Advances in Insect Physiology, Volume 12

Advances in Insect Physiology

Volume 12

This Page Intentionally Left Blank

Advances in Insect Physiology edited by

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

Volume 12

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

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

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

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

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

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

Contributors Fotis C. Kafatos

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

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

F. Poole’

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

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

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

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

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

This Page Intentionally Left Blank

Contents Contributors

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

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

v

. .

1

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

.

17

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

.

63

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

239

Subject Index

325

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

Cumulative List of Authors

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

345

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

347

Cumulative List of Chapter Titles

This Page Intentionally Left Blank

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

.

.

.

. .

.

1 2 4

.

5

*

.

. . .

9 11 12 12

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

2

FOTIS C. KAFATOS

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

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

CELLULAR POLYMORPHISM

3

nuclear branches, highly extended in the larval cell, are pulled together into a compact mass, surrounded by a thin layer of cytoplasm (Fig. 1 in Selman and Kafatos, 1974). Evidently, the pupal cells are at a developmental standstill. However, during the subsequent adult development they grow again and undergo a dramatic metamorphosis. First they secrete a thin (<1 pm), loosely fibrous cuticle, strikingly different from that in the larva. Then their mitochondria proliferate and become intimately associated with deep plasma membrane infolds which develop, first in the basal and then in the apical side of the cells. At or near the time of adult emergence, much of the cell consists of these mitochondria-membrane associations (Fig. 2 in Selman and Kafatos, 1974). This structural specialization corresponds to the new function of the cells: the gland now secretes copious amounts of essentially isotonic KHC03 solution (Kafatos, 1968). Physiological experiments suggest that active transport of K + by the cell, from the blood into the lumen, may drive the production of the secretion as a whole. The rate of secretion is very high: the volume of liquid produced per minute approaches the volume of the secretory cells. The copious secretion promotes the digestion of the cocoon by the enzyme cocoonase, in those species which lack an escape valve (Kafatos and Williams, 1964). The transformation of the larval, predominantly cuticle-producing cells into the predominantly salt-secreting cells of the adult is under hormonal control (direct or indirect). The transformation occurs during the metamorphic moults, which are triggered by ecdysone acting in the near or total absence of juvenile hormone OH). If JH is injected into the pupa at the very beginning of adult development (Hakim, 1972), the cells become pupaladult intermediates, with variable development of the mitochondriamembrane associations. A cuticle is always produced, whereas the pupal cells apparently lack a cuticle (Hakim, 1972). However, the cuticle in juvenile hormone-injected animals is usually considerably thicker than in normal adults. In fact, in some cases (including some glands forced by transplantation and J H injection to develop beyond the pupal state twice in the presence of JH), the ultrastructural appearance of the cuticle is somewhat reminiscent of that in the larval duct (Hakim, 1972). Formally, this cannot be considered a case of reversal of metamorphosis, since the pupa does not produce a cuticle. It can be argued that the information for producing a larval-type cuticle is included in the “pupal gene set”, but is normally not implemented because secretion of any cuticle is blocked by a mechanism which thereafter becomes insensitive to the presence or absence of the hormone. In any case, the abnormal cuticle and the unequal development of the adult specializations of the labial gland in different JH-injected animals (Hakim, 1972) suggest that the metamorphosis of the labial gland may not be a unitary process under all-or-none control by the

4

FOTIS C. KAFATOS

hormone. The period of JH-sensitivity is limited t o the beginning of adult development (Hakim, 1972). The cells of the labial gland are polyploid and thus do not undergo cell division. Later than the period of hormone sensitivity, and at about the time the mitochondria-membrane associations begin to appear, DNA synthesis does occur. This could be associated either with further growth of the cells which is occurring at that time, or with a necessity for new copies of the genome for “reprogramming” into the adult state of differentiation. The results of inhibitor experiments suggest that the former explanation may be correct: the DNA synthesis can apparently be inhibited without interfering with the metamorphosis of the cells (Selman and Kafatos, 1974). In conclusion, the labial gland cells of saturniids are an example of sequential cell polymorphism under hormonal control. The transformation into the adult state does not appear t o require DNA synthesis. The existence of pupal-adult intermediates suggests that several, partially dissociable processes may be involved in this transformation. Salt secretion, the function of the adult state, is certainly not unique t o these cells-but it is so extensively developed as t o constitute a state of differentiation as distinct as, say, that of the cecropia mid-gut (Anderson and Harvey, 1966), or the Malpighian tubules (Berridge and Oschman, 1969). 3 The sphingid labial gland: a more complex case of cellular metamorphosis In the sphingid, Munduca sextu, the duct of the larval labial gland again undergoes transformation during metamorphosis (Hakim, 1972; Hakim and Kafatos, 1976). In this case, the larval duct has a cuticle thinner than that of saturniids, but similar in construction. The secretory division produces a lubricant, rather than silk fibres, and the duct may play a more active role than in saturniids, since it has moderately prominent mitochondriamembrane associations (Figs 7-9 in Hakim and Kafatos, 1976). The secretory division of the larval gland degenerates during pupation, and the duct transforms into the complex salivary gland of the adult (Hakim and Kafatos, 1974, 1976). In contrast t o the uniform saturniid adult glands, each of the paired sphingid adult glands is divided into four distinct regions (Hakim and Kafatos, 1974). In a posterior t o anterior division, these are region I, which consists of cells specialized for the production of invertase; region 11, the c d s of which produce a K+-rich salt solution and are as rich in mitochondria-membrane associations as the saturniid adult gland; region 111, which contains squamous cells with sclerotized cuticle, apparently functioning in the forward conduction of the dilute invertase solution produced

