27(a2)) agrees rather well with location of the respective germ band segments when they become visible a day later. Schnetter then studied eggs ligatured at 1 2 h of development, but restricted his attention mainly to the posterior fragment-a restriction which severely limits the possibilities for interpretation, as pointed out above (section 4.5.2). He described two important results :

1. Posterior partial embryos from the 12-h egg comprise the gnathocephalon or the thorax either completely or not at all, i.e. the different segments of each body region appear together, or all fail t o appear. Schnetter concluded that at 1 2 h the different body regions were specified as “potency regions” (Potenzbereiche) (see Fig. 27(al )). The potency region was defined as a region “whose total or partial presence during differentiation of a body region (Orgunkreis) guarantees its completeness.” (M. Schnetter, 1934, p. 318, translation b y the present author). 2. The mean fragment lengths required for various body regions to form in posterior 12-h fragments do not coincide with the corresponding segment borders on the 24-h fate map. The posterior border of the potency region for abdomen is located anterior to the thoraco-abdominal border on the fate map, while the posterior borders of the other potency regions are found posterior t o the corresponding borders on the fate map (Fig. 27(a)). These results were taken to mean that in the early stages “potency regions” for the different body regions were assembled in or near a differentiation centre located between c. 70 per cent EL and 80 per cent EL, and subsequently spread from there until the proportions seen on the fate map were established (M. Schnetter, 1934; Seidel, 1936). Later investigations carried out on other strains of the honey bee did not support this notion. On trying to study the patterns formed in the anterior 12-h fragments neglected by Schnetter, Sauer-Loecher (1954) could not convincingly demonstrate the equivalent of potency regions. For instance, inclusion of the potency region for thorax (region anterior to border

KLAUS SANDER

186

marked t in Fig. 27(al ) did not at all guarantee completeness of the thorax; instead, the thorax formed only when the fragment comprised the anterior egg half or more (loc. cit., pp. 328-329). Maul (1970) ligatured honey bee eggs of three different age groups between 6-10 and 20-24 h. Even with the youngest stage used he obtained partial germ bands beginning with any thoracic segment, i.e. he could not reproduce Schnetter’s all-or-nothing effect for the thorax (neither could Sauer-Loecher, 1954, see loc. cit., p. 334). Moreover he found indications that the mean fragment length

(01)

(a2)

(bl)

(b,)

Fig. 27. “Potency regions” ( a l ) and fate map (a*) as established by ligaturing honey bee eggs at 12 h and 24 h respectively (after M. Schnetter, 1934, Fig. 33). The thin lines marking abdominal segments were added on the basis of statements by Schnetter (1934); full lines indicate direct data, broken lines were derived by interpolation. ( b l ) and (bz). Conjectural diagrams of eggs before and during ligaturing, to show how central structures (bold dots) could become translocated (arrow) during contriction while peripheric structures (black) would not (for corresponding measurements in leaf hopper eggs, see Sander, 1959, Fig. 4). Possible consequences of these differences are discussed in the text.

required for the four different sets of pattern elements studied by him decreases with advance in age at separation, as in other meroistic species (see e.g. Fig. 17(b), 25(a), 30(b)). A closer inspection of Schnetter’s data reveals that a given set of abdominal segments, too, was as a rule formed by shorter fragments with the 24-h stage than with the 12-h stage (loc. cit., p. 314) (see Fig. 27(a)). This apparent shifting of limits for formation of various partial germ bands is again comparable t o that observed in other meroistic species, although the distances traversed appear small when compared o n the basis of germ anlage length. The anterior fragments of honey bee eggs studied by Maul may also show the decrease in fragment

INSECT EMBRYOGENESIS

187

length required for certain sets of pattern elements, as found with anterior fragments of the above species (Maul, 1970, p. 58). The “gap” phenomenon which should result in this case (see section 4.4.4, Table 8) was probably observed by Schnetter himself because he states that loss of segments is greater after fragmentation at 1 2 h than 24 h (M. Schnetter, 1934, p. 314). It therefore appears that even the extreme long germ-type egg of Apis (as that of higher dipterans, see section 4.8) may have retained some traces of the anterior and posterior instructing influences on pattern specification inferred for other meroistic insect species. Two results seem to contradict this interpretation and to support Schnetter’s assumption that instructive influences spread from the differentiation centre. These are: the apparent anterior shifting of the more anterior borderlines in Fig. 27(a) between 12 and 24 h, and some events during development of “dwarf” embryos from rather short posterior fragments of the 12-h egg ( 279 per cent EL; Schnetter, 1936). For both, an explanation differing from Schnetter’s can be offered by taking into account two facts: (1) the increased mechanical stability of the 24-h egg, particularly in the superficial cell layer (Schnetter, 1936, pp. 83/84 and 88), and (2) the displacement of egg contents from the smaller into the larger fragment due to changes in egg geometry during constriction (Sander, 1959, Fig. 4). This displacement, which apparently was also observed by Schnetter (1934; p. 289: Lageverschiebung der Kerne), concerns predominantly the more central egg contents while components located immediately below the egg surface are less involved (Fig. 27(b)). These facts warrant the assumption that ligaturing at 24 h separates the instructed blastoderm without much dislocation, while early constriction forces considerable amounts of ooplasm from the anterior into the posterior fragment. If this ooplasm were involved in providing instructions for the blastoderm cells later on, it could account for the different results obtained with the two stages, and for the posterior displacement of the visible differentiation centre described by Schnetter (1936). The latter phenomenon would then correspond to the displacement of the lateral thickenings of blastoderm following transposition of posterior pole material in the leaf hopper Euscelis (see Sander, 1960, Fig. 17).

To sum up, the differences in pattern specification between the honey bee and the other meroistic insects discussed above may be differences in degree rather than in kind. Results obtained by ligaturing eggs of another hymenopteran, the ichneumonid wasp Pimpla turionellae L. (Achtelig and Krause, 197 1) indicate pattern specification by anterior and posterior influences comparable to those inferred for Euscelis and Bruchidius. Also, as in Euscelis, the number of pattern elements formed in an anterior fragment may be raised considerably b y indenting the egg from the posterior pole (and probably translocating material from there) before ligaturing. An influence on pattern specification exerted during cleavage by the posterior pole region was also inferred by Reith (1931) in the ant Camponotus.

KLAUS SANDER

188

4.6.2 Moths Investigations on lepidopteran eggs yielded results mainly of interest in connection with specification of the transverse pattern aspect (section 5.1). In Ephestia kuehniella Zell., Maschlanka (1938) cauterized anterior or posterior pole regions. She found that the posterior 20 per cent of the egg may be burned before or during the blastoderm stage without impairment to the final pattern. The prospective gnathocephalon appeared to react to anterior cautery during cleavage like a “potency region” (section 4.6.1)-i.e. all three segments were formed, or none. But head lobes, thorax and abdomen did not display this type of reaction. A similar result was obtained in Tineola biseliella Hum. by Luscher (1944) who irradiated transverse areas of 1/10 egg length with uv light of wavelengths above 280 nm. On irradiating various regions during cleavage he obtained the results set out in Table 1 1. This table shows that the frequency of complete TABLE 11 Effects of locally restricted uv irradiation (see text) on different regions of the germ band in the moth Tineola biseliella. (Data from Liischer, 1944, p. 569) Region afflicted Effect Procephalon Complete absence Partial absence

11 6

Gnathocephalon Thorax 19

9

2

7

Abdomen

3 6

suppression of a body region is inversely related to the length of that region on a “typical” fate map, and also on the fate map presented by Liischer for the germ anlage stage (loc. cit., Fig. 122, 16 h). This may mean that the high frequency of total elimination of the gnathocephalon in both moth species could be due t o the small size of its anlage rather than to a peculiar “potency region”-type of reaction. Luscher’s paper also has a bearing on the question of how early egg regions can be considered to be determined to form specific pattern elements. On discussing potency regions, he states: da/3 diese Orgunregionen sclzon im Keimhaiitblastem, also Plasma, festgelegt waren (Luscher, 1944, p. 611). But this statement, which would imply a considerable specificity of ooplasmic prelocalization (mosaicism), must be taken with caution. Of course the cytoplasm which later on should have provided the progenitor cells for the defective organ(s) must have been damaged by the uv irradiation, but whether this cytoplasm at the time of irradiation already

INSECT EMBRYOGENESIS

189

was instructed to form that organ is an open question. For the instructions to form the respective organ(s) might as well have arrived there after irradiation, with the irradiated area left incapable to respond to these instructions (see section 5.5.4). Therefore, Luscher’s conclusion (loc. cit., p. 624) that these early induced defects are proof of region-specific determination before the blastoderm stage is not compelling. The mode of pattern specification in Tineoh and in moths generally remains open under these circumstances. The defect maps which Luscher (1944) published for various stages must also be discussed; as just pointed out, these at best represent fate maps. They have been derived from the experimental results with considerable statistical effort (loc. cit., p. 596ff) and it may be doubted whether the changes observed from 8 h to 16 h (germ anlage stage) have any significance. The greater changes ensuing from 1 6 h onward correlate with contraction and flexure of the germ anlage and are in all probability due t o these deformations (Liischer, 1944, Figs 39-42 and p. 608). The maps up till 1 6 h may be compared to the results of Jung (1966) in the beetle Bruchidius (Fig. 25(c)), with the difference that mapping can be done before the nuclei arrive at the surface of the egg cell (which may be a question of dose). Luscher’s results from regional uv irradiation also resemble those which Heinig (1967) obtained by irradiating whole cricket eggs with X-rays (section 4.1.5). At first, large stretches of the germ band pattern become damaged. With somewhat later irradiations, the defects become restricted t o a few or even single segments, and shortly afterwards duplication of legs and abdominal parts may occur (Luscher, 1944, Fig. 124). This coincidence might be the expression of comparable steps finally leading to commitment of cells or regions to their specific tasks-an interpretation which apparently both authors favoured. A certain difference exists with respect t o stage: in the moth these reactions occur earlier than in the cricket. Imaginal structures, apart from the legs, can be influenced by uv irradiation only slightly later than the larval structures. Liischer concluded from this result that determination of the imaginal structures in Tineoh occurs shortly after larval determination, but this conclusion may be invalid due to a shielding effect (see section 4.8.4).

4.7

LOWER DIPTERANS-MIRRORING HEADS AND ABDOMENS

In 1960, Yajima described two striking types of aberrant patterns obtained by centrifuging cleavage stages of the harlequin fly Chironomus dorsalis: double heads and double abdomens (Fig. 28). They represent mirror-image duplications of the anterior head region consisting of head lobes and the mandible segment, or of the posterior 6-8 abdominal segments. The mirror symmetry is usually perfect with respect to segment sets formed, but primary germ cells are found only in the posterior partner, whether abdomen or head (Yajima, 1970). Yajima’s experiments were repeated on related species (Ch. thummi and Smittia sp.) where they led to the same types of aberrant pattern, albeit linked differently to the direction of centrifugal force (Gauss and Sander, 1966; Overton and Raab, 1967;

KLAUS SANDER

190

a& r

0.... ..... ......

~

& I

@-Q

..... .....

Fig. 28. Normal larva (top), double head (left) and double abdomen (right) from centrifuged eggs of Chironomus dorsalis (after Yajima, 1960). Stratified eggs demonstrate linking of results to direction of centrifugal force (arrow);this interrelation could not be established in other chironomids (see text). “Yolk zone” hatched, “clear zone” white, “oil zone” dotted; posterior egg pole marked by black pole cells.

Kalthoff, unpublished results); double abdomens were obtained by Yajima also in other chironomid species (Yajima, 1960, p. 203). Yajima d s o showed that the same types of aberrant pattern can be induced by local uv irradiation (Yajima, 1964). An extensive series of investigations on the egg of the chironomid midge Smittiu sp. by Kalthoff (Kalthoff and Sander, 1968; Kalthoff, 1971-1973) revealed that in this species only double abdomens can be induced by uv treatment (for segment numbers involved, see Sander, 1975a). Recently yet another way of inducing double monsters was found: Schmidt et ul. (1975) showed that in Smittiu removal of anterior pole material by puncturing may lead to formation of a double abdomen. The results of these experiments will be described in more detail and discussed below (section 7.5). The general conclusions drawn from experiments yielding double monsters are compatible with the general mode of pattern specitication inferred from results in leaf hopper and beetle (Bruchidius) eggs: Pattern specification in chironomid midges is apparently strongly influenced by terminal pattern elements (Yajima, 1960) and/or terminal egg regions (Kalthoff and Sander, 1968). The perfect pattern symmetry of double heads and double abdomens suggests that some continuum such as axial gradients might be involved in segment specification, as discussed previously in connection with the posterior mirror duplications obtained in the leaf hopper (section 4.4.3). Another parallel to the leaf hopper results is indicated by the patterns formed in egg fragments of Smittiu. The results obtained so far after early fragmentation reveal the “gap” in pattern (Table 8; Sander, 1975a), and fragments of a given size, as in Euscelis and Bruchidius, tend to produce more pattern elements when fragmented later in development (Sander, unpublished results). Some further results obtained by Yajima (1960) are very interesting but require more complicated interpretations. In Ch. dorsalis, length and/or mass of the blastoderm can be reduced by various manipulations during cleavage. These methods are: sharp centrifugation so as to separate proteid

INSECT EMBRYOGENESIS

191

yolk or lipid droplets completely from the bulk of the cytoplasm, constriction of centrifuged eggs to the same end, or removal of cytoplasm (and nuclei?) by puncturing the “cytoplasm” layer in the centrifuged egg. The blastoderm reduced by any one of these methods will form a double monster containing only the more distal elements of the double monsters produced by the complete blastoderm. The failure to produce the more proximal elements (gnathal segments in double heads, anterior abdominal segments in double abdomens) may be connected with difficulties in setting up sufficiently strong differences over short distances, but in any case the LL dominance” of the terminal pattern elements is stressed once more by these results. Eggs of Ch. dorsalis centrifuged twice and in opposite directions during cleavage produce the type of double monster usually linked to single centrifugation in the second direction. When the second centrifugation is applied during the syncytial blastoderm stage the resulting monster is of the type expected from the first centrifugation (performed during cleavage), but only one partner of the mirror duplication is formed while the ather apparently becomes suppressed by the mass of proteid yolk assembled centrifugally. It is thus possible to obtain posterior partial germ bands of inverted polarity in the anterior, and inverted anterior partial germ bands in the posterior egg half. The most intriguing-and with proper analysis most promising-results were obtained from oblique centrifugation. This treatment results in formation of asymmetric double monsters where the terminal pattern elements are said to be identical but the region in between is dominated by pattern elements related to only one of these. Such “asymmetric” pattern duplications cannot be understood on the basis of simple specifying gradients. At least some additional assumptions are required, such as differential timing of developmental processes in different egg regions. The key to further understanding might be provided by the fact that reasonable agreement exists between the number of pattern elements contained in the larger partner and the longitudinal extent of the clear plus lipid strata (Fig. 29). One possible explanation for this would be sequential specification of pattern elements, starting from both terminal regions and proceeding faster through clear than through yolky ooplasm. In any case, some novel and very useful information concerning pattern specification might be gained by studying the quantitative and temporal relationships involved in the formation of these aberrant patterns. Among lower dipterans other than chironomids, some Culicidae have yielded results of interest for pattern specification. Double abdomens were found by Price (1958) t o form spontaneously in a strain of Wyeomyia smithii (Coquillett). The ligation experiments performed on eggs of Culex pipiens L. by Idris (1960) were somewhat impaired by the fact that earlier stages rarely yielded definable sets of pattern elements. But the results were taken to indicate (1) that pattern specification in the middle region of the egg is under some posterior influence effecting “reduction of procephalic structures to

92

KLAUS SANDER

heir definite area” (translated by K.S.) (cf. section 4.5.1,) and (2) that an influence equired for gastrulation and segmentation in posterior egg regions spreads backwards lrom the region around 65 per cent EL during early development (Idris, 1960). The i o r m c ~conclusion is reminiscent of results from anterior egg fragments of other species ( x e Figs 17(b) and 25(a)), while the latter may indicate spreading of the capacity for \elf-differentiation from a differentiation centre (see section 4.9.3). Oelhafen (1961) I oncluded from regional uv irradiations that the late blastoderm (cephalic furrows 1 ormed) of Culex represents a mosaic of developmental subsystems. I

I ig. 29. Influence of plane of stratification on patterns formed in eggs of Chironomus #/orsalis(data from Yajima, 1960, text-fig. 3 ) . Lines in eggs indicate border between \ olk Lone and clear zone for various angles of centrifugation; the smaller part is the yolk /one. Lettering indicates pattern elements (between opposite arrows) constituting the Lirger partner in asymmetric double monsters resulting from oblique stratification. I ransverse stratification leads to symmetric double monsters with mirroring plane in qnathocephalon (Md?) or anterior abdomen (A*) (see Fig. 28); the bold arrows indicate Ilirection of centrifugal force for this type of stratification. ap, anterior; pp, posterior t gg pole: A2-A9, abdominal segments; Md, first segment of gnathocephalon (mandibuh ) ; l’, procephalon, TI, 11, first and second thoracic segments.

