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Advances in

INSECT PHYSIOLOGY

VOLUME 4

This Page Intentionally Left Blank

Advances in

Insect Physiology Edited by

J. W. L. BEAMENT, J. E. TREHERNE and V. B. WIGGLESWORTH Department of Zoology, The University, Cambridge, England

VOLUME 4

1967

ACADEMIC PRESS London and New York

ACADEMIC PRESS INC. (LONDON) LTD BERKELEY SQUARE HOUSE BERKELEY SQUARE LONDON, W.1

US.Edition published by ACADEMIC PRESS INC.

11 1 FIFTH AVENUE 10003

NEW YORK, NEW YORK

Copyright 0 1967 By Academic Press Inc. (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: 63-14039

Printed in Great Britain by William Clowes and Sons, Limited, London and Beccles

Contributors to Volume 4 D. J. AIDLEY, Department of Zoology, University of Oxford, England (p. 1)

E. BURSELL,Department of Biological Sciences, University of Rhodesia and Nyasaland, Salisbury, Rhodesia @. 33) L. I. GILBERT, Department of Biological Sciences, Northwestern University, Evanston, Illinois, U.S.A. (p. 69) A. C . NEVILLE, Department of Zoology, University of Oxford, England (p. 213) G. R. WYATT, Department of Biology, Yale University, New Haven, Connecticut, U.S.A. (p. 287)


Contents CONTRIBUTORSTO VOLUME4

.

V

EXCITATION OF INSECT

SKELETAL. D. J. AIDLEY

I. Introduction

.

11. The Resting Potential

MUSCLES

.

A. The effect of potassium ions . B. The effect of chloride ions . C. The effect of sodium ions . D. The effect of divalent cations . E. The effects of carbon dioxide F. Temperature dependence . 111. Neuromuscular Transmission . A. The innervation of insect muscle . B. Excitatory responses . C. The quanta1 release of transmitter substance . D. Inhibitory responses . , IV. The Electrical Excitability of the Muscle Fibre Membrane A. The electrical properties of electrically excitable responses B. The ionic basis of electrically excitable responses . C. Spontaneous activity . . V. The Excitation-contraction Coupling Process . A. Depolarization . B. The importance of calcium ions . C. The action of carbon dioxide . D, “Fast” and “slow” muscles . References .

.

1 2 2 4 5 6 6 6 7 7 8 15 17 20 20 21 23 23 23 24 26 26 27

THEEXCRETION OF NITROGEN IN INSECTS E. BURSELL

I. Introduction . 11. The Formation of Nitrogenous End Products A. The uricolytic pathway . . B. The uricotelic pathway . C. The formation of urea . D. The formation of ammonia . E. Amino acids . F. Miscellaneous materials . III. The Excretion of Nitrogenous End Products A. Collembola . B. Orthoptera . C. Odonata .

vii

.

33 34 36

40

.

41 42 43 44 44 45 46

47

viii

CONTENTS

D. Dermaptera

E. Hemiptera . F. Coleoptera G. Neuroptera . H. Hymenoptera I. Diptera . J. Lepidoptera 1V. Conclusions . References .

.

48 48 50 51 51 52 54 56 61

.

. .

LIPIDMETABOLISM AND FUNCTION IN INSECTS LAWRENCE I. GILBERT

I. Introduction . A. General . B. Definition and classification . 11. Lipid Content . A. Expression of data . B. Alterations during metamorphosis . C. Nature of insect lipids . III. Lipid Utilization . A. Digestion and absorption . B. Lipid release and transport . C. Extra-digestive lipases . D. Fatty acid catabolism . IV. Lipid Biosynthesis . A. General mechanism of fatty acid synthesis B. Fatty acid biosynthesis in insects . C. Phospholipid and triglyceride . D. Fatty acids in nutrition . E. Substrate interconversion . V. Hydrocarbons and Waxes . A. Cuticle B. Extra-cuticular . VI. Isoprenoid Compounds . A. Nutritional studies . B. Isoprenoid biosynthesis . C. Isoprenoid content . D. Sterol modification . E. Function F. Insect hormones . W.Conclusions . References . Addenda .

70 70 71 71 71 81 89 97 97 102 110 116 127 127 130 134 145 147 152 152 155 157 157 161 168 170 175 176 186 187 208

.

.

.

CHITIN ORIENTATION IN CUTICLE AND

ITS

CONTROL

A. C. NEVILLE

I. Introduction

.

II. Orientation and the Mechanicai Properties. of &tic& molecules

.

213

Macro:

217

CONTENTS

ix

. A. Parallel orientation . B. Crossed fibrillar orientation . C. Lamellar structure . D. Functional aspects . 1V. Orientation Control . A. Circadian organization . B. Metabolic oscillators and “switches” C. “Dermal” light sense . D. Implantation experiments . E. Nervous control . F. Discussion . V. Orientation Mechanisms . A. Primary orientation . B. Secondary orientation . C. Protein orientation . D. Hypothesis . M.Conclusion . References .

220 220 220 223 229 233 233 246 254 257 260 260 262 263 265 270 272 279 280

III. Types of Chitin Architecture

THEBIOCHEMISTRY OF SUGARS AND POLYSACCHARIDES IN INSECTS G. R. WYATT

1. Introduction . II. The Occurrence of Sugars in Insects . A. Glucose and reducing substances . B. Trehalose . C. Sugar content of insect hsmolymph . D. Sugar content of whole insects and insect tissues . 111. Intestinal Absorption and the Physiology of Hemolymph Sugar Levels . A. Absorption from the gut . B. Regulation of blood sugar . IV. Biosynthesis and Utilization of Sugars . A. Glucose . B. Use of monosaccharides other than glucose . C. Biosynthesis of trehalose . D. Cleavage and use of trehalose . E. Physiological roles of trehalose and trehalase . F. Dormancy and the properties of trehalose . .* . V. Glycogen . A. Glycogen in insects . B. Accumulation and conversion during growth and metamorphosis C. Glycogen in insect flight D. Metabolism of glycogen VI. Hormonal Effects on Carbohydrate Metabolism .

.

1.

. .

287 289 289 289 29 1 295 297 297 299 301 301 302 304 309 317 324 325 325 327 329 329 336

CONTENTS

X

W.Glycoproteins and Chitin

.

A. Glycoproteins in insects B. Metabolism of chitin . Vm. Glycerol and Sorbitol . . References

AUTHORINDEX .

SUBJECT INDEX

.

.

.

. .

340 340 341 345 347

.

361

. .

. 375

The Excitation of Insect Skeletal Muscles D. J. AIDLEY Department of Zoology, University of Oxford, England I. Introduction

.

11. The Resting Potential

1

.

A.

The effect of potassium ions B. The effect of chloride ions . C. The effect of sodium ions . D. The effect of divalent cations . E. The effects of carbon dioxide . F. Temperature dependence . UI. Neuromuscular Transmission . A. The innervation of insect muscle . B. Excitatory respomes . C. The quanta1 release of transmitter substance . D. Inhibitory responses . IV. The Electrical Excitability of the Muscle Fibre Membrane . A. The electrical properties of electrically excitable responses B. The ionic basis of electrically excited responses . C. Spontaneous activity . . V. The Excitation-contraction Coupling Process A. Depolarization . B. The importance of calcium ions . C. The action of carbon dioxide . D. “Fast” and “slow ” muscles References .

. . .

. . . . . . .

. . . . . . .

.

2 2 4

s

6 6 6 7 7 8 1s 17 20 20 21 23 23 23

. 2 4 26 . 26 . 27

.

I. INTRODUCTION

In the resting condition, muscle cell membranes ‘are electrically polarized so that the inside of the cell is a few tens of millivolts negative to the outside. Stimulation of the motor nerves supplying the muscle results in a reduction of this membrane potential, which is followed by contraction of the muscle. This article will attempt to view our knowledge of this chain of events in insect muscles mainly in the light of the much greater understanding of vertebrate muscles. I

2

D . J . AIDLEY

11. THERESTINGPOTENTIAL A . THE EFFECT OF POTASSIUM IONS

According to the theory developed by Boyle and Conway (1941) to explain the ionic distribution between the cell and the extracellular fluid in frog sartorius muscle, the distribution of potassium and chloride ions follows a Donnan equilibrium set up by the presence of indiffusible anions inside the fibre, the system being maintained in osmotic equilibrium by the presence of an effectively indiffusible cation (sodium) in the extracellular fluid. The resting potential at equilibrium (E) is then given by the equations

where R is the gas constant, T the absolute temperature, F is Faraday’s constant, and the subscripts and ,refer to the intracellular and extracellular media, respectively. These equations also define the potassium equilibrium potential, EK,and the chloride equilibrium potential, Eel. Changes in [Kl0 or [CI], result in movements of KCl across the membranes so as to restore the equilibrium condition given by eqs. (1) and (2). Since movement of a given quantity of KCI results in much greater relative changes in [CI], than in [K],, changes in [Kl0 cause much larger changes in membrane potential at equilibrium than do changes in [Cl],, although large transient changes in membrane potential may be produced by changes in [Cl], (Hodgkin and Horowicz, 1959). One method of testing the applicability of eq. (1) is to measure [K], and [K], and see if the membrane potential calculated from the equation agrees with that actually found. This was done by Wood (1963) for leg muscle fibres of Locusta, Periplaneta and Carausius after equilibration for four to six hours in salines of ionic composition approximating to that of the haemolymph, with [K], between 10 and 18 m ~ In. each case the actual resting potential was close to that predicted from eq. (1). A more usual method of testing the applicability of eq. (1) is by measuring membrane potentials at different values of [K],. It is necessary either to measure [K], at each value of [Kl0 (Conway, 1957), or to ensure that there is no change in [K], during the course of the experiment by maintaining the [K],[Cl], product constant or by eliminating chloride from the system by using extracellular solutions

THE EXCITATION O F INSECT SKELETAL MUSCLES

3

containing sulphate as the major anion (Adrian, 1956; Hodgkin and Horowicz, 1959). If [K]*is constant, eq. (1) predicts that the membrane potential should be related to [K], by means of a straight line with a slope (at 18") of 58 mV per unit change in log,,[K],. Hoyle (1953) studied the effects of substitution of potassium chloride for sodium chloride in the external saline solution on the membrane potential of fibres in the metathoracic retractor unguis of Schistocercu (Fig. 1). At concentrations greater than 10 mM, the relation between

Potassium concentration (mu)

RQ.1. The relation between potassium ion concentration and membrane potential in the metathoracic retracto: unguis muscle of Schisrocerca. Chloride ion concentration constant. (From Hoyle, 1953.)

membrane potential and log,,[K], was linear, with a slope of about 50 mV. Similar results have been obtained by Hagiwara and Watanabe (1954) from locust flight muscle (slope 25 mV) and Wood (1957) on the prothoracic flexor tibialis of Curuusius (slope 36 mv). In each case the slope of the straight line section of the curve was less than the 58 mV predicted by eq. (1). However, the method used in these experiments involves a change in the [K],[CI], product so" that the system will initially be displaced from equilibrium (so that EE is not equal to Ec3

4

D . J . AIDLEY

and can only reach a new equilibrium position by a change in [K],; both these effects will tend to reduce the slope of the line. When the external potassium ion concentration is low, the membrane potential is less than that predicted from eq. (I), so that the graph of membrane potential against log,,[K], tends to level out in this region (Fig. 1). This phenomenon is common to a number of types of cell, and can be ascribed to a constant small permeability of the membrane to sodium ions and a decrease in permeability to potassiuni ions when [K], is low (Hodgkin, 1951). The membrane potential can then be described by the equation

(Hodgkin and Katz, 1949), where PK,PNaand PClare permeability coefficients. There seems to be little doubt that this equation could be successfully applied to the results obtained from the experiments so far mentioned. There are, however, observations on some insects which are very difficult to reconcile with the potassium electrode hypothesis for the resting potential. Belton (1958, 1960) reported that the membrane potentials of muscle fibres of various Lepidoptera were unaffected by changes in [K],. Belton and Grundfest (1962) obtained similar results from Tenebrio larvae; here the membrane potential was not affected by changes in [K],up to about 100 m, but at higher concentrations there was a logarithmic relation between membrane potential and [Kl0with a slope of 58 mV. They also measured the potassium concentration of the muscle fibres (72 m) and the haemolymph (40 m),and pointed out that the value of EKcalculated from these figures (- 17 mv) is much less than the actual membrane potential (- 50 to - 60 mV in muscles bathed in haemolymph). B . T H E EFFECT O F C H L O R I D E IONS

The relationship between external chloride ion concentration and the membrane potential has been investigated by Wood (1965), in the leg muscle fibres ofbcusta and Periplaneta. In both insects there was an increase of a few millivoltsas [CI], was raised from 0 to 150mM. This implies that the membrane is permeable to some extent to chloride ions. At first sight, the fact that the membrane potential was not related to [Cl], by a straight line with a slope of 58 mV per unit change in logl0[C1], might be explained by movement of KCl out of the cell when [Cl], was less than about 100 mM. However, Wood found that [Cl], was almost independent

THE EXCITATION OF INSECT SKELETAL MUSCLES

5

of [Cl], in this range. He suggested that the results implied that the muscle

cell membrane is much less permeable to chloride than to potassium ions, with the possibility that there is some active transport of chloride across the membrane. Thus the insect muscle cell membrane would seem to be, in these respects, similar to the squid giant axon (Keynes, 1963) rather than the frog muscle fibre membrane, where the chloride permeability is high (Hodgkin and Horowicz, 1959). The importance of such a relatively low chloride permeability might be that it allows the membrane potential to be changed by increase of PCl during inhibition (see Section 111, D, below). However, Wood’s results cannot be taken as implying that the chloride conductance of the membrane is negligible. If this were so, it would be difficult to explain the departure of the slope of the relation between membrane potential and log,,[K], from the 58 mV predicted by eq. (1). A critical investigation of this problem would include measurements of the transient changes in membrane potential caused by changes in [Kl0 and [Cl],, such as were made by Hodgkin and Horowicz (1959) on frog muscle fibres. Usherwood and Grundfest (1965) noticed that transient depolarizations of 7 to 15 mV occurred when a chloride-free solution was substituted for the normal Ringer solution perfusing Romalea muscle fibres ;the reverse procedure caused transient hyperpolarizations (Usherwood, personal communication). C . THE EFFECT OF S O D I U M IONS

Wood (1961, 1963) showed that a decrease in external sodium ion concentrationresulted in a fall in the resting potential of muscle fibres of Lmusta, Periplaneta and Carausius. The tonicity of the low-sodium salines was maintained by substitution of sucrose or choline chloride for sodium chloride. In crustacean muscle fibres, Fatt and Katz (1953) observed a similar decrease in the resting potential when sucrose was substituted for sodium, but only a very small decrease when sodium was replaced by choline. In the case of sucrose substitution it is evident that the associated decrease in chloride concentration (and, probably, consequent movements of KC1) would be expected to lower the membrane potential, although it is not clear whether these effects would be sufficient to account for the magnitude of the observed changes. Obviously this explanation cannot be applied to the fall in membrane potential which occurs in insect muscles when choline chloride is used as a substitute for sodium chloride. Hence it seems (Wood, 1961) that there is some direct influence of sodium ions on the permeability of the resting muscle fibre membrane.

6

D. J. AIDLEY

There is at the time of writing no evidence for the existence of an active sodium extrusion mechanism in insect muscle fibres, but it would be most surprising if such a mechanism did not exist. D . T H E EFFECT O F D I V A L E N T C A T I O N S

It is well known that, in vertebrate muscles, decrease in the calcium ion concentration of the Ringer's solution causes a fall in resting potential, and that this fall can be prevented by the presence of other divalent cations (Jenden and Reger, 1963). This phenomenon has apparently not been looked for in insect muscles, but Wood (1957) showed that an increase in external calcium ion concentration caused an increase in the resting potential of Carausius muscle fibres ; increase in magnesium ion concentration did not affect the resting potential. High concentrations of barium ions cause a fall in the resting potentials of Romalea muscle fibres (Werman, McCann and Grundfest, 1961). E . T H E EFFECTS O F C A R B O N D I O X I D E

In Schistocerca spiracular muscle, introduction of carbon dioxide into the tracheal system causes a slight depolarization of the muscle fibre membranes, associated with a decrease in the membrane resistance (Hoyle, 1960). The ionic basis of this depolarization is not known, but an increase in sodium permeability seems to be the obvious candidate. Similar slight depolarization by carbon dioxide occurs in the flight muscles of the beetles Pissodes and Tenebrio, but very large depolarizations are produced in the flight muscles of the wasp Vespula and the fly Sarcophaga (McCann and Boettiger, 1961). In the pupal spiracular muscles of the silkmoths Hyalophora and Telea, application of carbon dioxide causes a hyperpolarization of the fibre membrane (van der Kloot, 1963). F . TEMPERATURE DEPENDENCE

Kerkut and Ridge (1 96 1) compared the immediate effects of a change in temperature from 15 to 25" on the resting potentials of muscle fibres of Carcinus, Rana and Periplaneta. The Qlo values over this range were 1.064 & 0.004(s.E.)for the crab, 1.060 2 0.004 for the frog, and 1.296 f 0.046 for the cockroach. These values are all significantly different from the value of 1.035 expected if the system obeys eq. (I), and the value for the cockroach is significantly higher than those for the frog and the crab. Kerkut and Ridge concluded that the resting potential is directly dependent upon metabolic processes. Since the measurements of membrane

THE EXCITATION O F I N S E C T S K E L E T A L MUSCLES

7

potential were made after about 1 min. at the new temperature, it is improbable that this effect is dependent upon changes in ionic concentrations in the cell. If there is a sodium extrusion mechanism which is not electrically neutral (for example, if there is no 1 :1 exchange with potassium ions) the resting potential will be dependent to some extent on the activity of the pump, being more negative when the pump is active (as it presumably is at higher temperatures within the physiological range). However, this is not the only possible interpretation of these results. As we have seen, eq. (3) gives a better description of the resting potential than does eq. (l), particularly at external potassium ion concentrations less than about 10 mM; in the salines used by Kerkut and Ridge the potassium ion concentration was approximately 5 mM. Now the Qlo of membrane potential given by eq. (3) will only be 1.035 for the range 15 to 25” if the Qlos of the permeability coefficients PK,P,, and Pcl are identical. In particular, if the Qlo of PNais less than those of PK and Pel, increase in temperature will result in a hyperpolarization greater than that predicted by eq. (1). It should be possible to distinguish between these two alternatives by measuring the effect of temperature changes on the membrane potential of fibres poisoned with some suitable metabolic inhibitor. 111. NEUROMUSCULAR TRANSMISSION A. THE I N N E R V A T I O N O F I N S E C T MUSCLE

This subject has been extensively discussed in a recent review by Hoyle (1965), and will therefore be but briefly examined here. Insect muscle fibres possess multiterminal innervation, there being numerous motor nerve endings on each fibre which are spaced at intervals of 30 to 80 p apart along the length of the fibre. Polyneuronal innervation also occurs, so that many muscle fibres are innervated by more than one motor axon. In these cases stimulation of the different motor axons causes different responses in the muscle fibre. The motor axons can be divided into two broad categories: excitor axons, which produce depolarization of the muscle fibre membrane leading to contraction of the fibre, and inhibitor axons, which produce hyperpolarization and can counteract, to some extent, the action of the excitor axons. In many cases fibres are innervated by more than one excitor axon, and stimulation of these axons produces electrical and mechanical responses of different magnitude; axons producing larger responses are then known as “fast” axons, and those producing,smallerresponses are known as ‘“slow” axons (Pringle, 1939; Hoyle, 1955a, b). Some muscles (such as the extensor tibiae of

8

D . J . AIDLEY

locusts) are innervated by only one set of axons, while others (such as the flexor tibiae of locusts) are innervated by a number of axons of each type. The latter may thus be said to possess a number of motor units, although it is obvious that the definition of a motor unit in a system in which there may be overlapping fields of motor innervation, between axons producing different types of response, is largely a matter of opinion. These points may be illustrated by reference to two muscles of the locust Schistocerca gregaria, in which the pattern of motor innervation has been closely examined. The metathoracic extensor tibiae (the jump ing muscle) is innervated by three motor axons (Hoyle, 1955a, b), a “fast” excitor, a “slow” excitor and an inhibitor. About 80% of the muscle fibres are innervated by the “fast” exciter, 20 to 30% are innervated by the “slow” excitor, and about 10% (all of them also innervated by the ‘‘slow’’ excitor) are innervated by the inhibitor (Usherwood and Grundfest, 1965). Thus some fibres are innervated by all three axons, some are innervated by the “ slow” excitor and either the inhibitor or the “fast” excitor, and the majority are innervated by the “fast” excitor only. In the meso- and metathoracic dorsal longitudinal flight muscles of Schistocerca, there are five motor units each separately supplied by one axon, the response to stimulation being of the “fast” type (Neville, 1963). It is probable that there is no “slow” or inhibitor innervation of these muscles. B. EXCITATORY RESPONSES

1. The general nature of the excitatory responses Stimulation of “slow” type excitatory axons causes a depolarization of the muscle fibre membrane, the postsynaptic potential (also known as the junction potential or end plate potential), which rises fairly rapidly to a peak and then falls more slowly (Fig. 2). The size of the response varies between different preparations and between different fibres of the same preparation; Hoyle (1957) gives a range of 2 to 50 mV for the size of the “slow” postsynaptic potential in the mesothoracic extensor tibiae of Schistocerca. Stimulation of “fast” type excitatory axons causes a larger electrical response (Fig. 3), which frequently overshoots the zero level of membrane potential so that the inside of the fibre becomes briefly positive to the outside. In many cases the “fast” response consists of two components, a postsynaptic potential and an electrically excited response


9

(sometimes called the “active membrane response”) caused by the depolarization constituting the postsynaptic potential (see Section IV below). This electrically excited component may be absent in some preparations, such as the metathoracic spiracular muscle of Schistocerca (Hoyle, 1959). There does not seem to be any fundamental difference between “slow” and “fast” responses. “ Slow” responses may summateto give a depolarization large enough to elicit an electrically excited response (Cerf et al., 1959),and “fast” responses lose their regenerativecomponents and look

FIG.2. The electrical response to stimulation of the “fast” axon in the mesothoracic extensor tibiae of Schistocercu. Intracellular records from three different fibres. The upper trace shows zero potential initially, then twitch tension.

just like “slow” responses when the muscle is subjected to low temperatures (del Castillo et al., 1953) or neuromuscular blocking agents (Hill and Usherwood, 1961; Hoyle, 1955~). 2. The ionic basis of excitatory postsynaptic potentials In their classic study of the postsynaptic potential in frog sartorius muscle, Fatt and Katz (1951) suggested that the chemical transmitter substance (acetylcholine in this case) released from the motor nerve ending causes a brief increase in the ionic permeability of the postsynaptic membrane, so that the resting potential is effectively shortcircuited at the motor end plate during the action of the transmitter

10

D. J . A I D L E Y

substance. In accordance with this hypothesis, it was found that the size of the postsynaptic potential increased proportionately when the membrane potential was increased by passing current through a second microelectrode inserted in the (curarized) muscle fibre. A similar effect

. . c-w-!=Qc

I 100 rnsec

FIG.3. Theelectrical response to stimulation of the “slow” axon in the mesothoracic extensor tibiae of Schistocerca. Intracellular records from four different fibres (a-d). Upper trace shows zero potential and stimulus monitor. Note reflex responses in c and summation in the right-hand record from ‘0.

was found by del Castillo et al. (1953) for the “fast” responses of locust leg muscles, the postsynaptic potential component of the electrical response being just over half the value of the membrane potential over the range - 50 to - 150 mV. Further confirmation of this view was provided by Cerf et al. (1959), who showed that depolarizations large enough to make the inside of the fibre positive to the outside resulted in a


11

reversal of the sign of the “slow” postsynaptic potential. Similarly, depolarization of the muscle fibre membrane by increasing the external potassium ion concentration causes a decrease in the size of the postsynaptic potential (Hoyle, 1955~;Wood, 1957). In frog muscles, voltage clamp studies have shown that sodium and potassium are the ions mainly involved in the increase in permeability during the postsynaptic potential (Takeuchi and Takeuchi, 1960). There is as yet no information of this kind for the excitatory postsynaptic potentials of insect muscles.