CELLULAR POLYMORPHISM

5

by regions I and 11; and region IV, which appears to function in the reabsorption of salts and the concentration of the saliva. In addition to the paired glands, a short common duct (region V) exists; it appears t o be derived from the larval common duct and t o be purely conductive in function. All four cell types of the paired glands are derived from the polyploid cells of the paired larval ducts (Hakim and Kafatos, 1976). During adult development the pupal cells, which were initially uniform in appearance, begin t o be distinguishable by their cytoplasmic growth pattern. Then they secrete distinctive cuticle, different for the various gland regions. Finally, they assume the cellular morphoIogy that corresponds to their respective adult functions. The origin of the adult regions from specific areas of the larval duct has been investigated by extirpation, transection and transplantation experiments (Hakim and Kafatos, 1976). While the cells are expressing their larval differentiation, they are already programmed fo\y specific adult states. At least in the fifth larval instar, particular areas of the duct are already determined to form specific adult regions. Preliminary experiments with earlier larval stages suggest that at least some determination may exist before the fifth instar. In the sphingid gland, sequential polymorphism is again punctuated by the moults of metamorphosis. The polymorphism is more dramatic than in saturniids, in that it results in the formation of several distinct cell types from the duct cells of the caterpillar. Moreover, one of these cell types secretes a digestive enzyme, invertase, which is undetectable in the larval gland. In this system, it is clear that a number of different (adult) determination states can coexist with the same (larval) state of overt differentiation. Since determination is the first step of differentiation, the results emphasize the temporal overlap between the states of sequential polymorphism,

4 The cocoonase organules of the silkmoth galea: multistage sequential polymorphism in epidermal derivatives Cocoonase is a trypsin-like proteolytic enzyme which digests sericin, loosening the silk fibres and permitting the moth t o escape from the cocoon (Kafatos and Williams, 1964; Kafatos et al., 1967a, b ; Hruska and Law, 1970; Hixson and Laskowski, 1970; Hruska et al., 1973; Felsted et QZ., 1973; Kramer et al., 1973). It is produced in the form of a zymogen, which is activated as it appears on the surface of the animal, one to two days before adult emergence (Berger et aZ., 1971; Berger and Kafatos, 1971a; Felsted et al., 1973). The cells which produce the zymogen are

6

FOTlS C. KAFATOS

lound in epidermal organules (Lawrence, 1966), in the lateral aspects of modified mouthparts, the maxillary galeae (Kafatos and Williams, 1964; Kafatos and Feder, 1968; Kafatos, 1970; Kafatos, 1972a; Selman m d Kafatos, 1975). The galeae first appear as two conical protuberances of the epidermis during the metamorphosis of larva into pupa. At that time they consist of a seemingly uniform population of epidermal cells, indistinguishable from those found in other parts of the integument. The cells produce the thick pupal cuticle and remain otherwise inactive throughout the pupal life. This epidermal cell population gives rise t o the zymogen-producing organules (luring adult development (the pharate adult stage). Formation of the organules presupposes action of ecdysone in the absence of JH: when sufficient JH is injected at the beginning of the moult cycle, so as t o cause I he development of a “second pupa” instead of an adult, formation of the Lymogen organules is blocked and the cells remain in the state characteristic of the pupa (unpublished observations). The period of JH sensitivity does not extend beyond the first two days of adult development. In gross terms, the sequential polymorphism is punctuated by the action of devclopmental hormones, and may seem t o be saltatory (from a cuticleproducing to a zymogen-producing state). Closer attention t o the process of zymogen organule formation reveals that this description is oversimplified (Kafatos, 1970; Kafatos, 1972a; Selnian and Kafatos, 1975). The hormonal state prevalent at the beginning of adult development does not bring about in one step the characteristic adult function (zymogen production), but instead initiates an entire sequence of “adult” developmental states, one of which corresponds t o production of Lymogen during a limited time period. This is particularly evident from the ultrastructural studies (Selman and Kafatos, 1975). Sequential polymorphism is saltatory in an overall sense, but gradual or multistage in detaiI. Early in adult development, the epidermis retracts from the pupal cuticle, leaving behind the moulting gel. Some cellular growth occurs, and then mitotic figures appear in the lateral epidermis of the galea. These are presumed t o be differential divisions, because of the vertical orientation of the spindle, which contrasts with the horizontal orientation in proliferative divisions of the insect epidermis (Lawrence, 1966). By day 5 of the three-week period of adult development, the epithelium is pseudostratified and several cell types begin t o be distinguishable. The organule forms from the specific association of three cells, which are probably derived from the differential divisions. Two of these cells become polyploid and are ultimately destined to synthesize zymogen (cells Z1 and Z 2 ) , and the third (cell D) forms a unicellular cuticular “main duct” which permits extrusion IWO

CELLULAR POLYMORPHISM

7

of the zymogen from the organule to the surface of the galea. These cells surround each other' in their apical region, forming three coaxial cytoplasmic layers, with the cytoplasm of cell D outermost and that of Z1 innermost; more basally, the coaxial arrangement is disrupted and the cells a e separate. The coaxial arrangement of the apical cytoplasmic regions is related to the morphogenesis of the main duct and of an attached cuticular valve, the end apparatus. The latter regulates extrusion of the zymogen into the main duct, from a storage cavity surrounded by cells Z, and Zz . Beginning with day 6, cells Z 1 and Zz retract from the surface, but during this process the intercellular junctions between Z 1 and Zz, Zz and D, and D and normal epidermal cells, respectively, remain intact. As a result, the formerly apical surface of cell D is drawn inwards and comes to line a narrow tubular extracellular channel; this surface then secretes a thin cuticular layer, forming the hollow tubular main duct. The formerly apical surfaces of Z1 and Zz secrete cuticular material, which makes up the end apparatus; cell Zz also contributes to the most basal part of the main duct, and to the joint between main duct and end apparatus. When completed, the main duct and the joint have a continuous epicuticular lining (cuticulin), and are thus presumably impermeable. By contrast, the end apparatus lacks such a lining and consists of fibrous cuticular walls, which are presumably permeable. The end apparatus includes a round ampulla and two long collecting ductules. The lumen of the ductules is free, whereas the ampulla is occluded by two dense fibrous cones, presumably preventing premature passage of the zymogen from the ductules into the main duct and thence to the surface of the animal. The respective cuticular structures are formed by the three cells of the organule on days 7 t o 9. The cuticulin layer, which is initially secreted in contact with numerous microvilli of cell D (Locke, 1969), constitutes a continuous barrier preventing occlusion of the duct by subsequently secreted cuticular material. By contrast, the material that will form the end apparatus penetrates freely into the long invaginated space created by the retraction of cells Z1 and Z2 away from the surface. Two long, microtubule-filled processes of cell Z1 (pseudoflagella) traverse this space and prevent its total occlusion by the cuticular material. The material is first deposited as a solid tubular mass, between the formerly apical surface of cells Z1 and Zz and the axial pseudoflagella. On day 9 this mass is suddenly reorganized into the loose fibrous wall of the end apparatus. The dense cones capping the ampulla are formed somewhat earlier, at the site occupied by the pseudoflagella, as the latter begin to degenerate. Further degeneration of the pseudoflagella creates the lumen of the collecting ductules.