!.8 HIGHER DIPTERANS: NO SPECIAL STATUS

I‘here has always been a tendency among developmental physiologists t o embryogenesis in the higher dipterans rather separately. Formerly, this was due t o the belief that these species were characterized b y an tkxtrrme degree of determinate development. More recently, progress made I,y employing genetic tools for the analysis of development in Drosophdu (section 6) has tended t o make results from other insect groups appear I I relevant t o higher dipterans. The subsequent paragraphs are intended t o disprove both beliefs and to link embryonic pattern specification in higher tlipttrans t o processes occurring in other insect groups as discussed in the preceding sections. Ireat

INSECT EMBRYOGENESIS

193

4.8.1 Pattern anomalies induced during oogenesis In Drosophila it is possible to influence embryonic pattern formation by experimental interference with oogenesis. Zimmermann (1954) was able to induce disorders of abdominal segmentation by exposing females t o heat shocks of 4 h duration from 12 t o 6 h before egg deposition. The defects in the survivors were essentially limited t o the dorsal side and therefore indicate problems arising during dorsal closure rather than during specification of the metameric germ band pattern. Mild centrifugation of females between 18 and 2 h before oviposition induces abnormal segmentation, with frequencies up t o 30 per cent of surviving larvae affected (Brown and Schubiger, personal communication). The sensitive period starts with vitellogenesis, i.e. no effects can be inducedsas long as the oocyte is free of proteid yolk. The abnormal larvae were in some cases affected on the ventral side; the abnormal segments always included at least one of the anterior abdominal segments. No defects were observed in the terminal segment bearing the spiracles and in the regions anterior t o the prothorax in 88 abnormal larvae scored. These results mean that during late oogenesis the oocyte can be influenced so that faulty patterns may arise in the middle region while the terminal regions of the pattern prove resistant t o changes in developmental programme. This is of interest in connection with the role in pattern specification ascribed to these regions in previous sections, and with the bicaudal mutant t o be discussed below (section 6.1). 4.8.2 Changes occurring between egg deposition and the blastoderm stage Illmensee (1972) demonstrated that in Drosophila lateral removal of cytoplasm from the equatorial egg region is tolerated much better at 2-10 min than at 37-40 min after egg deposition, with a gradual decrease in between. The percentage of adults obtained from eggs so treated is above 50 per cent for the 2-10 min interval, and below 20 per cent at 37-40 min. This result indicates that prior t o blastoderm formation the egg does not react like a strict developmental mosaic, but becomes increasingly sensitive / to localized removal of components. The idea of strict developmental mosaicism was also refuted by Howland and Sonnenblick (1936), Nothiger and Strub (1972), and Bownes and Sang (1974a, b) on the basis of results obtained after inflicting local damage with various methods (puncture, local cautery, local uv irradiation). Except in the uv results of Nothiger and Strub (1972), a reasonable correlation exists between the site damaged and the location of the ensuing deEect in the body pattern. According t o Illmensee (loc. cit., p. 275) this fact suggests that a “pattern of morphogenetic determinants exists even before the cleavage nuclei have immigrated into the cortical cytoplasm”. However, this conclusion is not compelling (see

i ~

i

’

194

KLAUSSANDER

also section 4.6.2). Illmensee himself states that formation and appearance of blastoderm cells are abnormal in the pricked egg region (loc. cit., p. 286). Therefore the ooplasm or the blastoderm cells forming in this region might have been unable t o convey or t o pick up instructing signals arriving or generated there subsequent t o pricking. We may therefore concur with Bownes and Sang (1974b) who suggest that “the egg is not mosaic, in the sense that determinants of differentiation are already patterned in some final form in the egg cortex”. The generalization from germ cell determinants to prelocalized determinants for various body regions, which Illmensee (loc. cit., p. 275) and others before him have suggested, is likely t o be untenable (Sander, 1975a). Differences in determination between germ cells and somatic pathways of development are for instance indicated by the finding that chironomid double monsters (section 4.7) contain germ cells only in the posterior partner, and independent of its developing into head or tail (Yajima, 1970; Sander, unpublished results). While all these results argue against strict mosaicism, they leave open to some degree the possibility that metameric organization before the blastoderm stage is represented by a pattern of “segment determining foci” with smaller or bigger spaces in between; an undisturbed body pattern could then result from those cases where only ooplasm located between such foci had been damaged or removed. This possibility was excluded for the blowfly egg by Nitschmann (1958) and Herth (1970) (Sander et al., 1970; Herth and Sander, 1973) on the basis of results from egg fragmentation at different stages. These results show quite clearly that under experimental conditions any given part of a broad equatorial region may be utilized to form a variety of different pattern elements other than head and terminal abdominal segments (Fig. 30(a) and (b)); in connection with this, certain other pattern elements are completely missing (Fig. 31), despite the absence of any visible structural damage which might have caused suppression of these pattern elements. The extensive series of pinching experiments described by Herth and Sander (1973) reveal reactions well known from other insect eggs: anterior as well as posterior fragments produce on the average more pattern elements after late than after early separation, and early fragmentation on the average causes a bigger “gap” in the pattern formed by an egg than does late fragmentation (Table 8). Whether and how long the anterior and posterior interacting influences indicated by this reaction (see section 4.4.5) reside in the plasmodia1 part of the system is not known. Unfortunately, experiments of the type performed by Hadorn and Miiller (1974) in Drosophilu, which might answer this question, can be carried out only after blastoderm formation, i.e. when specification of the segment pattern is probably complete.

INSECT EMBRYOGENESIS

195

70

70 .-•

-J

w

s 20

20 0

I

3

2

0

I

h

2

3

h

Fig. 30. Protophormiu sp., patterns produced in egg fragments separated with the pinching technique (condensed from Figs 7 and 8 in Herth and Sander, 1973). Curves represent mean fragment lengths required for various partial patterns. The numbers indicate the most posterior (left) or most anterior (right) denticle belt formed; belt no. 1 is located in anterior border of first thoracic segment. Stages between early cleavage (left) and blastoderm prior to formation of cephalic furrow (right). Fragmentations in dotted regions cannot be carried out with the technique used.

I00

4

8

50

5

6 35 O/O

q;; 7 53 ‘10 EL

10

0

OP

opm

yy

Fig. 31. Some typical results from fragmentation of Protophormiu eggs (from Herth and Sander, 1973). Op 111 corresponds to middle stage in Fig. 30; Op VII to the stage at right. After fragmentation at stage VII, the partial patterns formed in anterior and posterior fragments of individual eggs add up to the complete pattern (right). With earlier fragmentations, both anterior and posterior fragments have to be longer in order to produce the same partial patterns (centre). Consequently, the partial patterns produced after early fragmentation by individual eggs (left) do not add up to the complete pattern (“gap phenomenon”, see section 4.4.4).

KLAUS SANDER

196

Studying development in ligatured Calliphoru eggs, Alleaume (197 1) obtained data consistent with the “gap phenomenon”, as described by Kitschmann (1958). But she also obtained cases in which a fragmented egg produced more segments than normal (Table 12). Whether this result is due TABLE 12 requencies of segment numbers counted in ligatured Calliphoru eggs. The ligature was probably not quite tight in most cases. Segment number may exceed the real value by one, due to method of counting; the normal number is 12. (Data from Alleaume, 1971) 1

Number of segments (+1?) Stage at fragmentation

Lleavage Plasmodia1blastoderm

9

10

11

12

13

14

15

16

0 6

2 9

1 13

2 17

0 5

0

1

0

C.ellular blastoderm (;astrulation Relative frequencies

w

A I

3 7%

48 %

Relative frequencies

0

3

4

25

4 3

15%

0

0

0

3 ,oooJ ,005. 3 0% 84% 16%

t o an oblique arrangement of segment anlagen (loc. cit., p. 126), or to the

i x t that a cytoplasmic bridge persisted between both egg fragments (loc. (it., p. 37), or t o some other cause, in any case it demonstrates once more atid very clearly that the blowfly egg is not a developmental mosaic at c?viposition. Alleaume concludes that complete regional determination is not achieved before the blastoderm stage. The process of cellular commitment appears to be initiated at the cquatorial region and to spread from there in anterior and posterior directions-as in so many other species. However, this centre di’fbrenciateur i\ not located in the prospective prothorax but rather in the anterior al)dominal region-and it apparently becomes established by influences spreading from both egg poles (Fig. 32) (Alleaume, 1971, p. 39). These influences confer the capacity for self-differentiation on increasingly shorter anterior and posterior egg fragments, and the differentiation centre might ensue where tney first meet and interact (Fig. 32) (loc. cit., pp. 39 arid 123). Looked at in this way, the differentiation centre might be an zir tifact of interpretation: the process which appears to spread from this ( mtre would then actually represent two independent but overlapping spreading events. The results which Anderson (1960) obtained by ligaturing eggs of Dacus t ryoni indicate also a spreading of the capacity for independent differ-

INSECT EMBRYOGENESIS

197

entiation in anterior and posterior directions. However, issue must be taken with Anderson’s conclusions concerning pattern specification in Dacus. He inferred that determination ‘‘is completed at a very early stage since none of the developing parts here separated by ligature showed regulation” (loc. cit., p. 561). This argument contains a fallacy which is frequently encountered (Sander, 1971). Of course these egg parts did not “regulate” so as t o form complete larvae. But the observed failure of the fragments t o produce the complete pattern must not necessarily be due t o determination in mosaic-fashion prior t o fragmentation; it could as likely be due to

2

3 4 5 h Fig. 32. Limits of capacity for self-differentiation in Calliphora erythrocephala (data of Alleaume, 1971, plates 6-15). Posterior fragments shorter than indicated by broken curve, and anterior fragments shorter than indicated by solid curve are unable to reach larval level of differentiation (Lp, La). Embryonic differentiation may occur in somewhat smaller fragments with later stages (fragmentation levels in hatched areas; E,, Ea). MW, period of mitotic waves (see section 7.2). I, 11, 111, stages as in Fig. 25(a). 11-AS, blastodermal territories for second thoracic to 5th abdominal segments as established for Protophormia by Herth and Sander (1973) (see Fig. 30). Note that the centre diffkenciateur (DC) as defined by Alleaume (see text) is located in the anterior abdominal region (Alleaume, 1973). 0

I

prevention of subsequent interactions between various egg regions involved in epigenetic pattern specification, as shown beyond reasonable doubt in the leaf hopper Euscelis (section 4.4.2). Recent experiments with regional uv irradiation of Drosophila eggs before and during the blastoderm stage (Bownes and Kalthoff, 1974) failed . to yield the “double abdomen” pattern anomaly which can be induced by identical treatment in chironomid eggs (section 4.7), and also develops occasionally in eggs from Drosophila females of certain genetic constitutions (bicaudal factor; Bull, 1966; see section 6.1). However, some eggs irradiated in the prospective head region produced only a solitary abdomen, a pattern anomaly also found in eggs laid by bicaudal females.

198

KLAUS SANDER

4.8.3 The basic pattern at the blastoderm stage The partial patterns produced by blowfly egg fragments indicate that, with reference t o the segments of the larval body, the blastoderm may be considered a developmental mosaic. However, in Drosophila loss of a certain amount of cells can even then be tolerated, as was shown by development of viable flies after puncturing blastoderm eggs (Bownes, 1973). Illmensee (1973) was able t o demonstrate by transplantation of nuclei from early gastrula cells that at least some nuclei in various blastoderm regions are totipotent. Chan and Gehring (1971) on the other hand showed that with respect to adult structures the developmental programmes of anterior and posterior halves of the blastoderm are already restricted. It appears from these observations that in the blastoderm the areas which have to produce the different elements of the basic body pattern are fairly fixed. Minor defects can still be compensated for, and individual larval cells might not become committed to their definitive developmental pathways until well after the blastoderm stage. This is evidently so with the prospective imaginal disc cells which, although probably instructed with respect to segment specificity, are not yet fixed on any specific pathway within a disc (Bryant, 1974). 4.8.4 Embryonic pattern specification and imagznal discs in Drosophila The great advantages provided by studying imaginal discs have led t o a number of findings relevant to embryonic pattern specification. There is no reason t o doubt the assumption that the progenitor cells for imaginal discs as a rule are associated with the blastodermal anlagen for the corresponding larval segments (Postlethwait and Schneiderman, 19 73). For various discs, the location and number of these cells have been inferred from studies on mosaic flies (Ripoll, 1972; Bryant, 1974). Only two aspects of experiments concerning imaginal disc progenitors will be discussed in the present context: stage of determination and experimental alteration of disc specificity. Changes in disc specificity can be induced by mutant alleles (section 6.1) or by phenocopying treatment of eggs. In particular, transformation of dorsal metathoracic discs so as t o produce wing structures is rather easy to obtain. Recently, Capdevila and Garcia-Bellido (1974) found that ether fumes may cause such transformations when applied during cleavage, and not only during blastoderm stages as was formerly believed. The authors discuss the possibility that their phenocopying treatment might interfere with cytoplasmic signals for pattern specification. However, a specific effect on the cleavage nuclei which causes these t o misinterpret the signals conveying segment or disc specificity cannot be excluded at the moment.

INSECT EMBRYOGENESIS

199

The problem arising with the former hypothesis is that only patches of cells within the structures derived from a disc show the transformation. To account for this on the basis of a change induced in the specifying cytoplasm during cleavage would require a much finer pattern of instructing cues than indicated by other experiments quoted in previous sections. The time when disc specificity becomes determined is not known precisely but several observations may be relevant. The results of Chan and Gehring (1971) indicate regional specificity in the blastoderm but are not strict proof that progenitor cells were already committed t o form a particular disc. With somewhat later stages, duplication of legs or antennae (or fusion of the first pair of legs) may be obtained by X-raying whole eggs

E M

L

-/+

-

A + -

~

~ 12

-/+

-/+

-la

-

I

2

-I+

-

~

I

~

I1

El-- -- -- -- --12

4

21

Fig. 33. Stage-dependent patterns of adult defects produced by ventral uv irradiations of Drosophilu eggs (data of Geigy, 1932, pp. 440-441). 1-111, first to third leg; W, wing; A, abdomen. Symbols indicate complete absence (square filled completely), deformation or duplication (half filled) or normal appearance (white) of structure indicated. Abdomen may be defective (+) or not (-). Early, middle, late (E, M, L): stages from early germ band to hatching, not precisely determined. Figures indicate numbers o f cases in a sample stated by Geigy to be representative.

(Postlethwait and Schneiderman, 1973a). Ventral uv irradiation (253.8 nm) of whole eggs during stages from early germ band t o hatching (Geigy, 1932) may alter or suppress the adult derivatives of single imaginal discs. The staging for irradiation could not be done very accurately, but Geigy maintains that the spectrum of induced defects is stage dependent as shown in Fig. 33. With the earliest period after germ band formation, the dominant result was damage to the dorsal mesothoracic disc (defects in wing and thorax). Irradiation during the subsequent period damaged legs of all three or of the two posterior thoracic segments, while defects caused by later irradiation were restricted to metathorax and/or abdomen. The abdomen frequently also suffered from irradiation during the middle period. This spatio-temporal relation is reminiscent of that observed after exposing cricket eggs to X-rays (Table 4), but in the cricket the defects concerned larval segments and/or appendages. The cricket results were taken to indicate the timing of commitment for segment specific development (section 4.1.6.d). Geigy, too, linked his finding to

200

KLAUS SANDER

acquisition of an endgultiger, fixierter Determinationszustand. But his main argument (lot. cit., p. 443) actually referred to pattern specification within imaginal discs, and musc be considered untenable in the light of subsequent evidence. It was based on the fact that with his method adult duplications could no longer be induced some time after the onset of region-specific uv sensitivity. But this could be due to a shielding effect once the prospective imaginal discs have sunk below the surrounding epidermis; adult duplications can be induced much later using other methods (Postlethwait and Schneiderman, 1973a). So only the onset of regional uv sensitivity as observed by Geigy might be relevant, and it could at best be connected with disc-specific commitment of the progenitor cells, while instruction and commitment of that particular egg region to form the corresponding body segment (and consequently its discs) should have been completed at the blastoderm stage.

The spatio-temporal pattern described by Geigy for adult defects after ventral uv irradiation (Fig. 33) may reflect a process which spreads from the middle of the thorax. In other insects, events taking a similar spatio-temporal course have tentatively been linked t o acquisition of the capacity for self-differentiation (see sections 4.1.6.c and 4.4.3). I n this connection, a result of Hadorn et ul. (1968) may be of interest. They checked fragments of Drosophilu eggs aged 6 h or more at 25 "C for their c-apacity to produce adult structures after in vivo culture. Posterior Fragments isolated at 10.5 h frequently produced derivatives of the genital disc (located at the tip of the abdomen) but 6-h fragments practically never did. Hadorn et al. (1968) concluded that, under the conditions of the experiment, the genital primordia might reach the capacity for selfdifferentiation somewhat later than the more anterior imaginal anlagen. The changes in regional X-ray sensitivity observed during somewhat earlier \tages by Ulrich (1952. his Fig. 7) may perhaps also reflect the underlying basic process.

k.9

GENERALIZATIONS CONCERNING LONGITUDINAL PATTERN SPECIFICATION AND SOME DATA NOT COVERED BY THESE

In closing this section, an attempt will be made to piece together from \raric)us results a more or less coherent view of longitudinal pattern kpecification in insect embryogenesis. Results clearly not compatible with 1 his view will be indicated below, as will be the main differences t o previous qeneralizations. It is obvious that such an attempt is bound t o suffer from inany shortcomings; yet it is felt that a somewhat unifying view of pattern \pecification would be useful as a basis for further research, and perhaps ,tlso for teaching, where-to judge from recent textbooks-insect experiinental embryology is largely unknown as a subject. Two general modes for specification of the metameric longitudinal body

INSECT EMBRYOGENESIS

201

pattern are distinguished ,here-specification during proliferative growth of the germ anlage blastema, and in situ specification of the blastema, supposedly via instruction from the plasmodia1 parts of the system. Within the Pterygota, a tendency for progressive replacement of the former process by the latter during evolution is apparent. It is paralleled by transition from the short germ-type t o the long germ-type of development (Krause, 1939, 1961), and by changes in oogenesis permitting a considerable decrease in time required for embryogenesis (Bier, 1970). In contrast to this interpretation, Anderson (1972) favoured the intermediate germ-type as being the phylogenetically most primitive, but admitted that no selective advantage can be recognized which might have led to subsequent evolution of the short germ-type. By reversing the argument one might propose that the more primitive groups of Tracheata referred to by Anderson may have deserted the original budding mode of development long ago, and that this was one of the adaptations which enabled them to compete successfully with the pterygote insects to this day.