3. The blocking effect of tryptamine and similar compounds Hill and Usherwood (1961) showed that perfusion of the metathoracic flexor and extensor tibiae muscles of Schistocerca with solutions containing 1 to 10 DIM tryptamine diminished both the electrical and

FIG.4. The effect of tryptamine on the “fast” response of the metathoracicflexor tibiae of Schistocerca. The upper trace shows tension and, initially, zero potential. Time signal 500 c/s. Trace 1 shows the normal response. Traces 2-4 show responses at 5 sec. intervals after the application of 10 mM tryptamine. Traces 6-10 show responses at 5 sec. intervals after removal of tryptamine. (From Hill and Usherwood, 1961.)

mechanical responses to “fast” axon stimulation (Fig. 4). Similar results were obtained with 5-hydroxytryptamine, lysergic acid diethylamide, bromolysergic acid diethylamide, 5,6-dimethoxytryptamine and 3-(pyrrolidino-methyl)-thionapthene. These substances did not affect the conduction of action potentials in the crural nerve trunk, and neither tryptamine nor 5-hydroxytryptamineaffected the muscle fibre membrane resistance or the electrically excited responses of the membrane to depolarization via an internal electrode. It was concluded that these substances act as neuromuscular blocking agents, possibly by competing with the transmitter substance for receptor sites 04 the postsynaptic membrane.

12

D. J . A I D L E Y

Hill and Usherwood also found that tryptamine increases the synaptic delay, and that this effect appears to be more long-lasting than the blocking effect on the size of the post-synaptic potential. In the mesothoracic extensor tibiae of Schistocerca tryptamine also blocks transmission from the “slow” axon (D. J. Aidley, unpublished). 4. The nature of the excitatory transmitter substance A number of studies have been made on the effects of acetylcholine on

insect muscles. Harlow (1958) found that acetylcholine had no effect on resting locust leg muscles, and Wood (1958) obtained similar results for the prothoracic flexor tibiae of Carausius. These authors also reported that d-tubocurarine and adrenaline did not affect the responses to nervous stimulation. On the other hand, it seems that neurally evoked contractions can be increased in size in the presence of acetylcholine. This effect has been described by Harlow (1958) and Hill (1963) in locusts, and by Kerkut et al., (1965a) in the cockroach. The nature of the mechanisms behind this increase in contraction size is obscure; there is an urgent need for microelectrode studies on the form of the electrical responses to stimulation under these conditions. Similar increase in the contraction size is brought about by L-glutamic acid and, to a lesser extent, D-glutamic and L-aspartic acids (Kerkut et al., 1965a). The presence of glutamate in perfusates of the femoral muscles of Periplaneta has recently been demonstrated by Kerkut et al. (1965b). They found that the amount of glutamate produced was proportional to the number of stimuli to the motor nerves, and that the muscles contracted when perfused with low concentrations of glutamate, the contraction increasing in size as the glutamate concentration was increased. Similar results were obtained for crab and snail muscles. Usherwood and Grundfest (1965) state that glutamate causes a depolarization of insect muscle fibres. It is evident from these results that a number of substances have excitatory effects on insect muscles, although it is impossible at present to say which of them, if any, is the naturally occurring excitatory transmitter substance. Glutamate, or a glutamate complex, appears to have the strongest claims in this respect. It is possible that the “slow” and “fast” responses to nervous stimulation are brought about by different transmitter substances, but it seems more likely that “fast” responses are brought about by release of more transmitter substance per impulse than are “slow” responses. Qualitatively, the effects of blocking agents appear to be the same on

T H E E X C I T A T I O N OF I N S E C T S K E L E T A L MUSCLES

13

both systems. This opinion would have to be revised if it were shown that the reversal potentials for the two types of postsynaptic potential were different. 5. Calcium-magnesium antagonism Increase of the magnesium ion concentration of the external solution causes a diminution in the size of the “fast” postsynaptic potential in Locusta and Periplaneta (Hoyle, 1955~).This effect is antagonized by a corresponding increase in calcium ion concentration. “ Slow ” postsynaptic potentials are similarly affected @. J. Aidley, unpublished). These results are similar to those obtained by del Castillo and Engbaek (1954) from frog sartorius muscles. They concluded that the depressant action of magnesium occurs presynaptically, part of the evidence for this being that the very small postsynaptic potentials produced in high magnesium ion concentrationsshow quanta1fluctuations in size, thought to correspond with “packets” of acetylcholine released from the motor nerve ending (del Castillo and Katz, 1954). Similar fluctuations in postsynaptic potential size were observed by Usherwood (1963a) in Schistocerca leg muscles in solutions containing high magnesium ion concentrations, and hence it would seem probable that the site of this calcium-magnesium antagonism is presynaptic in locust muscle. The blood of some herbivorous insects may contain relatively large concentrations of magnesium ions (Duchilteau et al., 1953); in Carausius, for example, investigated by Wood (1957), the average magnesium concentration was 53 mM, whereas that for calcium was only 7.5 m. This situation is of some interest, for concentrations of this order are sufficient to block completely neuromuscular transmission in locusts and frogs. Wood found that the size of the “fast” postsynaptic potential decreased as the calcium ion concentration was reduced below its value in the blood, as in locusts and frogs. However, the relation which he found between the size of the total electrical response to stimulation of the “fast” axon (postsynaptic potential plus electricallyexcited response) and the magnesium ion concentration is unique among those preparations which have been investigated. The electrical response increased from a value of about 21 mV in the absence of magnesium ions to a maximum of about 43 mV at a concentration of approximately 75 mM, thereafter falling with increasing magnesium ion concentration to reach zero at about 200 m. This fall in the electrical response at very high magnesium concentrations could not be antagonized by calcium ions, and may imply, as Wood suggested, that a high”magnesiumion concentration reduces the sensitivity of the postsynaptic membrane to the

14

D . J. AIDLEY

transmitter substance, as appears to be the case in the frog sartorius muscle (del Castillo and Engbaek, 1954). It is not clear whether the increase in the size of the postsynaptic potential with increasing magnesium ion concentration over the range 0 to 75 mM is a presynaptic or a postsynaptic effect. 6. The action of carbon dioxide Postsynaptic potentials of Schistocerca metathoracic spiracular muscle (Hoyle, 1960) and the flight muscles of certain beetles and the bug Nazzara (McCann and Boettiger, 1961)are reduced in the presence of high concentrations of carbon dioxide. It is not clear whether the carbon dioxide acts presynaptically or postsynaptically in these cases.

7 . Time-dependentproperties of the postsynaptic potentials When the interval between motor nerve impulses is short enough, postsynaptic potentials readily summate so as to give a depolarization greater than that following a single impulse. In some cases, repetitive stimulation of the “slow” axon results in a “plateau” of depolarization whose degree is proportional to the stimulation frequency (Hoyle, 1955b; Wood, 1958). When the postsynaptic potential is accompanied by an electrically excited response (as in typical “fast” potentials) the situation is complicated by the presence of refractoriness in the electrically excited component, but it is sometimes possible to discern summation of the postsynaptic potential components when the stimulus interval is short enough (Wood, 1958). Some degree of facilitation is frequently present when the postsynaptic potentials are very small, but this phenomenon does not seem to be as well developed in insects as it is in crustacea. By estimating the quanta1 content of postsynaptic potentials in crustacean muscle fibres, Dude1 and Kuffler (1961) were able to show that facilitation is a presynaptic phenomenon, being caused by an increase in the amount of transmitter substance released from the motor nerve ending. It is probable that a similar mechanism occurs in insect muscles. Becht et al. (1960)claimed that, in some of the coxal muscles of the cockroach, the postsynaptic potential component of the second of two responses to “fast” axon stimulation showed the effects of refractoriness in that it was smaller than usual for about 15 msec. after the first response. The interpretation of this result is complicated by the presence of the electrically excited component associated with the first response, but it may well be that a process of depression (the reverse of facilitation) occurred.


15

C. THE Q U A N T A L RELEASE OF TRANSMITTER SUBSTANCE

1. Spontaneous miniature postsynaptic potentials If a microelectrode is inserted into a frog sartorius muscle fibre in the region of the end plate, small “miniature postsynaptic potentials” can be seen (Fatt and Katz, 1952). These are similar in their pharmacology to neurally evoked postsynaptic potentials, and are probably caused by the spontaneous release of acetylcholine in “quantal” units derived from the vesicles which occur in the presynaptic nerve ending (Katz, 1962). Usherwood (1961, 1963a) has observed similar spontaneousminiature potentials in the muscle fibres of Schistocerca, Blaberus and Periplanetu (Fig. 5). The amplitude and time course of these miniature potentials were more variable than in the frog sartorius, an effect which is almost certainly due to the multiterminal innervation of insect muscles, so that potentials originate at different distances from the recording site, with consequent differences in the degree of attenuation of the potentials. In accordance with this view, Usherwood showed that the frequency distribution of the amplitudes of the potentials was much more skew in fibres which were long in relation to their length constant than in short ones. Analysis of the frequency distribution of the time intervals between successive potentials in a long series showed that the discharge is a random process. The average frequency of occurrence of the spontaneous miniature potentials could be increased by raising the external calcium ion concentration, and decreased by raising the external magnesium ion concentration. Concentrations of magnesium greater than 10 m~ also reduced the amplitude of the potentials; it is thought that this effect is by means of a reduction in the sensitivity of the postsynaptic membrane to the transmitter substance. Increase in external potassium ion concentration caused an increase in the average frequency of the potentials, probably by depolarizing the motor nerve endings. Application of salines made hypertonic with sucrose caused a transient increase in the frequency of the potentials. In all these respects the miniature potentials are like those of vertebrate muscles, except that, as Usherwood points out, the insect system seems to be rather less sensitive to magnesium ions. Since there seems to be much evidence for the statement that changes in frequency indicate presynaptic actions whereas changes in amplitude indicate postsynaptic actions (Katz, i962), an examination of the effects of calcium and magnesium ion concentration on the

16

D. J . AIDLEY

frequency of discharge of miniature potentials in Carausius might give some indication of the site of action of these ions in this case. Curare, acetylcholine and prostigmine had no effect on the miniature potentials, but 5hydroxytryptamine reduced their amplitude, resulting in complete block at high concentrations. Thus the pharmacology of

FIG. 5. Spontaneous miniature potentials from six different muscle fibres of Bluberus, recorded on a free-running trace. Calibrations 1 mV and 400 c/s. (From Usherwood, 1961.)

the miniature potentials appears to be similar to that of the neurally evoked postsynaptic potentials. After denervation of locust muscles, “ giant’’ miniature potentials up to 10 mV in height are observed at about the same time that impulse transmission from the nerve to the muscle fails (Usherwood, 1963b).

T H E EXCITATION OF I N S E C T S K E L E T A L M U S C L E S

17

It seems likely that these are connected with a disorganization of the motor nerve endings.

2. The quantal nature of the postsynaptic potential The presynaptic nerve endings at chemically transmitting synapses contain numbers of small vesicles. These are thought to contain “packets ” of transmitter substance which are released spontaneously in small numbers (producing miniature postsynaptic potentials) and in large numbers in the depolarization of the presynaptic nerve membrane. According to this interpretation, the postsynaptic potential is composed of a large number of quantal units, each resulting from the action of one of these packets on the postsynaptic membrane. The evidence for this is mainly derived from the observations of the sizes of the reduced postsynaptic potentials seen in frog muscles after treatment with Ringer solutions containing high magnesium ion concentrations. These potentials fluctuate in size by amounts equal to those of single miniature potentials. Similar “quantal ” fluctuations have been observed by Ushenvood (1963a,b) in postsynaptic potentials of locust muscle fibres after treatment with high magnesium ion concentrations or following denervation of the muscle. Presynaptic vesicles occur at insect neuromuscular junctions (Edwards et al., 1958). From these results, it seems that the intimate nature of the transmission process at insect neuromuscular junctions is similar to that at other chemically transmitting synapses. D . INHIBITORY RESPONSES

1. Peripheral inhibition in insects

Hoyle (1955b) showed that the locust metathoracic extensor tibiae muscle is innervated by three axons, one of which produces a small hyperpolarization of the muscle fibre membrane on stimulation. He was, however, unable to show that stimulation of this axon produced any inhibitory effect and suggested that its function was to enhance the amplitude of the “fast” electrical response so as to produce a larger contraction. As a consequence, the existence of peripheral inhibition in insects was until recently considered doubtful. Ripley and Ewer (1951) and Becht (1959) showed that the contraction of certain muscles could be reduced by increasing the intensity of the stimulus applied to the nerve trunk supplying the muscle, although in the absence of electrical records from the muscle, explanations other than inhibition are possible (see Hoyle, 1955b). More recently, Ikeda and

18

D. J . AIDLEY

Boettiger (1965a,b) have demonstrated the presence of hyperpolarizing responses in the dorsoventral flight muscle of the bumble bee Bombus and in the basalar muscles of the rhinoceros beetle Oryctes, where it was shown that these hyperpolarizing potentials could cause attenuation of the excitatory postsynaptic potentials. The most complete study of this phenomenon is that performed by Usherwood and Grundfest (1964,1965) on the metathoracic extensor tibiae muscles of Schistocerca and Romalea, in which they were able to show both electrical and mechanical attenuation of the responses to excitatory stimuli; the rest of this section deals with their results. 2. Inhibitory postsynaptic potentials Stimulation of the inhibitory axon results in hyperpolarizing potentials with an amplitude of 1 to 25 mV (Fig. 6). These potentials summate on repetitive stimulation. In fibres in which the potentials are small in size, the potentials may show facilitation; in other cases this is not so.

IOOmsec

FIG.6. Intracellular records of the interaction of inhibitory and "slow" excitatory postsynaptic potentials in Schistocercu. The excitatory response was elicited at varying times after the inhibitory response. (From Usherwood and Grundfest, 1965.)


19

By applying brief current pulses across the membrane via an intracellular electrode during the inhibitory postsynaptic potential, it was shown that the membrane conductance rises sharply at the beginning of the rising phase of the potential and then falls less rapidly. The duration of the inhibitory potentials is variable (40 to 300 msec.), but always longer than the excitatory potentials (15 to 150 msec.) in any one fibre. 3. Pharmacology of inhibition y-Aminobutyric acid (GABA) reduces the membrane resistance, causes hyperpolarization, and diminishes the excitatory postsynaptic potentials of fibres which are innervated by the inhibitor axon. Hence it apparently produces the same effects on the muscle fibre membrane as the inhibitory transmitter substance. Picrotoxin reverses these effects, and also diminishes the size of the inhibitory postsynaptic potentials. In these respects the pharmacology of inhibition in insects is identical to that of the crustacea (van der Kloot, 1960). 4. The ionic basis of inhibitory postsynaptic potentials

As mentioned earlier, the inhibitory potential is associated with an increase in membrane conductance. If the muscle membrane is hyperpolarized slightly, the inhibitory potential decreases in size, and further hyperpolarization results in the inhibitory potential being a depolarization. The level of membrane potential at which this changeover occurs (the reversal potential) is usually about - 70 mV (range - 55 to - 75 mV) and is thus near the value of the equilibrium potentials for potassium and chloride ions. In experiments in which all of the chloride in the saline solution was replaced by propionate, so altering the equilibrium potential for chloride ions, the inhibitory postsynaptic potentials reversed in sign. The reversal potential for the changes in membrane potential produced by GABA could be estimated by passing square current pulses through the membrane and finding at what potential the current-voltage curves obtained in the presence and absence of GABA crossed. This potential was -68 mV in potassium-free solutions and -67 mV in solutions with a potassium ion concentration of 30 mM, indicating that the reversal potential for the action of GABA was not the potassium equilibrium potential. It was concluded from these experiments that the action of the inhibitory transmitter substance, and of GABA (which may be the same thing), was to increase the permeability of the

20

D. J. AIDLEY

postsynaptic membrane to chloride ions. Here, again, the insect inhibitory system is similar to that of crustacea (Boistel and Fatt, 1958).

5. Interaction of inhibitory and excitatory responses Fig. 6 shows the effects of an inhibitory postsynaptic potential on a subsequent “slow” excitatory postsynaptic potential. Marked attenuation of the latter occurs if it arises during the initial phase of the inhibitory potential. Similar results have been obtained by Ikeda and Boetigger (1965b). This rather precise dependence of the interaction of the excitatory and inhibitory potentials on the timing of the two potentials may explain Hoyle’s failure to observe the phenomenon (Usherwood and Grundfest, 1964). The tetanic tension produced by stimulation of the slow axon in Romalea could be reduced by stimulation of the inhibitor axon, the attenuation increasing with increasing frequency of inhibitory stimulation. Complete inhibition of the mechanical response to stirhulation of the “slow” excitor axon at 12 impulses/sec. was obtained by stimulation of the inhibitor at 200/sec. In Schistocerca, such complete inhibition was never obtained, since not all of the muscle fibres supplied with the “slow” excitor axon are also innervated by the inhibitor.

Iv. THE ELECTRICAL EXCITABILITY OF THE MUSCLE FIBRE MEMBRANE A . T H E E L E C T R I C A L P R O P E R T I E S OF E L E C T R I C A L L Y E X C I T E D RESPONSES

When depolarizing current pulses are passed through the muscle fibre membrane by means of an intracellular electrode, the voltage recorded by a second electrode shows a further depolarization beyond that attributable to the resting resistance of the membrane jdel Castillo et a/., 1953; Cerf et a/., 1959). This electrically excited response differs from the propagated action potentials of nerve axons and vertebrate “twitch” muscle fibres in being a graded phenomenon (i.e. the size of the response is dependent upon the size of the stimulus). As a consequence, it is not propagated without decrement along the length of the fibre, but dies away within a few millimetres (Cerf et al., 1959). A similar phenomenon is seen in the responses to stimulation of the “fast” excitor axon in many preparations (del Castillo et al., 1953). In this case the stimulus for the production of the electrically excited response is the postsynaptic potential, and the point at which the

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21

electrically excited response arises from the postsynaptic potential is given by the point of inflection on the rising phase of the response (Fig. 2). Here, again, the electrically excited component is a graded response, being proportional in size to the depolarization produced by the postsynaptic potential, as is evident in Fig. 4, where the size of the postsynaptic potential component is reduced in size by means of tryptamine. These electrically excited responses differ from the postsynaptic potentials in that they show refractoriness, so that there is a brief period after a response during which no second response can be elicited (the absolute refractory period) followed by a longer period during which the second response is reduced in size (the relative refractory period). Refractoriness occurs whether the electrically excited response is elicited by direct depolarization or by nervous stimulation (Cerf et al., 1959). The time course of recovery from refractoriness will probably be dependent on the size of the initial response, but this question remains to be investigated. If depolarizing pulses of long duration are passed through the membrane, the initial electrically excitable response is followed by a damped oscillation of the membrane potential (Cerf et al., 1959). B. T H E I O N I C B A S I S O F E L E C T R I C A L L Y E X C I T A B L E RESPONSES

The electrically excitable responses of insect muscle fibres are similar in type to the subthreshold “local responses” (Hodgkin, 1938) of nerve fibres. The well established theory of the ionic basis of the propagated action potential in squid giant axons (Hodgkin and Huxley, 1952) provides a convincing explanation of the mechanism of these responses. In a normal nerve action potential, depolarization causes an increase in the sodium conductance of the membrane which itself produces further depolarization, so that the membrane potential moves rapidly towards the sodium equilibrium potential. The return to the normal resting potential is brought about by two delayed consequences of depolarization: an increase in potassium conductance and a decrease in sodium conductance (the sodium inactivation process). Local responses arise because the increase in sodium conductance brought about by the subthreshold stimulus is too small and proceeds too slowly to counteract the effects of increase in potassium conductance. It seems very reasonable to suggest that the mechanism of the electrically excitable responses of insect muscle fibres is of the same type as the local responses of nerve axons (Hoyle, 1962). It is probable that the Hodgkin-Huxley equations would predict such responses if adjusted by

22

D . J . AIDLEY

suitable changes in the rate constants of the sodium and potassium activation processes. More specifically, it has been suggested that the increase in potassium conductanceis more rapid in the graded responses of insect muscles (Werman et ul., 1961). The presence of a sodium inactivation process operative at the normal resting potential seems to be implied by some results of Cerf et ul. (1959), in which the electrically excitable response arose at a much more negative membrane potential after the fibre had been hyperpolarized for a short time. While the general principles of this argument seem to be fairly sound, it is not so clear that the ion involved in the specific, regenerative increase in permeability (i.e. the ion responsible for the rising phase of the electrically excitable response) is sodium, or sodium alone. Wood (1963) has investigated the effects of sodium ion concentration in the size of the electrical responses to “fast” axon stimulation in the leg muscles of Locustu, Periplunetu and Curuusius, at the same time measuring the internal sodium concentrations of muscles subjected to similar treatments, so that estimates of the sodium equilibrium potential could be made. In Locustu, the peak of the response occurred at an average membrane potential of - 19 mV in a solution containing no sodium ions, rising to + 5 mV in a solution with a sodium ion concentration of 150 m ~The . change was rather less for PeripZunetu (the corresponding figures were - 15 and + 2 mv), and much less for Caruusius (-3 and +4.5 mv). The values obtained at nominally zero sodium ion concentrations are interesting in that they are more positive than the sodium equilibrium potential, even if the actual sodium ion concentration were as much as 5 m~ (which is most unlikely). Interpretation of these results is complicated by the fact that the quantity measured was total electrical responses to nervous stimulation, which would include (and in zero sodium salines may entirely consist of) a large postsynaptic potential component. Since sodium is the major cation in the haemolymph of Locustu and PeripZunetu and since the membrane potential at the peak of the response is moderately sensitive to the sodium ion concentration, it is probable that sodium is the major ion involved in the rising phase of the electrically excitable response, although participation of other cations, such as calcium, is not excluded. In Curuusius, however, where the haemolymph sodium concentration is only 15 m~ (giving a sodium equilibrium potential of about +4.6 mV; Wood, 1963) and the potential at the peak of the response is only slightly affected by the sodium ion concentration, it seems rather unlikely that sodium is the only, or indeed the major ion involved. In view of the dependence of the amplitude of the electrical response to stimulation


23

on the magnesium ion concentration, and the very high magnesium ion concentration in the haemolymph, it seems probable that magnesium carries the action current in this case (Wood, 1958). The observations of Treherne (1965) on nervous conduction in the abdominal nerve cord of Carausius are of interest in this connection; he found that nervous conduction ceased in the absence of either sodium or magnesium ions. If the conclusion that magnesium ions carry a major part of the action current in Carausius is correct, then there is a very high probability that an active transport system concerned with maintaining the magnesium ion concentration gradient across the cell membrane exists. C . SPONTANEOUS ACTIVITY

If locust muscle fibres are perfused with calcium- and magnesium-free saline solutions, there follows a brief period during which the membrane is hyperexcitable, producing repetitive spike-like depolarizations (D. J. Aidley, unpublished). Similar effects have been seen in frog skeletal muscle (Bulbring et al., 1956). Van der Moot (1963) has reported some interesting observations on the spiracular muscles of the pupae of the silkmoths Hyalophora and Telea. These undergo spontaneous activity which consists of slow depolarizations of the membrane (pacemaker potentials), each followed by a spike-like component. The pacemaker potentials were accompanied by an increase in membrane conductance. If the membrane potential was set to various levels by passing current through an intracellular electrode, the pacemaker potentials always reached a plateau at the same value, - 29 mV. This suggests that the potentials are produced by an increase in the membrane conductance for some ion which has an equilibrium potential at - 29 mV, but what this ion is is far from clear. If the membrane is depolarized beyond - 29 mV, the potentials reverse in sign; this is rather extraordinary, since it implies that the cyclic nature of the phenomenon is based on changes in membrane permeability which are not determined by changes in membrane potential. V. THE EXCITATION-CONTRACTION COUPLING PROCESS A . DEPOLARIZATION

The evidence that depolarization of the cell membrane is necessary for the contraction of the skeletal muscles of vertebrates (Kuffler, 1946; Sten-Knudsen, 1960) and crustacea (Orkand, 1962) is considerable. The same situation appears to exist in insect skeletal muscles. 2-kA.I.P.