8

FOTIS C. KAFATOS

As the cuticular mass secreted by cells Zl and.Z, transforms into the rid apparatus, these cells retract away, leaving an extracellular cavity 1)etween their apical surface and the apparatus. This cavity is now filled by ocoonase zymogen, which is synthesized and secreted by cells Z1 and Z 2 . \s zymogen accumulates, the cavity expands tremendously, reaching a ltmgth of 200 pm by day 15. It appears that the collecting ductules may c longate in paraIlel during this time, suggesting continued cuticle pro(luction, probably by cell Z 1 . Biochemical studies have revealed more definitively the time course of lymogen synthesis (Berger and Kafatos, 197 l b ; Kafatos, 1972a). The rate ( ) f S J nthesis is very low on days 7 and 8. Once the end apparatus and the \torage cavity have been formed, the rate begins t o increase substantially, t hanging from less than 1 per cent of total protein synthesis in the galea at the end of day 8 t o approximately 60 per cent of the total at the end of ctay 14. The absolute as well as the relative rate of zymogen synthesis increases by nearly two orders of magnitude during this &day period. Then, I he rate of synthesis decreases precipitously. The stored zymogen (nearly r ~ O p sper galea, or 2 ng per zymogen cell) is extruded through the end ipparatus and the main duct, and appears at the surface of the galea as tctibe cocoonase. The enzyme is used shortly thereafter, in conjunction tvith the KHC03 solution of the libiaI gland, t o open a hole in the cocoon I t the time of adult eclosion. The organules regress and remain functionless I hroughout the brief life of the adult moth. With respect t o zymogen production, the main period of differentiation lor cells Z1 and Z2 is day 9 t o day 14. We have called this period of rapid \ynthesis “full differentiation” or phase I1 (Kafatos, 1972a). The period of linite but low zymogen synthesis preceding it is phase I and the period of \hut-off is phase I11 (protodifferentiated and modulated states, respectively, ~ccordingto Rutter et al., 1968). The mechanisms regulating the transitions 1)etween the states are unknown. However, the kinetics of change in the tat? of zymogen synthesis during phase I1 are generally consistent with a bimple transcriptional control model (Kafatos, 1972a, b; Kafatos and Gelinas, 1974). According t o this model, at the beginning of phase I1 iranscription of the zymogen gene is set at a high, nearly maximal rate, and 1 e m i n s so throughout phase 11. The zymogen mRNA produced accumulates continuously because of its high stability and is translated at a constant efficiency, thus supporting an ever-increasing rate of protein bynthesis. Although the information necessary for testing this model in detail is not available, the key postulates of the model appear t o be ;enerally valid for highly specialized cells having a proIonged phase I1 (long time constant systems: Kafatos and Gelinas, 1974). These postulates are: a ,ingle copy of the specific gene per genome, high and essentially constant t

t

CELLULAR PO LY MORPH ISM

9

transcription of this gene during phase 11, high stability of the mRNA, and constant efficiency 'of translation (polypeptides produced per mRNA molecule per minute). With respect to sequential polymorphism, it is noteworthy that zymogen production is not the only differentiated function of cells Z 1 and Z 2 . At the beginning of adult development, the cells of the galea behave like ordinary epidermal cells, undergoing apolysis and secreting moulting gel. Following the mitotic divisions, cells Z1 and Z2 become distinguishable and their apical cytoplasmic regions undergo an intricate morphogenesis. Then these cells secrete specialized cuticular products. At the same time the ceIls begin production of zymogen at a low rate (phase I). When the cuticular valve is in place, zymogen production shifts to the fully differentiated state (phase 11); a low level of cuticle production may persist, at least for cell Z 1 . The end of zymogen production coincides with unknown cellular activities, leading t o zymogen extrusion and activation and t o the regression of the cells. Except for the initial hormonal requirements, nothing is known about the mechanisms controlling the progression through this sequence of distinct, temporally overlapping functions. One suspects that the timing controls are internal t o the organule, rather than external hormonal changes. Only development of the gland in organ culture could answer this question.

5 The follicular epithelial cells of silkmoths; biochemically defined sequential polymorphism

The follicular epithelium in silkmoths consists of a single layer of cells surrounding an oocyte and the seven associated nurse cells. The entire complex is a follicle. A linear array of attached progressively more mature follicles constitutes each of the eight ovarioles in the ovaries of a developing adult. The formation and early functions of the follicle have been reviewed recently (Telfer, 1975). In response t o the hormonal conditions which trigger adult development, the follicles begin their growth, emerge in sequence from the sheath of the germarium, and enter vitellogenesis. During vitellogenesis, intercellular channels open u p among the follicular epithelial cells, permitting access of the blood proteins to the oocyte surface. There, specific uptake of vitellogenin takes place, leading t o formation of yolk spheres within the oocyte. During this period, the follicular epithelial cells appear to help vitellogenesis not only by the formation of channels, but also by secretion of a histidine-rich product, probably a glycoprotein, which finds its way through the channels into the oocyte; this product may aid the specific