4.9.1 Pattern specification in the short germ-type of development In the short germ-type of development, the body pattern appears t o become specified essentially after germ anlage formation, and by processes probably linked t o proliferative growth. A terminal “progress zone” model, similar to that inferred by Summerbell et al. (1972) for the chick wing, might be applicable at least for the regions behind the gnathocephalon and possibly for all metamers (see section 4.3). In forms where the visible segment borders appear in strict antero-posterior sequence (see Anderson, 1972), this sequence may depend directly on the spatio-temporal course of previous pattern specification. Visible segmentation from a thoracic differentiation centre (Krause, 1939), on the other hand, might reflect a corresponding spatio-temporal course of regional commitment rather than pattern specification (see section 4.9.3). The plasmodial part of the system in the short germ-type of development apparently does not directly take part in specification of the metameric body pattern. It does, however, appear t o provide some preconditions for germ anlage formation, such as cues leading t o local accumulation of nuclei or cells, and probably conveys longitudinal polarity on the ensuing germ anlage. Some of these indispensable functions may be restricted t o certain regions of egg cell and yolk plasmodium, so that n o germ anlage will form if these regions are prevented from functioning properly. Such “activation centre” effects can be seen after early elimination of the posterior egg pole (e.g. Tachycines, Schistocerca) or after interference with some more anterior region (Atrachya). The functions initially restricted t o these regions may be found somewhat outside with advancing development.

202

KLAUS SANDER

In less extreme instances of short germ development (e.g. in Tenebrio) the posterior part of the ooplasmodium may play some more direct role in pattern specification. However, this role must be secondary or subordinate because the germ anlage, after removal of its posterior half by cautery, as a rule still’produces the complete segment pattern (Fig. 25(d)).

1.9.2 Pattern specification in the intermediate and long germ-types o f development The eggs of dragonflies and crickets, which represent the semi-long germ-type of development in panoistic insects, react t o early elimination of critical posterior region like short germ eggs. If they form a germ anlage at all, then this is capable of producing the whole longitudind pattern. However, experiments from mid-cleavage onward locally reveal the presence or generation in the ooplasm of instructing signals specifying the pattern elements of head and thorax. These instructions are not prelocalized in those egg regions which later on produce these pattern elements, but appear t o move anteriorly in an ordered sequence under some influence from the posterior pole region; their apparent movement could be due to establishment of a specifying gradient by some process in the pole region (see Fig. 23). The abdominal segments, on the other hand, are apparently being specified during proliferative growth, as in the short germ-type of development. In meroistic species representing semi-long germ development (e.g. Euscelis, Necrobia), additional anterior influences prerequisite for specification of some or most segments anterior to the abdomen become evident. These apparently move backwards during and after cleavage, again in an orderly fashion which might be due t o a specifying gradient. A given segment may form only when the respective anterior and posterior prerequisites are present. In normal development, the signal instructing the overlying germ anlage cells t o produce that particular segment will be provided where an appropriate combination of anterior and posterior instructing influences becomes established (Fig. 34). T h e t o their different positions at the beginning of embryonic development, anterior and posterior prerequisites for a given pattern element (or series of pattern elements) may be separated from each other by ligaturing during early stages. The resulting exclusion of some anterior prerequisites from the posterior fragment, and vice versa, leads to failure of the corresponding pattern elements to become properly specified in either fragment. The result is a “gap” in the segment pattern of eggs fragmented early. The number of segments which fail to be formed decreases with increasing egg age at fragmentation, until a state of segmental mosaicism results, where all segments not formed by one fragment are formed by the

INSECT EMBRYOGENESIS

203

other. This type of “mosaic reaction” is reached at the blastoderm stage in Dipterans (Protophormia), but much later in a beetle (Bruchidius) and in the leaf hopper Euscelis (Table 8) (see Herth and Sander, 1973, for discussion). In the extreme long germ-type of development (e.g. Apis), the germ anlage becomes subdivided into the various body regions and segments without differential proliferative growth. Posterior and anterior instructing influences can still be traced. Their apparent relative movements are less obvious than in the intermediate and the less extreme long germ-types, but certainly still extensive enough to exclude the possibility that the longitudinal pattern could be preformed during oogenesis as a mosaic of prelocalized segment determinants. STEP

LOCATION

Visible d i f f e r e n t i a t i o n

t t lnstruc t ng signal (s)

Cells or cell groups

Cellulor commitment i

Anterior Poster tor Instructing influences (prerequisi tesl

J

Plosmodial system

Fig. 34. Hypothetical steps in specification and differentiation of elements of the basic longitudinal body pattern (see text).

4.9.3 Differential timing of commitment to segment-specific pathzoays of development SO far, the discussion in this chapter has dealt with the processes which instruct various regions of the developing system as t o their specific courses of development. Many results indicate that these processes, after having established the status of segmental mosaicism as defined in the preceding section, are followed b y some other processes which are also essential for the formation of visible pattern, but display different spatio-temporal characteristics. The latter processes start apparently somewhere in the middle’ of the prospective germ band pattern and spread from there in anterior and posterior directions. Indications for this are the acquisition by egg fragments of the capacity for self-differentiation (Figs 26 and 32), the spatio-temporal pattern of regionally increased radiation sensitivity (Fig. 25(b), (c), and Fig. 33; Table 4), and sequential loss of the capacity

204

KLAUS SANDER

for bilateral duplication (see section 5.1). The processes underlying these and some other phenomena may tentatively be linked t o commitment of the germ anlage cells t o carry out the specific instructions provided to them. Region-specific commitment would then represent the first event to be initiated in a differentiation centre and t o spread from there in a way foreshadowing the spreading of subsequent events in embryonic differentiation such as gastrulation, visible segmentation, and histodifferentiation (see section 2). The antero-posterior spreading of these events may have been retained phylogenetically from pattern specification during proliferative growth as found in the short germ-type of development (and with the abdomen in the intermediate germ-type). However, here this spreading effect would no longer be due t o the spatio-temporal course of specification but rather to the spreading of some process which commits the germ anlage cells to produce pattern elements specified by other means (e.g. b y interacting terminal influences originating from the plasmodia1 part of the system). A comparable course of regional commitment can be demonstrated more directly with respect t o the elements of the transverse body pattern (section 5.1). 4.9.4 Essential differences t o former interpretations As stated above, one of the aims of this review was t o arrive at a hypothetical framework which would encompass most observations. This necessarily implies contradicting some well-established interpretations in the literature which were based on more limited evidence. Five major points of disagreement with earlier interpretations will be discussed here. These concern instruction versus commitment, the supposed mosaicism of some insect eggs, the role of the differentiation centre, the concept of potency regions, and the activation centre. a. Instruction and commitment considered as separate steps in embryonic determination The splitting-up of embryonic determination into two formally distinct steps was suggested by experimental evidence, and has provided possibilities for reconciliation of strongly conflicting views (see e.g. section 4.2.3, 4.5.3 and 4.5.5). Thinking in terms of economy as a basic principle in evolution it might nevertheless appear improbable that two steps instead of one should be employed to programme embryonic cells at the outset. However,it is only by postponing commitment until proper spatial distribution of different instructions has been achieved that the system becomes flexible enough to cope with some potentially adverse consequences of biological variability and changing environment. Immediate commitment of the cells involved in pattern specification would rule out any subsequent corrections and thereby render the nascent pattern unable

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to adapt t o individual variations in egg size, width of germ anlage, local mitotic activity, etc. b. Mosaic development The idea that the insect egg represents a preformed mosaic of cytoplasmic determinants for the different elements of the basic body pattern, refuted for more primitive insects long ago (Seidel, 1926), appears discounted by now also for the most determinate types of insect development. Even the eggs of the higher dipterans must be viewed as systems which epigenetically generate the definitive arrangement of pattern elements, starting from a more or less complicated network of spatial cues in the ooplasm which somehow interact with each other and/or with the ensuing cells (section 4.8). Specification of a developmental pathway via prelocalized specific determinants, as observed in germ cell development (Illmensee and Mahowald, 1974; Okada et al., 1974a), represents the exception rather than the rule in insect embryogenesis. Arguments t o the contrary are either based on the unfounded assumption that failure of egg fragments to “regulate”, i.e. to produce the complete pattern, must necessarily be due t o mosaicism (see section 4.8.2), or on the equally untenable assumption that defective patterns obtained after damaging an egg region b y uv irradiation or puncture prove a “determined” status for that region at the time when the damage was inflicted (see sections 4.1.6.b, 4.6 and 4.8.2). c. Differentiation centre As pointed out in section 3, the differentiation centre was introduced orginally on a purely descriptive basis which is beyond any dispute. However, the physiological role ascribed later on t o this centre has t o be modified in the light of recent experimental evidence. There is no stringent proof, comparable t o the evidence from transposing posterior pole material in the leaf hopper Euscelis (section 4.4.2), for any ‘“instructing” role of this centre in pattern specification; Haget’s claim to the contrary will be refuted below (section 6.3). On the other hand, what has emerged more clearly than before is the fact that not only visible steps of embryonic differentiation occur in this region first, but also some preceding physiological changes, as revealed, e.g. b y increased sensitivity t o X-rays in the cricket, and b y acquisition of the capacity for self-differentiation in various species. Whether other regions in order t o undergo the same changes have t o be physically connected with such a centre, because some sort of indispensable stimulus spreads from there, is at present a question without a simple answer. Egg or germ anlage fragments which appear t o require stimulation from the centre are either relatively small (Acheta, Tuchycines), or come from eggs where this centre appears to be located near the equatorial region (Leptinotarsa, Dacus, Calliphora). Therefore, the possibility must be considered that the effects ascribed t o such a

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centre might actually result because these fragments fall short of a (stagedependent) threshold of a critical length (or mass); what is lacking might then not be the specific stimulus from a centre endowed with special capabilities, but just a bit more material which does not contain a specific quality per se. This caution is prompted by the observation that in Leptinotarsu the region of the “differentiation centre” itself is incapable of differentiation when contained in too small an egg fragment (Haget 1953, p. 193). That some differentiation centre effects might simply be due t o overlapping of processes spreading from both pole regions in opposite directions has already been pointed out (section 4.8). d. Potency regions The concept of “potency regions” for procephalon, gnathocephalon, thorax and abdomen introduced by M. Schnetter (1934) was not confirmed by more recent investigations (section 4.6.1). The nearest recent approximation t o this concept is represented b y the results which Kuthe (1966) obtained by uv cautery of Dermestes eggs at 6.5 h after egg deposition. He found that germ band parts missing after this treatment always corresponded t o the whole abdomen o r t o the whoIe gnathocephalon plus procephalon; in either case the thorax was complete. In other species studied recently, no indications whatsoever of potency regions appeared; the concept therefore certainly cannot claim general validity. If future experiments should reveal reactions as described originally by Schnetter, this would be of considerable interest in connection with the compartmentalization phenomena recently inferred from clonal analysis (section 6.3). e. Activation centre Besides the differentiation centre, two other centres important for insect embryogenesis have previously been established, the cleavage centre (Krause, 1938a) and the activation centre (Seidel, 1929a, 1961). The cleavage centre need not be discussed here because apparently it has no direct influence on pattern specification (Krause and Sander, 1962). The role of the activation centre according to Seidel’s final definition (section 4.2.2) is t o activate more anterior egg regions for germ anlage formation. The results obtained since then show that an activation centre not involved in pattern specification may exist in short germ-type eggs (Tachycines, Schktocerca), although not necessarily located close t o the posterior egg pole (Atrachya, see section 4.5.1). In intermediate germ-type eggs, the activation centre effect, if observed at all, cannot be separated from instructing influences which specify the anterior elements of the body pattern and spread forwards during the same period of development. This is ohvious from the partial patterns formed in anterior egg fragments of the cricket (Fig. 6(b), Table 1); the few pertinent results published for Platycnemis (Fig. 12) point in the same direction, as noted originally by

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Seidel (1929a). With transition from the panoistic to the meriostic type of ovarioles, the “activation” aspect diminishes altogether because the instructions for procephalon formation apparently become lodged rather anteriorly during oogenesis, and no forward spreading of the capacity to form germ anlage is noted (Fig. 1 7 ) . However, the linking of what looks like an “activation centre effect” to pattern specification can be demonstrated in the leaf hopper Euscelis by supplying instruction for some elements of the body pattern to an egg region which otherwise would yield only serosa : anterior egg fragments separated above 35 per cent EL during cleavage will nearly always fail to form any kind of germ anlage, but more than 50 per cent of comparable fragments supplied with posterior pole material become “activated” to form a germ anlage-and some, but frequently not all, elements of the body pattern (Table

13). TABLE 13 The influence of posterior pole material on germ anlage formation in anterior egg fragments of the leaf hopper Euscelis plebejus. Fragments without visible germ anlage showed extraembryonic development or produced a small cell aggregate of uncertain status. (Data from Sander, 1959, Fig. 22b) Germ anlage Anterior egg fragments (36-45% EL) Visible With posterior pole material Without posterior pole material

21 1

Not visible

22 32

4.9.5 Results not covered by the generalizations proposed Many earlier results, although interpreted differently at that time, appear tolerably well compatible with the concepts developed above. However, two sets of well-documented results cannot be fitted into the general hypothesis. The apparent movements of instructions for various body regions which Kuthe (1966) inferred from uv irradiation of Dermestes eggs (Fig. 25(c)) are without parallel, particularly since it is claimed that they could not be caused by movements of blastodermal cells. This special reaction might be due t o technical peculiarities. Kuthe’s uv irradiations denatured the yolk right through to the central axis, and were done stepwise: one stretch of roughly 12 per cent EL length was added t o the next, probably beginning from the egg pole. Since each step lasted 50 seconds, some kind of contraction (W. Schnetter, 1965) in neighbouring regions is conceivable which-if stage dependent (see Bruhns, 19 74)-could

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account for distortion of the defect map. Also the remote possibility exists that the irradiated region exerts some rather general influence on pattern specification, comparable t o that of uv irradiation of an egg pole in chironomid midges (section 4.7); this effect again would have t o be stage dependent to some degree in order t o account for the observed results. The other set of results comes from chironomid midges where the asymmetric double monsters obtained by Yajima (section 4.7) and recent results with temporary fragmentation (Sander, 1975a) require at least some additional assumptions. Like so much other evidence, these results point towards a crucial role in pattern formation for the terminal egg regions, yet these may act here by initiating rather fast signalling processes within periplasm or blastoderm. These results emphasize that one of the urgent tasks ahead is t o establish the components of the developing system which constitute and/or convey the interacting terminal influences on pattern specification inferred for several species (see sections 4.1.6.b and 4.5).

5 Specification of the transverse bilateral body pattern As pointed out in section 2, the transverse body pattern appears t o be specified according t o formal principles which clearly differ from those established for longitudinal pattern specification (see preceding section). Comparable differences are known from other animal groups, e.g. the Amphibia, and may be inherent in the difference between symmetric and asymmetric patterns, respectively. Such fundamental differences are not apparent with subsequent steps in pattern formation, e.g. those linked to the differentiation centre (section 4.9.4.b). These may spread simultaneously in transverse as well as longitudinal directions and thereby “stabilize” the two-dimensional body pattern which was previously specified along its two axes by different mechanisms.

5.1

PATTERNS FORMED BY LONGITUDINAL HALVES OF THE BLASTODERM OR GERM ANLAGE

In most insects, bilateral symmetry is already evident in late oocyte stages. Yet the bilateral (transverse) aspect of the basic body pattern becomes established epigenetically as was shown in a variety of species ranging from cricket to moth. This may be the general rule because stages prior to the blastoderm even in the most determinate egg types display only very vague cytological indications of bilateral organization (e.g. Mahowald, 1963; Davis, 1966). It is usually not before germ anlage formation or gastrulation that the future median plane can be recognized in all regions.