4

24

D. J. A I D L E Y

If the amplitude of the electrical response to nervous stimulation is reduced by means of neuromuscular blocking agents, the twitch tension is also reduced, as is shown in Fig. 4. Perfusion of a muscle with salines containing high potassium ion concentrations causes depolarization and contraction. In the metathoracic spiracular muscle of locusts (Hoyle, 1961), the threshold for the development of this contracture occurs at a potassium ion concentration of about 30 m~ (membrane potential - 34 mv) ; contracture tension increases with increasing potassium ion concentration to reach the maximum level at about 70 m~ (membrane potential - 18 mv). A similar result has been obtained from the mesothoracic extensor tibiae of Schistocerca (Aidley, 1965a). A more subtle influence of membrane potential on tension development is seen in frog “ twitch ” fibres, where the potassium contracture lasts only for a few seconds (Hodgkin and Horowicz, 1960). If the membrane is then repolarized by perfusion with a solution containing a lower potassium ion concentration, a restorative (“priming”) process occurs, so that the fibre is again able to contract on depolarization. The extent and rapidity of this “priming” process increases with increasing (more negative) membrane potentials. Potassium contractures of a similar short duration occur in the metathoracic extensor tibiae (Hoyle, 1961) and flexor tibiae (D. J. Aidley, unpublished) of Schistocerca, and it is not unlikely that a “priming” process similar to that described by Hodgkin and Horowicz could be demonstrated in these and some other insect muscles. B. THE I M P O R T A N C E O F C A L C I U M IONS

If the mesothoracic extensor tibiae of Schistocerca is perfused with a calcium-free solution containing the chelating agent ethylenediaminetetra-acetate (EDTA), it will no longer contract when depolarized by an isotonic solution of potassium chloride (Aidley, 1963, 1965a). In this condition, contraction can be elicited by addition of calcium chloride to the potassium chloride solution bathing the muscle (Fig. 7). The effect of solutions containing various concentrations of calcium ions (in the absence of EDTA) is slower, but it can be shown that the maximum contracture tension is related to the external calcium ion concentration, with a threshold at about 0.03 m~ and a saturation value in the region 2 to 4 mM. Thus it seems that insect muscles are similar to vertebrate muscles (Frank, 1960; Niedergerke, 1956; Edman and Schild, 1962) in requiring calcium ions for contraction. In vertebrate muscles, the presence of calcium ions in the sarcoplasm appears to be necessary for the interaction of actin, myosin and adeno-

-

T H E E XC I T A T I O N O F I N S E C T SKELETAL MUSCLES

25

sine triphosphate so as to produce contraction, and the calcium ion concentration is probably controlled by a “relaxing factor” system composed of vesicular elements derived from the endoplasmic reticulum (see, for example, Needham, 1960, and Weber et al., 1964). A relaxing factor whose action is antagonized by calcium ions has been isolated from locust skeletal muscles by Tsukamoto and his colleagues (quoted in Maruyama, 1965). The contraction of glycerinated fibres from the

A -

C

-

7 ]log

5 min

FIG.7. The effects of treatment with EDTA and subsequent addition of calcium chloride on the potassium contracture of the mesothoracic extensor tibiae of Schistocerca. The horizontal line under each record shows the duration of perfusion with KCl solution. Before each perfusion with KCl solution the muscle was treated as follows: (A) perfusion with a standard Ringer solution (4 mM CaCI2); (B) perfusion for 40 min. with a Ca-free Ringer containing 4 m~ EDTA; and, (C) perfusion for a further 60 min. with standard Ringer. The plateau of a tetanus produced by stimulation of the motor nerves is shown at the beginning of record (A). (From Aidley, 1965.)

fight muscles of the giant water bugs Lethoceros and Hydrocirius in the presence of adenosine triphosphate is dependent upon the presence of calcium ions (Jewell et al., 1964). This contractile action of calcium ions appears to be rather specific, in that strontium and a number of other divalent cations do not form adequate substitutes for calcium in the promotion of potassium contracture (Aidley, 1965b). Application of solutions containing strontium ions to calcium-depleted muscles depolarized by potassium chloride solution results in submaximal, phasic contractures;but successive$responses to depolarization decline in the continued absence of calcium

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D . J . AIDLEY

ions, suggesting that the small contractions mediated by strontium ions are brought about by a displacement of calcium ions from some intracellular store. Similar results have been obtained by Frank (1962) on frog toe muscles. C . THE ACTION OF CARBON DIOXIDE

Hoyle (1961) has shown that application of carbon dioxide to the metathoracic spiracular muscle of Schistocerca causes relaxation when the muscle is in potassium contracture, although the membrane potential of the depolarized fibres was unaffected. This result implies that carbon dioxide has some direct effect on the coupling process in this muscle in addition to its effects on the resting membrane and the size of the postsynaptic potential. Contracture tension of the spiracular muscle is maintained for many hours in isotonic solutions of potassium chloride or sulphate, but falls to zero in about half an hour in isotonic potassium bicarbonate (Hoyle, 196 1). The mechanisms of the relaxations occurring in the presence of bicarbonate or carbon dioxide are not known, although it is conceivable that bicarbonate acts by reducing the calcium ion concentration. Further evidence for a direct action of carbon dioxide on the coupling process of the locust spiracular muscle has been adduced by Hoyle from experiments in which he measured the effects of a brief “pulse” of carbon dioxide on the size of the postsynaptic potentials of individual fibres and the twitch tension of the whole muscle (Hoyle, 1960). It was found that a “hysteresis” effect occurred, in which the size of the postsynaptic potential fell and rose more rapidly than the twitch tension. These experiments cannot be considered conclusive, however, since the whole cycle of depression and recovery lasted only a few seconds, a time which is probably comparable with the diffusioii delay which must occur between the carbon dioxide affecting fibres in different parts of the muscle. D.

“

FAST

”

AND

“

SLOW

”

MUSCLES

Skeletal muscle fibres of frogs can be divided into two clearcut types, known as “twitch” (or “fast”) and “slow” fibres. Some muscles, such as the sartorius, are composed entirely of “fast” fibres, others, such as the iliofibularis and rectus abdominis, contain a high proportion of “slow” fibres (Kuffler and Vaughan Williams, 1953). “Slow” fibres differ from “fast” fibres in that they are multiterminally innervated, electrically inexcitable, contract at much slower velocities and do not


27

relax after a few seconds when immersed in solutions containing high potassium ion concentrations. These distinctions cannot be usefully applied to insect muscles, which in many cases can be classified as either “fast” or “slow” according to the criteria used. For example, if we compare the metathoracic and mesothoracic extensor tibiae muscles of locusts, we find that the fibres of both muscles are electrically excitable (as in “fast” muscles), that they are both multiterminally innervated (as in “slow” muscles), and that the potassium contracture is brief (as in “fast” muscles) in the metathoracic muscle whereas it is maintained (as in “slow” muscles) in the mesothoracic muscle (Hoyle, 1961; Aidley, 1963). Becht and Dresden (1956) suggested that the coxal muscles of the cockroach could be divided into two types (termed “fast” and “slow”) on the basis of their mechanical responses to stimulation. However, later investigations using more sophisticated techniques failed to uphold this view (Becht et al., 1960; Usherwood, 1962). It is clear that there are differences in the mechanical properties of various insect muscles, but a clearcut division into two types is not at present possible. I am very grateful to Dr. P. N. R. Usherwood and Dr. A. C. Neville for their comments on an early draft of this article. REFERENCES

Adrian, R.H. (1956). The effect of internal and external potassium concentration on the membrane potential of frog muscle. J. Physiol. 133, 631-658. Aidley, D. J. (1963). Influence of calcium ions on potassium contracture in an insect leg muscle. Nature, Lond. 198,591-592. Aidley, D.J. (1965a). The effect of calcium ions on potassium contracture in a locust leg muscle. J. Physiol. 177, 94-102. Aidley, D.J. (1965b). The effects of strontium and other divalent cations on potassium contracture in a locust leg muscle. J. Physiol. 177, 103-111. Becht, G. (1959). Studies on insect muscles. Bijdr. Dierk. 29, 5-40. Becht, G. and Dresden, D. (1956). Physiology of the locomotory muscles in the cockroach. Nature, Lond. 177,836-837. Becht, G., Hoyle, G. and Usherwood, P. N. R. (1960). Neuromuscular transmission in the coxal muscles of the cockroach. J. Insect Physiol. 4, 191-201. Belton, P. (1958). Membrane potentials recorded from moth muscle fibres. J. Physiol. 142,20P. Belton, P. (1960). Effect of ions on potential in lepidopteran muscle fibres. Biol. Bull. mar. biol. Lab., Woods Hole 119, 289. Belton, P.and Grundfest, H. (1962). Potassium activation and K spikes in muscle fibres of the mealworm larva (Tenebrio molitor). Am. J. Physiol. 203, 588594.

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Boistel, J. and Fatt, P. (1958). Membrane permeability change during transmitter action in crustacean muscle. J. Physiol. 144, 176-191. Boyle, P. J. and Conway, E. J. (1941). Potassium accumulation in muscle and associated changes. J. Physiol. 100, 1-63. Bulbring, E.,Holman, M. and Lullman, H. (1956). Effects of calcium deficiency on striated muscle of the frog. J. Physiol. 133, 101-1 17. Cerf, J. A., Grundfest, H., Hoyle, G. and McCann, F. V. (1959). The mechanism of dual responsiveness in muscle fibres of the grasshopper Romalea microptera. J . gen. Physiol. 43, 377-395. Conway, E. J. (1957). Nature and significance of concentration relations of potassium and sodium ions in skeletal muscle. Physiol. Rev. 37, 84-132. del Castillo, J. and Engbaek, L. (1954). The nature of the neuromuscular block produced by magnesium. J. Physiol. 124, 370-384. del Castillo, J. Hoyle, G. and Machne, X. (1953). Neuromuscular transmission in a locust. J. Physiol. 121, 539-547. del Castillo, J. and Katz, B. (1954). Quanta1 components of the end-plate potential. J. Physiol. 124, 560-573. Duchgteau, G., Florkin, M. and Leclercq, J. (1953). Concentrations des bases fixes et des types de compositions de la base totale de l’hemolymph des insectes. Arch. int. Physiol. 61, 518-549. Dudel, J. and Kuffler, S. W. (1961). Mechanism of facilitation at the crayfish neuromuscular junction. J. Physiol. 155,530-542. Edman, K. A. P. and Schild, H 0. (1962). The need for calcium in the contractile responses induced by ecetylcholine and potassium in the rat uterus. J. Physiol. 161,4244ll. Edwards, G. A., Ruska, H. and de Harven, E. (1958). Neuromuscular junctions in flight and tymbal muscles of the Cicada. J. biophys. biochem. Cytol. 4,251-255. Fatt, P. and Katz, B. (1951). An analysis of the end-plate potential recorded with an intracellular electrode. J. Physiol. 115,320-370. Fatt, P. and Katz, B. (1952). Spontaneous subthreshold activity at motor nerve endings. J. Physiol. 117, 109-128. Fatt, P. and Katz, B. (1953). The electrical properties of crustacean muscle fibres. J. Physiol. 120, 171-204. Frank, G. B. (1960). Effects of changes in extracellular calcium concentration on the potassium-induced contracture of frog’s skeletal muscle. J. Physiol. 151, 518-538. Frank, G. B. (1962). Utilization of bound calcium in the action of caffeine and certain multivalent cations on skeletal muscle. J. Physiol. 163,254-268. Hagiwara, S. and Watanabe, A. (1954). Action potential of insect muscle examined with intracellular electrode. Jap. J. Physiol. 4, 65-78. Harlow, P. A. (1958). The action of drugs on the nervous system of the locust (Locusta migratoria). Ann. appl. Biol. 46,55-73. Hill, R.B. (1963). The effects of acetylcholine on twitches in the locust leg. Comp. Biochem. Physiol. 10, 203-208. Hill, R. B. and Usherwood, P. N. R. (1961). The action of 5-hydroxytryptamine and related compounds on neuromuscular transmission in the locust, Schistocerca gregaria. J. Physiol. 153,393-401. Hodgkin, A. L. (1938). The subthreshold potentials in a crustacean nerve fibre. Proc. R. SOC.B 126,87-121.


29

Hodgkin, A. L. (1951). The ionic basis of electrical activity in nerve and muscle. Biol. Rev. 26, 339-409. Hodgkin, A. L. and Horowicz, P. (1959). The influence of potassium and chloride ions on the membrane potential of single muscle fibres. J. Physiol. 148, 127-160. Hodgkin, A. L. and Horowicz, P. (1960). Potassium contractures in single muscle fibres. J. Physiol. 153,386-403. Hodgkin, A. L. and Huxley, A. F. (1952). A quantitative description of conduction and excitation in nerve. J. Physiol. 117, 500-544. Hodgkin, A. L. and Katz, B. (1949). The effect of sodium ions on the electrical activity of the giant axon of the squid. J. Physiol. 108,37-77. Hoyle, G.(1953). Potassium ions and insect nerve muscle. J. exp. Biol. 30,121-135. Hoyle, G . (1955a). The anatomy and innervation of locust skeletal muscle. Proc. R. SOC.B 143,281-292. Hoyle, G. (1955b). Neuromuscular mechanisms of a locust skeletal muscle. Proc. R. SOC.B 143,343-367. Hoyle, G. (195%). The effects of some common cations on neuromuscular transmission in insects. J. Physiol. 127, 90-103. Hoyle, G. (1957). Nervous control of insect muscles. In “Recent Advances in Invertebrate PhysiQlogy ” (B. T. Scheer, ed.), pp. 73-98. University of Oregon Publications. Hoyle, G. (1 959). The neuromuscular mechanism of an insect spiracular muscle. J. Insect Physiol. 3, 378-394. Hoyle, G.(1960). The action of carbon dioxide gas on an insect spiracular muscle. J. Insect Physiol. 4, 63-79. Hoyle, G.(1961). Functional contracture in a spiracular muscle. J. Insect Physiol. 7, 305-314. Hoyle, G . (1962). Comparative physiology of conduction in nerve and muscle. Am. Zoologist 2, 5-25. Hoyle, G. (1965). Neural control of ske!etal muscle. In “The Physiology of Insects" (M. Rockstein, ed.) vol. 2, pp. 407-449. Academic Press, New York and London. Ikeda, K. and Boettiger, E. G. (1965a). Studies on the flight mechanism of insects. 11. The innervation and electrical activity of the fibrillar muscles of the bumble bee, Bombus. J. Insect Physiol. 11, 779-789. Ikeda, K. and Boettiger, E. G. (1965b). Studies on the flight mechanism of insects. 111. The innervation and electrical activity of the basalar fibrillar flight muscle of the beetle, Oryctes rhinoceros. J. Insect Physiol. 11, 791-802. Jenden, D. J. and Reger, J. F. (1963). The role of resting potential changes in the contractile failure of frog sartorius muscles during calcium deprivation. J. Physiol. 169,889-901. Jewell, B. R., Pringle, J. W. S. and Riiegg, J. C. (1964). Oscillatory contraction of insect fibrillar muscle after glycerol extraction. J. Physiol. 173,6-8P. Katz, B. (1962). The transmission of impulses from nerve to muscle, and the subcellular unit of synaptic action. Proc. R. SOC.B 155, 455-479. Kerkut, G. A. and Ridge, R. M. A. P. (1961). The effect of temperature changes on the resting potential of crab, insect and frog muscle. Comp. Biochern. Physiol. 3, 64-70.

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Kerkut, G. A., Leake, L. D., Shapira, A., Cowan, S. and Walker, R. J. (1965b). The presence of glutamate in nerve-muscle perfusates of Helix, Carcinus, and Periplaneta. Comp. Biochem. Physiol. 15,485-502. Kerkut, G. A., Shapira, A. and Walker, R. J. (1965a). The effect of acetylcholine, glutamic acid and GABA on the contractions of the perfused cockroach leg. Comp. Biochem. Physiol. 16, 37-48. Keynes, R. D. (1963). Chloride in the squid giant axon. J. Physiol. 127, 90-103. Kuffler, S. W. (1946). The relation of electrical potential changes to contracture in skeletal muscle. J. Neurophysiol. 9, 367-377. Kuffler, S. W. and Vaughan Williams, E. M. (1953). Properties of the “slow” skeletal muscle fibres of the frog. J. Physiol. 121, 318-340. Maruyama, K. (1956). The biochemistry of the contractile elements of insect muscle. In “The Physiology of Tnsecta” (M. Rockstein ed.) Vol 2, pp. 451-483. Academic Press, New York and London. McCann, F. V. and Boettiger, E. G. (1961). Studies on the flight mechanism of insects. I. The electrophysiology of fibrillar flight muscle. J. gen. Physiol. 45, 1 25-1 42. Needham, D. M. (1960). Biochemistry of muscular action. In “The structure and function of muscle” (G. H. Bourne, ed.) Vol. 2, pp. 55-104. Academic Press, London and New York. Neville, A. C. (1963). Motor unit distribution of the dorsal longitudinal flight muscles in locusts. J. exp. Biol. 40, 123-136. Niedergerke, R. (1956). The potassium chloride contracture of the heart and its modification by calcium. J. Physiol. 134, 584-599. Orkand, R. K. (1962). The relation between membrane potential and contraction in single crayfish muscle fibres. J. Physiol. 161, 143-159. Pringle, J. W. S. (1939). The motor mechanism of the insect leg. J. exp. Biol. 16,220-23 1. Ripley, S. H. and Ewer, D. W. (1951). Peripheral inhibition in the locust. Ncture, Lond. 167, 1066. Sten-Knudsen, 0. (1960). Is muscle contraction initiated by internal current flow? J. Physiol. 151, 363-384. Takeuchi, A. and Takeuchi, N. (1960). On the permeability of the end-plate membrane during the action of transmitter. J. Physiol. 154, 52-67. Treherne, J. E. (1965). Some preliminary observations on the effects of cations on conduction processes in the abdominal nerve cord of the stick insect, Carausius morosus. J. exp. Biol. 42, 1-6. Usherwood, P. N. R. (1961). Spontaneous miniature potentials from insect muscle fibres. Nature, Lond. 191, 814-815. Usherwood, P. N. R. (1962). The nature of “slow” and “fast “ contractions in the coxal muscles of the cockroach. J. Insect Physiol. 8, 31-52. Usherwood, P. N. R. (1963a). Spontaneous miniature potentials from insect muscle fibres. J. Physiol. 169, 149-160. Usherwood, P. N. R. (1963b). Response of insect muscle to denervation. 11. Changes in neuromuscular transmission. J. Insect Physiol. 9, 81 1-125. Usherwood, P. N. R. and Grundfest, H. (1964). Inhibitory postsynaptic potentials in grasshopper muscle. Science, N. Y. 143, 817-818 Usherwood, P. N. R. and Grundfest, H. (1965). Peripheral inhibition in skeletal muscle of insects. J. Neurophysiol. 28, 497-518.


31

van der Kloot, W. G. (1960). Picrotoxin and the inhibitory system of crayfish muscle. In “Inhibition in the nervous system and y-aminobutyric acid” (E. Roberts, ed.) pp. 409-412. Pergamon Press, New York and Oxford. van der Kloot, W. G. (1963). The electrophysiology and the nervous control of the spiracular muscle of pupae of giant silkmoths. Comp. Biochem. Physiol. 9,317-333.