10

FOTlS C. KAFATOS

upt .ike of vitellogenin (Anderson and Telfer, 1 9 6 9 ) . The epithelium also appears t o produce a thin vitelline membrane between itself and the ooc yte. ,\t the end of vitellogenesis secretion of the histidine-rich product stops, tht. intercellular channels close up, and a substantially thicker vitelline membrane is deposited by the follicular epithelial cells. During this time, thc oocyte swells considerably by uptake of water (terminal growth phase; Anderson and Telfer, 1969). It is not known what, if any, contribution the epithelium makes t o the uptake of water. Following terminal growth, the epithelium secretes the largely proteinaceous eggshell or chorion. This last tie\ elopmental stage, choriogenesis, has been studied extensively io recent ) e m (reviewed in Kafatos et al., 1976; and Kafatos, 1975). Upon its conclusion, the follicular epithelium is shed (ovulation), and the oocyte, SUI rounded b y the fully formed shell, is ready for passage into the ovidict, tei tilization, and oviposition. rhe chorion consists of four size classes of proteins (A, B, C and D, re\pectively, in order of increasing molecular weight; Paul et al., 1972). O\ erall, the proteins are unusually small and very distinctive in amino acid c o inposition (e.g. 35 per cent glycine, but essentially histidine-free; comIwye the follicular cell product synthesized during vitellogenesis). At least 1 5 to 20 chorion polypeptide species can be distinguished by electrophoresis on SDS-polyacrylamide gels, and more than double that number on isorlectric focusing gels (Regier, 1975; Kafatos et al., 1976). Within each \i/r class the individual components are closely similar to each other; valuericing studies on A proteins suggest that the similarity is due t o Iiomologous amino acid sequences (Regier, 1975). At the outset of choriogenesis, the synthesis of proteins other than c:hurion declines precipitously. The various chorion proteins then begin to I N synthesized in sequence. There are gross changes in terms of synthesis of p'trticular size classes; thus, C proteins are produced early and their \>nthcsis is soon overtaken by A and B synthesis. In addition, there are numerous changes in the synthesis of various subclasses and of individual ptllypeptides. The absolute or relative rates of synthesis of a large number o I chorion components wax and wane individually during choriogenesis. Phese temporal changes could be described by a series of overlapping curves (svnthetic rate versus time), differing in shape, height, width, and time of aiiainment of the maximum value. Based on the pattern of chorion pioteins synthesized at any one time, eighteen distinct stages of choriog( nesis can be identified in Antheraea polyphemus (Paul and Kafatos, 107 5). Each lasts approximately three hours, and thus choriogenesis rc,quires approximately two days. An important observation is that progression through this sequence of dc velopmental stages is autonomous. When follicles are placed individually

CELLULAR POLYMORPHISM

11

in organ culture, in a completely defined medium, they proceed through the normal sequence of stages at the same rate as they would in vivo (Paul and Kafatos, 1975). Moreover, follicles in terminal growth can enter choriogenesis in culture. Thus, the sequential polymorphism of choriogenesis is not under the immediate control of external hormones. An initial hormonal stimulus is necessary t o initiate the process of follicular maturation during adult development, but the programme of developmental changes which is thus set in motion becomes independent of external cues at some point. Our long-term goal is t o understand the mechanisms internally regulating the temporal sequence of stages in choriogenesis. The mRNAs for chorion proteins have been identified (Gelinas and Kafatos, 1973), and resolved electrophoretically into a number of bands, after removal of their poly(A) (Vournakis et al., 1975); the poly(A) causes smearing of the intact messages, presumably because it is variable in length (Vournakis et al., 1974). mRNA labelling and cell-free translation studies (Kafatos et al., 1976) suggest that the messages are synthesized throughout choriogenesis, and accumulate in a state suitable for cell-free translation only during this time. Higher molecular weight mRNAs appear t o be produced in the early chorionating stages, in parallel with production of the higher molecular weight C proteins. It remains t o be determined whether the detailed programme of sequential polymorphism evident at the level of chorion protein synthesis is paralleled in equal detail and is driven by a corresponding transcriptional programme. 6 General discussion In the four systems discussed in this paper, sequential polymorphism is evident at a morphological, functional, or biochemical level. Since we are dealing with insects, in all cases the hormonal conditions of metamorphosis play a role in permitting the unfolding of this polymorphism. However, the polymorphism is not a simple binary alternative under the immediate control of the hormones. Detailed studies indicate that even in the simplest case, the saturniid labial gland, the “adult state” as it develops can be distinguished into a series of phases (e.g. growth, cuticle formation, and elaboration of mitochondria-membrane associations). It is a matter of perspective whether these phases will be considered distinct stages of differentiation, or will be subsumed under a single temporal programme. The multistage or progressive nature of polymorphism is most clearly evident when this process can be defined biochemically, as in the follicular epithelium. In that case, we can ultimately look forward to understanding polymorphism not only in terms of what triggers it but also in terms of how it proceeds in detail: we can hope to understand the continuing temporal controls on the expression of identifiable genes. The

12

FOTIS C. KAFATOS

con(ro1s need not be hormonal, and in fact in the follicular system they apptbar to be internal to the system itself. *Zs we learn more about a system, our perspective shifts from the o \ ~ i a l lgated process to the continuous changes which make it up. For convenience, we may still describe the continuous changes in terms of a succession of stages. This perspective of development as sequential polymotphism, as a temporal progression through distinct states of gene qxpression, a progression which has strong elements of self-regulation, is widely applicable. One only needs to reflect on the sequential production and disappearance of specific enzymes during slime mould development, or 011 the sequence of rapid changes occurring during early embryogenesis, even in the absence of a functioning nucleus. Having used the term sequential polymorphism to make the connection with classical studies on inscct development, I should now say that i t is important to view insect deLelopment with the modern perspective of cell differentiation in all orgmisms. Once used, sequential polymorphism might best be replaced by thr term temporal programming. Insect development offers a number of opportunities for studying temporal programming a t this deeper level-and in fact offers the po5sibility of studying this process directly at the level of gene activity. An ex
Foundation, and Harvard University. I thank my secretary, Linda S. Lawton, my assistants, P. B. Moore and M. Koehler, and my students and co I leagues whose contributions are identified in the References. References Anderson, E. and Harvey, W. R. (1966). Active transport by the cecropia midgut. 11. Fine structure of the midgut epithelium.]. Cell Biol. 31, 107-134. Anderson, L. M. and Telfer, W. H. (1969). A follicle cell contribution to the yolk spheres of moth oocytes. Tissue and Cell, 1, 633-644. Ashburner, M., Chihara, C., Meltzer, P. and Richards, G. (1974). Temporal control of puffing activity in polytene chromosomes. Cold Spring Harb. S y n p . quant. BioZ. 38, 655-662.