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The gradual establishment of bilateral embryonic organization is evident from results of unilateral uv irradiation during different stages. In the cricket (G. Sauer, 1961a, b), elimination of nuclei (and at least some components of cytoplasm) located superficially o n one lateral egg half can be completely compensated for until 42 h after egg deposition, and by some eggs as late as the 53rd hour. The other eggs irradiated from 42 h onward show various unilateral defects. These always concern the procephalon and extend backwards from there to varying degrees. The abdomen, however, is always of bilateral organization, probably because it arises from an as yet unorganized bud subsequent t o the stages irradiated. When in addition t o one lateral egg half a dorsolateral strip of the other half is irradiated (G. Sauer, 1962), the results are similar. When the additional irradiation eliminates the ventrolateral half o r three-quarters of the other egg side (i.e. the germ anlage itself, Fig. 9 ) , the capacity for compensation persists even longer. Sauer linked his results t o the possibilities left for cell movements to occur after irradiation. He felt that the lateral half of a germ anlage could re-establish symmetry as long as it had a chance to broaden sufficiently. However, in other insects comparable results were obtained in the absence of any large-scale cell movements. In Dermestes, Kuthe (1966) found that unilateral superficial uv irradiation is tolerated until shortly before cellularization of the blastoderm (6.5 h, see Fig. 25(c)), while irradiation of the cellular blastoderm led t o suppression of one lateral half of the germ bmd-excepting again the abdomen which always was bilateral as in the cricket. In Leptinotursu (Haget, 1953), lateral egg halves may form bilateral germ bands (lacking only the terminal segments of abdomen) even if separated at the cellular blastoderm stage, b u t egg halves cut shortly before the germ anlage stage produce only half-embryos (Fig. 35, I). The results -+. from beetle eggs thus show that otherwise identical unilateral regions of the blastoderm when separated early may organize themselves symmetrically so as to form a bilateral pattern, but fail to do so if separated later r>n. This result must be due to some kind of progressive specification and/or commitment of cells or blastema t o form their “proper” share of the transverse pattern, in this case one lateral half. Since the same conclusion must be drawn from results obtained in a gryllid orthopteran (Tuchycines, see below), it appears reasonable to assume similar processes in the cricket as well, and t o regard the concomitant changes in motility of germ anlage cells observed by Sauer (1961a, b) as epiphenomena. This interpretation is compatible with the observation that in the cricket the capacity to produce bilateral germ bands is retained longer in the dorsolateral (mostly extraembryonic) cells than in the germ anlage blastema proper (see above). This was also observed in the bruchid beetle Cullosobruchus where a bilateral

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germ band may arise on each side of the original germ anlage when the median region is destroyed by KCN treatment (Brauer, 1938). Epigenetic establishment of bilateral organization is documented in a

I 24

m

Fig. 35. Results from longitudinal and oblique cutting of Leptinotarsa eggs at various blastoderm stages (data of Haget, 1953); arabic numerals indicate age (h at 24 " C ) at the time of cutting. Surviving egg part is dotted, restituted part of pattern is hatched. For interpretation see text. I, Median cutting may be followed by resymmetrization if carried out at 18 h, but not at 24 h. 11, Obliquely cut anterior fragments. Note progressive loss of capacity for resymmetrization, and increase of germ band region representing left half. 111, Anterior oblique cut combined with contralateral cautery (dotted) of gnatho-thoracic region (see text).

particularly drastic way by formation of duplications (duplicitas parallela of Krause, 1953). In experiments primarily aiming at the longtudinal pattern aspect, such duplications have been obtained in a dragonfly (Seidel, 1929b), and possibly in beetles (Brauer and Taylor, 1936; Jung, 1966). In more controlled ways, they were produced experimentally in species

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21 1

ranging from primitive orthopterans t o moths. The most detailed analysis was carried out by G. and J. Krause on the camel cricket Tuchycines (for reviews see Krause, 1958a, b). They showed that in this species the two halves of a germ anlage cut medially may establish bilaterality by two different modes, depending on the stage of cutting (Fig. 36). When separated early, each half will form a bilateral germ band from the germ anlage blastema proper, and gastrulate in the new midline. After later separation, the cut part of the germ anlage blastema more or less follows its normal course of development; in this case, bilateral organization is regained by induction of neighbouring amniotic material so as t o form the missing half of the pattern. The capacity to become bilateral after longitudinal cutting is first lost in the prothoracic differentiation centre

Fig. 36. Stage-dependent ways of re-establishing bilaterality in cut halves of the germ anlage of Tuchycines (from Krause, 1952). Diagrams represent transverse sections (see Fig. 16): amnion black, mesoderm dotted. Top row: resymmetrization of halves after early separation. Bottom row: induction (curved arrows) of the lacking half-pattern in amniotic material joined to the germ anlage at the cut after late separation.

and subsequently in more anterior and posterior regions (Krause, 1934). In the leaf hopper Euscelis, both parts of an egg pinched longitudinally during cleavage may form complete and bilaterally organized germ bands, even when the egg was separated into a ventral and a dorsal fragment (Sander, 1971). Partial or complete twins were obtained after KCN treatment in the beetle Cullosobruchus (see above). As in Tuchycines, these reveal a spatio-temporal course of commitment t o definitive bilaterality beginning in the prospective thorax and spreading from there in anterior and posterior directions (Brauer, 1938). Twin-like structures in Lepidopterans were obtained by uv irradiation of a median strip of germ anlage in Tineolu (Luscher, 1944), and by dorsal centrifugation of Bombyx eggs before or during cleavage (Miya, 1956). Again the best documented results are those of Krause and Krause (1965) who in vitro or in ouo cut the germ anlage of Bombyx mori L. into lateral halves. With early cutting, each half may gastrulate along a newly established midline of its own, while after later separation symmetry is not regained. Commitment to form specific elements of the transverse pattern again appears to occur first in the thoracic

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region: this region sometimes failed t o become,bilateral when procephalon and abdomen still did. A peculiar way of re-establishing bilaterality was observed in vitro in some halves where the mesoderm was segregated at the medial margin (i.e. in the correct region of the normal pattern) but then migrated t o a new midline appearing in the ectodermal half-blastema which meanwhile had acquired a bilateral shape.

5.2

DIFFERENCES BETWEEN TRANSVERSE AND LONGITUDINAL PATTERN SPECIFICATION

These results, especially from cutting blastoderm or germ anlage, clearly show that in these species the bilateral transverse aspect of the body pattern may become established by mechanisms which on d formal level are quite different from those which specify the longitudinal pattern aspect. The latter are characterized b y specific terminal regions indispensable for pattern specification, whether by budding or by interacting terminal influences (section 4). The bilateral pattern, on the other hand, can apparently be set up by (and in) any longitudinal and not too narrow strip of blastoderm. The midline of the pattern becomes established somewhere between the margins, and probably on the basis of signals emitted by these. Specification of the pattern elements between midline and both margins may be a consequence of further interaction. Irreversible commitment to specific pathways of development may occur some time after specification, and in mediolateral sequence-as shown by the prolonged capability of more lateral areas t o produce bilaterally symmetrical patterns. Commitment frequently becomes evident first in the thoracic region, as does commitment for the longitudinal pattern (section 4.9.3) with which it may be identical. The cues indicating in normal development the margins of the prospective pattern are not known and may vary with different groups. In the cricket and similar species, the drop in cell density (number of nuclei per unit area) caused at the border between germ anlage and serosa by pecularities of periplasmic streaming may be the triggering signal, while in forms with less extensive peripheral movements the necessary cues might be prelocalized more directly in the ooplasm.

5.3

THE DIFFERENTIATION CENTRE REVISITED

The results obtained by cutting Leptinotarsa eggs in the median plane (section 5.1) provide the basis for re-evaluation of experiments thought by Ilaget (1953) to prove pattern specification via inductive influences from a prothoracic differentiation centre (see also Krause, 1958b; Counce, 1972).

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The first of these experiments,consisted in cutting the egg diagonally in a dorsoventral plane. Typical results obtained by cutting from 75 per cent EL at the right t o 25 per cent EL at the left-hand side during different stages are set out diagrammatically in Fig. 35,II. Anterior fragments of eggs cut diagonally at 18-20 h yielded bilaterally symmetrical anterior partial germ bands terminating somewhere behind the prothorax (frequently in a

G 2 G 3 T

I

T I I

T

N e w /Original task Fig. 37. Haget’s interpretation of his result shown in Fig. 3 5 , I I , at 20 h (modified from Haget, 1 9 5 3 ) ; left and right sides have been interchanged in this diagram as compared to Fig. 35. The posterior flap which turned anteriorly and fused there is presumed to become reprogrammed so as to produce gnathal instead of abdominal structures. This assumption is unnecessary (see text and Figs 3 5 and 38). P, procephalon; GI-G3, segments of gnathocephalon; T 1-111, segments of thorax; A 1-5, segments of abdomen.

fused segment or appendage), while fragments from later stages produced asymmetric anterior partial germ bands. Haget (1953) maintained that the result from cutting at 18-20 h was due t o a change of developmental programme (including reversal of polarity) in the posterior flap of blastoderm shown at the left, which he thought was utilized for right-hand side gnathal and anterior thoracic structures instead of forming left parts of posterior thorax and anterior abdominal segments (Fig. 37). He ascribed this change t o

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inductive influences from the left gnathal and thoracic regions and proceded to show by another experiment (Fig. 35,111) that these influences were exerted by a differentiation centre located in the prospective prothorax. However, the results from median cutting (Fig. 35, I) suggest quite a different approach, and recent experiments (Sander, unpublished) tend to abolish Haget’s interpretation.

Fig. 38. Photograph of Leptinotarsa egg fragmented obliquely with the pinching technique (anterior pole at top). Note that the posterior flap did not turn anteriorly as in Fig. 37, and that the blastoderm (dark contour) has healed straight along the line of separation. Such fragments may produce anterior partial germ bands organized bilaterally from procephalon to prothorax. Bright areas at the sides represent light reflected by the surrounding paraffin oil.

A series of 30 Leptinotarsa eggs fragmented obliquely with the pinching technique yielded 3 anterior partial germ bands organized bilaterally back till at least the maxillary, labial and first thoracic segments (one case each). Yet, in this type of experiment the flap of blastoderm cannot (and did not) turn anteriorly as in Haget’s experiment; rather, the blastoderm healed straight along the cut within minutes after the operation (Fig. 38).

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21 5

The following interpretation could account for this as well as for Haget’s result. As shown by median cutting at 18-h (Fig. 35, I), the full bilateral pattern can be established if at least half of the original circumference of the blastoderm is available. In Fig. 35, XI, the egg regions where this much is left are those above the point where the cut crosses the midline; in Fig. 35, 111, those below this point. Since in Fig. 35, 11, the cut crosses the midline at 50 per cent EL, i.e. in the prospective prothorax, no additional material from the posterior flap is needed for all structures which showed bilateral development at 18-20 h. Bilateral fusion of the more posterior regions, as frequently observed, was probably due t o lack of material, a condition known t o cause fusion, e.g. in head development of amphibians. The most posterior segments failed to form altogether (as after median cutting; Fig. 35, I, 18 h), probably because the blastoderm left there was too narrow. These considerations (and the experimental results) show that supplementation of the anterior blastoderm by a flap of posterior blastoderm is not required, and that alternatives t o Haget’s interpretation exist. A detailed consideration of Haget’s Figs 40 and 41 (loc. cit.) will, moreover, reveal some geometric inconsistencies speaking against his interpretation. If the posterior flap reached as far anterior as indicated in Haget’s Fig. 40/2, and if the prospective mesothorax (located at c. 35-45 per cent EL; loc. cit., p. 198) really became lodged in the most posterior region after healing, then the flap must have been a narrow tongue rather than the broad wedge depicted. One may therefore assume that the blastoderm flap pulled anteriorly during healing in Haget’s experiments either did not become functionally integrated there, or (less probably) it must have been so narrow as t o be of no importance. The results obtained with later stages (Fig. 35, 11, 22 h and 26 h) differ from the 20 h results by an increase of segments at the left, and by a decrease at the right. The latter is to be expected if the capacity t o become symmetrical is lost during these stages starting from the thoracic region (see Fig. 35, I, and section 5.1). The former difference could be due t o forward spreading of segmental instructions as in other beetles. Eggs of Bruchidius fragmented at c. 30 per cent EL during comparable stages would, in close agreement with Fig. 35, 11, yield partial germ bands terminating in prothorax, metathorax and the 6th abdominal segment, respectively (Fig. 22 in Jung, 1966, stages Pz, Bl(m), and VKa). This interpretation seems especially warranted because Bruchidius represents the Euscelis mode of pattern specification (section 4.4) which W. Schnetter (1965) favoured also for Leptinotarsa. The other experiment claimed b y Haget (1953) to demonstrate pattern specification via inductive influences from a prothoracic differentiation

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21 6

centre is rather complicated. The eggs were burnt at the right side near the equator, and then cut obliquely at the left in the head region (Fig. 35,111). The typical result from 18-h operations was a defect in the right gnathocephalon and thorax with a more or less fused procephalon carrying a right larval eye. The defects at the right were ascribed t o elimination of the differentiation centre, and the formation of left gnathal segments to inductive influences from the left differentiation centre. As pointed out above, median cutting (Fig. 35, I) shows the latter assumption to be superfluous, and the defects at the right may as well be due t o inactivation of the blastoderm cells required for formation of the absent half-segments. The inactivation probabIy did not extend into the procephaIic region, and this is why the eye formed at the right and not at the left as it should if it were “induced” from the intact half of the prothorax. Elimination of the right prothorax might be the cause for the observed defects in so far as stimuli required for cellular commitment and/or self-differentiation normally spreading from there could have failed t o reach the gnathal region (as assumed for the “polarized” effects of transverse uv barriers in the cricket germ anlage; section 4.1.4). To sum up, it may be stated that Haget’s results do not prove an “instructing” influence of the prothoracic differentiation centre in specification of the longitudinal pattern, and that the alternative interpretations offered here for his results are much more compatible with experimental data obtained since then in Leptinotarsa and other beetles. 6 Genetic tools in the study of pattern specification Ontogenesis can be considered as the transformation of genetic information into three-dimensional structure. From this point of view, the previous chapters were concerned with releasing systems which serve t o initiate the proper spatio-temporal order of gene utilization in insect embryogenesis. The present section is intended t o review some genetic evidence possibly related t o these ooplasmic releasers, and t o assess some recently developed methods. It may safely be stated that the wealth of information potentially avaifable from mutants influencing embryonic development (see Wright, 1970; Bakken, 1973; Postlethwait and Schneiderman, 1973b) for the study of pattern specification remains t o be retrieved. 6.1

MUTANTS TRANSFORMING PATTERN ELEMENTS

Mutants interfering with embryonic pattern formation may either be maternal effect mutants which due to faulty oogenesis affect development in the offspring, or zygotic mutants (Gehring, 1973). With respect to the

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21 7

type of anomaly, mutants where pattern elements develop abnormally for secondary causes must be distinguished from “transforming” or homeotic mutants. The latter affect basic steps in pattern formation, with the result that pattern elements are formed in atypical regions of the system or, as viewed more commonly, that some pattern elements differ from those typically occupying the respective places in the pattern. With the generalizations outlined above for nonproliferative pattern formation (section 4.9.2 and Fig. 34), transformation of pattern elements may be caused either by altering instructing signals, or by false interpretation of correct signals by the reacting cells. A good example of the former category is the bicaudal factor in Drosophilu (Bull, 1966) which by way of a maternal effect produces the “double abdomen” type of pattern anomaly (section 4.7). This type of mutant might also be called a “coordinate mutant” because it clearly affects the spatial coordinates providing the “reference points” (Wolpert, 1969) for pattern specification. Considering the mode of pattern specification by terminal influences postulated in section 4, at least two classes of longitudinal coordinate mutants could be expected: mutants lacking anterior pattern elements and mutants lacking posterior pattern elements. Duplication of the remaining elements, e.g. in the double abdomens, might be a secondary effect, as is indicated by “single abdomens” in the offspring of bicaudal mothers (Bownes, 1973). A third, less easily recognized class of maternal effect mutants might produce the correct pattern, but from an atypical region of the oocyte. The class of “misinterpreting” mutants should include the E-series in Bombyx (Tazima, 1964) and the bithorax alleles of Drosophilu (Lewis, 1963: Kiger, 1973; Garcia-Bellido, 1975; Sander, 1975b); the assumption of Capdevila and Garcia-Bellido (1974) that changes in instructing signals, rather than in cellular interpretation, should be involved in phenocopying the bithorax effect has been discussed above (section 4.9.4). The fact that in Drosophila so few pattern transforming mutants of the maternal effect type are known may be significant in itself. If there were as many different ooplasmic determinants as required for a mosaic-type system of pattern specification, mutations altering one or the other of these should have been found by now; particularly since they have been searched for with some effort, using imaginal discs as criteria (Bryant, 1974). Yet the best that has come up so far was a temperature-sensitive lethal mutant (mut(3)3 of Rice and Garen, 1975) which at the permissive temperature causes restricted adult defects of rather erratic distribution. All thoracic or abdominal structures may be affected, individually and in various combinations. Since occasionally patches of the blastoderm fail t o cellularize in these eggs (T. Rice, personal communication), the mutant need not necessarily affect pattern specification but might also prevent subsequent

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ieali/ation of the properly specified pattern. A maternal effect mutant whicli possibly affects the transverse pattern aspect in Bombyx is Izi (‘Taz;ma, 1964). Eggs of monozygous ki females produce purely ectodermal germ bands, but it is not known whether this is due t o nonspecification of mesoderm, failure t o gastrulate, and/or decay of mesoderm cells. In other species, several types of pattern aberration are known which may be due to mutation. These are the double abdomens observed in the inoscluito Wyeomyia (Price, 1958) (section 4.7) the cricket germ bands

1 ig. 3$!. Parthenogenetically developed larva of the migratory locust Locusta migmtoriu. .\bdolliinal segments in the region between bars have been transformed to resemble \egnit llts of the thorax (after a more detailed drawing kindly supplied by Dr M. Verdier, I’ms)

(oiis1,ting only of the most anterior and posterior pattern elements (Sander rjt a / . , 1970), and the thoracoid transformations of anterior abdominal iegnlrnts found in the offspring of a parthenogenetic Locusta migratoria m’p-~~torzozdes R. and F. female by Verdier (1961) (Fig. 39). Nonformation o f posterior body segments is rather more frequent (e.g. Verdier, 1961; Caritl(.r et al., 1970) but is probably due in most cases to phenotypic [nodI rications of oogenesis, or injury during oviposition. Maternal effect niut,lnts of this type would be highly interesting because the effect then >hould be due t o an insufficient supply of posterior ooplasmic instruction (sect 10114.4.2).