Weber, A., Herz, R. and Reiss, I. (1964). The regulation of myofibrillar activity by calcium. Proc. R. SOC.B 160, 489-499. Werman, R., McCann, F. V. and Grundfest, H. (1961). Graded and all-or-none electrogenesis in arthropod muscle. I. The effects of alkali-earth cations on the neuromuscular system of Romalea microptera. J. gen. Physiol. 44, 979-995.

Wood, D. W. (1957). The effect of ions upon neuromuscular transmission in a herbivorous insect. J. Physiol. 138, 119-139. Wood, D. W. (1958). The electrical and mechanical responses of the prothoracic flexor tibialis muscle of the stick insect. J. exp. Biol. 35, 850-861. Wood, D. W. (1961). The effect of sodium ions on the resting and action potentials of locust and cockroach muscle fibres. Comp. Biochem. Physiol. 4, 42-46.

Wood, D. W. (1963). The sodium and potassium composition of some insect skeletal muscle fibres in relation to their membrane potentials. Comp. Biochem. Physiol. 9, 151-159. Wood, D. W. (1965). The relationship between chloride ions and resting potential in skeletal muscle fibres of the locust and the cockroach. Comp. Biochem. Physiol. 15, 303-312.

2+


The Excretion of Nitrogen in Insects E. BURSELL Department of Biological Sciences, University College of Rhodesia and Nyasaland, Salisbury, Rhodesia I. Introduction . 11. The Formation of Nitrogenous End Products A. The uricolytic pathway . B. The uricotelic pathway. . C. The formation of urea D. The formation of ammonia . E. Amino acids . F. Miscellaneous materials . LU. The Excretion of Nitrogenous End Products A. Collembola . B. Orthoptera . C. Odonata . D. Dermaptera . E. Hemiptera F. Coleoptera G. Neuroptera . H. Hymenoptera . I. Diptera . J. Lepidoptera . N. Conclusions . References .

.

. .

33 34 36

.

41 42 43

. 4 0

. .

. 4 4 .

. . . . . . . . . . . .

4

4 45 46 47 48 48 50 51 51 52 54 56 61

I. INTRODUCTION The occurrence of uric acid in the excreta of insects was established before the turn of the century and its presence has been confirmed subsequently by a large number of investigators (see reviews in Wigglesworth, 1950; Prosser, 1952; Roeder, 1953; Chauvin, 1956). Earlier findings were generalized in statements such as those of Needham (1935): “. . . in the insect, excretion of uric acid as the main end product of nitrogen metabolism is widespread, if not universal”, and of Florkin (1945): “I1 est bien Ctabli que chez les insectes adultes, l’acide urique reprksente le terme pridominant du catabolisme protidique”. On this basis the insect was assessed as being predominantly uricotelic in a general classification of excretory metabolism (Needham, 1950). Uricotelism was seen as an adaptation to a terrestrial mode of life; the 33

34

E. B U R S E L L

excretion of toxic end products, like ammonia, or of soluble end products, like urea, was seen as being militated against by the shortage of water. This view of the insects as a class still prevails (e.g. reviews by Craig, 1960; Stobbart and Shaw, 1964), subject to certain exceptions. In recent years, however, data have accumulated which suggest that nitrogenous end products other than uric acid may figure largely in the excreta of a variety of insects; the exceptions are becoming more numerous, and the validity of the earlier generalization appears to be threatened. In the following account, an attempt has been made to summarize the available information, and, in this way, to provide some basis for a reassessment of the situation. 11. THE FORMATION OF NITROGENOUS END P R O D U C T S The metabolic interrelationships of the main nitrogenous end products in insects, and the sources from which they derive, are summarized in Fig. 1. Two essentially opposing metabolic pathways appear to be involved.

(a) One has its origin in the nucleic acids, and may be termed the uricolytic pathway. It involves deamination and oxidation of the purine raw material (adenine and guanine) followed by the breakdown of the ring system to progressively simpler nitrogenous waste products. The last steps are thought to play little, if any, part in the excretory metabolism of insects, and they have been indicated by dotted lines. (b) The other has its origin in the proteins and their constituent amino acids, and may be termed the uricotelic pathway. It involves the synthesis of purines from amino acid nitrogen. In some insects the uricotelic pathway appears to be inoperative, and amino acid nitrogen is excreted as ammonia. In many others, urea appears as a minor constituent of the excreta, being apparently derived from protein nitrogen. As a third possibility, in certain insects specific amino acids are excreted as such; these alternative pathways are indicated in the diagram. Values for the nitrogen content and the solubility of nitrogenous end products have been included in Fig. 1. Ammonia is highly soluble, but a most efficient vehicle for the removal of waste nitrogen, with a nitrogen content of more than 80%; urea contains substantially less nitrogen and is of the same order of solubility as ammonia. The purines and their near derivatives contain between 30 and 40% nitrogen, but are

35

THE EXCRETION O F NITROGEN I N INSECTS

very much less soluble than ammonia and urea, while two of the amino acids listed have a nitrogen content not much lower than the purines, but are relatively soluble. Further reference to these differences and

I Nucleic Acid I

260

33

6

Allantoin

35

60

Allantoic Acid

32

slightly soluble

46

119,300

82

89,000

32 27 6

15,000 soluble

Uric Acid

’’ Arginine Histidine Cystine

Solubility mg/lOO ml

37

Xanthine

I Protein HAmino Acids I

% Nitrogen

Ammonia

.

FIG. 1. Nitrogenous end products and their metabolic interrelationships. (Data from “Handbook of Chemistry and Physics” ed. Hodgman, C. D., Chemical Rubber Publishing Co., Cleveland, Ohio.)

their implications in relation to nitrogenous excretion will be deferred until the excretory metabolism of different insects has been reviewed. The pyrimidines constitute another nitrogenous component of nucleic acid, but little information is available concerning their metabolism in insects. Recent studies (Bruno and Cochran, 1965) suggest

11

36

E. B U R S E L L

that it may conform in part to the vertebrate pattern, but further work will need to be done before their possible role as nitrogenous end products can be assessed. A . THE URICOLYTIC P A T H W A Y

The starting material for this reaction chain is nucleic acid, arising in part as a product of digestion, and in part from processes of tissue

Adenine

Hypoxanthine

1

(xanthine dehyhogenase)

Xanthine

Guanine

1

(xanthine dehydrogenase)

I

Uric Acid

maintenance and repair. The primary breakdown products are the nucleosides adenosine and guanosine, and these may be deaminated directly under the influence of the corresponding enzymes, adenosine deaminase and guanosine deaminase. In the presence of purine nucleosidases, the ribose sugar is then split off to liberate the purine bases.

T H E EXCRETION OF N I T R O G E N I N INSECTS

37

Enzymes responsible for such actions have been demonstrated in the cockroach (Cochran and Bruno, 1963; Cordero and Ludwig, 1963), and adenosine deaminase activity has been established in extracts of the larval fat body of blowflies (Desai and Kilby, 1958a) and in homogenates of Drosophila (Wagner and Mitchell, 1948). On the other hand, Duchlteau et al. (1940) were unable to show the presence of adenosine or guanosine deaminase in certain insects, and it is possible that in such species deamination occurs after hydrolysis of the nucleosides, as shown opposite. Adenase and guanase, the enzymes responsible for deamination of the free purine bases, have been shown to occur in a number of different insects (Duchfiteau et al., 1940; Desai and Kilby, 1958a; Prota, 1961). In extracts from the fat body of the silkworm the activity of guanase is much greater than that of adenase (Hayashi, 1961), and this appears to be so in other insects as well (Anderson and Patton, 1955). The deaminated purines, xanthine and hypoxanthine, are oxidized to uric acid under the influence of a single enzyme, xanthine oxidase, or xanthine dehydrogenase as it should more properly be called in view of the investigations of Irzykiewicz (1955); this author has shown that, unlike its mammalian counterpart, the insect enzyme cannot use molecular oxygen, but can reduce methylene blue. It is possible that nicotinamide-adenine dinucleotide (NAD) may act as the hydrogen acceptor for this reaction under normal conditions. Early workers failed to find evidence of the oxidation of xanthine in insects (Truszkowski and Chajkinowna, 1935), but xanthine dehydrogenase activity has subsequently been demonstrated in a variety of species, and the enzyme appears to be widely distributed throughout the class (Duchlteau et al., 1940; Florkin and Duchlteau, 1941; Anderson and Patton, 1954; Desai and Kilby, 1958b; Lisa and Ludwig, 1959; Ursprung and Hadorn, 1961; Hayashi, 1962; Cordero and Ludwig, 1963; Keller and Glassman, 1963; Parzen and Fox, 1964). In fact, there are indications that there may be more than one molecular form of the enzyme in Drosophila (Keller et al., 1963; Smith et al., 1963). The uric acid formed under the influence of xanthine dehydrogenase may be subjected to a series of degradative reactions in the presence of uricolytic enzymes as summarized overleaf. The first step is the oxidation of uric acid to allantoin under the iduence of the enzyme uricase. This enzyme has long been known to be active in insects (Leifert, 1935; Truskowski and Chajkinowna, 1935;Brown, 1938a; ROCCO, 1938),and more recent work has confirmed

38

E. BURSELL

its wide distribution within the class (Razet, 1952,1953, 1954,1956,1957, 1961;Lisa and Ludwig, 1959; Corder0 and Ludwig, 1963; Nelson, 1964), though it has been reported absent in some species (Florkin and DuchC teau, 1943; Razet, 1961). In Popillia japonica uricase is present in extracts of the embryo and of larval stages, but absent from the prepupa and pupa (Ross, 1959); a similar loss of uricolytic activity during pupal development has been reported for the blowfly Lucilia (Brown, 1938a).

I I

Allantoic acid O=C

(uricase)

(allantoinme)

YH2 COOH I \,cH. H

N\H2

c=o

N’

H

1

(allanroicme)

COOH

I

CHO

/NH2

-t-2O=C,

Glyoxylic acid

NH, Urea

I

I

(weme)

2 C 0 2 + 4NHs

T H E E X C R E T I O N OF N I T R O G E N I N I N S E C T S

39

The hydrolysis of allantoin to allantoic acid, which constitutesa further breakdown of the purine ring system, is catalysed by the enzyme allantoinase. Active extracts of this enzyme have also been obtained from a variety of insects (Rocco, 1936 and 1938; Manunta, 1948 and 1949; Razet, 1952-1957), but it appears to be somewhat less widespread than uricase (Razet, 1961). The next step in the sequence of uricolytic breakdown is the hydrolysis of allantoic acid under the influence of allantoicase, leading to the formation of urea and glyoxylic acid. This enzyme appears to have a very restricted distribution. It has been reported for larval stages of Bombyx (Manunta, 1948), but Razet (1961) failed to confirm its presence in this species. Feebly active preparations have been made from insects belonging to a number of different orders, but only from two of the many species tested by Razet (1961) have highly active extracts been obtained, namely from Xenylla wekhii (Collembola) and Achetu domesticu (Orthoptera). Since urea is absent from the excreta, or present in very small amounts, even in those insects which possess an active allantoicase, it seems safe to conclude that uricolytic breakdown beyond the stage of allantoicacid is of little significancein the excretorymetabolism of insects. The last in the chain of uricolytic enzymes is urease, which catalyses the hydrolysis of urea to ammonia and carbon dioxide. The presence of this enzyme has not been unequivocally established in insects. It has been reported to occur in the meat-eating larvae of certain Diptera (Tomita and Kumon, 1936; Baker, 1939; Robinson and Baker, 1939; Robinson and Wilson, 1939), and in a small number of other insects (Razet, 1961), but the methods used for its detection have not been above reproach, and the evidenceshould be accepted with caution. Lennox (1941) failed to find trace of its existence in a careful study of the enzymes responsible for ammonia production in larvae of the blowfly Luciliu cuprina. Even if the presence of urease were accepted for the species concerned, it is doubtful if it could be considered as a uricolytic enzyme, since earlier members of the chain are missing (Razet, 1961). A considerable amount of work has been done on the localization of enzymes involved in purine metabolism. The activity of xanthine dehydrogenase is high in the fat body of Peripluneta (Anderson and Patton, 1954 and 1955), and in larvae of Drosophila the fat body is more active than the Malpighian tubules (Ursprung and Hadorn, 1961). In Tenebrio the enzyme appears to be more concentrated in extracts from the mid gut and fat body than from other tissues (Irzykiewicz, 1955). Uricase has also been shown to occur in the fat body ofinsects (Leifert, 1935; Lisa and Ludwig, 1959; Pierre, 1964), but in the species studied by Razet

40

E. BURSELL

(1961) highest activities were usually found in extracts of the Malpighian tubules, with substantially lower concentrations occurring in mid gut and fat body extracts; and in Musca domestica, too, uricase activity is concentrated in the Malpighian tubules (Nelson, 1964). In insects which possess an active allantoinase, the enzyme distribution appears to be similar to that of uricase, with highest activity in the Malpighian tubules, followed by fat body and mid gut (Razet, 1961). B . T H E URICOTELIC P A T H W A Y

While the breakdown of uric acid in insects has been the subject of extensive investigations during the last decade, its synthesis has received relatively little attention. It has long been realized that the large amounts of uric acid which occur in the excreta of insects cannot derive solely from nucleic acid sources; the bulk of it must originate from protein nitrogen, but the synthetic pathways involved have not yet been fully elucidated. An early suggestion was made that arginine and histidine might be involved as intermediaries in uric acid synthesis (see Baldwin, 1948; Hoskins and Craig, 1935),but the existence of such a pathway has not been confirmed. The possibility of identifying precursors of uric acid in insects has been investigated both in vivo (Brighenti and Colla, 1940) and in vitro (Leifert, 1935;Anderson and Patton, 1955; Desai and Kilby, 1958b), and various non-purine substances including urea, malonate, ammonium salts, monoethyloxaloacetate and 4-amino-5-imidazole carboxamide riboside have been reported to stimulate the production of uric acid in certain cases. A more direct approach to the problem was made by McEnroe and Forgash (1957 and 1958) who investigated the fate of radioactive formate in Periplaneta umericana.Their results suggest that the pathway of uric acid synthesis may be the same as that in birds (Buchanan, 1951), where it involves a building up of the purine ring system on a basis provided by ribose sugar phosphate, by successive reaction with glutamine, glycine, formate, glutamate, carbon dioxide, aspartate and formate, as shown schematically opposite (from Baldwin, 1963). This view receives some support from the work of Heller and Jezewska (1959) who show that uric acid synthesis in Antherea may be stimulated by the simultaneous addition of precursors like formate, ribose-5phosphate, glutamate and aspartate, in the presence of added adenosine triphosphate (ATP). ATP is required at nearly every step of purine synthesis, so that the process is an expensive one in terms of metabolic energy. But upon completion of the ring system, ribose-5’-phosphate is split off to


41

liberate hypoxanthine, and this is subsequently oxidized first to xanthine and then to uric acid. In view of the demonstration that a dehydrogenase system is involved (Irzykiewicz, 1955), it may be that a substantial proportion of energy is recaptured at this stage. The recent work of Ito and Mukaiyama (1964) provides confirmation of the role of hypoxanthine and/or xanthine as intermediaries in the metabolism of protein nitrogen. These authors show that an increase in the protein intake of silkworms causes an increase in the activity of .*

(carbon dioxide).; * a .

(formare)

8

O= -pyrophosphate

xanthine oxidase. On the basis of these results, and of the earlier findings of Hayashi (1961) that adenase activity of silkworms extracts is low compared with the guanase activity, they suggest that a pathway involving guanine and xanthine may be of importance in protein catabolism, but this does not seem to follow. It should be emphasized that evidence for the existence of a reaction sequence in insects similar to the one which has been established for birds is far from complete. Only a few of the steps have been unequivocally confirmed, and some of the results obtained (e.g. the stimulating effect of malonate and urea) cannot easily be interpreted on this basis. The possibility cannot at present be excluded that different metabolic pathways may be operative in the synthesis of uric acid by insects. C . THE FORMATION O F UREA

Urea has been reported as a minor constituent of the haemolymph (see review by Buck, 1953), of the tissues (e.g. Leifert, 1935; Brown, 1938b) and of the excreta (e.g. Powning, 1953; Razet, 1961) of many insects. Since none of the species investigated in this respect possess an active allantoicase, it seems unlikely that the urea could represent a purine breakdown product, nor can it be considered to derive from the diet in all of the species concerned. The question of its metabolic derivation in insects must therefore be considered.

42

E. B U R S E L L

One possibility is that it might be produced from amino acid nitrogen through the mediation of the ornithine cycle (see Gilmour, 1961), which has been shown to be operative in other uricotelic groups like the birds and reptiles (Cohen and Brown, 1960). Arginine, citrulline and ornithine occur as intermediaries in this reaction sequence, and all of these amino acids have been detected in certain insects (Garcia et al., 1956a); and arginase, the enzyme responsible for the hydrolysis of arginine to form ornithine and urea, has been detected in several species (Kilby and Neville, 1957; Garcia et al., 1956b; 1957 and 1958; Szarkowska and Porembska, 1959). On the other hand, arginase appears to be absent from extracts of fat body of Calliphora (Desai and Kilby, 1958a), and in Celerio euphorbiae, the ornithine cycle has been shown to be incomplete, despite the presence of its three constituent amino acids (Porembska and Mochnacka, 1964). It is natural to associate the presence of arginase with the operation of an ornithine cycle, but this may well involve a misinterpretation, as has been pointed out by Kilby (1963); it is quite possible that the function of arginase in insects is not the production of urea, but the supply of ornithine “for some as yet unknown purpose”. Direct biochemical evidence in favour of the existence of the ornithine cycle is thus very inconclusive, and the results of relevant nutritional studies are hardly less equivocal. Hinton (1955) showed that Drosophila has a developmental requirement for arginine, and that arginine can be partially replaced by citrulline, but not at all by ornithine; similar results have been obtained by Davis (1962) with larvae of Oryzaephilus. The question of the synthesis of urea in insects must clearly be left open. D. THE FORMATION OF AMMONIA

Ammonia occurs as a minor excretory product in many insects, but it is only in aquatic forms (cf. Staddon, 1955) or in meat-eating fly larvae (Brown, 1938b) that it constitutes a substantial proportion of the nitrogenous waste. The manner of its formation has not yet been satisfactorily established. Where it occurs in small quantities it is possible that it may derive entirely from nucleic acid breakdown products, by deamination of purines under the influence of adenase, guanase or adenosine deaminase, all of which enzymes have been found in insects (see above). But in species which excrete large amounts of ammonia, the nitrogen must be assumed to derive from protein sources. Attempts to demonstrate deamination of amino acids by homogenates of dipteran larvae have not been successful, nor does a diet of

THE EXCRETION OF N I T R O G E N IN INSECTS

43

free amino acids lead to an increase in ammonia production by them. A protein diet, on the other hand, causes a substantial increase in ammonia output, and homogenates are capable of causing the liberation of ammonia from various peptone mixtures (Brown and Farber, 1936; Brown, 1938b). It would seem, therefore, that the ammonia produced by feeding larvae results from a deamination of proteins or higher breakdown products, but neither the mechanism nor the precise substrate have been identified. In other insects it is likely that ammonia arises in part by direct deamination of amino acids (Bheemeswar, 1959), since amino acid oxidases have been demonstrated in the fat body of a number of insects (Kilby and Neville, 1957; Desai and Kilby, 1958a; Auclair, 1959). Alternatively, amino acids may undergo transamination with a-ketoglutaric acid as the amino acceptor, and the glutamic acid formed may be deaminated under the influence of glutamic dehydrogenase : L-amino acid a-keto acid

x

NH3

a-ketoglutaric acid

.

glutamic acid

Y (tmnsaminase)

:

NAD .H NAD

Y (gluramic dehydrogenase)

Glutamic dehydrogenase activity has been demonstrated in the cockroach Periplunetu (McAllan and Chefurka, 1961b) and in extracts from the fat body of locusts (Kilby and Neville, 1957), and the presence of transaminases has been detected in a variety of insects (Kilby and Neville, 1957; McAllan and Chefurka, 1961a, b; Zandee et al., 1958; Murphy and Micks, 1964; Chen and Bachmann-Diem, 1964; Emmerich et ul., 1965). Clearly there are a number of ways in which ammonia may be formed from protein nitrogen in insects ; but what contribution the different reactions make to the total ammonia output of a given insect cannot be decided on the basis of available evidence. E. AMINO ACIDS

Small quantities of a variety of amino acids have been found in the excreta of different insects (e.g. Harrington, 1961; Mitlin et ul., 1964), but it is possible that their presence should in many cases be interpreted as a loss of amino acid rather than as an excretion of amino nitrogen. In plant-sucking Hemiptera (e.g. Mittler, 1958) the quantities involved

44

E. B U R S E L L

are substantial in relation to the total nitrogen output, but in these insects the amino acids should perhaps be regarded as in the nature of a faecal material. In a few insects, however, large quantities of particular amino acids are excreted (Powning, 1953; Bursell, 1964a), and here there can be little doubt that a disposal of metabolic waste is involved. F. MISCELLANEOUS MATERIALS

A number of other nitrogen-containing substances have been identified as components of insect excreta; for instance, creatine has been identified in the excreta of Luciliu (Brown, 1938a) and of Rhodnius (Wigglesworth, 1931), and haematin in that of the tsetse fly (Bursell, 1964b); but in view of the diet of these insects, it seems likely that the substances represent products of digestion rather than of nitrogenous metabolism. Pteridines have been identified in the excreta of the milkweed bug, Oncopeltus fusciutus (Bartel et ul., 1958) and has been shown to make up over 5% of excretory nitrogen (Hudson et ul., 1959); but little is known of the metabolism of these substances in insects, and they will not be further discussed.