CELLULAR POLYMORPHISM

13

Berger, E. and Kafatos, F. C. (1971a). Immunochemistry of an insect protease, cocoonase and its zymogene. Immunochemistry 8, 391-403. Berger, E. and Kafatos, F. C. (1971b). Quantitative studies of prococoonase synthesis and accumulation during development. Deul Biol. 25, 377-397. Berger, E., Kafatos, F. C., Felsted, R. L. and Law, J. H. (1971). Cocoonase 111. Purification, preliminary characterization and activation of the zymogen of an insect protease. J. biol. Chem. 246, 4131-4137. Berridge, M. J. and Oschman, J. L. (1969). A structural basis for fluid secretion by malpigian tubules. Tissue and Cell, 1 , 247-272. Felsted, R. L., Kramer, K. J., Law, J. H., Berger, E. and Kafatos, F. C. (1973). Cocoonase. IV. Mechanism of activation of prococoonase from Antheraea p o l y phemus. J. biol. Chem. 248, 3012-3020. Gelinas, R. E. and Kafatos, F. C. (1973). Purification of a family of specific messenger ribonucleic acids from moth follicular cells. Proc. natn. Acad. Sci. U.S.A. 70, 3 7 64-37 68. Hakim, R. S. (1972).The growth and differentiation of lepidopteran labial glands. Ph.D. Thesis, Harvard University, Cambridge, Mass. Hakim, R. S. and Kafatos, F. C. (1974). The structure and salivary function of the labial gland in adult Manduca sexta. Tissue and Cell, 6 , 729-750. Hakim, R. S. andKafatos, F. C. (1976). Cellular metamorphosis. 11. The larval labial gland duct andits prospective adult fates in the tobacco hornworm. Devl. Biol. 49, 369-380. Hixson, H. F. and Laskowski, M. (1970). Evidence that cocoonase and trypsin interact with soybean trypsin a t the same reactive site. Biochem. (Easton, Pa.) 9, 166-1 70. Hruska, J. F. and Law, J. H. (1070).Cocoonases. In “Methods in Enzymology” (Eds G. E. Perlmann and L. Lorant), vol. 19, pp. 221-226. Academic Press, New York andLondon. Hruska, J. F., Felsted, R. L. and Law, J. H. (1973). Cocoonase of silkworm moths: catalytic properties and biological function. Insect Biochem. 3, 31-43. Kafatos, F. C. (1968). T h e labial gland: a s a l t secreting organ of saturniid moths. J. exp. Biol. 48, 435-453. Kafatos, F. C. (1970). Cocoonase synthesis: cellular differentiation in developing silkmoths. In “Problems in Biology: RNA in development” (Ed. E. W. Hanly), pp. 111-140. University of Utah Press, Salt Lake City, Utah. Kafatos, F. C. (1971). Cellular metamorphosis. I. The labial gland of Antheraea pernyi. Endocr. Exp. 5 , 101-107. Kafatos, F. C. (1972a). The cocoonase zymogen cells of silk moths: a model of terminal cell differentiation for specific protein synthesis. In “Current Topics in Developmental Biology” (Eds A. A. Moscona and A. Monroy), vol. 7, pp. 125-191. Academic Press, New York and London. Kafatos, F. C. (1972b). mRNA stability and cellular differentiation. Acta endocr. Suppl. (Copnh), 168, 319-345. Kafatos, F. C. (1975). In “Control Mechanisms in Development: Activation, Determination and Modulation of the Genetic Program” (Eds R. Meints and E. Davies), vol. 62, 103-121. Plenum Press, New York. Kafatos, F. C. and Feder, N. (1968). Cytodifferentiation during insect metamorphosis: the galea of silkmoths. Science, N. Y. 161, 470-472. Kafatos, F. C. a n d Gelinas, R. E. (1974). In “MTP International Review of Science-

FOTIS C. KAFATOS

14

Biochemistry of Differentiation and Development” (Ed. J.,Paul). vol. 9,pp. 223-264. Medical and Technical Publishing Company, Oxford. K.iFatos, F. C. and Williams, C. M. (1964). Enzymatic mechanisms for the escape of certain moths from their cocoons. Science, 146,538-540. L f a t o s , F. C., Tartakoff, A. M. and Law, J. H. (1967a). Cocoonase I. Preliminary characterization of a proteolytic enzyme from silkmoths. J. biol. Chem. 242, 1477-

1487. k‘ifatos, F. C., Law, J. H. and Tartakoff, A. M. (1967b). Cocoonase 11. Substrate specificity, inhibitors and classification of the enzyme.J. biol. Chem. 242, 1488-1494. h‘rfatos, F. C., Regier, J. C., Mazur, G . D., Nadel, M. R., Blau, H., Petri, W. H., Wyman, A. R., Gelinas, R. E., Moore, P. B., Paul, M., Efstratiadis, A., Vournakis, J. N., Goldsmith, M. R., Hunsley, J. R., Baker, B. and Nardi, J. (1976).The eggshell of insects: differentation-specific proteins and the control of their synthesis and accumulation during development. In “Results and Problems in Cell Differentiation” (Ed. W. Beermann), Biochemical Differentiation of insect glands. In press. SpringerVerlag, Berlin, Heidelberg, New York. hramer, K. J., Felsted, R. L. and Law, J. H. (1973).Cocoonase. V. Structural studies on an insect serine protease. J. biol. Chem. 248,3021-3028. I.awrence, P. A. (1966). Development and determination of hairs and bristles in the milkweed bug, Oncopeltus fasciatus (Lygaeidae, Hemiptera). J. Cell Sci. 1, 475-498. I ocke, M. (1969). The structure of an epidermal cell during the development of the protein epicuticle and the uptake of molting fluid of an insect. J. Morph. 127,7-39. Lucas, F., Shaw, J. T. B. and Smith, S. G. (1958).The silk fibroins. Adv. Protein Chem. 13, 107-241. I’aul, M. and Kafatos, F. C. (1975).Specific protein synthesis in cellular differentiation. 11. The program of protein synthetic changes during chorion formation by silkmoth follicles, and its implementation in organ culture. Deul Biol. 42, 141-159. I’aul, M., Goldsmith, M. R., Hunsley, J. R. and Kafatos, F. C. (1972).Specific protein synthesis in cellular differentiation. Production of eggshell proteins by silkmoth follicular cells. J. Cell Biol. 55, 653-680. Kegier, J. C. (1975).Ph.D. Thesis, Harvard University, Cambridge, Mass. iiutter, W. J., Kemp, J. D., Bradshaw, W. S., Clark, W. R., Ronzio, R. A. and Sanders, T. G. (1968). Regulation of specific protein synthesis in cytodifferentiation. J. Cell. Physiol. 72, 1-18. \elm:tn, K. and Kafatos, F. C. (1974). Transdifferentiation in the labial gland of silk moths: is DNA synthesis required for cellular metamorphosis? Cell Differentiation, 3 , 81-94. Selman, K. and Kafatos, F. C. (1975). Differentiation in the cocoonase producing silkmoth galea; ultrastructural studies. Devl B i d . 46,132-150. relfer, W. H. (1975). Development and physiology of the oocyte-nurse cell syncytium. In “Advances in Insect Physiology” (Eds J . E. Treherne, M. J. Berridge and V. B. Wigglesworth), vol. 11, pp. 223-319.Academic Press, London and New York. Vournakis, J. N., Gelinas, R. E. and Kafatos, F. C. (1974). Short polyadenic acid frequencies in insect chorion messenger RNA. Cell, 3,265-273.