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6.2 MAPPING OF DEVELOPMENTAL FOCI Several methods of studying insect development with the aid of genetic mosaics have been conceived recently. Among these are attempts to locate developmental foci. Fausto-Sterling (1971) studying gynandromorph offspring from fu5 and r9 mothers found no flies which in head or thorax carried spots hemizygous for these alleles. She concluded that inclusion of sizeable numbers of mutant cells in anterior egg regions must be lethal. Bryant and Zornetzer (1973) studied zygotic lethal mutants by a similar technique. They recognized some of these as “locally active lethals” and proceded t o establish their foci on blastoderm fate maps constructed by the method of Sturtevant (Garcia-Bellido and Merriam, 19 69; Hotta and Benzer, 1972). These foci should be the regions where absence of the wild-type allele cannot be tolerated. As yet, no foci for embryonic lethals have been mapped, and the technique will need considerable improvement if it is t o reach a useful level of resolution. 6.3

CLONAL ANALYSIS AND DEVELOPMENTAL COMPARTMENTALIZATION

Another approach employing genetic mosaicism has led to discovery of the phenomenon called developmental compartmentalization by GarciaBellido et al. (1973). The authors found that in Drosophih genetically marked clones of epidermal cells do not transgress certain borderlines, even if the genetic constitution of the cells in these clones raises their mitotic rate far above that of the surrounding epidermis. Such clones can be induced by X-irradiation of suitable genotypes. This permits correlations to be established between stage of irradiation and borders respected by the ensuing clones. In the structures derived from the dorsal mesothoracic disc, the borderline separating anterior from posterior regions is respected even by clones induced around the blastoderm stage (see Fig. 2 in Garcia-Bellido et al., 1973), which probably means that from then on cells located in either “developmental compartment” do not mingle any more, and stick t o compartment-specific pathways of development. With respect to pattern specification, the most interesting aspect of these findings is the progressive subdivision into daughter compartments, for which a “splitting mechanism segregating different groups of cells from a previously homogeneous and contiguous cell population” has been postulated by GarciaBellido et al. (1973). In the wing disc, this mechanism must act mainly during larval life, but it might have counterparts involved in specification of the basic body pattern. The “potency region” effect described by M. Schnetter (1934) could reflect such a mode of pattern specification but, as pointed out in section 4.6, more recent experiments have failed t o substantiate this effect.

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Compartmentalization during formation of the ,basic body pattern has ixen studied in the bug Oncopeltus fusciatus Dallas by Lawrence ( 1 9 7 3 ) . IIe found that X-ray-induced pigment anomalies appear t o be inherited c lonally. Abdominal clones induced from the blastoderm stage onward are usually restricted t o a single segment half, while most earlier induced clones spread over several segments. The switch t o single-segment clones is not linked to a drop in clone size such as to make inclusion into more than one \egment improbable. Lawrence ( 1 9 7 3 ) therefore concluded that “demarcation of segments coincides with formation of the blastoderm”. This interpretation taken at face value poses some dilemma because in the leaf hopper Euscelis and in beetles-the closest “relations” to Oncopeltus discussed in section 4-longitudinal pattern specification is thought not t o he completed until shortly before the germ anlage stage (sections 4.4 and 4.5) which in Oncopeltus is reached some hours later. Perhaps one method \ields the earliest and the other the latest estimate for the same process. I Iowever, the clonal data would need re-interpretation if proliferation rates lor segmental and intersegmental cells should turn out t o differ strongly in the period between specification and visible segmentation. With proper cytological backing, clonal analysis should prove even more valuable as a method in the study of pattern specification.

7 Cytological and molecular aspects of embryonic pattern specification in insects I leeper understanding of experimental results described in the previous chapters can only come from cytological and molecular data. For these to Ix collected and evaluated in a useful way, some conceptual framework at the level of embryology is needed. An attempt has been made in previous bections t o provide such a framework. Cytological and biochemical data on c,arly insect embryogenesis, although appearing at an increasing rate, are .till very unsatisfactory from the point of view of pattern formation. We \hall therefore discuss only selected data which are relevant t o some key topics. On the cytological level, these topics are cytoarchitecture of the I )ocyte, mitotic waves prior to germ anlage formation, functional differentiaI Lon of nuclei, and roles of cell boundaries. Finally, the prospects of studying I he molecular mechanisms involved in pattern specification will be cliscussed.

7.1

CYTOARCHITECTUREOF THE OOCYTE

‘l‘he cytoarchitecture of the mature oocyte must contain all spatial cues I equired for the body pattern to be formed. Considering the complexity ( ) f this pattern, the paucity of visible regional differences is impressive

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(Mahowald, 1973; Engels, 1973). Structural asymmetries, if visible at aI1, are of a quantitative rather than qualitive character. The only prominent structures differing qualitatively (at least in the light microscope) from their surroundings may be found near the posterior egg pole, and are known as polar granules or oosome. Long considered to be germ cell determinants (Illmensee and Mahowald, 1974; Okada et al., 1974a) they may yet exert different morphogenetic functions in some species (Krause and Sander, 1961; Meng, 1968); but no influence on specification of the basic body pattern has been demonstrated so far (Achtelig and Krause, 1971). With this dearth of apparent regional differences in cytoarchitecture it might be worth while considering the possibility that egg shape as such provides spatial cues for pattern specification. This possibility seems remote in short germ eggs, particularly when egg shape may greatly vary according t o space available for oviposition (e.g. Atrachya, Dermestes) (Kuthe, 1966; Miya, 1965). But it should be noted that in the double abdomens of Smittia the plane of mirroring symmetry initially becomes established half-way between poles (Kalthoff and Sander, 1968), so that the initial antero-posterior polarity in both partners coincides with tapering in egg shape. Thus, while the anterior or posterior character of an egg region clearly must be determined by some other means, the polarity of the pattern t o be formed could be specified more or less directly via egg shape. All these observations certainly mean that no evidence in favour of a preformed mosaic determining the elements of the body pattern can be derived from oocyte architecture, or from the experimental results described in previous sections. Considering the fact that even in eggs belonging to the extreme long germ-type the cytoarchitecture may become strongly disrupted during oviposition (Went and Krause, 1974), epigenetic modes of pattern specification based on rather simple spatial cues appear probable . 7.2

MITOTIC WAVES DURING BLASTODERM FORMATION

The cleavage nuclei have been considered equipotent since the early days of experimental insect embryology (Counce, 1972; Okada et al., 1 9 7 4 ~ )After . their arrival in the periplasm, they sooner or later undergo visible changes which may be connected with acquisition of developmental specificity (see below). In several species, these changes are preceded or accompanied by so-called mitotic waves (e.g. Bergerard and Maisonhaute, 1967; Stanley and Grundman, 1970; Alleaume, 1971; see Counce, 1972). These represent the earliest regional differences at the nuclear level (apart from pole cell nuclei) and therefore have been credited with a role in regional determination (e.g. Agrell, 1961). This idea has met with increased interest since mitotic waves

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as observed in a blowfly (Agrell, 1961) were linked by Kauffman (1973) to a model interpreting the transdetermination data of Hadorn and co-workers (Hadorn, 1966) in terms of bistable memory circuits. A particularly detailed description of mitotic waves has been given for the Colorado beetle by Bergerard and Maisonhaute (1967). Since this species is amenable to a variety of experimental procedures (Haget, 1953; W. Schnetter, 1965,1967; see also Fig. 38) a comparison of the effects of these procedures on mitotic waves and on the ensuing body pattern might be very rewarding.

7.3

FUNCTIONAL DIFFERENTIATION OF NUCLEI

Functional changes occurring in the nuclei are first indicated by the appearance of nucleoli and changes in size and shape (H. W. Sauer, 1966; Grellet, 1971; Fullilove and Jacobson, 1971; Schwalm and Bender, 1973), accompanied probably by changes at the level of chromosomal proteins (Pietruschka and Bier, 1972). Yet the capacity of germ anlage nuclei to support development of different elements of the body pattern need not become restricted by these changes, as shown for Drosophilu by the nuclear transplantation experiments of Illmensee (1973). The failure of W. Schnetter (1967) t o obtain normal development after transplantation of early germ anlage nuclei (or rather cells) may be due t o difficulties in method (loc. cit., p. 499). Thus, the nuclear activities indicated by these changes probably have a bearing on general processes of development rather than on regional specificity. In particular, this appears true for early transcription, as shown, e.g. by actinomycin treatment of Leptinotursu eggs at different stages (Maisonhaute, 1971). Regional differences between nuclei are visible during germ anlage formation in some hemimetabolans. In the cricket, the prospective germ anlage nuclei shrink (H. W. Sauer, 1966) while the prospective serosa nuclei increase in volume and become polyploid (Grellet, 1971). It is interesting t o note that these differences are reflected by changes in X-ray sensitivity. With a suitable dose, the germ anlage nuclei can be eliminated in the cricket while the prospective serosa nuclei survive (Schwalm, 1965). In Kulotermes (Truckenbrodt, 1965), germ an Iage nuclei become incapable of further division after irradiation with 6000 r while cleavage nuclei may still undergo further mitoses after 35 000 r. None of these data indicate an active role of nuclei in pattern specification, comparable to that demonstrated by Seidel (1932) for the cleavage energid reaching the “activation centre” in Platycnemis. However, it remains to be shown whether a reaction between this centre and the nucleus itself is necessary for subsequent specification of pattern in this species (see section 4.2.2).

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7.4 BLASTODERMAL CELL BOUNDARIES Cell boundaries are likely to play important roles in pattern formation. Various authors have concluded that the cortical region of the oocyte is of predominant importance in embryonic pattern formation (Counce, 1972). It is not known how much of this importance, and particularly of prelocalized cues for pattern specification, is connected with the actual oocyte membrane or oolemma-a structure of interest because of the possibility of “cortical inheritance” (see Gehring, 1973, for discussion). But this structure no doubt plays a crucial role by initiating functional separation of different nuclei b y cell boundaries, a process which most likely is a prerequisite for initiation of differential pathways of development. The membranes which cut in between the future blastoderm nuclei originate as extensions from the original oolemma (see Wolf, 1969). Regional failure in boundary formation is followed by pattern defects (W. Schnetter, 1965). This observation and the fact that cell membrane formation must largely depend on preformed molecules (Fullilove and Jacobson, 1971; Schwalm and Bender, 1973) may provide an explanation for the erratic location of defects in the Drosophilu mutant isolated by Rice (section 6.1) and in flies derived from eggs irradiated very early with uv (Nothiger and Strub, 1972). Both mutant effects and irradiation could reduce the pool of cell wall precursors, with chance deciding afterwards the exact region where cell separation remains incomplete due t o lack of precursors. Cell boundaries are also required t o separate the blastoderm cells from the yolk plasmodium. This process is considerably delayed in some species (Schwalm and Bender, 1973), and may occur in the germ anlage later than in the extraembryonic blastoderm (W. Schnetter, 1967)-an interesting observation in view of the instructing influences ascribed t o the underlying yolk plasmodium (section 4.5). Failure of the blastoderm cells t o separate from the yolk plasmodium is thought t o initiate abnormal development in the selected line of the beetle Dermestes studied by Ede (1964).

7.5 PROSPECTS

FOR A MOLECULAR APPROACH TO PATTERN SPECIFICATION IN THE

INSECT EMBRYO

To understand embryonic pattern specification at the molecular level may, for two reasons, prove next t o impossible in species where the basic body pattern becomes specified during proIiferative growth (section 4.3). The reasons are the small numbers of cells in a “bud”, and the specific programming of only a few cells at a time which seems t o occur with this type of pattern specification. With pattern specification via graded proper-

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ties in the ooplasm (see sections 4.4.2, 4.4.4 and,4.9), there may be some hope for identifying structures or molecules which provide the instructing cues. But this can be done only by applying biochemical, topohistochemical and/or autoradiographic methods t o systems where a specific pattern anomaly can be induced by either genetic or physical techniques, and to individuals where this anomaly actually had been induced. Among the many biochemical investigations on insect embryos published in recent years (Duspiva, 1969; Counce, 1972; Brahmachary, 1974; also Ernst et ul., 1975; Schneider, 1975; W. Schnetter, 1975), only the paper of HansenDelkeskamp (1968) on rRNA synthesis in embryoless cricket eggs met the latter condition-albeit without demonstrating for the stages involved in pattern specification any differences between normal and embryoless eggs. 'I'his result is a reminder that it might be wise before embarking on any biochemical analysis of pattern specification t o consider carefully the possibility that specifying cues might exist which are not amenable t o biochemical analysis. For instance, the germ anlage in Kulotermes nearly always forms underneath the micropylar openings in the chorion (Truckenbrodt, 1971); therefore, before searching the ooplasm for molecules triggering germ anlage formation, the possibility should be tested that the actual cue t o which the cells react is provided by structural pecularities in the chorion. Another possibility t o be considered is preformed differential orientation or arrangement of identical (and therefore chemically indistinguishable) molecules, e.g. actomyosin-like protein complexes (Moser et al., 1970). Such differential orientation could cause the regionally different vectors of cytoplasmic movements which shape the germ anlage in the cricket, and thereby possibly play a key role in transverse pattern specification (section 5.1). With histochemical and autoradiographic methods, a few regional peculiarities have been found which might be related t o pattern specification or cellular commitment. But again no attempts have been made t o apply these methods to eggs actually about to produce an aberrant pattern, and to search for correlations between chemical events and patterns formed. Two findings might permit such studies: the increased incorporation of amino acids in the posterior pole region of Muscu domesticu L., which might be linked to specifying influences exerted by that region (Pietruschka and Bier, 1972; section 4.8), and the restricted distribution of alkaline phosphatase in the Drosophilu embryo which may reflect the activities of a differentiation centre (Yao, 1950). Approaching the problem from the other side, i.e. looking for predictable pattern alterations which might be suitable for molecular analysis of pattern specification, some hopes are raised by the double abdomens of Drosophilu (section 7.1) and chironomids (section 4.7), and by the aberrant

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germ band patterns produced by transporting posterior pole material in Euscelis (section 4.4.2). Bicaudal or “dicephalic” Drosophila mutants with high penetrance would be ideal but have not been isolated so far. The possibilities provided by transposition of pole material in Euscelis have not yet been seriously exploited. However, it is obvious that the morphogenetically active material must adhere closely t o the symbiont mass or, more correctly, t o the egg cortex enveloping it; the symbionts themselves in all probability do not exert the decisive effect (Sander, 1968, 1974). The posterior pole material needs to be located rather close t o the egg cortex ( 6 30 pm) in order to be effective after transposition (Sander and Franz, unpublished), and is fully effective through bottle-necks where the egg diameter had been reduced to less than half of its original value (Sander, 1959). These and other observations put certain restrictions on the molecular mechanisms possibly involved, but much more could be learned by injection of fractionated egg contents or defined substances into egg fragments devoid of pole material. The operations leading t o double monsters in chironimids apparently act by triggering in the wrong place a strictly channelled reaction which leads to formation of head instead of tail, or vice versa. The reaction implies instruction for the region-specific character of the pattern elements t o be formed, and for antero-posterior polarity of the ensuing series of pattern elements. The switch from head t o abdomen in Smittia occurs also in partially irradiated anterior egg fragments, i.e. in the absence of the signals normally specifying abdomen (Sander, unpublished results). That this switch is largely preprogrammed in the system and just needs to be triggered is evident from the range of manipulations which produce double monsters. In Smittia, double abdomens may be induced by irradiation of the anterior pole region (Kalthoff and Sander, 1968), by anterior or posterior centrifugation (Kalthoff, unpublished results), and by puncturing the anterior egg pole (Schmidt et al., 1975). An equally broad array of treatments may cause a switch back to head specification in irradiated Smittia eggs: reverting effects are known from subsequent irradiation with light of longer wavelengths (photoreversal; see Kalthoff, 1973), elevation of rearing temperature (Kalthoff, 1971a), and irradiation of the posterior egg pole with uv before or after irradiation of the anterior egg pole (Kalthoff, 1971b). By looking for the common denominator of different treatments yielding the same type of result it should be possible t o pin down the triggering agent(s). It will be much more difficult to recognize the physiological and molecular reactions which constitute the ‘(channels” ultimately leading t o formation of the anterior or posterior pattern sets observed. The uv-induced double abdomens of Smittia at present provide probably

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t h e nio\t promising system for molecular studies on pattern specification in

i I I sect embryogenesis. With suitable experimental conditions, virtually

ed t ly

all thr eggs treated will produce double abdomens-a yield much superior t o that obtained so far in any other chironomid. This high yield could be due i o the tiny size of the egg which may require exclusion of all but the mosl c ssential egg components, and thereby minimize secondary reactions le idin L: t o less well-defined results. The uniformity of patterns produced ~1ouLtlqualify the Smittiu egg for a direct biochemical approach, but its low r n m cloes not encourage this. Therefore, mainly biophysical studies have been arried out so far. These demonstrated that the uv targets are located I n t hc peripheral ooplasm, and that their distribution there-or .their el Tic i t ncy in the irradiated state-follows an antero-posterior gradient (Kaltlioff, 1 9 7 1 ~ ) The . action spectra for induction of double abdomens, ‘ind t lie conditions under which photoreversal of the uv effect is possible, p o i i ~towards ~ nucleic acid-protein complexes as targets (Kalthoff, 1973). Xlitot tiondria, ribosomes and masked messengers are among the structures ieprc \enting such complexes. Mitochondria cluster in the region most sensitive I O uv light (Zissler and Sander, 1973) and become damaged visibly in the radiated region (Zissler, unpublished); yet the idea that they might i n f l u c nce pattern specification via metabolism (Sander, 1975a) was so far ~ p p )rted c neither by measurements of oxygen consumption and ATP c o i i i ( nt, nor by application of mitochondrial inhibitors and uncouplers of oxid.itive phosphorylation (Kalthoff and co-workers, unpublished). Masked Incsvmgers as targets would of course be a rather attractive idea, but it shoiild be born in mind that these in a11 probability could not be specific incsengers for different pattern elements, but must be involved in gener‘itiny a general “anterior” quality. The attempt t o “rescue” irradiated eggs h y iiijection of intact cytoplasm (cf. Garen and Gehring, 1972; Okada et d., 1974b) o r fractions thereof has so far not been successful. 11