111. THEEXCRETION OF NITROGENOUS END P R O D U C T S The data presented in Section I1 have shown that enzymes capable of catalyzing the formation of a variety of end products are widely distributed among insects. But the occurrence of an enzyme which catalyses a given reaction does not necessarily mean that the product of that reaction constitutes an important component of nitrogenous excretion. Many of the substances considered may have specific parts to play in the general metabolism of the insect, and this could account for the occurrence of the corresponding enzymes. It is only by an analysis of excretory material that the contribution of different metabolic pathways to the disposal of nitrogenous waste materials can be accurately evaluated. A great deal of information is available on this point, but unfortunately it is seldom complete. The aims of investigators in this field have differed greatly. Some have been concerned simply with demonstrating the presence or absence of a specific substance in excretory material; some have focused their attention on certain classes of substance, purine degradation products for example, to the neglect of others, and so on. It is only in very few cases that the excretory material of a given insect has been accurately partitioned among all the possible nitrogenous waste products. But even with the somewhat fragmentary information which is available, it is possible to get a

THE EXCRETION OF NITROGEN I N INSECTS

45

reasonable indication of the tremendous diversity of excretory patterns which exists within the class, and the picture that emerges is not at first sight easy to reconcile with the traditional view of insects as a uricotelic group of animals. In view of the large number of species which have been investigated during recent years, it will be convenient to give a separate account of each of the orders whose members have been studied, and the classification adopted by Imms (1958) will be used for this purpose. The proportion of nitrogenous waste in the faeces of insects varies enormously, depending to a large extent on feeding habits. In herbivorous species, for instance, the nitrogen load may be light and undigested plant material often makes up the bulk of faecal matter, so that uric acid may comprise no more than 0.1% of the dry weight (e.g. Razet, 1961); in blood-sucking insects, on the other hand, the nitrogen load is heavy, there is little indigestible material, and uric acid may account for as much as 60% of the dry weight of faeces (e.g. Bursell, 1964a). In order to facilitate comparison of values which may differ by several orders of magnitude, the quantity of nitrogen excreted in different nitrogenous end products will be expressed as a proportion of nitrogen in the predominant end product. For instance, an insect which excretes 100 mg of uric acid, 10 mg of urea and 2 mg of ammonia, which would be 33.3 mg of uric acid nitrogen, 4.6 mg of urea nitrogen and 1.6 mg of ammonia nitrogen, will be listed as: Uric acid Urea Ammonia 1-00 0.14 0.05 showing that for every 100 mg of uric acid nitrogen excreted, 14 mg of urea nitrogen and 5 mg of ammonia nitrogen are disposed of. It must be emphasized that values of this kind indicate only the proportionate composition among the substances assayed. In some cases it is possible that unidentified substances, or substances not quantitatively estimated, may exceed the listed products in importance. Furthermore, values quoted for different species in the tables which follow should be regarded as no more than generally indicative of the proportionate composition in different species. Variability within species is known to be great, and in many cases determinations have been based on single samples (Razet, 1961). A . COLLEMBOLA

In view of the occurrence in a member of this order of three of the uricolytic enzymes, uricase, allantoinase and allantoicase (Razet, 1961),

46

E. BURSELL

it is a pity that no data are available on the composition of excreta, so that the extent to which degradation of uric acid is a feature of nitrogenous excretion must remain in doubt. B . ORTHOPTERA

Some of the available information concerning the nitrogenous end products in members of this order has been summarized in Table I. A number of other species have been investigated (Razet, 1961), but in TABLE I Excretory products in the Orthoptera, Odonata and Dermaptera Uric Allantoic Amino acid Allantoin acid Urea Ammonia acids

Author

Order ORTHOPTERA Schistocerca gregaria 1.00

0.00

0.00

+

+

Locusta migratoria

1-00

0.00

0.10

-

-~

MeIanopIus bivittatus

1.00

-

-

0.07

053

Acheta dornesticus

1.00

0.01

0.01

0.00

-

Mantis religiosa Periplaneta americana BIatta orientalis Carausis morosus

1-00

0.01

0.00

-

-

1.00

0.00 0.64

0.00

-

__

-

0.64 0.69

1.00

-

-

- Razet, 1961

0.44

-

-

--

Razet, 1961

Order ODONATA Aeshna cyanea (larva)

0.08

-

-

Staddon, 1959

Order DERMAPTERA ForficuIa auricuIaria 1.00

-

1-00

_.

0.00

-

1.00

-

-{

Chauvin, 1941 Razet, 1961 Razet, 1961 Nation and Patton, 1961

0.20 Brown, 1937

Razet, 1961 Nation and Patton, 1961 - Razet, 1961

-{

Razet, 1961

- Razet, 1961

the interests of brevity a selection, illustrative of the range of variation within the order, has been made for this group, as for most of the others. Uric acid is clearly the predominant end product in many Orthoptera, with other components appearing in relatively small amounts. Blattu orientalis, however, excretes most of its nitrogen as allantoic acid, while the stick insect, Curuusis morosus, has a preponderance of allantoin in its excreta. Such differences do not seem to be correlated at all


47

closely with the presence of the corresponding uricolytic enzymes. Achetu has been shown to possess active uricase, allantoinase and allantoicase,yet it excretes virtually none of the correspondingproducts; Cuurausishas a highly active allantoinase (Poisson and Razet, 1952), yet allantoin predominates in its excreta. Nor is there any apparent correlation with feeding habit, since uric acid predominates in the excreta of herbivorous, and omnivorous as well as carnivorous species. In fact, consideration of just this one order illustrates the sort of diversity in respect of nitrogenous excretion which succeeding pages will show to characterize the class as a whole. The deposition of uric acid in cells of the fat body seems to be a feature of many orthopteran species, particularly among the Blattidae (e.g. Srivastava and Gupta, 1960), and uric acid may comprise as much as 10% of the total dry weight of such insects (Razet, 1961). Haydack (1953) has shown that the amount of uric acid stored in the fatty tissues of the American cockroach is greatly affected by the level of protein intake. On a high protein diet the fat body becomes enlarged, and is filled with white deposits of uric acid. If such insects are transferred to a low protein diet the uric acid deposits largely disappear. The readiness with which uric acid may be mobilized from fat body deposits in these, as in other, insects, has led to the suggestion that it may serve in part as a reserve of nitrogen for synthetic purposes (Ludwig, 1954; Ross, 1959), and there is some indication that endocrine control mechanisms may be involved (Bodenstein, 1953). Recent work by Roth and Dateo (1964 and 1965) has shown that in males of certain cockroaches the accessory sex glands play a special role in the storage of uric acid, and that as much as 5% of the live weight of males may be attributed to uric acid deposited in the utriculi majores of these glands. Most of this uric acid is poured over the spermathecae during copulation, and the sex glands may thus be said to serve as accessory excretory organs. C . ODONATA

Excretion in the aquatic larva of Aeshna cyuneu has been investigated by Staddon (1959). In starving animals most of the excretory nitrogen appeared as ammonia, with uric acid making up less than a tenth of the ammonia nitrogen (see Table I). When the larvae were fed on protein (heat-coagulated egg white) the output of ammonia increased greatly, while uric acid excretion remained constant. It would seem that ammonia is the main end product of protein catabolism, while uric

48

E . BURSELL

acid represents the end product of purine metabolism, and is therefore unaffected by protein intake: this is in accord with the absence of uricolytic enzymes in the species investigated by Razet (1961). D . DERMAPTERA

The excreta of Forjicula has been shown to contain a high proportion of uric acid (see Table I), and despite the presence of an active allantoinase, no allantoic acid could be demonstrated (Razet, 1961). E . HEMIPTERA

1. Sub-order Heteroptera The excretory material of a number of plant bugs has been investigated, and some of the results are summarized in Table 11. Allantoic acid has not been recorded for any member of this group, and this is in TABLE I1 Excretory products in the Heteroptera Uric Allantoic Amino acid Allantoin acid Urea Ammonia acids

____

Micrelytra fossularum Verlusia rhombea Palomena prasina Rhaphigaster griseus Dysdercus fasciatiis Rhodniusprolixus

0.65

0.02 1.00 1.00

0.00 0.00

1.00 1.00

1.00 0.87

1 .oo

0.00

~

0.00 0.00 0.00

0.00 ~~

-

0.26 0.03

Author -

.__-

-

Razet, 1961 Razet, 1961 Razet, 196:

- Razet, 1961 0.24 Berridge, in press trace Wigglesworth, 1931 Harrington, 1956 and 1961

accord with the apparent absence of the corresponding enzyme, allantoinase, in the large number of species examined by Razet (1961). Allantoin, on the other hand, occurs in all species, and is the predominant end product in several, which relates well to the widespread presence of a highly active uricase among members of this suborder (Poisson and Razet, 1953; Razet, 1961). Allantoin is the predominant end product in Dysdercus, but substantial quantities of amino nitrogen have been recovered from the excreta, and urea is also an important component (Berridge, 1966);


49

in view of the general absence of allantoinase and allantoicase which characterizes the group as a whole, the urea is unlikely to represent a product of uricolysis, and the possibility of faecal contamination can be excluded in this insect, since the alimentary canal is discontinuous. Urea has also been found in the excreta of Oncopeltus fasciatus, but in this species uric acid constitutes the predominant end product (Nation and Patton, 1961). The blood-sucking hemipteran RhochiusproZixus is a member of the suborder, and its excretory metabolism has been the subject of several studies (Wigglesworth, 1931; Harrington, 1956, 1961). Uric acid is the main end product, but urea accounts for 2.3% of the dry weight of excreta, and a number of amino acids have been detected together making up 0.2% of the total dry weight, with histamine and histidine predominant among them. In view of the fact that the food of this insect is composed almost entirely of amino acids, arising by hydrolysis of blood proteins, the appearance of such very small quantities of amino acid nitrogen in the excreta should perhaps be interpreted as a loss, rather than as an excretion, of amino acids. 2. Sub-order Homoptera Investigations of excretory metabolism in this group of insects has centred upon the occurrence of large amounts of amino acid in the honeydew of various aphids and coccids (see review by Auclair, 1963). These substances account for 13.2% of the dry weight of honeydew in Myzus circumflexus (Maltais and Auclair, 1952) and make up the bulk of nitrogenous material also in Brevicoryne brassicae (Lamb, 1959). The pattern of amino acids in honeydew is qualitatively similar to that of the plant juices ingested, though the concentration may be slightly lower due to absorption of nitrogenous material (Mittler, 1958 with Tuberolachnus salignus), or somewhat higher (Auclair, 1958 with Acyrthosiphon pisum) possibly due to a concentration of fluids in the insect associated with evaporative losses of water. But it would seem that the insects are ingesting nitrogenous substances in excess of their requirements, and that this excess is voided as a faecal material. If this is the correct interpretation then such amino acids have not been subjected to metabolic manipulation, and should hardly be considered as end products of nitrogenous metabolism. Gray and Fraenkel(l954) have reported the occurrence of uric acid in Pseudococcus citri on the basis of qualitative, colour reactions, but Mittler (1958) failed to find evidence of uric acid or of ammonia in Tuberolachnur.In Breuicornis neither uric acid nor urea, allantoin or

50

E. BURSBLL

allantoic acid could be detected (Lamb, 1959), but ammonia was found to constitute 0.5% of the total nitrogen in honeydew. In view of the rapid throughput of liquid in these insects it is not impossible that even such low concentrations of ammonia may represent a high proportion of truly excretory nitrogen. Uricase has been demonstrated in homogenates of Aphis brassicae, but other members of the group which have been investigated appear to be devoid of uricolytic enzymes (Razet, 1961). F. C O L E O P T E R A

Table I11 summarizes results of investigations of the excretory metabolism in beetles. Uric acid is the predominant end product in three of the species examined, and in these uricolytic activity has been shown to be absent or feebly developed (Razet, 1961). In Chrysobothris TABLE I11 Excretory products in the Coleoptera and Neuroptera Uric Allantoic Amino acid Allantoin acid Urea Ammonia acids Author Order COLEOPTERA 1.00

0*00

0.00

-

-

-

1.00 1.00

0.01 0.10

0.03 0.00

-

-

- Razet, 1961 Razet, 1961

0.83

1.00 -

0.00 -

-

0.72

1.00

0.57

- Razet, 1961 0.50 Pomhg, 1953

Order NEUROPTERA Uroleon nostras (larva) 1.00

0.03

0.00

-

-

Melolontha vulgaris Chaetocarabus intricatus Procrustes coriaceus Chrysobothris afinis Attagenuspiceus

Razet, 1961

-

- Razet, 1961

the quantity of allantoin recovered in excreta exceeds that of uric acid, which suggests the presence of an active uricase, but this has not been directly verified. In the carpet beetle, Attagenus, urea is the predominant end product identified, substantial quantities of ammonia are excreted, and the presence of the sulphur-containing amino acid, cystine, accounts for a considerable proportion of excretory nitrogen (Pawning, 1953); its occurrence is correlated with the high cystine content of keratin, which forms the major component of diet in this species, and it is possible that this amino acid should be considered to represent a faecal material rather than an excretory product. It must be noted


51

that the nitrogenous components listed for this insect make up only a proportion of the total nitrogen excreted, and some unidentified excretory products must be involved. The excreta of the mealworm beetle, Tenebrio molitor, contains mostly uric acid, but the presence of urea and allantoin has been established (Nation and Patton, 1961) although none of the uricolytic enzymes could be demonstrated in this species (Razet, 1961). Urea and ammonia have been found as constituents of the excreta of other members of the order (Delaunay, 1931) and ammonia has been identified in the excreta of Ephestia kuhniella (Payne, 1936). The excreta of the boll weevil, Anthonomus grandis has been examined for the presence of non-protein amino acids, and these were found to constitute 3.2% of the total nitrogen, with about a third attributable to free amino acids (Mitlin et al., 1964). Storage of uric acid in cells of the fat body has been reported for one of the species of beetle examined by Gupta and Sinha (1960), but storage excretion does not appear to be a general characteristic of the group, to judge by the generally low uric acid contents recorded by Razet (1961). G . NEUROPTERA

Data for the larva of Uroleon have been included in Table 111. Since the alimentary canal is discontinuous, excretory material could be collected free of faecal contamination. 33% of the dry weight was uric acid, and only a small quantity of allantoin was present; allantoic acid could not be detected. In the larva of Chrysopa carnea the deposition of uric acid in cells of the fat body has been reported (Spiegler, 1962). The presence of large quantities of ammonium bicarbonate in excretory fluids of the aquatic larva of Sialis lutaria was demonstrated by Shaw (1955). The excretory metabolism of this insect was further investigated by Staddon (1955) who showed that 86% of the nitrogen output of starving animals appeared in the form of ammonia. H . HYMENOPTERA

The excreta of certain herbivorous larvae belonging to this group has been investigated by Razet (1961), and some of his results are summarized in Table IV. Here again some species excrete predominantly uric acid, in others allantoin or allantoic acid may be important constituents, and in some they achieve dominance. The differences between

52 E. BURSELL species cannot be correlated with differencesin host plant, nor do they accord with the distribution of uricolytic enzymes. Uricase is poorly developed in both the genera listed; and Pteronidea sulicis, which has a highly active allantoinase, excretes a very small proportion of allantoic acid, while in P. ribesi allantoinase activity is slight, but allantoic acid forms a high proportion of the excretory material. TABLE IV Excretory products in the Hymenoptera and Diptera Uric Allantoic Amino acid Allantoin acid Urea Ammonia acids Author Order HYMENOPTERA Hemichroa alni

1.00

0.00

0.00

- Razet, 1961

1.00

0.03

0.06

- RBWt, 1961

0.78

1.00

0.90

- R m t , 1961

1.00 1-00 1.00

0.04

0.30 0.30

0.00 0.62

0.05

1.00

(larva) Pteronidea salicis

(larva) Pteronidea ribesi

(larva) Order DIPTERA Compsilura concinnata Tipulapaludosa Lucilia sericata Lucilia sericata Oarva) Lucilia sericata

(pupa) Bibio marci (larva) Aedes aegypti Anopheles guadrimaculatus Culex pipiens Glossina morsitans

I

0.45

1*0° 1.00

-

-

0-30

0.02

-

1.00

0.00 1.00

-

0.15

0-36

-

0.22

+

0.18

- Razet, 1961 - X m t , 1961

-

-{

Brown, 1936; 1938a and b

-

- Razet, 1961

0.11 Irrevere and Terzian, 1959 0.22 Bursell, 1964b

I . DIPTERA

A number of insects belonging to this order have been studied, and results are included in Table IV. Most of the adults examined are predominantly uricotelic, though in Tipula, where active uricolytic enzymes have been demonstrated (Razet, 1961), substantial amounts of allantoin and allantoic acid are present in the excreta. In the sheep ked, Melophugus ovinus, uric acid is the main end product, but quantities of xanthine and hypoxanthine may appear in the excreta of pregnant females (Nelson, 1958).

53 The excretory metabolism of several blood-sucking members has been the subject of detailed investigation. Uric acid is the dominant excretory product in mosquitoes, but urea and ammonia occur in fairly high concentration, and amino acids make up a substantial proportion of total nitrogen; there is little difference between the three species examined, and in Table IV results have been averaged. The effect of nutrition on the excretion of Aedes aegypti has been studied by Terzian and his colleagues (1957), who show that on a diet of sugar there is a decrease in the nitrogen output, and the proportion of uric acid in the excreta falls to about 4%. Following a blood meal the nitrogen output increases greatly, and the proportion of uric acid rises to SO%, thus confirming that uric acid is the end product of protein metabolism. In the tsetse fly also, the predominant excretory product is uric acid; urea occurs in trace amounts, and a substantial proportion of nitrogen is excreted in the form of histidine and arginine. These two amino acids, which together make up about 10% of the protein amino acids in human blood, are particularly rich in nitrogen (see Fig. 1). It would presumably be uneconomical to deaminate materials of this kind, since any benefit which the insect might derive from deamination products would be offset by metabolic losses involved in uricotelic detoxication of the nitrogen they contain. In view of this, a quantitative elimination of ingested arginine and histidine might be expected, and this is roughly in accord with observation (Bursell, 1964b). There is some evidence that all of the amino acids liberated during digestion are absorbed from the midgut, and that arginine and histidine are subsequently excreted, so that they cannot be regarded simply as faecal materials. Indeed, recent work has shown that arginine becomes rapidly labelled following injections of C14-glutamate(E. Bursell, unpublished), and it is possible that this substance may play a more active role in excretion than was originally envisaged. The occurrence of small quantities of ammonia has been reported in the excreta of adult Diptera by a number of workers (Brown, 1938b; Lennox, 1940; Hitchcock and Haub, 1941; Sedee, 1958), but it is only in the meat-eating larvae of species like Calliphora and Lucilia that this product becomes dominant (Brown, 1936; 1938a, b; Robinson, 1935; Robinson and Baker, 1939). Allantoin and uric acid are present in relatively small amounts in the larval excreta of Luciliu (see Table IV), and the proportion between them appears to depend in part on larval diet; with larvae fed on casein allantoin predominates, but on a meat diet relatively little allantoin is produced (Brown, 1938a). THE EXCRETION O F NITROGEN I N INSECTS

54

E. B U R S E L L

Allantoin is an important constituent of excreta in larvae of Bibio, and allantoic acid occurs as well, in accord with the presence of an active allantoinase in larval extracts (Razet, 1961). The excretory products which accumulate in the meconium during pupal development have been examined by Brown (1938a, b) in Lucilia. A considerable amount of ammonia was found, but uric acid constitutes the bulk of excretory nitrogen and no allantoin could be detected. The absence of allantoin is correlated with a loss of uricase activity at this stage of the life history; the enzyme disappears suddenly when larvae leave the meat, to reappear immediately after emergence from the pupa. The results of investigations of dipteran excretion provide a striking illustration of the lability which appears to characterize this aspect of metabolism, with the distribution of nitrogenous end products varying widely from stage to stage in the life history of a single species. J. LEPIDOPTERA

A great deal of information is available concerning the uricolytic metabolism of this group, thanks to the work of Razet (1961). A selection of results has been summarized in Table V, where the proportions of uric acid, allantoin and allantoic acid in the excreta of different stages in the life history of members of the order have been set out. Variability within the group is considerable; many of the adults are predominantly uricotelic, but allantoin and allantoic acid occur as minor components in several species, and in some allantoic acid is the predominant end product. In pupal stages uric acid is predominant in all the species listed, including those whose adults show a preponderance of allantoic acid; but the proportions of allantoic acid and allantoin are often appreciable, and in some species exceed those found in adult excreta (e.g. Aglais urticae). In larval stages the three earcretory products are more evenly distributed in the excreta of different species; where allantoic acid is the end product, uric acid occurs in substantial proportion, and vice versa. But there is little correlation between adult and larval excretory metabolism, for some of the species whose adults are predominantly uricotelic show a preponderance of allantoin in larval excreta, while some of the species whose adults have allantoic acid as the main end product have uricotelic larvae. In certain species it has been demonstrated that larval diet may have an important effect on excretory metabolism. For instance, with

55


larvae of Lasiocampa trifolii fed on leaves of Hordeum murinum the predominant excretory product is allantoic acid, while a diet of Betulus aZba produces a predominance of uric acid. Such effects cannot be attributed to the pre-existence of nitrogenous end products in the diet, since of all the host species examined only one, the cabbage Brassica TABLE V Excretory products in the Lepidoptera (Razet, 1961) Uric acid

1. ADULTS Mammestra brassicae Agritos comes Aglais urticae Trigonophora meticulosa Pieris brassicae Vanessa atalanta Conistra vaccinii 2. PUPAE M . brassicae A. comes A . urticae T. meticulosa P. brassicae V. atalanta C. vaccinii

3. LARVAE M . brassicae A. comes A. urticae T. meticulosa P. brassicae V. atalanta C. vaccinii

1.00 1.oo 1.oo 1.00 1.00 0.89 0.12

1.00 1.00 1.00 1.00

Allantoin -

0.00 0.01 0.04 0.14 -

-

0.29 -

1.oo 1.00 1.00

0-03 0.14 0.04

0.49 0.67 0.56 1-00 0.28 1.oo 1.00

0.11 0.02 0.18 0.02

0.16 0.26 0.06

Allantoic acid 0.00 0.01

0.06 0.05 0.01

1.00 1.00

0.03 0.01 0.43 0.19 0.05 0.29 0.04 1.00 1.00 1.00 0.12 1.00 0.20 0.05

oleracea, could be shown to contain small quantities of allantoin and allantoic acid. The occurrence of uricolytic products in the excreta of different members of the order is in general accord with the presence, in most species, of the corresponding uricolytic enzymes, but in point of detail there is little agreement. In some species where enzymatic activity is 34-A.I.P.