CE LLU LA R POLY MOR PH ISM

15

Vournakis, J. N., Efstratiadis, A. and Kafatos, F. C. (1975). Electrophoretic patterns of deadenylylated chorion and globin mRNAs. Proc. natn. Acad. Sci. U.S.A. 7 2 , 2959-2963. Wigglesworth, V. B. (1954). “The Physiology of Insect Metamorphosis”. 151 pp. Cambridge University Press, Cambridge. Yamanouchi, M. (1922). Morphologische Beobachtung uber die Seidensekretion bei der Seideraupe.J. Coll. A p i c . Hokkaido I m p . Univ. Sapporo, 10(4), 1-49.

This Page Intentionally Left Blank

The Extraction and Determination of Ecdysones in Arthropods E. David Morgan and Colin F. Poole Department of Chemistry, University of Keele, Keele, Staffordshire, England

1 Introduction 2 Large-scale extraction . 2.1 Initial extraction 2.2 Concentration of material into an active fraction 2.3 Isolation of ecdysones from concentrate . 3 Microdetermination of ecdysones 3.1 Thin-layer chromatography. 3.2 Bioassay . 3.3 Optical spectroscopy. 3.4 Radioimmunoassay . 3.5 Gas-liquid chromatography . 3.6 Mass fragmentography . 3.7 High-pressure liquid chromatography 4 Summary of analytical methods. 5 Conclusion Acknowledgements References

.

.

.

. .

.

.

. . .

. . . .

.

. . .

. . . . .

17 20 24 25 26 29 29 33 35 36 38 49 50 53 56 56 56

1 Introduction

The foundations of the study of the control of moulting in insects were laid on the pioneering work of KopCc, Wigglesworth and Fraenkel before 1939. In the mid-1960s the subject advanced into a new and active stage with the identification of the first hormones controlling moulting. Ten years later the volume of research in this area has expanded many times and yet a number of important points remain unresolved or unexplored. Growth and development in arthropods (insects, arachnids and crustaceans) are dependent upon a periodic shedding of the hard exoskeleton, through a complex process of new cuticle formation, apolysis, and ecdysis, 17

18

E. DAVID MORGAN AND COLIN F. POOLE

and this can be further complicated by metamorphosis from juvenile to pupal or adult forms. That the initiation of moulting was under hormonal control was first suggested by KopPc (1922) and proven by the work of Wigglesworth (1934) on Rhodinus prolixus. The description of a potential bioassay 6y Fraenkel (1935) fulfilled one necessary condition for an attack o n the chemical isolation of such hormones. The systematic search for a chemical hormone was taken up by Butenandt and Karlson in the early 1940s and their work culminated in the isolation, from the pupae of the silkworm Bombyx mori, of crystalline material named ecdysone (Butenandt and Karlson, 1954) which was later identified as a pentahydroxysterol (Huber and Hoppe, 1965; Karlson et al., 1965). Within the past ten years, with the advantages of modern shemical methods for dealing with small quantities of materials, eight ecdysones from arthropods have been isolated and identified. Their structures, are given below. In a brief period of activity covering a few years, over forty ecdysone-like compounds (referred to as phytoecdysones) have been isolated from higher plants; their function there is still unknown. This part of the subject has been reviewed by Horn (1971), Nakanishi (1971) and Rees (1971).

flHf OH

HO

HO

OH

HO

H

OH

OH HO

0

H O

Ecdysone (a-ecdysone) (1)

(2) Ecdysterone (crustecdysone, Pecdysone

20-hy droxy ecdysone)

OH

OH

HO-

(3)

26-Hydroxyecdysone

I1

(4)

0

20,26-Dihydroxyecdysone

ESTIMATION OF ECDYSONES

19

OH

A

HO

0

HO”

(5) 2-Deoxyecdysterone (2-deoxy-20-hydroxyecdysone)

0 (6) 3-Epi-ecdysterone (3-epi-20-hydroxyecdysone)

OH

(7) Inokosterone (25-deoxy-20,26-dihydroxyecdysone)

(8) Makisterone A (20-hydroxy-24-methylecdysone)

The naming of ecdysones is unsystematic and sometimes confusing. Ecdysone ( 1 ) is systematically 2P,3/3,14a,22R,25-pentahydroxy-5P-cholest7-en-6-one, though the name ecdysone provides a convenient generic name by which to name its relatives. It is also known sometimes as a-ecdysone. Compound (2) should be 20-hydroxyecdysone, but is variously known as 0-ecdysone (which suggests it is an epimer or isomer of a-ecdysone, which it is not), ecdysterone, or crustecdysone (which has the claim of primogeniture); this confusion is a legacy of being identified and described by different workers at about the same time. The name ecdysterone is used arbitrarily here as being short and less misleading than P-ecdysnne. The names inokosterone (7) and makisterone A (8) are derived from the Japanese names of the plants from which they were first isolated. The physiological role of ecdysones in insects has been reviewed recently (Gilbert and King, 1973; Burdette, 1974; Slama et al., 1974); the essential details are known but hedged with uncertainties. Prompted by unknown stimuli, a proteinaceous brain hormone activates the prothoracic gland which secretes a substance, which is converted t o ecdysone,