8 Concluding remarks

Tht, problem of pattern specification in early insect embryogenesis has been tre.1 ted in the preceding sections essentially on the formal level characteristic of “classical” developmental physiology. This limitation was enforced by the fact that little is known beyond. However, it is hoped that critical rc.\ iewing of the formal aspects may prepare the ground for future attempts tci cackle pattern specification in more molecular terms. The emphasis, po\iiblv ill-founded, placed on the common aspects of data from different s p cies and groups as practised in this review may have helped t o provide a r,iiher comprehensive view, but it did so at the expense of the group- or spc cies-specific pecularities of early development which may determine

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success or failure of a,research project. To disregard these peculiarities is a real danger in insects where they appear to be more prominent than in other animal groups, or at least have been very prominent in promoting knowledge: a consideration of sections 3-5 will show that certain crucial experiments, such as result, e.g. in longitudinal mirroring and polarity reversal, apparently can be carried out successfully only in certain groups, and the results may at best be linked conceptually t o results from other groups via reactions common to both groups, as, e.g. the “gap” phenomenon. Hopefully it is for trivial reasons that at times comparison of results even from very similar experiments carried out in different species appears next to impossible. The generalizations proposed in the preceding sections need checking by further experiments in order t o rest on a firm basis, and to be amended where required; those results which apparently are not compatible with these generalizations (e.g. sections 4.1.7 and 4.9.4) merit special attention. Beyond this, much of the data reviewed here indicates that further research on embryonic pattern specification should be carried out predominantly on species amenable to both experimental manipulation and genetic analysis of early embryonic development. Drosophila, although ideal for the latter approach, is not so for the former. A more optimal combination of possibilities might be found in some beetles which also permit the use of different genotypes (see Sokoloff, 1966, 1972), but do not represent the extreme long germ-type of development. In the genetic approach, a first step might be to intensify the search for maternal effect mutants affecting embryonic pattern formation. On the experimental side, the relative contributions of the plasmodial and of the cellularized components of the developing system to pattern specification should be more closely assessed with different methods. Last but not least, the understandable but unhealthy tradition of experimental insect embryologists never to repeat other author’s experiments using the same species should be abolished. Some deviations from this tradition have recently been observed and it is hoped that this trend will continue at least with easily tractable species and types of experiment. Yet there must also always be some attempts to explore the possibilities offered ‘by seemingly esoteric species. For, to quote Spemann (1938), “the path of the possible through the vast field of the desirable is but narrow”, and one might miss crucial turns of this path by studying only those systems which guarantee safe returns for the labours invested. Acknowledgements

The author is deeply indebted to M. Berridge, K. Kalthoff, P. A. Lawrence and H. Vollmar who read drafts of this review and made many useful comments. Research by the author and his collaborators was supported by

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Garcia-Bellido, A. (1975). Genetic control of wing disc development in Drosophih. In “Cell Patterning”, Ciba Foundation Symp. 29, (new series), 161-182. Garcia-Bellido, A. and Merriam, J. R. (1969). Cell lineage of the imaginal discs in Drosophila gynandromorphs. J. exp. 2001. 170, 61-76. Garcia-Bellido, A., Ripoll, P. and Morata, G. (19 73). Developmental compartmentalisation of the wing disk of Drosophila. Nature New Biol. 245, 251-253. Garen, A. and Gehring, W. (1972). Repair of the lethal developmental defect in deep orange embryos of Drosophila by injection o f normal egg cytoplasm. Proc. natL Acad. Sci. U.S.A. 69, 2982-2985. Gauss, U. and Sander, K. (1967). Stadienabhangigkeit embryonaler Doppelbildungen von Chironomus th. thummi. Naturwissenschaften, 53, 182-183. Gehring, W. J. (1973). Genetic control of determination in the Drosophih embryo. Symp. SOC.DevlBioL 31, 103-128. Gehring, W. J. (1972). The stability of the determined state in cultures of imaginal disks in Drosophila. In “The Biology of Imaginal Disks” (Eds H. Ursprung and R. Nothiger), pp. 35-58. Springer-Verlag, Berlin, Heidelberg, New York. Geigy, R. (1932). Erzeugung rein imaginaler Defekte durch ultraviolette Eibestrahlung bei Drosophih melanogaster. Wilhelm Roux. Arch. EntwMech. Org. 125, 406-447. Grellet, P. (1971). Variations d u volume et teneur en ADN des noyaux de Scapsipedus marginatus Afz. et Br. (Orthoptkre, Gryllide) au cours de l’embryogenise. Wilhelm Roux Arch. EntwMech. Org. 167,243-265. Hadorn, E. (1966). Konstanz, Wechsel und Typus der Determination und Differenzierung in Zellen aus mannlichen Genitalanlagen von Drosophih melanoguster nach Dauerkultur in vivo. Devl Biol. 13, 424-509. Hadorn, E., Hiirlimann, R., Mindek, G., Schubiger, G. and Staub, M. (1968). Entwicklungsleistungen embryonaler Blasteme von Drosophih nach Kultur im Adultwirt. Revue suisse Zool. 75, 557-569. Hadorn, E. and Miiller, G. (1974). Has the yolk an influence on the differentiation of embryonic Drosophila blastemas? Wilhelm Roux Arch. EntwMech. Org. 174, 333-335. Haget, A. (1953). Analyse expkrimentale des facteurs de la morphogenkse embryonnaire chez le colkopt2re Leptinotarsa. Bull. biol. Fr. Belg. 87, 123-217. Hansen-Delkeskamp, E. (1968). Synthese ribosomaler RNS in normalen und embryolosen Keimen von Acheta domestica L . Wilhelm Roux Arch. EntwMech. Org. 161, 23-29. Heinig, S. (1967). Die Abanderung embryonaler Differenzierungsprozesse durch totale Rontgenbestrahlung im Ei von Gryllus domesticus. Zool. Jb. (Anat.) 84, 425-492. Herth, W. (1970). Analyse des ooplasmatischen Reaktionssystems der cyclorrhaphen Dipteren. Das segmental gegliederte Cuticulamuster aus vorderen und hinteren Eifragmenten von Protophormia spec. (Calliphoridae). Anhang: Vorversuche an Eiern von Drosophih. Diploma thesis, Freiburg i. Br. Herth, W. and Sander, K. (1973). Mode and timing of body pattern formation (Regionalization) in the early embryonic development of cyclorrhaphic dipterans (Protophormia, Drosophila). Wilhelm Roux Arch. EntwMech. Org. 172, 1-27. Hotta, Y. and Benzer, S. (1972). Mapping of behaviour in Drosophih mosaics. Nature, Lond. 240, 527-535.

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an endoparasitic hymenopteran as a result ,of natural or imitated oviposition. Wilhelm Roux Arch. EntwMech. Org. 175, 173-184. Winter, H. (1974). Ribonucleoprotein-Partikel aus dem telotroph-meroistischen Ovar von Dysdercus intermedius Dist. (Heteroptera, Pyrrhoc.) und ihr Verhalten im zellfreien Proteinsynthesesystem. Wilhelm Roux Arch. EntwMech. Org. 175, 103-1 27. Wolf, R. (1969). Kinematik und Feinstruktur plasmatischer Faktorenbereiche des Eies von Wachtliella persicarzae L. (Diptera). I. Das Verhalten ooplasmatischer Teilsysteme im normalen Ei. Wilhelm Roux Arch. EntwMech. Org. 162, 121-160. Wolpert, L. (1969). Positional information and the spatial pattern of cellular differentiation. /. theor. Biol. 25, 1-47. Wright, T. R. F. (1970). The genetics of embryogenesis in Drosophila. Adu. Genet. 15, 261-395. Yajima, H. (1960). Studies on embryonic determination of the harlequin-fly, Chironomus dorsalis. I. Effects of centrifugation and of its combination with constriction and puncturing. J. Embryol. exp. Morph. 8 , 198-215. Yajima, H. (1964). Studies on embryonic determination of the harlequin-fly, Chironomus dorsalis. 11. Effects of partial irradiation of the egg by ultra-violet light. J. Embryol. exp. Morph. 12, 89-100. Yajima, H. (1970). Study of the development of the internal organs of the double malformations of Chironomus dorsalis by fixed and sectioned materials. J. Embryol. exp. Morph. 24, 287-303. Yao, T. (1950). Cytochemical studies on the embryonic development of Drosophila melanogaster. 11. Alkaline and acid phosphatases. Q. Jl Microsc. Sci. 91, 79-88. Zimmermann, W. (1 954). Ueber genetisch und modifikatorisch bedingte Storungen der Segmentierung bei Drosophila melanogaster. Z. VererbLehre, 86, 327-372. Zissler, D. and Sander, K. (1973). The cytoplasmic architecture of the egg cell of Smittia spec. (Diptera, Chironomidae). I. Anterior and posterior pole regions. Wilhelrn Roux Arch. EntwMech. Org. 172,175-186.

Hormonal Control of Metabolism in Insects J. E. Steele Department of Zoology, University of Western Ontario, London, Ontario, Canada

1 Introduction 2 Thehormones 2.1 Moulting hormone (MH) . 2.2 Juvenile hormone UH) . 2.3 Brain hormone (BH) 2.4 Diapause hormone (DH) . 2.5 Bursicon 2.6 Hyperglycaemic hormone (HGH) . 2.7 Adipokinetic hormone (AKH) and hypolipaemic factors 2.8 Biogenic m i n e s . 3 Control of carbohydrate metabolism 3.1 Moultinghormone 3.2 Juvenile hormone . 3.3 Diapause hormone 3.4 Hyperglycaemic hormone 3.5 Medial neurosecretory cell (MNC) hormone 3.6 Octopamine 3.7 5-Hydroxytryptamine 4 Control of lipid metabolism 4.1 Juvenile hormone 4.2 Diapause hormone . 4.3 Hyperglycaemic hormone . 4.4 Adipokinetic hormone 5 Control of amino acid metabolism . 5.1 Moulting hormone . 5.2 Juvenile hormone . 5.3 Bursicon . 5.4 Control of nitrogen metabolism by the corpora cardiaca . 6 Effect of hormones on respiration 6.1 Endocrine control of respiration is isolated tissues 6.2 Endocrine control of mitochondria1 respiration 7 Conclusions Acknowledgements . References

.

1

. .

.

.

.

.

. . .

.

239

.

240 241 . 241 . 243 244 245 * 246 246 . 246 . 247 . 247 * 247 249 254 * 259 * 268 . 269 . 270 270 . 271 . 281 282 . 283 286 287 . 288 291 . 294 . 294 301 303 305 . 307 . 307

.

. .

-

i

-

.

. . . . . . .

240

J. E. STEELE

1 Introduction The postembryonic development of insects is characterized by a series of synchronized events controlled by the moulting and juvenile hormones. In the adult insect, tissue maintenance, homeostasis and reproduction are also under hormonal control. The needs of these processes are met by anabolic or catabolic metabolism. Since the requirement for amino acids, carbohydrate and lipid used in the synthesis of structural elements and the energy required for endothermic reactions varies during different stages of development and under different environmental conditions, a mechanism of metabolic control seems imperative. The choice of pathway by which an intermediate is metabolized, and its rate of flux through the pathway, is an important aspect of homeostasis. Therefore, the possibility that metabolism is controlled by hormones is an attractive one. Indeed, the idea of a “metabolic” hormone has long been popular with insect endocrinologists although definitive proof for the existence of such a hormone has not been forthcoming. In this paper I will take the view that the effects which many hormones have on metabolism are of a secondary nature, and thus the words “effects on” in the title might well substitute for “control of”. It is disconcerting t o find that reviews of insect endocrinology seldom devote more than a few pages t o hormonal control of metabolism; the same is true of biochemical reviews devoted to insects. The fact that important advances have been made during the past three decades in this aspect of vertebrate endocrinology leads one t o conclude that insects have not been regarded as suitahle material for the study of endocrine control of metabolism. The reason for the present state of affairs can be attributed to the fact that only two insect hormones are readily available in a pure state. The difficulties associated with the elucidation of the effects on metabolism of a single hormone used in the presence of other biologically active factors (as is the case with gland extracts) are practically insurmountable and have inhibited development in this field. Notable exceptions t o our general ignorance are those aspects of metabolism controlled b y moulting hormone and juvenile hormone. Because of unique effects on development and their potential use as insect control agents much effort has been expended in attempting t o understand their mode of action. It is clear that these hormones play an important role in the regulation of protein synthesis, both qualitatively and quantitatively, although their role in the metabolism of other classes of compounds is less obvious. Much has been written on the effects of moulting hormone and juvenile hormone on nucleic acid and protein synthesis and the subject has been reviewed frequently. It is unnecessary to repeat this material and these topics will not be covered here. The reader interested in the role of hormones in the

HORMONAL CONTROL OF METABOLISM IN INSECTS

24 1

control of nucleic acid and protein synthesis should consult the reviews of Wyatt (1972), Doane (1973), Gilbert (1973); Ilan and Ilan (1973); Price (1973); and Sekeris (1974). Endocrine control of carbohydrate and lipid metabolism in insects has never been reviewed in detail although aspects of the problem have been considered by Goldsworthy and Mordue (1974). Present knowledge showing that protein synthesis undergoes cyclical change during development suggests that energy metabolism associated with carbohydrate and lipid synthesis must be regulated to provide precisely the right amount of ATP at the appropriate time. The reproductive processes as well as a variety of homeostatic mechanisms in insects are also dependent o n a source of energy which is, to a large extent, obtained from carbohydrate and lipid. Important advances in further understanding these mechanisms demands that we become more informed about the complex relationship that exists between metabolism and physiological function. It is hoped that this review will act as a simulus for further studies. It is freely admitted that many of the statements made are of a highly speculative nature, but no apology is felt necessary if they lead to further study and discussion of these problems.

2 The hormones 2.1

MOULTING HORMONE (MH)

Moulting is one of the most distinctive features of insect development. The appeal of this phenomenon to the insect physiologist is attested t o by the fact that one of the earliest studies showed the origin of MH t o be the prothoracic gland (Fukuda, 1940). The hormone has since been isolated (Butenandt and Karlson, 1954) and its structure determined (Karlson et al., 1965; Huber and Hoppe, 1965). In the early purification procedures two fractions showing biological activity were obtained. The more abundant fraction was termed a-ecdysone and the other fi-ecdysone. Their structure is shown in Fig. 1. Both hormones are C2 sterols of remarkably high polarity due to the predominance of hydroxyl groups. P-Ecdysone (20-hydroxyecdysone: ecdysterone), isolated by Hocks and Weichert (1966) is more polar and generally more active than a-ecdysone. On instruction from the brain the prothoracic gland (PTG) initiates events leading to the synthesis and release of a-ecdysone (King et al., 1974; Chino et al., 1974) which is transported through the haemolymph to the target organs, probably bound t o a protein (Emmerich, 1970a, b). The view that the prothoracic gland is the only source of ecdysone has recently been questioned. King and Siddall (1969) have shown that ligated and isolated

242

J. E. STEELE

blowfly abdomens lacking ring glands are able t o convert a- to 0-ecdysone; larval fat body of M Q ~ ~ Usextu C U can accomplish the same conversion (King, 1972). The occurrence of a-ecdysone as well as the more active hydroxyhted derivative P-ecdysone suggests that a-ecdysone is a prohormone which is converted into the physiologically effective form in the target organ. In support of this idea King and Marks (1974) have shown that a-ecdysone occurs in subphysiological concentrations in cockroach haemolymph just prior t o the adult moult while P-ecdysone is present in excess.

HO

HO a-Ecdysone

QH OH :

0-Ecdy sone Fig. 1.

The structure of (Y- and 0-ecdysone.

The initial response to ecdysone is apolysis or separation of the epidermal cell layers from the cuticle. The induction of apolysis by P-ecdysone in epidermal fragments of diapausing pupae of the rice stem borer Chilo suppressalis in vitro shows that it is a direct response to the hormone (Agui et al., 1969). Apolysis is followed by cuticle deposition and wax secretion, events which are not solely under the control of ecdysone but are also under the control of neuroendocrine factors. In late intermoult development of fifth instar Culpodes, peak prothoracic gland activity coincides with a sharp increase in the rate of endocuticle deposition (Locke et al., 1965). Wax secretion is also under the control of the prothoracic gland.