4

56

E. BURSELL

relatively high the corresponding end product is poorly represented in the excreta (e.g. allantoinase in adults of Aglais urticae), while species with relatively low enzymatic activity may show a preponderance of the reaction product in their excreta (e.g. Conistra vaccinii). The excretory metabolism of lepidopteran larvae at different stages of larval life has also been investigated by Razet (1961), and the results serve further to emphasize the lability of this function within the life history of the individual, for the ratio of allantoin to uric acid may fluctuate enormously from day to day. In most of the species studied there is a general tendency for uric acid to become increasingly important with larval age. A number of end products other than these three have been assayed in the excreta of different members of the order. In Bombyx mori, amino acids have been demonstrated in excretory material of the larva (Yoshitaka and Aruga, 1950); uric acid is the predominant end product in this species, and the quantity exsreted has been shown to increase on a diet rich in protein (It0 and Mukaiyama, 1964). In larvae, of Corcyra cephalonica uric acid is again predominant in the excreta, but small quantities of urea and of xanthine have been demonstrated (Srivastava, 1962) ; both xanthine and hypoxanthine have been identified in the excreta of the wax moth, Galleria melonella (Nation, 1963; Nation and Patton, 1961), with hypoxanthine making up as much as 10% of the total; ammonia has also been identified in the larval excreta of this species (Zielenska, 1952). In the clothes moth, TineoZa bisselliella, uric acid also predominates, but for every 100 mg of uric acid nitrogen excreted, 24 mg of ammonia nitrogen and 10 mg of urea nitrogen are disposed of. In addition, the excreta of this species contains a substantial amount of amino nitrogen in the form of cystine, which correlates with its diet of keratin (Waterhouse, 1952; Powning, 1953). I V . CONCLUSIONS The preceding pages have given some indication of the tremendous progress which has been made in the field under review during the last 15 years. A number of uricolytic enzymes have been investigated in tissue homogenates, their distribution among Werent tissues has been established and some of their properties have been described. A great deal has been discovered about xanthine oxidase, or xanthine dehydrogenase as it should more properly be called, and here studies have advanced to the stage of enzyme purification. But unfortunately there remain several gaps in our knowledge of nitrogenous metabolism

THE EXCRETION OF N I T R O G E N I N INSECTS

57

at the level of enzyme studies. The contribution made by different reactions (adenase and guanase deamination, oxidative deamination of amino acids, the glutamic dehydrogenase system) to the appearance of ammonia remains under dispute. The origin of urea is still uncertain, and, above all, the precise pathway of uric acid synthesis has not been unequivocally settled. The evidence concerning these aspects of nitrogenous metabolism is often contradictory, and the problems stand in urgent need of resolution. The application of radioactive tracer techniques, coupled wit% the use of insects whose nutrition, in terms of protein and nucleoprotein intake, can be rigidly controlled, would seem to offer prospects of rapid advance in this field. The role of urea in nitrogenous excretion has never been fully understood; it is present in the excreta of many insects, but usually forms a rather minor component. It cannot be regarded as a purine degradation product, since so far no insect has been shown to possess the enzymes required for the degradation of uric acid to this stage. The presence of arginase in many insects suggests that it may originate by hydrolysis of arginine, but evidence for the existence of the complete omithine cycle is weak. It seems possible that arginine may be the raw material for its production, but that its occurrence in insects should be seen as a reflection of the use of arginine as a phosphagen in this class of animals (Razet, 1964) ;the precise implications of this suggestion have, however, yet to be worked out. The data summarized in Section I1 of this review give some indication of the complexities involved in the excretory metabolism of insects. In the face of such complexities one may be forgiven for looking back with a degree of nostalgia to the early generalization-that insects as a class are uricotelic, and that the production of uric acid as the main end product of nitrogenous metabolism can be seen as a reflection of the shortage of water to which most members of the group are subject. This shortage of water would make toxic substances, like ammonia, and%oluble substances, like urea, unsuitable as waste products. It was possible to accommodate certain exceptions within the general framework of this generalization; for instance, the predominance of ammonia as an excretory product of aquatic larvae, of certain dipteran larvae and perhaps of certain plant-sucking bugs, could be interpreted on the basis of the ready availability of water associated with these modes of life. Similarly, the excretion of substantial proportions of specific amino acids could be seen as an adaptation to special diets; insects which ingest a lot of keratin excrete a lot of the sulphur-containing amino acid which this diet contains; blood-sucking insects, whose

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diet contains a high proportion of nitrogen-rich amino acids, may eliminate these unchanged, though why this should occur in the tsetse fly and to some extent in mosquitoes, but not in Rhodnius, is unclear. Up to this point it would appear permissible to retain the view of uricotelism as an insect characteristic. But what investigations of the last ten years have brought to light is the high proportion of purine derivatives other than uric acid that occur in the excreta of many insects. These include not only degradation products like allantoin and allantoic acid, but also some of the reduced derivatives like xanthine and hypoxanthine. Since these substances may occur in the excreta of insects in amounts far exceeding any that could be attributed to nucleic acid catabolism, they must derive largely from amino acid nitrogen. The situation, therefore, seems to be that amino acid nitrogen is detoxicated by incorporation in a purine ring system at the expense of considerable amounts of metabolic energy; and that the purine ring system is then broken down in a sequence of hydrolytic reactions which are unproductive of metabolic energy, and lead to the formation of reaction products that differ appreciably from uric acid neither in respect of solubility nor in respect of nitrogen content. Coupled with the apparent lack of correlation between the proportion of degradation products in the excreta of different insects and their mode of nutrition and way of life, this provides small basis for an interpretation in terms of water balance or of excretory efficiency. In seeking for an alternative basis of interpretation, perhaps the most sigmficant finding is the localization of enzyme activity in cells of the Malpighian tubules and of the midgut, both of which are regions which have been implicated in the elimination of excretory material. It seems possible that the degradation of the purine ring may in some way be associated with the transfer of material across a secretory epithelium (M. J. Berridge, in the press), perhaps by some form of facilitated diffusion. The ability to break the purine ring to the stage of allantoic acid appears to be widely, and maybe universally, distributed within the class. It is true that negative results of enzyme assays have been reported for a number of insects (Razet, 1961), but this may mean not that the enzymes are absent, but that they are present in concentrations too low to be detected by the methods used. The nature of the results obtained by Razet does not allow a great deal of reliance to be placed on negative results. In many cases the quantity of substrate broken down is inversely related to the amount of enzyme present, and Razet suggests the possibility that inhibitory factors may be involved, coming into play at high homogenate concentrations. Similar effects could be involved in

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extracts from species showing no detectable activity; indeed, the development of more refined methods led to the detection of uricase in species which had previously been thought devoid of activity (Razet, 1964). It would seem that investigations with dialysed enzyme preparations would be required for unequivocal demonstration of the absence of uricolytic activity. These considerations suggest that the development of uricotelism in insects has not been associated with a loss of uricolytic enzymes. The ability to synthesize these enzymes appears to have been preserved in the genotype, perhaps because certain of the reaction products have special parts to play in general metabolism. It is the degree to which this ability is reflected in the phenotype which is subject to immense variation from order to order within the class, from species to species within the orders, from stage to stage in the life history of a species, and from time to time within a given stage. The main problem which confronts the investigator today is to discover what is the biological significance of these variations, and with what aspect of species biology they are correlated. The demonstration that different nitrogenous end products may achieve dominance in the excretory metabolism of different insect species calls, however, for some reassessment of the generalization that terrestrial insects as a group are uricotelic. Either this statement must be qualified to take account of recent discoveries, or it must be abandoned in favour of a more complex statement of the situation. The second alternative is the one favoured by P. Razet (in the press) in his recent review of nitrogenous metabolism in insects. He proposes that insects should be assigned to one or other of a number of different categories depending on the nature of the predominant excretory end product. The terms uricotelic, ureotelic and ammonotelic would be retained to indicate a preponderance in the excreta of uric acid, urea and ammonia respectively, and two new categories would be added : allantoinotelic for insects which excrete mainly allantoin, and allantoicotelic for insects which excrete mainly allantoic acid. Apart from the rather cumbersome nature of these new epithets, a classification of this sort does not seem very helpful at the present stage. In the first place it bears a largely random relation to the generally accepted taxonomy of the class, and representatives of a given category may occur equally among primitive and advanced members. Secondly, the proposal to classify species on the basis of the predominance of a given end product would run into difficulty in the case of certain Diptera in which the larva is ammonotelic and the adult uricotelic; or

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certain Lepidoptera, which may be uricotelic in the larval stage and allantoicotelic as adults, or whose larvae may switch from one category to another depending gn age. What is described would here cease to be a species characteristic; the terms would become descriptive simply of a particular moment in the life history, and would thus be deprived of their generally accepted connotations. Even if it should be found desirable to institute a new system of classification of the kind proposed by Razet, the time to apply it to the orders of insect would not be now. For while the information available is reasonably extensive in respect of certain groups (e.g. the Lepidoptera), it is very meagre in respect of others (e.g. Collembola; Dermaptera), and these latter might have to be assigned to their place in classification on the basis of a single species. In view of the wide spectrum of end product predominance discovered in all the groups which have been thoroughly studied, it seems certain that any scheme proposed would be shortlived and subject to continual adjustment in the light of new information. For these reasons it seems to the present reviewer that some attempt should be made to retain the older generalization for the time being, if this can be done by suitable qualification. Granted the complexities reviewed in Section 11, it still remains true to say that uric acid is an important excretory product in nearly all terrestrial insecfs, and to this limited extent the statement that uricotelism is a characteristic of the class remains valid. It is not always the predominant excretory product, but where it is not, its place seems invariably to be taken by allantoin or allantoic acid, as far as present information goes. Since these substances differ from uric acid in neither of the properties which are chiefly relevant to the disposal of nitrogenous waste, namely nitrogen content and solubility, it would seem permissible to regard them all as belonging, to a single class of excretory substance, and to widen the definition of uricotelism accordingly-to regard an animal as uricotelic if it excretes uric acid or one of its primary degradation products, allantoin or allantoic acid, or some mixture of these three substances, as the predominant waste product. With such a qualification the view of insects as a uricotelic class, in line with thsir terrestrial mode of life, remains tenable, and the centre of interest in the field of nitrogenous metabolism then shifts from further cataloguing of the relative proportions of these end products in the excreta of insects to what seems at present a more rewarding problem, namely a study of the biological significance of such differences in the proportion of end products, possibly in relation to the mechanism of excretion. The most promising approach to this study, and through it to the general problem of the distribution of


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nitrogenous end products, would seem to lie with the species which show a pronounced shift in excretory metabolism during the course of their life history. Here there would be some possibility of relating observed changes in the proportion of different end products to the level and type of nutrition and to the availability of water, and in this way to gain some insight into the significance of such changes. The work of the last decade has adequately demonstrated the diversity and complexity of excretory metabolism in the insects as a whole. What seems to be required now are intensive studies of selected species in which the problem of excretory metabolism can be set squarely in the context of species biology.

ACKNOWLEDGEMENTS My sincere thanks are due to Dr. P. Razet for his kindness in presenting me with a copy of his thesis, and for allowing me to read and to refer to his review of nitrogenous metabolism before its publication.

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Srivastava, P. N. (1962). Physiology of excretion in the larva of Corcyra cephaIonica Stainton (Lepidoptera, Pyralidae). J. Insect Physiol. 8, 223-232. Srivastava, P. N. and Gupta, P. D. (1960). Excretion of uric acid in Periplaneta americana L. J. Insect Physiol. 6, 163-167. Staddon, B. W. (1955). The excretion and storage of ammonia by aquatic larvae of Sialis lutaria (Neuroptera). J. exp. Biol. 32, 84-94. Staddon, B. W. (1959). Nitrogen excretion in nymphs of Aeshna cyanea (Mull.) (Odonata, Anisoptera). J. exp. Biol. 36, 566-574. Stobbart, R. H. and Shaw, J. (1964). Salt and water balance: excretion. In “Physiology of Insecta”, (M. Rockstein, ed.) Academic Press, New York and London. Szarkowska, L. and Porembska, Z. (1959). Arginase in Celerio euphorbiae. Acta biochim. pol. 6,273-276. Terzian, L. A., Irreverre, F. and Stahler, N. (1957). A study of nitrogen patterns in the excreta and body tissues of adult Aedes aegypti. J. Insect Physiol. 1, 221-228. Tomita, M. and Kumon, T. (1936). Zur Chemie der Fliegen-larven. HoppeSeyler’s Z. physiol. Chem. 238, 101--104. Truszkowski, R. and Chajkinowna, S. (1935). Nitrogen metabolism of certain invertebrates. Biochem. J. 29, 2361-2365. Ursprung, H. and Hadorn, R. (1961). Xanthinedehydrogenase in Organen von Drosophila melanogaster. Experientia, 17, 230-23 1. Wagner, R. P. and Mitchell, H. K. (1948). Enzymic assay for studying the nutrition of Drosophila melanogaster. Archs Biochem. 17, 87-96. Waterhouse, D. F. (1952). Studies on the digestion of wool by insects. IV. Absorption and elimination of metals by lepidopterous larvae, with special reference to the clothes moth, Tineola bisselliella (Humm.). Aust. J. scient. Res. (B), 5, 143-168. Wigglesworth, V. B. (1931). The physiology of excretion in a blood-sucking insect, Rhodnius prolixus (Hemiptera, Reduviidae). J . exp. Biol. 8, 41 1-451. Wigglesworth, V. B. (1950). “Principles of Insect Physiology.” Methuen, London. Yoshitaka, N. and Aruga, H. (1950). Studies on the amino acids in the silkworm. IV. On the free amino acids in the silkworm faeces. J. seric. Sci., Tokyo 19, 536. Zandee, D. L., Nijkamp, H. J., Roosheroe, L., De Waart, J., Sedee, P. D. J. W. and Vonk, H. J. (1958). Transaminations in invertebrates. Archs int. Physiol. Biochim. 66, 220-227. Zielenska, Z. M. (1952). Studies on the biochemistry of Galleria melonella. Nitrogen metabolism of the larva. Acta Biol. exp. Vars. 16, 171-186.


Lipid Metabolism and Function in Insects LAWRENCE I. GILBERT Department of Biological Sciences, Northwestern University, Evanston, Illinois, U.S.A. I. Introduction . A. General . B. Definition and classification 11. Lipid Content . A. Expression of data . B. Alterations during metamorphosis . C. Nature of insect lipids . III. Lipid Utilization . A. Digestion and absorption . B. Lipid release and transport C. Extra-digestive lipases . D. Fatty acid catabolism . IV. Lipid Biosynthesis . A. General mechanism of fatty acid synthesis B. Fatty acid biosynthesis in insects C. Phospholipid and triglyceride . D. Fatty acids in nutrition . E. Substrate interconversion . V. Hydrocarbons and Waxes A. Cuticle B. Extra-cuticular . VI. Isoprenoid Compounds . A. Nutritional studies . B. Isoprenoid biosynthesis . C. Isoprenoid content . D. Sterol modification . E. Function . F. Insect hormones VII. Conclusion . References .

.

.

. . . . . . .

.

. . . . . . . . . . . . . . . . . . . . . .

70 70 71 71 71 81 89 97 97 102 110 116 127 127 130 134 145 147 152 152 155 157 157 161 168 170 175 176 186 187

In the course of this paper, the reader will encounter the following abbreviations: ADP-adenosine diphosphate; ATP-adenosine triphosphate; CoA or COASHcoenzyme A; Co@-coenzyme Q ; CTP4ytidine triphosphate; DGLAiglyceride; FAD-flavine adenine dinucleotide; FFA-free or unestersed fatty acids ; MGLmonoglyceride; NAD-nicotinamide adenine dinucleotide; NADP-nicotinamide PTC-phosphatidylcholine; adenine dinucleotide phosphate; PL-phospholipid; FTE-phosphatidylethanolamine ;TGL-triglyceride. 69

70

LAWRENCE I . GILBERT

I. INTRODUCTION A . GENERAL

The awarding of the Nobel prize in medicine to Bloch and Lynen for their contributions to the study of lipid metabolism is a heartening occurrence for many biologists and biochemists. This act pointed out once again the importance of the study of lipids, their synthesis and catabolism. It was in fact in this research area that the first definitive data were obtained on the dynamics of any protoplasmic constituents when Schoenheimer and Rittenberg (1936) studied fatty acid turnover utilizing heavy water. One prime impetus to the study of lipid biochemistry in insects in recent years has been the finding that many, if not all of the insect growth hormones, pheromones and sex attractants are lipoidal (cf. Gilbert, 1964). An understanding of the biosynthetic pathway and means of catabolizing these humoral and air-borne messengers necessitates a vigorous experimental approach to the fields of insect lipid chemistry and biochemistry. As will be discussed subsequently, lipids are also of vital importance to many insects as substrates for embryogenesis, metamorphosis and flight. And several members of this large category of biochemical compounds are without peers as vital nutritional growth factors. The purpose of this review is not so much to tabulate all knowledge of insect lipid biochemistry as to point out the gaps in our knowledge, in an attempt to attract others to this increasingly important research area. When this paper was begun, the last tome on insect lipids was more than a decade out of date (Scoggin and Tauber, 1950). In recent years however, several reviews on aspects of insect lipid chemistry and biochemistry have appeared (Clayton, 1964; Fast, 1964; Gilby, 1965 and Tietz-Devir, 1963),while other reviews that consider the metabolism of lipids along with other biochemicals have also been published (Chefurka, 1965; Gilbert and Schneiderman, 1961a; Gilmour, 1961; Kilby, 1963). Numerous papers on the biochemistry of lipids in microorganisms, fowl and mammals have appeared in the recent past and will be alluded to when considered important to the present discussion. It is of interest to note that several journals dealing exclusively with lipid biochemistry have appeared in the last eight years (Journal of Lipid Research, Lipid section of Biochimica Biophysica Acta, Steroids, as well as Advances in Lipid Research) but one has difficulty finding more than a dozen publications considering insect material in the cumulative

L I P I D METABOLISM A N D F U N C T I O N I N I N S E C T S

71

editions of these journals. It may be that as many reviews have appeared on this subject in recent years as breakthroughs in this research area. We shall concentrate on developments within the past ten years but will discuss older literature when applicable to the historical development of the topic or when it buttresses the argument. However, not all of the older literature will be discussed. Some aspects of the topic will be treated in a cursory manner, some omitted and others dwelled upon at length. The hierarchy of discussion is in part due to the author’s own interests and it is possible that topics not treated in depth (e.g. nutrition) are of more importance than those treated extensively. A final point worthy of note is that few generalizations can be made regarding all members of this largest animal class. In most cases, sophisticated experiments have been conducted on only a few species of “domesticated” insects and one cannot extrapolate the results to all insects. The multitude of different ecological niches and behavioural characteristics are no doubt reflected in a great number of metabolic variations on perhaps more than one basic theme. Notwithstanding the above, the past five years have brought us a clearer understanding of at least some aspects of the lipid biochemistry of insects. Before considering these advances it is of importance to agree on a vocabulary. B. DEFINITION A N D CLASSIFICATION

There has as yet been no rigorous definition of the term “lipid” that has been accepted categorically by all workers in the field. Generally, all compounds that are insoluble or only sparingly soluble in water and are extractable with, or soluble in, organic solvents such as diethyl ether, chloroform, acetone, etc., are termed lipids. For our present purposes, “lipids” will be defined as all compounds falling into the classification outlined in Table I, which is adapted and modified from similar tables by numerous authors, but notably Deuel (1951) and Strickland (1963). 11. LIPIDCONTENT A . EXPRESSION OF D A T A

One of the fundamental questions concerning the role of lipids in the physiology of the insect concerns the quantity of lipid contained upon, and interior to, the rigid exoskeleton. The exact amount will of course vary with physiological state, but also appears to vary according to the method of extraction and whether the resqlts are expressed in terms of dry weight, wet weight or lean dry weight. At first glance it

72


would appear most desirable to express the quantity of lipid in terms of dry weight of the insect or tissue since this value can be obtained easily by drying the tissue to constant weight at about 100” and subsequently extracting the lipid. Unfortunately this procedure usually TABLE I Classification of lipids

A. Simple Lipids 1. Glycerol esters of fatty acids (mainly long-chain fatty acids; commonly called “neutral fat ”; includes monoglycerides, diglycerides and triglycerides) 2. Esters of long chain monohydric alcohols (commonly called “waxes”) 3. Sterol esters (sterol plus fatty acid) 4. Glycerol ethers (containing palmityl, stearyl or oleyl alcohols) B. Compound Lipids (esters of fatty acids and alcohol containing one or more additional groups) 1. Phosphoglycerides (commonly called phospholipids; esters of phosphate and free a hydroxyl of an a, 8, diglyceride or monoglyceride) (a) phosphate-monoester (phosphatidic acid) (b) phosphate-diester (e.g. phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine) (c) phosphate-triester (complexes of phosphatidylethanolamine or phosphatidylserine) 2. Sphingolipids (sphingosine containing N-fatty acyl group) (a) sphingomyelin (containing phosphorus and choline) (b) cerebrosides (containing sugar; usually galactose) (c) sulfatides (cerebroside plus sulfate residue) (d) gangliosides (commonly called mucolipid; contains sialic acid) C. Derived Lipids (products of A. and B. still possessing lipid characteristics) 1. Fatty acids 2. Long-chain alcohols 3. Sterols 4. Terpenes 5. Hydrocarbons 6. Sphingosine D. Complex Lipids 1. Lipoprotein 2. Proteolipid (lipid plus peptide) 3. Phosphatide peptide (contains sphingosine, inositol, phosphorus and nitrogen)

leads to profound changes in the nature of the lipid (e.g. oxidation) and the lipid becomes virtually useless for further analysis. It is therefore not unusual to express the lipid content in terms of fresh or wet weight. This procedure can lead to large errors due to the fluctuations in the

LIPID METABO LISM A N D F U N C T I O N I N I N S E C T S

73

water content of the insect. From our experience, lean dry weight is a more constant value and is obtained by extracting the tissue with organic solvents (ethanol-ether or chloroform-methanol) and weighing the air-dried residue. The lipid can then be treated gently (e.g. evaporation under nitrogen) and used further for identification of its individual components. The lean dry weight then is the dry weight minus the weight of the lipid. For reviews of lipid extraction procedures see Haahti (1961) and Horning (1964). As Table I1 demonstrates, there is a wide variation in lipid content of insects of different orders and even within a single family. This is not difEcult to understand when one considers the large diversity of insects, TABLE I1 Lipid content of various insect species ~~

Insect COLEOPTERA Bruchidae Pachymerus bactris Callosobruchus chinensis Laria irresecta Buprestidae Eurythyrea marginata Carabidae Pterostichus vulgaris Pterostichus nigrita Pseudophonus pubescens Harpalus pubescens Harpalus griseus Cebrionidae Cebrio gigas Cerambycidae Ergates faber Rhagium inquisitor Plagionotus arcuatus Cerambyx scopolii Chrysomelidae Leptinotarsa decemlineata ¶¶ ¶¶

,,

¶¶ ¶¶ ¶¶

Galerucella luteola Colaspidema atrum

Stage

Per cent lipid

41 D 33-2W 23.6W 12.2w

57w 4.8W 28*4D 4.8W 6.0W 144W 13.0W 7.0W 14.4W 8.5W 4.2W 3.4w 13.4W 39.6D 7.0W 3.9w continued

74


Insect Aulacophora fumolaris Cistelidae Heliotaurus menticornis Curculionidae Balaninus elephas Rhynchophoruspalmarum Anthonomus grandis

,,

Dermestidae Dermestes sp

,,

9,

Dytiscidae Dytiscus marginalis Lampyridae Luciola vitticollis Meloidae Mylabris pustulata Lytta vesicatoria Scarabaeidae Oryctes nasicornis Melolontha hippocastani Melolontha vulgaris Cetonia aurata Phyllophaga rugosa Geotrupes stercoralis Popillia japonica 99

9, 99

9,

9,

,,

Tenebrionidae Tenebrio molitor 9,

,,

9,

3,

,,

,?