20

E. DAVID MORGAN AND COLIN F. POOLE

or is ecdysone itself., which in turn is transformed into ecdysterone

somewhere in the abdomen, probably the oenocytes, and then travels to the epithelium, possibly by direct action on chromosones t o cause “puffing’’ and synthesis of mRNA for specific proteins and enzymes for ecdysis (Karlson, 1974). However, by no means all experimental evidence leads t o the same view (cf. Vedeckis et al., 1974; Romer et al., 1974; Hsiao et al., 1975). An experiment by Carlisle (1965) showing that insect and crustacean moulting hormones were cross-active was confirmed a year later by the isolation of ecdysterone from an insect and a crustacean (Horne et al., 1966). That these hormones are apparently found not only in arthropods, but in nematodes and molluscs (see Table 1) adds t o their interest t o evolutionists as well as endocrinologists. This review is concerned with the methods employed in the isolation and dctermination of ecdysones in arthropods, where most of the work thus far has been directed. The physiological concentration of ecdysones in insects is of the order of lo-’ t o lo-’ g per g tissue, or less in crustaceans, and therefore the microdetermination of hormones presents special problems. Much of the work t o now has relied upon bioassays (see section 3.1) or the addition of exogenous radio-labelled hormone; unfortunately there is evidence that exogenous hormone can be metabolized differently from endogenous hormone (Nakanishi e t al., 1972; Clever e t al., 1973). lt‘is generally accepted that ecdysones are derived from dietary cholesterol or related phytosterols, but by no means all the steps along the biosynthetic rotite have been established. There is also a conflict of views on the ultimate excretory product of the hormones; and why ecdysones accumulate in the body of the adults of some lepidoptera remains unexplained. Yet the efforts placed in this area and the advances being made may result in this area of hormone research leading t o a more fundamental understanding of how hormones act at the molecular level than has yet been achieved by over forty years of investigation of mammalian hormones.

2 Large-scale extraction The isolation of the moulting hormone from an insect source became possible with the development of a suitable bioassay t o monitor separated fractions. Butenandt and Karlson (1954) isolated ecdysone from the pupae of the silkworm moth Bombyx mori using ligated abdomens of Calliphora erythrocephala (Karlson, 1956a) for the bioassay. Since this first extraction, eight ecdysones have been isolated and identified from thirteen arthropod species (Table 1). Of those species investigated, the amount of hormone found in crustacea is demonstrably lower than that in insecta. The

TABLE 1 The concentration of ecdysones isolated from Metazoa

Species

Stage

Weight extracted kg

Ecdysone isolated

Weight mg

rn

v)

d Concentration mg kg-'

Reference

5

0

z

n

INSECTA

Antheraea pernyi (saturniid oak silkmoth)

Pupae

31

B o m b y x mori (silkworm moth)

Pupae

500 1000 (dried) 3000*

2200t

Calliphora erythrocephala

Pupae

15

(blue blowfly)

Ecdysterone

0.2

0.006

Horn et al. (1966) Butenandt and Karlson (1954) Karlson et al. (1963) Hocks and Weicheit (1966) Hocks and Weichert (1966) Hoffmeister (1.966), Hoffmeister and Grutzmacher (1966)

Ecdysone

25

0.05

Ecdysone Ecdysone

250 206

0.25 0.068

Ecdysterone

48

0.016

Ecdysterone

9

0.004

Ecdysone Ecdysterone

0.075 0.005 Not determined

Karlson (1956b) Karlson (1956b)

Calliphora stygia (brown blowfly)

hepupae Pharate adults

1.87 1.795

Ecdysterone Ecdy sterone

0.19 0.17

0.1 0.095

Galbraith et al., (1969b) Galbraith et al. (196913)

Calliphora vicina

Prepupae

0.5

Ecdy sterone

0.043

0.11

Galbraith et al. (196913)

Egg

5

26-Hydr0~y ecdysone Ecdysterone

26.5

5.3

Kaplanis et al. (1973)

0.5

0.1

Kaplanis et al. ( 197 3)

(blue blowfly) Manducta sexta (Tobacco hornworm)

c!

N h)

TABLE 1-continued

Species

Stage

Weight extracted kg

Pupae

12.7 40.2

Meconium fluid

12.5 litres

Ecdysone isolated

Weight mg

20,26-Dihydroxyecdysone 1.5 Ecdysone 1.2 Ecdysterone 3.7 Ecdysone 4.4 20,26-Dihydroxy3.0 ecdysone 3.6 Ecdysone 3-Epi-ecdy1.7 sterone

Concentration mg kg-'

Reference

0.3 0.24 0.287 0.32

Kaplanis e t al, Kaplanis et al. Kaplanis et al. Kaplanis e t al.

0.075 0.08

Thompson et al. (1967) rn Thompson et al. (1967) . CJ D Thompson e t al. (1974) 5 CJ Morgan e t al. (1975a, b)

-

Schistocerca gregaria (desert locust)

Late 5th instar

32.0

Ecdysterone

Dociostaurus maroccanus (moroccan locust)

Adult

10

Ecdysone

11.0

1.1

Ecdysterone

13.0

1.3

1.9

0.06

(1973) (1973) (1966b) (196Gb)

Stamm (1958)

2

U

0

CRUSTACEA

Crangon vulgarti (shrimp)

5 0

Balanus balanoides (acorn barnacle)

Intermoult

1500

Ecdysterone Ecdysone

3000

Not identified

1.2 0.009

8.0 x l o 4 6.0 x lo-'

Bebbington (1975) Eebbington (1975) Karlson and Schmialek (1959)

5 0 r m

m

--3I v)

b

-I 0

Callinectes sapidus (shore crab)

PremouIt “green”

25

Inokosterone

0.125

0.005

Premoult “peeler”

25

Premoult “soft shell”

25

Inokosterone Ecdysterone Ecdyst erone Makisterone A

0.5 0.1 7.0 0.6

0.02 0.004 0.28 0.024

Faux et al. Faux et al. Faux et al. Faux e t al.