HORMONAL CONTROL OF METABOLISM IN INSECTS

243

The mechanism whereby ecdysone stimulates protein synthesis is unknown. Two hypotheses have been proposed t o explain the mode of action of ecdysone: one suggests the genome to be the site of action, the other the nuclear membrane. Karlson and his colleagues are the chief protagonists of the genic action of ecdysone. They have proposed that in the absence of the hormone the genes are repressed b y a protein and unable to express themselves (Karlson, 1974). Ecdysone is thought t o bind with the repressor protein and by steric interaction cause derepression of the gene resulting in the synthesis of mRNA specific for certain proteins. The alternative argument (Kroeger, 19 63) postulates that ecdysone acts on the nuclear membrane t o alter its permeability t o Na+ and K+, the synthesis of mRNA at a particular time during development being dependent on the relative and absolute concentrations of Na+ and K + present in the nucleus. 2. 2 JUVENILE HORMONE (JH) Juvenile hormone, acting in conjunction with MH, determines the form of the insect at all times during its development. Decapitation experiments performed on Rhodnius, in which the corpora allata (CA) were either left intact or removed, showed that the quality of the moult is controlled by the CA (Wigglesworth, 1936). Metamorphosis ensues when moulting occurs in the absence of JH. This is shown most dramatically in Bombyx where allatectomy leads t o pupation at the succeeding moult and subsequent formation of diminutive adults (Bounhiol, 1938). Metamorphosis is more spectacular in holometabolous than in hemimetabolous insects. Both the timing of release and titre of JH is important in controlling the degree of differentiation occurring at each moult (Wigglesworth, 1952). S'ince new cuticle can be laid down only after exposure of the epidermal cells t o MH it follows that this hormone is necessary for JH to express itself in that tissue. There are other well-established functions of JH which apparently do not require the intervention of MEI. These include the control of accessory glands, ovarian function and vitellogenin synthesis in the adult female. Most workers believe that the active substance produced by the CA in the adult insect is identical t o that regulating morphogenesis during larval development (Bowers et al., 1965). This is an excellent example of an organism using the same chemical substance for different physiological functions. The fortuitous discovery by Williams (1956) that the abdomen of the adult male Cecropia moth contains large reserves of JH, which is readily extractable with ether, resulted in intense efforts to isolate and identify the hormone. The hormone, first isolated by Roller et al. (1967) from Cecropia oil, was identified as the acyclic sesquiterpene methyl-10-epoxy 7-ethyl-3, ll~dimethyl-2,6-tridecadienoate ( C , 8 , JH I). Meyer et al. (1968) using the

244

J.

E. STEELE

same hormone source showed that an active analogue of JH I was also present but in lower concentration. It has been designated JH I1 and has a methyl instead of an ethyl group at C-7. Prior to the isolation of the Cecropia JH Bowers et al. (1965) had shown that 10, 11-epoxy-farnesoic acid methyl ester possessed high JH activity. Recently it has been confirmed as a naturally occurring JH (JH 111) in Manduca sexta (Schooley et al., 1973), Schistocerca (Judy et al., 1973; Pratt and Tobe, 1974), Melolontha (Truatman et at., 1974) and PerlPlaneta (Miiller et al., 1974). The structures of the three naturally occurring juvenile hormones are given in Fig. 2.

CllJH or JH I

C1,JH or JH I1

C JH or JH 111 Fig. 2. The structure of juvenile hormones.

Like ecdysone the site and mode of action of JH is unknown. The suggestion by Wigglesworth (1957) that it may alter the permeability of membranes is supported by the work of Baumann (1968, 1969) showing that cecropia oil and active JH analogues depolarize the cell membrane of the wax moth salivary gland. These results are particularly interesting in view of the effects that Na' and K + have on the puff response of Chironornus salivary gland chromosomes (Lezzi and Gilbert, 1970).

2. 3

BRAIN HORMONE (BH)

The brain has a permissive effect on the last larval moult in Lymantria (Kopec, 1922). This was the first organ to which an endocrine function was attributed with certainty in the insects. In Rhodnius, moulting is initiated by a factor produced in that part of the brain containing the neurosecretory cells (Wigglesworth, 1940). In a classical series of experiments,

HORMONAL CONTROL OF METABOLISM IN INSECTS

245

Williams (1952) showed that the brain is responsible for activating the prothoracic gland and initiating moulting. The first change noted in the prothoracic gland of pupal silkmoths is an elevation in the rate of RNA synthesis (Oberlander et al., 1965). An increase in the absolute amount of RNA has been observed in prothoracic glands of Periplaneta after implantation of brains or injection of partially purified “activation hormone” (BH) (Gersch and Sturzebecher, 1970). Early attempts to isolate the brain hormone gave false leads in that they suggested it might have a steroid nucleus (Kirimura et al., 1962), but this was soon shown t o be incorrect. The active factor obtained from Bombyx brains, although resistant to pepsin and trypsin, is inactivated by Pronase and is nondialysable and water soluble indicating a possible protein nature (Ichikawa and Ishizaki, 1963). Recent studies have shown that biological activity is associated with three fractions corresponding to molecular weights of 9000, 1 2 000 and 31 000 when chromatographed on Sephadex G-100 (Ishizaki and Ichikawa, 1967). The different molecular forms are possibly the result of degradation of the parent protein molecule during extraction or binding of the unaltered active molecule t o proteins of different molecular weight. Yamazaki and Kobayashi (1969) also obtained an active fraction from Bornbyx brains but having a molecular weight in the range of 20 000. The presence of glucose in the active fractions led these authors to suggest that the hormone is a glycoprotein. Gersch and Sturzebecher (1968) have also obtained a protein fraction from Per@Zaneta brains (MW 20 000-40 000) having BH activity. Williams (1967) suggested that the active factor from the brains of Antheraea pernyi was a mucopolysaccharide because of its insensitivity t o pepsin, trypsin and chloroform-amyl alcohol mixtures and lack of absorbance at 280 nm. However, these properties are not all shared by the factor isolated by the other workers. It seems that the brain hormone is a protein, possibly conjugated with another molecule.

2. 4

DIAPAUSE HORMONE (DH)

Eggs of the silkworm Bornbyx mori undergo a period of facultative diapause if the so-called diapause hormone (DH) is present during the period of oogenesis. The silkworm is the only insect in which a diapuse hormone has been identified. DH is produced by the suboesophageal ganglion (SOG) of the mother but synthesis is dependent on the particular photoperiod and temperature conditions she was exposed t o as an egg (Hasegaea, 1957). The release of the hormone is controlled by the brain via nerves in the circumoesophageal connectives (Fukuda, 1962). The hormone has not been characterized.

246

2. 5

J. E. STEELE BURSICON

Tanning of the cuticle in newly moulted insects, as in the puparium of Diptera, is under hormonal control. Newly emerged blowflies have a soft white cuticle in which tanning is prevented if a ligature is placed around the neck. The hormone reponsible for this effect was first described by Cottrell (1962a, b) and Fraenkel and Hsiao (1962, 1963) and has been given the name “bursicon”. Bursicon is produced by the neurosecretory cells in the brain and is also present in the compound ganglion in the thorax (Fraenkel and Hsiao, 1965). Ligature experiments in cockroaches have shown that. the hormone is released from the terminal abdominal ganglion but is also present at a number of other sites in the nervous system (Mills, 1965). Bursicon has been partially purified and characterized as a protein. The blowfly hormone is not dialysable, is inactivated by TCA, acetone, alcohol, trypsin and Pronase, and precipitated by ammonium sulphate (Fraenkel and Hsaio, 1965). Both the Sarcophaga (Fraenkel et al., 1966) and Periplaneta (Mills and Nielsen, 1967) hormones have a molecular weight in the range of 40 000. The blowfly hormone is more resistant to heat denaturation than is that from cockroaches. 2. 6

HYPERCLYCAEMIC HORMONE (HGH)

The hyperglycaemic hormone elevates the level of trehalose in haemolymph. Although it is probable that its function is to maintain an adequate level of trehalose in the haemolymph the relationship of sugar levels t o specific physiological functions is unknown. The hormone was first described in the cockroach Periplaneta (Steele, 1961) where it is concentrated mainly in the corpora cardiaca (CC). It has been suggested that it may originate in the neurosecretory cells of the brain (Mordue and Goldsworthy, 1969). Electrical stimulation of the CC induces release of the hormone, suggesting release in vivo may be under nervous control. The hormone is stable t o brief boiling in 0.06 N HC1 but is destroyed by incubation with trypsin (Natalizi and Frontali, 1966) indicating a peptide structure. Its emergence slightly behind the void volume on a Sephadex G-25 column suggests that it is probably a small protein or large peptide (Natalizi and Frontali, 1966). 2.7

ADIPOKINETIC (AKH) AND HYPOLIPAEMIC FACTORS

The corporal cardiaca contain factors that raise the concentration of diglyceride in locust haemolymph (Mayer and Candy, 1969; Goldsworthy et al., 1972a) and decrease lipid levels in cockroach haemolymph (Downer

HORMONAL CONTROL OF METABOLISM IN INSECTS

247

and Steele, 1969, 1977). The interesting observation that cockroach CC extract has the effect expected of locust extract when assayed in locusts and locust extract acts in a manner similar to cockroach extracts in Peripluneta (Downer, 1972) raises the interesting possibility that the factors may be identical. Mayer and Candy (1969) have shown that the adipokinetic hormone is heat stable but destroyed by a number of proteolytic enzymes. Goldsworthy (unpublished observations cited in Goldsworthy and Mordue, 1974) reported that a purified preparation of the hormone exhibited little absorbance at 280 nm and concluded that it was a peptide structure with a low aromatic amino acid content. The hypolipaemic factor has not been characterized. 2. 8

BIOGENIC AMINES

Two amines known to occur in insect tissues and demonstrated to have an effect on metabolism will be discussed. 2.8.1 5-Hydroxytryptumine (5-Hi7 5-HT was first demonstrated in a number of insects by Welsh and Moorehead (1960), a finding later confirmed by Gersch et ul. (1961) and Colhoun (1963) for nervous tissue. Colhoun (1963) also observed that Periplaneta brain and to a lesser extent other nerve tissue was able to synthesize the amine. The function of 5-HT in insect nerve tissue is unknown. 2.8.2 Octopamine The recent demonstration that octopamine (DL;D-hydroxyphenylethanolamine) is present in cockroach nervous tissue (Robertson and Steele, 1973a) is of great physiological interest but of unknown significance. Brain, with approximately 4 pg g-' octopamine contained the highest concentration while the remainder of the nerve cord contained about one-quarter this amount. Similar concentrations of octopamine occur in the nervous system of Schistocercu greguriu (Robertson, 1975). The demonstration that nerve cords can synthesize this amine from tyramine in vitro (Robertson, 1970) suggests that it may be of functional significance in the tissue. 3 Control of carbohydrate metabolism

3. 1 MOULTING

HORMONE

The role of MH in the promotion of growth and development is well known. Since growth is synonymous with protein synthesis it is proper to

248

J. E. STEELE

enquire whether the hormone directing the synthesis of protein also controls the activity of other pathways associated with growth. Much of the ATP required for the synthesis of protein is derived from the oxidation of carbohydrate while 5-carbon sugars utilized in the synthesis of RNA are made available from the same source. Apart from the spectacular effects which it has on the outward appearance of the insect, MH also stimulates the synthesis of a number of proteins in the fat body (Neufeld et al., 1968; Arking and Shaaya, 1969; Sahota and Mansingh, 1970). The observation by Chen and Levenbook (1966) that the concentration of protein in Phormia regina haemolymph increases 24-fold between the second and fifth day of larval life, attaining a concentration of 20% (w/v) just prior t o pupation, suggests that the effect of MH at this site alone will have considerable impact on energy metabolism. However, there is, as yet, no evidence that MH acts directly on carbohydrate metabolism. Ligation of Bombyx mori pupae at the metathoracic level prevents utilization of abdominal glycogen (It0 and Horie, 1957). In normal pupae the glycogen level decreases from 1 2 mg g-' to 2 mg g-' during the 11 days of pupal development while that of ligated pupae remains constant at the higher concentration. Furthermore, the isolated abdomens do not undergo further development. Since implantation of prothoracic glands was known to initiate adult development (Fukuda, 1941) it was concluded that MH produced by the glands was responsible for the activation of glycogenolysis. It has been shown recently that injection of ecdysone into dauer (brainless) pupae of Bombyx results in a stimulation of trehalose synthesis and decrease in glycogen synthesis as measured by the distribution of 6-' C-glucose previously injected into the pupae (Kobayashi and Kimura, 1967). One hour after the injection of labelled glucose the ecdysone-treated pupae had incorporated 315 per cent more label into blood trehalose than had control insects, whereas the incorporation of glucose into glycogen in the hormone-treated pupae was only 18.8 per cent of that in untreated pupae. It is particularly important to bear in mind the following facts when interpreting these observations. The pupae were injected with ecdysone at least 18 hours prior to the injection of the labelled glucose; thus the various morphogenetic events had already been initiated at the time of glucose injection. In effect the activity of ecdysone was being measured 1 8 hours after treatment of the insect suggesting that the effect of the hormone is probably a chronic rather than an acute one. This idea is supported by the earlier demonstration that in vivo the presence of the prothoracic gland is associated with a decline in glycogen that extends over a period of days (It0 and Hone, 1957). Although the rate of synthesis of trehalose was increased by ecdysone the concentration of sugar in the haemolymph did not change (1.18 mg ml-' for ecdysone-treated as compared with 1.10 mg ml-' in

H O R M O N A L CONTROL OF METABOLISM IN I N S E C T S

249

control pupae). Furthermore, ecdysone did not have a significant effect on the glycogen content of the fat body (49 mg g-' in ecdysone-treated pupae as compared with 51 mg g-' in control pupae), further strengthening the idea that the effect of ecdysone on glycogen metabolism is a chronic one manifesting itself only over a period of days (Kobayashi and Kimura, 1967). A stimulatory effect of ecdysone on trehalose synthesis has also been demonstrated in diapausing brainless pupae of Samia Cynthia pryeri (Kobayashi et al., 1967; Kobayashi, 1967). A highly purified extract of BH was also tested and found to have activity similar t o ecdysone which is hardly surprising since the function of BH is to activate the prothoracic glands. On the basis of present evidence one can conclude that the effect of ecdysone on carbohydrate metabolism is secondary t o its effect on morphogenesis and protein synthesis. However, Kobayashi and Kimura (1967) have suggested that the increase in trehalose synthesis and inhibition of glycogen synthesis induced by ecdysone are direct effects on each of the biosynthetic pathways. If this were so one would expect an accumulation of trehalose in the hormone-treated pupae. This does not occur (Kobayashi and Kimura, 1967). An increase in the rate of trehalose synthesis is most readily explained by assuming a primary morphogenetic effect by the hormone, the energy requirements of which are met from the haemolymph pool of trehalose. Since an increase in trehalose synthesis may merely reflect a greater utilization of haemolymph trehalose by the peripheral tissues the findings of Jungreis and Wyatt (1972) that 0-ecdysone makes thecell membrane more permeable t o trehalose may be of considerable importance. The rate of penetration of trehaIose into isolated fat body taken from diapausing pupae of H. cercropiu rose from 3 per cent of that present in the medium 18 hours after treatment with ecdysone to 53 per cent 42 hours later. It seems probable that facilitation of trehalose entry into the cell coupled with enhanced utilization due t o morphogenesis would tend to deplete the haemolymph pool of trehalose and lead t o increased synthesis through feed-back control.

3.2

JUVENILE HORMONE

3.2.1 Glycogen synthesis Allatectomy has frequently been shown t o cause an excessive accumulation of glycogen in the fat body (See Table 1). With the exception of the mosquito, where removal of the CA has no effect on fat body glycogen (Van Handel and Lea, 1970), only one instance of JH acting t o promote the synthesis of glycogen has been recorded. Implantation of CA, together

N

cn

0

TABLE 1 Control of glycogen metabolism by juvenile hormone Species

Stage

Operation

Effect

Reference

Carausius morosus

Nymphs and adults

Allatectomy

Increase in whole body glycogen

L’Helias (1953)

Pytrhocoris apterus

Adult 9

Allatectomy

Whole body glycogen increases from 0.5 mg to 1.5 mg 15 days after operation

Janda and S l h a (1965)

Calliphora ery thro cephala

Adult 9

Allatectomy

Accumulations of much glycogen in the fat body I week after operation

Thomson (1952)

Phormia regina

Adult 9

Allatec tomy

Total fat body glycogen increases from 65 pg to 327 pg 6 days after operation

Orr (1964)

Musca domestica

Adult 9

AIIatec tomy

Total fat body glycogen increases from 68 pg t o 237 pg 4 days after operation

Liu (1974)

Aedes taeniorhyncus

Adult 9

Allatec tomy

No effect

Van Handel and

Drosophila melanogaster

Adult d

Implantation of CA + CC

30% increase in fat body glycogen 8 days after operation. Similar effects obtained with synthetic JH

Butterworth and Bodenstein (1969)

Lea (I 9 70)

c rn

Yni m r m

I 0

n I

TABLE 2 Control of protein synthesis by juvenile hormone

0

z Species

Sex and stage

Operation

Effect

Reference

B

I-

0

Leucophaea maderae

Nauphoeta cinetea

Peripkzneta americana

Periplaneta americana

Adult 0

Adult 0

Adult 0

Adult ?

Allatectomized. CA implanted into experimental insects

Incorporation of ''~-leucine into serum proteins increased 100% in presence of CA. Similar effect with synthetic JH.

Engelmann (19 7 1)

Decapitated. CA implanted into experimental insects

Four days after operation incorporation of l4 C-alanine into fat body protein is increased %fold by CA.

Luscher (1968)

Allatectomized 2-4 days prior t o experiment

Incorporation of labelled amino acids into haemolymph protein decreased 33%

Thomas and Nation

Allatectomy

Whole body protein decreased from 637 mg per 100 g to 560 mg per 100 g in 35 days. Controls increase 13% during same period. Rate of incorporation of l4 Clabelled protein hydrolysate into proteins of fat body, haemoIymph and ovary lower at all times up to 24 days after operation.