LEPIDOPTER A Arctiidae Arctia caja Estigmene acraea Bombycidae Bombyx mori ,¶

99

,, 9,

Stage

Per cent lipid 22

w

6 W 28 W 22.3W 12.8W 35 D 47 D 21 D 6.2W 7.3w 4.8W 12.5D 13-5D 15.0W 6.1W 16.9W 1.9W 9.0 7.7 4.0W 3.2W 3.8W 4.0W 3.8W 17.4W 154W 14.9W 12.9W

4.1W 13.OD 12 w 343W 24.4D continued

75

LIPID METABOLISM AND FUNCTION IN INSECTS

Insect Bombyx mori 99

Carposinidae Carposina niponensis

,,

,,

9)

3,

9,

,,

Citheroniidae Citheronia regalis Eacles imperiaris

27.6W

Gelechiidae Pectinophora gossypiella

,, ,, ,, ,,

99

,, ,,

9,

Hesperidae Acentrocneme hesperiaris Lasiocampidae Malacosoma franconicrim Malacosoma americanum

,,

99

Lithosiidae Asura conferta Lymantridae Lymantria dispar Euproctis chrysorrhoea (Linn.) Noctuidae Alabama argillacea Heliothis armigera Prodenia ornithogalli Laphygma frugiperda Lycophotia margaritosa Euxoa segetum Agrotis segetum 99

¶3

9)

9)

27*1W 85.4D 35-7w 81.6D 17.4W 55W 6*0W

Cossidae Cossus ligniperda Danaidae Danaus plexippus

99

Per cent lipid 5.9w 9.1W

99

99

Stage

Laphygma exempta solitary

23 W 12 w 15.3W 44.8D 16.7W 50.5D 16-1W 33*4D 10

w

1.9w 82 D 24.7D 1.ow 1.5w 4-9w 24.4D 20.9D 18.1D 13.1D 0.9W 6.4W 23.1D 30.1D 13.5W 15W continued

76


Insect Laphygma exempta gregarious Spodoptera abyssinia solitary Spodoptera abyssinia gregarious Spodoptera abyssinia solitary Spodoptera abyssinia gregarious Acronycta rumicis Colocasia coryli Daseochaeta at'pium Scoliopteryx libatrix PIusia gamma Notodontidae Pha lera bucephala Nymphalidae Vanessa urticae Olethreutidae Carpocapsa pomenella Papilionidae Papilio troilus Papilio turnus Papilio zolicaon Pieridae Pieris brassicae 99

>¶

Pieris napi 9)

Y9

Pieris rapae 9,

99

,,

,,

9,

99

9,

$9

,I

Y9

Psychidae Thyridopteryx sp. Pyralidae Galleria mellonella 99

>,

Myelobia smerintha Ephestia figulilella Loxostege similalis Loxostege sticticalis

Stage

Per cent lipid 2.7W 3.1W 4.9w 7.1 W 9.3w 25.5D 29.5D 12.5D 18.2W 334W 34.8W 10.3W 3.7w 44.2D 3-3w 4.6W 7.6W 10.8W 1.9w 6.0W 2.2w 3.6W 16.0d 7.7D 2.3W 12.1D 6.4W 26.1D 11.4W 29.7D 5-6W 9.1w 56-2D 21-0w 22 w 21-6W 18.8D 29.7D

continued

LIPID METABOLISM A N D FUNCTION I N INSECTS

Insect Loxostege sticticalis

,,

Y9

,,

9)

,,

9,

Pyrausta nubilalis 3,

YY

99

9,

Stage

77

Per cent lipid 5-9w 7.5w 30.2D 96W 3.2W 26.5W 9-7w 143W

Saturniidae Saturnia pyri Antheraea pernyi Y9

9,

Y9

,,

*Antheraea mylitta *Antheraea polyphemus *Antheraea roylei *Hyalophora cecropia *Hyalophora euryalus *Actias luna Adelocephala heiligbrodtii *Attacus atlas

*Attacus canningii *Samia Cynthia Automeris io

* Callosamia promethea * Calosaturnia mendocino *Rothschildiaforbesi

*Rothschildia orizaba

4.0W 1.ow 4*6W 14.8D 17.8D 4-OW 6.0W 21.6W 6-1W 38.3W 5.ow 216W 4-2W 22.8W 4.4w 17.6W 1*9W 2.8W 9-9w 41.3W 8.3W 40-OW 8.0W 36-7W 9.8W 18.2W 2.9W 13.4W 2.9W 22.8W 294W 3.7w 32.2W

Sphingidae Deilephila sp. Sphinx elpenor

35w 4.9w continued

78


Insect

Stage

Sphinx ebenor Sphinx ligustri

,, 39

6-1W 7.8W 3.8W 6.1W 2*8W

9,

9,

Sphinx euphorbiae Thaumetopoeidae Thaumetopoea pityocampa

35w

HEMIPTERA Aphididae Aphis rumicis Aphis rosae Aphis fabae Hyalopterus pruni Coccidae Coccus cacti Eriosomatidae Pemphigus utricularias Jassidae Euttetix tenellus

7.5 w 6.5W 12 w 6.2W 8 W

20

9,

,,

9,

36.3D 11-6W 5-2W 6-2W 6-2W 32.7D

ORTHOPTERA Acrididae Acrida bicolor Anacridium aegyptium Orthocanthacris aegyptium Dociostaurus maroccanus Oxya japonica Locusta migratoria 9s

,,

Locusta migratoria solitaria

iAi ,, Locusta migratoria migratorioides ( A ) (A) Locusta migratoria solitaria (N) 9)

99

99

99

w

34.2D 11.5D

Lygaeidae Blissus leucopterus Oncopeltus fasciatus Notonectidae Notonecta glauca Pyrrhocoridae Pyrrhocoris apterus 99

Per cent lipid

,

9,

I

3.2W 3-8W 2.6W 3.3w 3 w 24W 2.8W 2.8W ll*ID 3-6W 14.0D 7.5D continued

79

L I P I D METABOLISM A N D F U N C T I O N I N INSECTS

Insect

Stage

Locusta migratoria migratorioides

(N) ?(A) $.(A) (A) (A) (A) (A) (A)

,>

99

Locusta pardalina solitary Locusta pardalina solitary Locusta pardalina gregarious 9,

,,

9,

Per cent lipid 10-7D 10.4W 147W 2.9W 12.8D 3.7w 14-6D 10 w 5.3D 9.3D 3.8W 3-2W 12 w 3.5w 25w

Schistocerca gregaria Schistocerca gregaria solitary (N) Schistocerca gregaria gregarious (N) Pezotettix giornae ?(A) Melanoplus atlanis (A) Melanoplus differentialis (E) 99 (A) Sphenarium purpurescens (A) Bacunculidae Dixippus morosus (A) Blattidae BIatta orientalis (A) ,* (A) BlattelIa germanica (N) ,, ?(A) d(A) (A> Periplaneta americana ?(A) $.(A) (N) ?(A) $.(A) Tettigoniidae Tettigonia uiridisima (A) 9,

2*4W 4.3w 3.2W 5.7w 4.8W 1-7W 17.1D 13.9D 14.5D 7.7w 8.9W 7-1W

9,

9

9,

9,

9,

73

3.8W

ODONATA Aeschnidae Aeschna sp. ISOPTERA (unspecified)

25w

(N)

6.1W

(L)

5-6W

(L) ?(A) $.(A)

30-OD 3.3w 34w

DIPTERA Anthomyiidae Pegomyia ulmaria Calliphoridae Calliphora erythrocephala

,,

')

continued

80


Insect Calliphora erythrocephala Calliphora vomitoria Lucilia sericata

,.

9,

99

9,

,,

99

Phormia terraenovae Phormia regina 99

3,

9,

,,

Chironomidae Chironomus sp. Tanytarsus lewisi Culicidae Culex pipiens 3,

9,

Y9

99

Anopheles atroparuus Anopheles stephensi Anopheles gambiae

Glossinidae Glossina palpalis Glossina morsitans

Muscidae Musca domestica

Oestridae Gastrophilus intestinalis Syrphidae Eristalis tenax

Stage

Per cent lipid 12.7D 7.8D 19 D 31 D 27 D 20 D 12.2w 8.9W 6.8W 52W 8.3W 11.7D 27.9W 3-7w 32*7D 4.0W 5-9w 57w 8.4W 15-OD 9.8D 22.7D 21.9D 16.8D 352? 2.7W 7.1W 6-3W 4.2W 2.5W 4.9w 4.2W 2.6W 2*9W

5w 1.8W continued

LIPID METABOLISM A N D F U N C T I O N I N INSECTS

Insect

Stage

81

Per cent lipid

HYMENOPTERA Apidae Apis dorsata Apis melrifera workers

,, ,, ,, 9,

,9

,, ,9

,,

9,

,, ,, ,, ,, ,, ,, ,, ,,

7,

),

,, 77

,, 9,

Y,

,, 9,

,, drones 79

freshly hatched wax producing forager winter worker Queen worker Queen summer winter

Formicidae Camponotus vagus Cremastogaster scuiellaris Tenthredinidae Croesus septentrionalis Vespidae Vespa cincta

(N) (P) (A) (PI (A) (A) (A) (A) (A) (L) (L) (P) (P) (A) (A)

4.7w 4.1W 0.9w 6.1W 1.5W 9.4D 10.2D 95D 7.3D 3-7w 4.9w 3.7w 5-3w 13.OD 12*2D

(L)

(A)

2.4W 10.9W

(PP)

26.OD

(I-)

67W

(E)egg; (L)larva; (N)nymph; (PP)prepupa; (P)pupa; (A) adult; (W) wet weight; (D) dry weight; * abdomens only. Modified from review by Fast (1964); consult Fast (1964) for original references and other data.

their modes of living and varying habitats. In most insects the female contains more lipid than the male, as lipid is a most efficient substrate for egg development. However, the reverse may be true for many species and this is especially evident when we consider the Lepidoptera (Table 11). This must be borne in mind since most of the author's research has utilized the American silkmoth Hyalophora cecropia, and this insect w ill be used to exemplify many points in this review. B . ALTERATIONS D U R I N G METAMORPHOSIS

In the course of a study on the alterations in juvenile hormone content during the life cycle of H.cecropia, we noted a sexual dimorphism in lipid content in the adult stage, and conducted a number of experiments to determine its basis (Gilbert and Schneiderman, 1961b). By preparing lipid extracts of insects at various stages in the life cycle the observations in Table I11 were made. Lipid content per individual larva

00 h)

TABLE I11 Lipid content of H. cecropia from egg to adult emergence

Stage

Number of animals

unfertilized eggs embryos 1st instar larvae 2nd instar larvae 3rd instar larvae 4th instar larvae 4th instar larvae 5th instar larvae 5th instar larvae 5th instar larvae prepupae Diapausing pupae 3 Diapausing pupae 9 Chilled pupae d Chilled pupae d Chilled pupae 0 Chilled pupae 0 Developing adult 3

750 1000 200 20 17 6 6 1 4 3 3 20 20 20 2 20 2 4

Time sacrificed freshly laid 7 clays

Fresh weight (€9

Lipid Lipid Dry Percent Grams as % as % weight dry lipid: fresh dry (g) weight individual weight weight

3.80 4.30

0.00025 0.00019

4.94 4.30

0.00012 0.00109 0.00922 0.0204 0-0416 0-0460 0-1189 0.2538 0-3723 0.2890 0.3150 0.3970 0.27 18 0.2435 0.2135 0.3100

4-53 2.27 0-98 1.34 1-28 1.40 1.63 1.86 5-68 7-34 5.57 8.68 6.44 5.43 4-24 8.67

0.516 newly hatched 0.957 early 15.879 late 9.122 early 19.463 late 3.285 early 29.233 mid 40-976 late shortened body 19.6648 1 month old 78.8 113.1 1 month old 6O, 6 months 9 1-50 6O, 6 months 8.439 6O, 6 months 91.50 6O, 6 months 10.067 day 2 14.30

0.125 0-131 2.316 1~207 2-300 0.334 5.141 6-690 5.361

24.22 13-69 14.59 13-23 11.82 10.17 17.59 16-33 27.26

-

-

2.209

26.19

2-485

24.69

-

-

-

Comment

includes chorion includes chorion and yolk 18.72 whole animal 16.56 whole animal 6.77 whole animal 10.13 whole animal 10.85 whole animal 13.77 whole animal 9-25 whole animal 11.38 whole animal 20.84 whole animal - whole animal whole animal whole animal 24-59 whole animal whole animal 17.18 whole animal - abdomens only and pupal cuticle

I 2

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I


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83

84


was observed to increase with age and size as expected, but the rate of increase varied in the various instars when compared to the net increase in non-lipid constituents. The newly hatched larva contains an appreciable amount of lipid, but this percentage decreases to a more or less constant value in the fourth and fifth instars (1-3-1.9% fresh weight; 9-3 to 13.8% dry weight), although the lipid content increases as the larva grows. The prepupa loses water rapidly and lipid content increases, so that at pupation lipids constitute between 5 and 7% of the wet weight of the insect. At this time a sexual dimorphism in lipid content is evident; the male pupa contains 50% more lipid than the female per gram fresh weight. This dimorphism also exists in pupae chilled for 6 months at 6". During the pupal-adult transformation this dimorphism increases markedly until in the adult moth the tissues of males contain about 5 times as much lipid per gram fresh weight as the tissues of the female. During adult development, the female apparently utilizes far more lipid than the male (see also Domroese and Gilbert, 1964). A 5 g previously chilled male pupa contains about 360 mg of lipid while the newly emerged adult moth contains approximately the same amount. For the female, the picture is quite different. A similar 5 g female pupa contains about 230 mg of lipid. The correspondingnewly emerged adult with eggs contains about 185 mg. In short, during adult development females appear to use more lipid than males. Because the abdominal tissues of female moths have a greater water content than those of males, the sexual dimorphism in lipid content is not as impressive when calculated on a dry weight basis. The results of a typical series of extractions reveal that during the first week of adult life the abdomens of males contain from 51-79% lipid per gram dry weight while the abdomens of females contain from 19-25%. After the sixth day of adult life there appears to be a rapid utilization of lipid in the males indicating a significant increase in catabolism at this time. The same is true of female moths, with the most rapid utilization occurring after the tenth day of adult life: senile females may contain as little as 8% lipid per gram dry weight as contrasted with about 20% in young females. To determine the extent of the lipid metabolism associated with egg development during adult development, a series of female pupae were , castrated and allowed to develop. Two days after eclosion the moths were extracted and the lipid content determined. The abdomens of castrated females contained about twice as much lipid as normal eggless female abdomens but still less than as much as male abdomens (less

+


85

than 3% on a fresh weight basis). Thus, although egg development involves some utilization of lipids, it cannot by itself account for the sexual difference in lipid content between males and females. It also appears that during adult development both normal and castrated females utilize far more lipid than males. Castration of males had no effect on their lipid content. When ovaries were implanted into male pupae, the adults which emerged contained large masses of eggs. Extraction revealed that these egg-filled male abdomens contained less lipid than normal abdomens but twice the amount of those of castrated or normal females. This result implies that egg development involves lipid catabolism but that certain non-ovarian tissues of the developing female utilize more lipid than corresponding male tissues. Parabiotic union of male and female pupae resulted in parabiotic moths. This technique resulted in the elevation of the lipid content of the female but a normal content in the male. What conclusions can we draw from this data? As was expected, the lipid content of individual larvae increased with size and age reflecting the synthesis of lipid from leaf material. Since no attempt was made to sex the larvae, nothing can be said regarding any sexual dimorphism in lipid content at this stage. The 50% decrease in lipid content per individual between egg and first instar larva indicates lipid utilization during embryogenesis. Although we shall return to this point later, this does confirm observations made on insects of other orders (Rothstein, 1952)and on B. mori (Niemierko et al., 1956), although the lipid content is much lower in H. cecropia eggs (as per cent fresh weight) than in the diapausing egg of Bombyx. This could be due to both the greater weight of the chorion of the Cecropia egg and to greater substrate storage in the diapause egg which supports a living embryo over winter. In the fifth instar larva when the water content reaches the extraordinarily high level of about 90% of the total weight, the animal begins to synthesize lipid at a high rate. The fifth instar larva has about five times as much total lipid as the early fifth and a lower water content. During spinning, when a large quantity of protein is used for constructing the cocoon, there is a further increase in total lipid and a loss of water. Whether this large increase in lipid during spinning reflects a release of lipids firmly bound to the proteins used in construction of the cocoon or whether the increase is a result of enhanced lipid synthesis has not been definitely answered. The fact that male pupae and adults contain more lipid than females seems peculiar to Lepidoptera. In most insects, the female usually contains more lipid (Table 11). Our results are consistent with the findings of

86

L A W R E N C E I . GILBERT

Niemierko et al., (1956; see also Vaney and Maignon, 1906; TimonDavid, 1930; Yamafuji, 1937) in Bombyx mori, and Demyanovsky and Zubova (1957) in Antheraea pernyi. Niemierko and his colleagues found that the lipid content of newly emerged male Bombyx was 48% per gram dry weight while that of the female was only 25% (17.5% in males and 6% in females on a fresh weight basis). The females used 50% of their stored lipid between spinning and adult emergence while the males utilized only 30%. In A . pernyi, Demyanovsky and Zubova found the sexual dimorphism in lipid content evident as early as the fifth instar larva and it continued until adult emergence. Our results (Gilbert and Schneiderman, 1961b) extend this dimorphism to a total of seven families of Lepidoptera. The above results indicate that lipid is used during the pupal-adult transformation of female Lepidoptera but that if any lipid is used for energy to underwrite the many syntheses occurring during construction of the male adult, other substrates are presumably being converted to lipid. Biochemical systems that accomplish such conversions have already been established (see Section IV). Why is there a lipid-sparing system operating during adult development of male Cecropia? The higher concentration of lipid in the male moth is most likely correlated with mating behaviour, since the male moth flies relatively great distances in search of the virgin female. As we shall see subsequently (Section 111) the male appears to utilize lipid as the primary substrate for flight. The monarch butterfly is a continental migrant and also utilizes lipid during flight. Prior to migration this specieslays down a store of fat as do migratory birds (Beall, 1948). Since its only food is nectar which does not contain lipid, it evidently converts carbohydrates to lipid during periods of repose and then uses it during flight. In migratory moths, it appears that there is no sexual dimorphism in lipid content (Williams, 1945) since both sexes migrate and presumably use equal amounts of lipid. The fact that the female Cecropia moth utilizes a significant portion of her pupal lipid during adult development and contains much less than the male, can be explained in part by her adult role. First, the female converts a large percentage of her endogenous substrate into the egg (yolk, cytoplasm, chorion, etc.). Secondly, the female does only limited flying after emergence and therefore needs little flight fuel. A by-product of the female’s lipid utilization during adult development is metabolic water, which is reflected in the greater water content of the female as compared to the male. During adult life, lipid appears to be the favored substrate of both


87

sexes. The utilization of lipid at this stage when the animal has left its desiccation-proofcocoon and heavy pupal cuticle, provides these moths with enough metabolic water to offset desiccation. It is possible that the male converts protein to lipid during the first week of adult life when the lipid content drops only slightly, since there is very little carbohydrate present at this time (Domroese and Gilbert, 1964). Perhaps these non-feeding adults ultimately die of lipid depletion. What is the basis of the sexual dimorphism in lipid content in these Lepidoptera? Our data reveal that egg development can explain in part

0.72

1

0.68 I

l

4

l

I

6

l

l

8

l

l l 10

l , I2

l

l 14

Day

l

l

16

l

l 18

I

, 20

I

t

1

I

P

3

I

5

Adult emergence

FIG. 1. The respiratory quotient of male H. cecropia during adult development and adult life. Determinations were made on five animals, each designated by a different symbol. (From Domroese and Gilbert, 1964.)

the low lipid content of female moths. However, the surgical operations performed never reversed or even equalized the lipid differences between the sexes. Since this dimorphism exists in the pupal and even in the larval stages (Demyanovsky and Zubova, 1957) we believe it to be a genetic difference which may find its first phenotypic expression during embryonic life. The above experimental findings were based almost entirely on direct lipid extraction of animals at different stages of development. To explore this sexual dimorphism further, we (Domroese and Gilbert, 1964) determined the respiratory quotients (RQ) of both sexes of H . cecropia during pupal-adult development. The results of these determinations show a differencein pattern between males and females (Figs. 1,2). In the female, kA.1.P.