Thomas and Nation

Locusta migratoria

Adult ?

Allatectomy

L o custa m igrato rM

5th instar nymph

Allatectomy

Increase in total body protein depressed in operated insect.

0

f 5

0

I-

% 3 rn

(1966a)

k 3

z

(1966b)

5

m

n

d

Minks (1967)

Goltzene-Bentz et al. (1972)

N

2

TABLE 2-continued Species ~~~

Sex and stage

Operation

Effect

Reference

~

Schistocerca gregaria

Adult

Oncopeltus fasciatus Caliiphora erthrocephala

0

Allatectomy

Incorporation of l4 C-glycine into fat body protein decreased by 5% 8 days after operation.

Hill (1965)

5th instar nymph

Injection of JH (2.5 P d

Doubling of 3H-leucine incorporation at mid-instar.

Bassi and Feir (1971)

3rd instar larva

Ligature behind CA

Normal decline of body protein prior to pupation accelerated

Price (1968)

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253

with the CC, into Drosophila increased the total glycogen content of fat body by 30 per cent (Butterworth and Bodenstein, 1969). That this effect is not due t o the CC was shown by other experiments where injection of synthetic JH elicited an effect similar t o the glands. It is perhaps worth noting that female glands which may differ from male glands were used as implants in the male hosts. 3.2.2 Protein synthesis The reduction in glycogen utilization following allatectomy indicates a decrease in the activity of an energy-dependent process. It is interesting therefore, but hardly surprising, that allatectomy has a consistent inhibitory effect on the rate of protein synthesis while treatment of the insect with purified hormone has the opposite effect. A number of studies ihstrating this effect are presented in Table 2. Although parallel determinations of glycogen levels were not performed in these studies it seems likely that the degree to which protein synthesis is affected is sufficiently great t o be detected as a change in the concentration of glycogen; assuming, of course, that the change in glycogen is secondary t o the decrease in protein synthesis and not an independent effect. 3.2.3 Haemolymph trehalose levels The increase in total fat body glycogen following allatectomy of female houseflies (Table 1) is accompanied by a rise in haemolymph trehalose from 33 pg ml-' t o 486 pg ml-' four days after the operation (Liu, 1973). Undoubtedly this condition arises from reduced utilization elsewhere in the body since the trehalose synthetase mechanism is not inhibited by the operation (Liu, 1974). 3.2.4 Phosphorylase activity Some authors have attempted t o show that allatectomy has a direct effect on carbohydrate metabolism. Treatment of pupae of the stable fly, Stomoxys calcitrans, with the synthetic JH analogue (E)-4- [ (6,7-epoxy-3ethyl-7-methyl-2-nonenyl)oxy] -1,2-(methylenedioxy) benzene results in the formation of pupal-adult intermediates and prevents the decrease in glycogen which normally occurs during the first six days of pupal life (Wright and Rushing, 1973). Phosphorylase, the rate-limiting enzyme controlling glycogen degradation, is unaffected b y t h e JH analogue (Wright, et al., 1973). The cessation of adult differentiation coincident with the decrease in glycogen utilization emphasizes the fact that the effect of JH on carbohydrate metabolism is probably a secondary one. The absence of an effect by J H analogues on phosphorylase activity in stable fly pupae (Wright et al., 1973) contrasts with the observations of Liu

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(1974) showing that allatectomy caused. not only the total amount of fat body phosphorylase to decrease but also the proportion of enzyme present in the active form. These results are strikingly similar to those obtained by Goldsworthy (1970) in Locusta after removal of the CC and probably explain the results obtained by Liu who also removed the CC along with the CA. It is possible that the decrease in glycogen synthetase activity described in the same study may be due to removal of the CC rather than the CA. 3.3

DIAPAUSE HORMONE

The silkworm Bombyx mori overwinters as an egg in a state of diapause induced by the DH originating in the SOG. Diapause in these eggs is characterized by a high concentration of sorbitol and glycerol (Chino, 1958) as well as negligible phosphofructokinase (PFK) activity (Kageyama and Ohnishi, 1971). The sorbitol and glycerol are derived from glycogen laid down in the egg during oocyte development and are reconverted to glycogen in the developing embryo when diapause is completed. 3.3.1 Trehalose and glycogen levels

It was recognized as early as 1957 that ovaries producing diapause eggs had a higher glycogen content than those producing nondiapause eggs (38.6 mg g-I compared with 24.4 mg g-' ) (Chino, 1957; Yamashita and Hasegawa, 1964). The difference in glycogen content appears related to the titre of DH, since removal of the SOG from female pupae that would have produced diapause eggs led t o a reduction in ovary glycogen from 20.1 mg g-' to 14.5 mg g-' (Hasegawa and Yamashita, 1956). This decrease can be accounted for by the elevation of haemolymph trehalose (from 461 mg per 100 ml t o 627 mg per 100 ml) and an increase in fat body glycogen (from 5.1 t o 8.2 mg g-I). That fat body is not the site of action for the hormone is shown by the observation that SOG removal was without effect on haemolymph trehalose or fat body glycogen following ovariectomy. The direct action of DH on the ovary has been confirmed by implanting SOG and ovaries into a male host. In this situation the synthesis of glycogen by the ovarian implant only occurs in the presence of the SOG showing unequivocally that the ovary is the target for DH. 3.3.2 Stage-dependent glycogen accumulation It is the oocytes and not the nurse cells, follicle cells or other tissue of the ovary that respond t o the DH by synthesizing glycogen. Yamashita and Hasegawa (1970) have shown that the oocytes will respond to DH only at a

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specific time coinciding wit,h the degeneration of the nurse cells and the encirclement of the oocyte by the follicle cells. This is at a time when the follicle cells are engaged in active vitellogenesis, suggesting that they play an important role in the synthesis of glycogen. The synthesis of glycogen in the oocyte, as determined by the direct measurement of glycogen and the rate of incorporation of U-' C-glucose into glycogen, is most rapid when the oocyte has reached the physiological age represented by a weight of 500 pg. This is the physiological age at which the oocyte is most responsive t o DH. Below 250 pg and above 750 pg the uptake of labelled glucose is very low. On reaching 750-800 pg the synthesis of glycogen ceases and, although it cannot be determined with certainty, may coincide with the degeneration of the follicular cells. Since DH is effective only at a specific time in oocyte development and because ovaries contain eggs at all stages of development, the effect of SOG removal cannot be reflected in a time-dependent effect on total ovary glycogen or haemolymph trehalose. This has been confirmed by Yamashita and Hasegawa (1966). 3.3.3 Ovarian trehalase activity The decline in ovarian glycogen levels caused by removal of the SOG occurs concomitantly with a corresponding change in trehalase activity (Fig. 3) but it is important t o note that the decrease in enzyme activity anticipates the decline in glycogen by one day (Yamashita and Hasegawa, 1967; Yamashita et al., 1972). Similarly, the injection of DH extracts into female pupae having low levels of ovarian trehalase activity because of SOG removal increased the activity of the enzyme t o a detectable level in 3

Pupal age (days)

Fig. 3. Change in trehalase activity and glycogen content of pupal ovaries of B o m b y x mori from which the suboesophageal ganglion had been removed on the day of pupation. (After Yamashita et nl., 1972.)

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hours and maximally in 1 day. Because the hormone was ineffective in activating the enzyme in ovary homogenates and because of the timedependent response of the enzyme in vivo it seems likely that the enzyme is synthesized de novo in response t o the hormone. It is unfortunate that no measurements of trehalase activity have been made on ovaries of developing pupae destined to produce nondiapause eggs. Presumably these ovaries contain some trehalase activity since glycogen is being synthesized. Ideas on the role played by trehalase in the synthesis of glycogen are, at best, speculative. Since the appearance of the follicular cells is temporally associated with the synthesis of oocyte glycogen the trehalase may arise in these cells. If the enzyme were located in this position hydrolysis of trehalose could lead t o high local concentrations of glucose in or at the surface of the follicular cell and thus through the establishment of appropriate concentration gradients facilitate the entry of glucose into the oocyte. In the oocyte the rate and therefore the amount of glycogen synthesized would then depend on the concentration of glucose which in turn would be a function of the concentration gradient and trehalase activity . 3.3.4 Phosphofructokinase (PFK) and polyol formation The observation that PFK is absent in diapause eggs may well prove to be the significant feature in explaining polyol formation during diapause. At the time of oviposition both diapause and nondiapause eggs are without detectable PFK but three days later the enzyme makes its appearance in the nondiapause eggs (Kageyama and Ohnishi, 1971). It does not appear in diapause eggs until diapause has been completed. Apart from modifications in the electron transport system the absence of PFK appears t o be the only biochemical lesion associated with the synthesis of polyols during diapause. Kageyama and Ohnishi (1973) have analogized the accumulation of sorbitol and glyceroi during diapause t o that occurring during anaerobiosis in nondiapause eggs. The accumulation of polyols induced experimentally by anaerobiosis in early egg development is shown in Fig. 4. It must be kept in mind that the anaerobic imitation of the diapause condition can be induced only during the first three days following oviposition since PFK makes its appearance at this time (Kageyama and Ohnishi, 19 7 1). Under anaerobic or dhpause conditions the electron transport system is inoperative and the glycolytic pathway becomes the main source of ATP (Chino, 1963). The source of NAD making this possible is the reduction of pyruvate to lactate as indicated by the accumulation of the latter (Fig. 4). The oxidation of glyceraldehyde-3-phosphate to pyruvate (which includes the ATP yielding reactions) is aided by the conversion of dihydroxyacetone phosphate t o a-glycerophosphate since this provides an additional source of NAD for the

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oxidation of glyceraldehyde-3-phosphate. The a-glycerophosphate arising from the reduction of dihydroxyacetone phosphate is dephosphorylated by an acid phosphatase (Chino, 1961) and the free glycerol that results accumulates in the egg. The key t o understanding the accumulation of sorbitol in the diapause eggs appears on the one hand to be the presence of polyol dehydrogenases requiring NADPH (Chino, 1960) and on the other the absence of PFK. The poIyol dehydrogenases, in the presence of NADPH, will convert glucose and fructose-6-phosphate to sorbitol and sorbitol-6-phosphate respectively. However, these enzymes are also present in nondiapausing eggs during the first three days of development when PFK is absent (Kageyama and Ohnishi, 1973). In the absence of PFK the glucose-6-phosphate resulting from the phosphorolytic cleavage of glycogen will be directed through the hexose monophosphate shunt (pentose pathway) before being readmitted to the glycolytic pathway. This would lead t o the synthesis of a large pool of NADPH that is then available for the polyol dehydrogenases. The suggested flow of metabolites is outlined in Fig. 5. Chino (1961) has shown 30

\ \

\

0

H (controi) H ( A ) Days after oviposition

Fig. 4. Change in glycogen, polyols and lactate content of nondiapausing eggs incubated in air or nitrogen. 0, nondiapausing eggs incubated in air; nondiapausing eggs incubated in nitrogen. Nz time of transfer of eggs to nitrogen. Air (A), Air (B), Air (C), times of recovery from nitrogen. H (continued), H (A), H ( B ) , times of hatching. Polyol values are the summed value of sorbitol and glycerol. (After Kageyama and Ohnishi, 1973.)

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that sorbitol-6-phosphate is dephosphorylated by the same acid phosphatase that dephosphorylates a-glycerophosphate. It is interesting t o speculate on the reason for the absence of PFK in the diapause egg:One possibility is that DH acts as a repressor for PFK synthesis and only after diapause has been completed and the hormone metabolized can the genome for PFK express itself. Another aspect of diapause in the egg of Bombyx not so readily explained is the rapid conversion of glycogen to sorbitol and glycerol. The short period over which polyol concentrations increase suggests that the conversion is “triggered” in some fashion. The GIycogen p Glucose

, Sorbitol NADPH Sorbitol-6-P

f

NADH 1 I

2ADP 2ATP

!

I

I I

FAT

Fig. 5. Metabolic pathway thought to be operating in the nitrogen-incubated nondiapause eggs and similar to that in diapause eggs. (After Kageyama and Ohnishi, 1973.)

mechanism has all the earmarks of phosphorylase activation, possibly by the DH, although activation of the enzyme could also be accounted for in other ways, e.g. an increase in the level of 5’-AMP. Since phosphorylase in the diapausing pupa of H. cecropia has been shown t o be cold activated (Ziegler and Wyatt, 1975) it would be interesting t o know if a similar mechanism occurs in the diapause egg. It is difficult to generalize and care should be taken in extrapolating from diapausing Bombyx eggs to other developmental stages in the same physiological condition. Possibly the different species have evolved different metabolic patterns superimposed on a similar diapause condition in order to adapt to particular environmental conditions. Whether or not the

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absence of PFK is a constant feature of the diapause state remains to be seen. The observations' by Wyatt and Meyer (1959) that different species, even closely related species, do not show a similar pattern of polyol accumulation would suggest that it may not be so. 3.4

HYPERGLYCAEMIC HORMONE

More than a decade has passed since HGH was first described (Steele, 1961) yet its physiological role is as uncertain today as it was then. The principal blood sugar in most insects, and that which responds to HGH, is the nonreducing disaccharide trehalose (Wyatt and Kalf, 1957). The biosynthesis of this sugar is illustrated in Fig. 6. On the basis of experiments with Periplaneta in which CC extracts were shown to give a strong hyperglycaemic response Steele (1963) suggested that the function of the hormone was to maintain an adequate supply of haemolymph trehalose in the face of intense metabolic or physical activity. However, as Goldsworthy and Mordue (1974) point out it is difficult to visualize in the cockroach such periods of sustained vigorous exercise capable of bringing about the release of HGH. This view is reasonable since a maximum response to the hormone is attained only after 3-5 hours (Steele, 1963). Furthermore, the

I

Glycogen

PDA- ' - i ; G. . iI- P

Glucose

-

G-6-P

? UDP

ATp

Trehalose - 6 - P

J.

Trehalose Fig. 6. The biosynthesis of trehalose.

maximum effect of the hormone in vitro, as measured by the rate of trehalose released from the fat body, is reached only after a period of 75 minutes (Wiens and Gilbert, 1967a). Unfortunately the lack of information on conditions governing the release of the hormone from the CC and their relationship to the physiological state of the insect hampers any real understanding of its role. 3.4.1 Effect o n haemolymph trehalose concentration The cockroach Periplaneta americana is an ideal insect in which to study the effect of the hormone because of its low threshold (0.002 gland

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equivalents) t o the hormone and pronounced response (300 per cent increase with 1 gland equivalent) (Steele, 1961, 1963). The results obtained with Periplanetu have been confirmed by Ralph and McCarthy (1964) while similar observations in a variety of other insects have been made by other authors. CC extracts elevate the level of haemolymph trehalose in the cockroach Bluberus discoidulis by 100 per cent (Bowers and Friedman, 1963). Other insects in which a hyperglycaemic response has been reported are Phormiu reginu ( Friedman, 1967), Curausius (Dutrieu and Gourdoux, 1967), Culliphora ( N o r m a n and Duve, 1969), and Locustu (Goldsworthy, 1969). It is important to note that Periplunetu is sexually dimorphic with respect to the hyperglycaemic response (Ralph and McCarthy, 1964). Whereas haemolymph trehalose in males was raised 300 per cent b y 0.5 pair CC the comparable figure for females was only 100 per cent. This finding has been confirmed in our laboratory (Steele, unpublished observation). 3.4.2 Effect o n f a t body glycogen Different lines of evidence suggest that trehalose appearing in the haemolymph following treatment with CC extract is derived from fat body glycogen. Since a main site of trehalose synthesis is known t o be the fat body (Candy and Kilby, 1961) and fat body glycogen is also known t o serve as a precursor to haemolymph trehalose in the blowfly (Clegg and Evans, 1961) it seems likely that fat body is the source of the additional trehalose present in haernolymph after treatment of the insect with HGH. That fat body is the origin of the additional trehalose has been demonstrated in Bluberus (Bowers and Friedman, 1963), Periplunetu (Steele, 1963) and Locustu (Goldsworthy, 1969). The relationship between hormone-stimulated trehalose synthesis and depletion of glycogen in the fat body can be seen in Fig. 7. In passing it should be noted that the other major source of glycogen in the insect, the thoracic musculature, is unaffected b y HGH. This specificity for the target organ by the hormone is reminiscent of the action of the vertebrate hormone glucagon (Rall and Sutherland, 1958) which has a glycogenolytic effect in liver but not in muscle. In certain insects an excess of glycogen or depletion of reserves may prevent the HGH from expressing its hyperglycaemic effect. Phormiu must be starved for at least 24 hours before the CC extract is effective (Friedman, 1967). It is suggested that under optimal nutritional conditions the fat body is geared t o produce trehalose at a maximal rate determined by the level of glycogen in the tissue. Only when the concentration of glycogen falls below a certain level and is limiting can the hormone express itself, presumably by increasing the activity of the enzymes that degrade the glycogen. In Locustu Chalaye (1969) reported that CC extract was

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without any effect on blood trehalose, yet the same extracts were able t o increase haemolymph trehalose in Periplaneta by 72 per cent. Goldsworthy (1969) has claimed that negative results obtained with Locusta were due to a lack of glycogen in the fat body, a common condition in laboratory-fed locusts. Although starving the locusts prevented the CC extract from having its effect, attributing the lack of response t o an absence of glycogen cannot be the whole story. Goldsworthy (1969) has shown that a hyperglycaemic effect was obtained in males only during days 3-6 of adult Iife, yet fat body glycogen increased in a more o r less regular manner from 75 mg per fat body t o 447 mg per fat body during the first 10 days of adult life.

,