4

88


the RQ decreases from higher values early in development to values between 0.75-0.80 shortly after the first week of development. The RQ remains at this characteristic level throughout the rest of development and in adult life. In terms of the role of lipid as an energy source for development, the RQ pattern suggests that after the first week of development lipid provides a greater portion of the energy expended in the female. In the male, the RQ is maintained for the most part at a level well above 0.80 until the seventeenth day of development, at which time it falls to levels indicative of greater lipid utilization. The RQ persists at this lower 1.00

8

0.92

c

o*68 4

6

8

10

I

12

I

I 1 1 14 16

I

I I8

I

1

1

I

2 0 t l

1

1

3

1

I 5

Adult

Day

emergence

FIG.2. The respiratory quotient of female H. cecropia during adult development and adult life. Determinations were made on five animals, each designated by a different symbol. (From Domroese and Gilbert, 1964.)

value during the remainder of adult development and in adult life. This drop in RQ characteristically occurred simultaneously with the appearance of the large dark pigmented wing spot on the seventeenth day of development. This RQ pattern as well as the data previously cited indicates that the male uses more of the non-lipid substrates for energy during adult development with a conservation of the lipid stores for use during adult life. In Section 111, we will discuss in detail the role of lipid in the adult life of the male moth. Among the various deductions that can be made regarding the above/ one fact is quite clear. The amount of lipid varies considerably during the life of an insect as well as between various species. The difference in the quantity of lipid expressed as per cent fresh weight has been reported to


89

vary from 1% in some Lepidoptera to 50% in the beetle Puchyrnerus (Niemierko, 1959). Examples of the high lipid content of some insects can be seen in the older work of Maciuca (1935) who showed that in the fall, Pyrrhocoris upterus contains 32.7% lipid (dry weight) while Fulton and Chamberlain (1934) noted that the lipid content of E. tenellus may rise as high as 42.2% of the dry weight of the female. Sinoda and Kurata (1932) showed that ether extracts of Dermestes larvae account for as much as 47% of the dry weight of the insect. As Scoggin and Tauber (1950) point out, many factors influence the lipid content of insects including stage of development, nutrition, environmental temperature, sex, starvation, diapause, cold hardiness, whether migratory or not and finally the systematic position of the organism under study. Rather than pursuing the most laborious task of discussing the lipid content of diverse insect species, let us now turn to the qualitative nature of insect lipids. C . NATURE OF INSECT LIPIDS

1. Glycerides In general, almost all of the compounds listed in Table I have been identified in one insect or another. These will be discussed in some detail throughout the paper, but there is no doubt that the major lipid component in insects as in other animals, is triglyceride (TGL) : the glycerol esters of long chain fatty acids. We can think of triglycerides as a secondary energy source since they must be hydrolyzed before energy can be made available to the cell. This results when the individual fatty acids cleaved from the glycerol moiety undergo B-oxidation within the mitochondria (see Section 111). In general, these TGL form the greater part of the lipid content of the insect at all developmental stages (Fig. 3). In Muciosiphium burri, about 80% of the extractable lipid is TGL and less than 3% exists as unesterilied fatty acid (FFA) (Strong, 1963a, b). This predominance of neutral lipid has been demonstrated in many insects, both in total body extracts (cf. Fast, 1964) and isolated fat body (Chino and Gilbert, 1964, 1965a; see also reviews of Kilby, 1963 and Gilby, 1965). Large quantities of FFA have been reported in the older literature (cf. Scoggin and Tauber, 19Sl) but in many cases one can “ see” the esterified lipids hydrolyzing before one’s eyes while reading the methods sections of these papers (see, however, Albrecht, 1961). In a study of the lipid of adult Anthonornus grundis by silicic acid chromatography, Lambremont et ul. (1964) showed that newly emerged

90

LAWRENCE I . G I L B E R T

adult boll weevils had 2 4 % body lipid of which only 2% was triglyceride. After 2-3 weeks of feeding, non-diapausing adults contained 6 1 0 % lipid (40-60% of which is TGL), whereas diapausing adults had 18-25% body lipid containing 75-85% TGL. It thus appears that lipid serves as the energy source for this diapausing insect which must last over winter, a suggestion made earlier by Braze1 and Newsom (1959).

Tube no.

FIG.3. Column chromatography of lipids from male H. cecropia moths. The column support was Florisil and elution was according to Chino and Gilbert (1965a). Tubes 12 to 22 represent triglycerides. (From Domroese and Gilbert, unpublished observations.)

Lambremont and his colleagues also demonstrated that the major fatty acids in the boll weevil were oleic and palmitic acids, and that the diet of the adult was the determining factor controlling the type of fatty acids incorporated into TGL.

2. Fatty acids (a) General characteristics. Protoplasmicfatty acids usually vary from C2to C,,in chain length and are most commonly composed of an even number of carbons, with oleic acid being the most abundant in nature. Of the many unsaturated fatty acids, the majority have a double bond between Cgand Clo (Fig. 4). Isomers of any particular fatty acid differ from one another by the position of the double bond, cis-trans isomerism, or both. There is a large variety of fatty acid types including


91

branched, unsaturated (up to six double bonds), hydroxy saturated or unsaturated, and cyclic. Saturated fatty acids having ten or less carbon atoms are liquid at room temperature and are thus easier to handle in experimental situations although more prone to auto-oxidation. All fatty acids with the exception of acetic acid have a density of less than one. The most common form for fatty acids in living things is as constituents of glycerides which are usually mixed (i.e. contain more than one variety of fatty acid per molecule). Although we speak of unesterified fatty acids as free fatty acids, in the cell or in the hemolymph they are usually in the form of the cationic salt (e.g. sodium palmitate) and can be referred to as palmitate or oleate rather than palmitic acid or oleic acid. 0

1I

CH3(CH2)7CHaCH2(CH&C--OH I. Stearic acid

0

It

CH~(CH&CHL-CN(CH~)~C-OH 11. Oleic acid (AS:l0) 0

CH3(CHa)rCHSHCHaCH=CH(CH& !! -OH 111. Linoleic acid (Aa:loJ2:13 )

i

CH3CH2CHSHCHaCH=CHCH2CH=CH(CH2)7 -OH IV. Linolenic acid (A9:10.12:13.15:16 1

FIG.4. The structure of some important C,, fatty acids.

(b) Fatty acid composition. The fatty acid composition of numerous insect species has been reviewed by Fast (1S64), and only the more recent work will be discussed here. The older literature suffers from a lack of sophisticated analytical tools (i.e. gas chromatography) and in many cases the unsaturated fatty acids were handled without respect, undoubtedly leading to oxidative deterioration in many cases. In a study of the lipids of homopterous insects, Strong (1963a) determined the fatty acid composition of 21 species of aphids and 6 species of leafhoppers. The percentage of the various fatty acids varied quite a bit between groups and even between species of a single group. In general, the aphids had a relatively large percentage of C14saturated fatty acid while the leafhoppers had a predominance of C18(especially

92

L A W R E N C E I . GILBERT

C,,,,) fatty acid. Strong concludes that “the fatty acid composition of an aphid appears to be a species characteristic not especially influenced by the host plant.” Honeydew secreted by Myzus persicae contains for the most part free sterols and fatty acids and appears to originate from the phloem sap (Strong, 1964). It seems from Strong’s work that the fatty acid composition of a species is rather specific and if this is true, it may be that gas chromatography is the “ultimate weapon” for the taxonomist. In examining the fatty acid content of the boll weevil, Lambremont and Blum (1963) identified 23 fatty acids varying in chain length from six to twenty carbons. Of these, 8 fatty acids (myristate, palmitate, palmitoleate, heptadecanoate, stearate, oleate, linoleate, linolenate) constitute 98% of the total, with 62% of the fatty acids containing at least one point of unsaturation. Palmitate makes up about 31% of the total while oleate constitutes about 30% of the total. Of the eight predominant fatty acids in the boll weevil, all but palmitoleate, heptadecanoate and stearate were identified in Drosophila melanogaster (Anders, 1960). It is possible that Anders’ paper chromatographic system was too insensitive for detection of the other three. Fawzi et al. (1961) have also shown that palmitate and oleate are the most common fatty acids in the TGL of Locusta migratoria. Almost all of the studies on fatty acids in insects utilized extraction of whole insects and generally palmitate and oleate predominate. The situation in the arachnids seems to be similar (Blum et al., 1963). Other more exotic fatty acids have been identified in insects and insect products, but are the exception rather than the rule. In bees for example, hydroxy fatty acids are common in queen bee larvae and royal jelly. Royal jelly is of course the nutrient fed to young larvae causing them to develop into queens. In 1957, Butenandt and Rembold identified 10-(OH)-decenoic acid in royal jelly. Weaver and Law (1960) studied the heterogeneity of fatty acids in this most interesting nutrient and showed the presence of: decandioic acid; monohydroxymonocarboxylic acids ; dihydroxymonocarboxylic acids. Weaver et al. (1964) reported that 9-13% of the dry weight of royal jelly is lipid of which 90% is FFA. Pain and her colleagues (1962) had demonstrated previously that royal jelly contains adipic, pimelic and suberic acids. Brown and his group (Brown and Felauer, 1961; Brown et al., 1961) showed the presence of 9-(OH)-dec-Zenoic acid, lO-(OH)-dec-2-enoic acid, 10-(OH)-decanoic acid, dec-Zendioic acid, p-hydroxybenzoic acid and sebacic acid. The presence of these acids is now known but their function is still a matter of speculation. They may have an anti-


93

bacterial action (cf. Slepecky and Gilbert, 1962) or may have morphogenetic effects in inducing those larvae fed on royal jelly to develop into queens. Several of these fatty acids and their derivatives have been identified in the products of hydrolysis of queen bee larval lipid (Pain et al., 1962). Identified by the French group in larval lipids were myristate, palmitate, dec-Zenedioic acid, suberic acid and trans-10(OH)-dec-Zenoic acid. This latter acid accounted for about 20% of the total fatty acids present but its function is unknown. The wax of Apis mellifera was thought to be composed largely of fatty acids and alcohols containing the even numbered homologues of CZ4to C34fatty acids (Chibnall et al., 1934) but the use of gas chromatography has revealed the presence of some odd numbered fatty acids as well (Downing et al., 1961). The cuticular wax of B. mori is composed for the most part of fatty acid-alcohol esters with fatty acid chain lengths of from c16 to C,, (Amin, 1960; Bergmann, 1934; Shikata, 1960). The most complete survey by one individual of the fatty acid constitution of insects is that of Barlow (1964), who by gas chromatography analysed 30 species of insects (Table IV). Unfortunately, only one analysis was conducted on most of the species due to lack of material. Barlow’s findings indicate that the fatty composition is characteristic of the species. For the family aphidoidea (Homoptera) for instance, more than SO% of the fatty acid was saturated C14 whereas in most species outside of this group, this fatty acid only constituted about 15% (19 to 60%) of the total. All the Diptera had a high proportion of while most other species yielded no more than about 2.2% of this acid. That some insects can synthesize this c16:1is no longer in doubt since A . aflnis contained a high concentration of this fatty acid even when grown on a diet devoid of it. In general, Barlow’s work suggested that the stage of the insect had little effect on the fatty acid composition (see, however, the subsequent discussion of the work of Herodek and Farkas). [The new rash of papers on fatty acid composition of insects (and other living things) is due in great part to the classic efforts of Martin and Synge (1941) who first described the concept of gas chromatography in which gas is the transient phase rather than a liquid (as in paper or column chromatography).] A relationship appears to exist between the temperature to which a tissue is constantly exposed and the degree of unsaturation of the fatty acids present in the tissue. This appears to be true of all animals and has been demonstrated in insects. Newly emerged female Culex tarsalis for example possess a greater percentage of unsaturated fatty acids

TABLE IV Total fatty acid composition of some insect lipid

% fatty acid1 Stage

Insect Coleoptera Calosoma calidum (Fabricius) Harpalus caliginosus (Fabricius) Sitona scissifrons (Say) Leptinotarsa decemlineata (Say) Trirhabda virgata Lec. Pyropyga decipiens Harris Tetraopes tetraophthalmus (Forster) Neuroptera Corydalus cornictus (Linnaeus) Trichoptera Unidentified Homoptera-Aphididae Dactynotus ambrosiae (Thomas) S.L. Aphis pomi de Gier Acyrthosiphon pisum (Harris) Tuberolachnus salignus (Gmelin) Pemphigus populicaulis (Fitch) Homoptera-o ther Cicadidae unidentified Campylenchia latipes (Say) Stictocephala diceros (Say) Philaenus spumarius (Linnaeus)

12

14

16:O

16:l

18 13 10 11 9 11 8

5

2 3 1 1 1 2

adult adult adult Pupa adult adult adult

&r

Amount % ctslmin % ctslmin % Amount % recovery recovery (mg) fresh (mg) fresh wt. Wt.

s

I -

ctslmin

%

ctslrnin

recovery

%

m

recovery

0

479 471

-

113 115

0.01 0.01

8 7

0.00 0.00

78 65

-

2,550 2,700

0.25 0.27

-

-

364 366 291 342 332 349 341 249 334 349 316 273

7.8 7.7 6.5 5.7

2,410 2,115 4,250 3,006 23,760 21,055 17,830 17,840 22,9W 19,840 25,500 23,900

0.24 0.21 0.43 0.30 2.4 2.1 1.8 1.8 2.3 2.0 2.6 2.4

162 150 200 242 303 382 328 249 194 270 348 273

0.02 0.02 0.02 0.02 0.03

101 49 31 57 48 40 87

2.2 1.0 0.5 0.9 1-2

120,800 75,400 118,040 107,400 52,550 33,000 64,300 34,700 4,730 13,730 78,780 58,900

12.1 7.5 11.8 10.7 5.2 3.3 6.4 3.4 0.47 1.4 7.9 5.9

47,470 51,620 37,460 36,550 122,530 142,750 108,490 109,900 335,180 239,980 300,330 230,950

4.7 5.2 3-7 3.7 12.3 14.3 10.8 11.0 33.5 24.0 30-0 23.1

8.0

8.1 5.4 5.9 8.6 8.4 4.7 4.2

0.04

0.03 0.02 0.02 0.03 0.03 0-03

44 2 4 30 45

0.9 1.6 1.0 0.04 0.08 0.05 0.8

2 W

t:

-

~

E

~-

From Chino and Gilbert (1965~). Animals were sacrificed one day after injection of loEctslmin. The control consisted of injecting the ['*C]glucose into a pupa and sacrificing the animal immediately by freezing in a bath of dry ice and acetone. These animals were then analysed in an identical manner to the experimentals.

2 2 cl

=] 2

5

.-cl 2 CI

P

W

150


The radioactivity found in the long chain FFA fraction was low but significant. Although a considerable amount of the label from [“CIpalmitate was incorporated into glycerides, we could detect no sign%cant incorporation into glycogen (Table XIII). Similar results were obtained with labelled acetate; that is, high incorporation into lipid, a large quantity combusted to C02, but no appreciable amount found in carbohydrate. These results support our previous contention that when FFA are synthesized they are readily incorporated into glycerides ; but more relevant to the present section is the demonstration that Cecropia can convert carbohydrate to lipid, but not the reverse. There have been several suggestions that insects possess the mechanism for converting lipid to carbohydrate, but for the most part this evidence has been circumstantial or only suggestive (Nair and George, 1964; Hitchcock and Haub, 1941 ; Levinson and Silverman, 1954). As far as this author is aware, the conversion of lipid to carbohydrate has not been unequivocally demonstrated in any metazoan although it is a reality in plants and certain microorganisms. In a careful study of the larval-pupal moult in Cecropia, Bade and Wyatt (1962) found that it was unnecessary to invoke a lipid to carbohydrate conversion to explain the changes in nutrient reserves during pupation, although by examining the changes in substrate one may have assumed such a conversion. It appears that the seeming increase in carbohydrate can be explained by a transfer of material from the cuticle to the interior. In fact, the glyoxylate cycle could not be demonstrated in Cecropia and this is prerequisite for such a substrate interconversion (Bade, 1962). It should be noted however, that isocitric lyase activity has been detected in pre-pupae and young pupae of Prodenia (Carpenter and Jaworski, 1962). According to these authors, the enzyme which converts isocitrate to succinate and glyoxylate is most likely maximally active at those developmental stages when the insect is consuming its lipid store. It may be that the past failure to demonstrate this enzyme in insect material is due to utilization of the wrong stage in the life history of the insect. The complete glyoxylate cycle still remains to be demonstrated in insects. Taking a teleological view, one can find a number of reasons why an insect, or any animal for that matter, would find it more beneficial to convert carbohydrate to lipid rather than the converse. The reasons have been stated by many and include the fact that lipid contains more energy per unit weight and yields more metabolic water than carbohydrate. Only if the insect lives in an environment of low oxygen tension or is dependent on anaerobic metabolism, would carbohydrate be

+?

21

7

Irr

r

P

U

TABLEXI11

z1

The fate of (l-14C)palmitate injected into pupae a n d developing adults of H. cecropia Glycerides Stage

Sex

Control (chilled pupa) Chilled pupa

Developing adult (3-5 days) Developing adult (1517 days)

Male Female Male Male Female Female Male Male Female Female Male Male Female Female

i

I {

From Chino and Gilbert (196%).

Amount % (mg) fresh wt. 403 336 386 371 298 333 349 439 274 247 345 356 201 232

8.7 8.0 5.5

5.8 7.1 8.6 4.8 4.7 8.8 9.2 4.3 4.2

ctslmin

)

% recovery

2,635 1,805 346,400 345,000 277,500 323,000 352,700 377,500 379,000 379,800 278,100 382,300 467,000 322,300

coz

Glycogen

A

i

U

0.03 0.02 34.6 34.5 27.8 32.3 35.3 37.8 39.9 38.0 27.8 38.2 46-7 32.2

zm

A

I

Amount % (mg) fresh wt. 85 98 76 84 85 120 55

107 60 59 0 0 21 53

-

1.7 1.8 1.5

2.0 1.4 2.0 1.0 1.1 0.0 0.0 0.45 1.0

cts/min

%

j-2

recovery 1,824 1,955 1,210 1,025 850 1,170 525 1,320 845 810 350 645

0.18 0.19 0.12 0.10 0.09 0.12 0.05 0.13 0.08 0.08 0.03 0.06

recovery 0

!2

89,930 76,620 . 55,230 76,090 56,790 63,170 61,790 80,720 194,000 148,900 226,400 138,900

-

-

8.9 7.7 5.5 7.6 5.7 6.3 6.2 8.1 19.4 14.9 22.6 13.9

E 9

1:

z

2 cI

2: c1

1: 0 +I vl

152


preferred. Glycolysis, although inefficient, will yield enough energy for life processes to continue up to a point, but the oxidation of fatty acids will not occur in the absence of oxygen. Perhaps an intensive investigation of insects inhabiting an ecological niche where the pOz is low would uncover instances of a complete glyoxylate cycle and demonstrate lipid to carbohydrate conversion. Until such data are forthcoming, we must assume that this conversion does not take place in the Insecta. Thus far we have not discussed waxes, hydrocarbons, or the so-called cuticular lipids. These compounds do not really fit into the topic under consideration but are still lipids and are vital to the existence of the insect.

V. HYDROCARBONS A N D WAXES A . CUTICLE

It is now taken for granted that one or more layer of lipid on the exterior surface of the cuticle is in part responsible for the resistance of the insect to desiccation. This relationship between the cuticular lipid and its role in minimizing transpiration was demonstrated more than thirty years ago (Ramsay, 1935). In his review of the insect cuticle, Beament (1964) notes that “organized lipid plays the most important role in the passive regulation of water movement and in modifying the adhesion of water to the surface of cuticular membranes” (p. 68). According to Beament, the physico-chemical properties of the cuticular lipid layer suggest that the lipid acts as a “valve” or “rectifier”. He visualizes the organization of the lipid of the cockroach (P. americana) cuticle as consisting of a tightly packed single monolayer of lipid molecules up against the cuticular surface. Overlaying this monolayer is a “grease” twenty to thirty times as thick as the monolayer but arranged in a random manner. It is the monolayer, however, that is more impermeable to water. This arrangement probably varies in different insects but little research effort has been dedicated to the problem in insects other than cockroaches. In 1955, Beament suggested that shorter chain molecules may act as solvents and promote the tightly packed arrangement of the waterproofing layer. Unfortunately, very little is known of the biochemistry of this lipid, although it is a most promising area of investigation. There have however, been several chemical analyses of insect cuticular lipids and some may shed light on the mechanism by which this material is synthesized. The total hydrocarbon content of M . domestica is composed of unsaturated, cyclic and straight chain compounds (Louloudes

L I P I D METABOLISM A N D FUNCTION I N INSECTS

153

et al., 1962). These range in chain length from cl6 to CS5and are composed of both odd and even numbers of carbons although the odd numbered compounds predominate. This appears to be true for the Mormon cricket (Anabrus simplex) as well (Baker et al., 1960). The cuticular hydrocarbons of Musca differed from the total body hydrocarbons by having a higher concentration of lower molecular weight compounds. This finding appeared to corroborate Beament’s (1955) suggestion regarding the presence of “solvents ” in cuticular lipid. In 1963, Gilby and Cox reported on their analysis of the wax of the cast cuticle of P . americana (the same insect investigated by Beament). By utilizing column and gas chromatography as well as infra-red spectrometry, they demonstrated that hydrocarbons compose 75% of the cuticular wax. Fatty acids, esters and aliphatic aldehydes are present in roughly equal amounts and together with a trace of sterol account for the remaining 25%. The major component was identified as a C2, unconjugated diene, probably heptacosa-9,18-diene (see also Baker et al., 1963). The largest percentages of saturated hydrocarbons were C25and c 2 6 although most of the hydrocarbons were unsaturated. Table XIV reveals that the main fatty acids present are c16 and C18 while the predominant aldehydes are C14 and CI6. Of great interest is the absence of alcohols, except for sterols that comprise less than 1% of the ‘total wax. This finding negates the argument that the cuticular lipids are similar to other waxes. The results are remarkably similar to those reported for the Mormon cricket (Baker et al., 1960). The main points of difference are that the cricket wax contained more varieties of hydrocarbons and these were saturated; the cricket wax contained polyunsaturated C18 fatty acids whereas they are absent from the cockroach wax; the cricket wax was devoid of aldehydes but contained polymeric resins. As Gilby and Cox suggest, it is possible that these cricket resins are derived from aldehydic precursors. In 1962, Gilby reported the absence of natural volatile substances in the cockroach wax and suggests (Gilby and Cox, 1963) that the fluidity of the “grease” can be accounted for by involatile liquid constituents of the hydrocarbons and free acid fractions. Although the cuticular wax hardens when removed from the cuticle, it remains fluid in vivo. The Australian investigators propose that in vivo, an inhibitor prevents the chemical reaction leading to wax hardening or that physical factors are responsible. As noted previously, Beament suggested that a monolayer was largely responsible for prevention of water loss. If alcohols are absent from the wax, what molecules are present that would form a monolayer with

154


TABLE XIV Composition of the cuticular wax of Periplaneta americana Chemical Class

% of wax

Components of class

Hydrocarbons

75-77

C27 n-alkene (heptacosa-9, 18diene) C25, C26, C27 n-alkanes C17-C20, C28, C29 n-alkanes C14, C16, C18 n-alkanoic acids C13, C15, C17 n-alkanoic acids C15, C16, C17 branched alkanoic acids C18 n-alkenoic acid (octadec-9enoic acid) unknown unsaturated C14, C15, C16 n-alkanals C13, C17-C25 n-alkanals C16, C18 branched alkanals C18 n-alkenal (octadec-9-enal)

Fatty acids

Aldehydes

Esters Sterols

7-1 1

8-9

Relative abundance of components weight % of class 66 15, 14, 12 each < 1 5 , 24, 23 each ,1 each 1-2 35 6 16, 10, 14 each 2-8 0.5, 3 3

3-5

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