Gagosian et al. (1974)

Faux et al. (1 969) (1969) (1969) (1969) (1969)

2

$1 rn 0

0

2 2 rn v)

Homarus americanus

Post moult

5

Ecdy sterone

0.09

0.006

Intermoult

1000

Ecdysterone

2.0

0.002

3000

2-Deoxyecd ysterone

0.2

Ecdysterone

0.0045 3.0 x l o 4

(lobster) Jams lalandei (crayfish)

Hampshire and Horn (1966) 7.0 x 1 0 - ~ Galbraith et al. (1968a)

NEMATODA

Ascaris lumbricoides (parasitic nematode)

Adults

15.5

~

Hornet al. (1974)

~~

Ecdysones incompletely identified in the nematode Haemonchus contortus juveniles (approximately 0.5 pg b y bioassay in 1 g freezedried tissue, Rogers (1973)) and in the mollusc Mytilus edulis (edible mussel) (approximately 50 ng to 500 pg kg-’, Takemoto et al. (1967b)). * 2.8 tons. t 2.0 tons. N

w

E. DAVID MORGAN AND COLIN F. POOLE

24

concentration of ecdysones varies with the stage of the life-cycle so that the amount of hormone extracted is very much dependent on the age of the juvenile selected for study. The separation and identification of ecdysones from biological material can be considered as a three-part operation. 1. The initial extraction of polar material and partial purification by removal of less polar material (neutral lipids, sterol esters, etc.) and then removal of material of higher polarity. 2. The concentration of ecdysones into an active fraction. 3. The separation and determination of ecdysones in the active fraction.

2.1

INITIAL EXTRACTION

The initial extraction of ecdysones is achieved by blending the insect material in methanol, aqueous methanol, aqueous ethanol or acetonemethanol mixtures (2-7 cm3 g-' ). Alcoholic solvents are not only efficient in extracting ecdysones but also act as a preservative when insect samples have t o be stored (Karlson, 1956b). No special precautions are needed with the purity of solvents at this stage. The insoluble residue is removed by filtration and re-extracted with solvent. The filtration equipment used is determined by the scale of the operation. Buchner funnels (12 in. diameter) with vacuum are sufficient for kilogram amounts but hydraulic presses and process plant equipment are required for handling megagramme amounts. Solvent from extracts is removed by vacuum distillation under nitrogen at tcmperatures less than 40 "C to avoid decomposition of the hormone. The methanolic extract is reduced t o an aqueous concentrate which is partitioned with butanol and the butanol residue is redissolved in aqueous methanol and partitioned with hexane. This is the order of operations described by Butenandt and Karlson (1954) and has been followed b y most subsequent workers. However, there are advantages to be gained by extracting the aqueous concentrate with hexane first. The butanol-water partition is often made difficult by emulsion formation and during reduction of the butanol phase in uacuo it often froths badly unless lipids, etc., have been removed with hexane. The aqueous concentrate from the alcoholic extract is usually diluted with methanol (to 30% v/v) before partition with hexane t o avoid emulsion formation. The extracted phases are back-washed with the counter solvent (usually twice) and combined. The washing of the butanol phase by a series of acid and base washes as used by Karlson (1956a, b) can lead t o a serious loss of the more polar hormone ecdysterone (Horn et al., 1968). This step has generally been omitted by later workers. In the extraction of crayfish waste by Horn et al. (1968), a mixture of hexane and n-propanol (1:3 v/v) was used t o

ESTIMATION OF ECDYSONES

25

extract the aqueous concentrate as propanol is more readily removed by low-temperature distillation. Addition of ammonium sulphate was required to promote phase separation. By the use of a series of highly efficient extraction steps, the ecdysonecontaining fraction is reduced to a size which can be conveniently handled at the laboratory bench. An indication of the purification obtained is given in Table 2. TABLE 2 Purification factors for the preliminary extraction of ecdysones and their concentration into an active fraction

taken

Weight of butanol residue

kg

g

Weight

Species

Balanus balanoides Schktocerca gregarin Manducta sexta Jasus lalandei Calliphora sty@

2.2

1500 32 12.7 1000 1.87

798 96 4.5 400 4.4

Purification factor of stage

Weight of active fraction

1880 333

2800 2500 425

g

8.5 1.47 0.074 12.0 0.049

Purification factor of stage

94 65 61 33 90

CONCENTRATION OF MATERIAL INTO AN ACTIVE FRACTION

Usudy at this stage the concentration of ecdysones is such that positive bioassay results are achieved. The choice of further techniques to purify the extract depends on the weight of material to be processed. Adsorption chromatography is the method of choice provided that the extract is not too bulky, as columns overloaded with adsorbed material give poor resolution. Associated with adsorption chromatography is the need for a large number of bioassays. If many fractions are t o be tested, then a shortage of suitable test animals can be a problem. Generally if the amount of material at this stage is large (>20 g) then further partition systems are used to reduce the bulk of the ecdysone-containing residue before chromatography. Partition procedures also require fewer bioassay tests by comparison with column chromatography as the number of fractions generated is far fewer. Suitable partitioning solvent systems are given in Table 3. With the exception of the water-butanol system the low value of the distribution coefficient means that countercurrent techniques are required for highyield separations. The addition of potassium bicarbonate to the aqueous

26

E. DAVID MORGAN AND COLIN F. POOLE TABLE 3 Partition functions for ecdysones in various solvent systems* ~~

Partition coefficient Solvent system Ecdysone

Ecdysterone

~~~

0.16 0.52 0.06

Cyclohexane-butanol-water(6:4: 10) Cyclohexane-butanol-water(5:5: l o ) ? Ethyl acetate-water (1: 1) Butanol-water (1: 1) Ethyl formate-butanol-water (9: 1: 10) Chloroform-methanol-water(2: 1: 1) Chloroform-ethanol-water(1: 1: 1) ~

~

=10

0.75 2.0 -

1.27 3.54 0.32 5.3 0.2 5.0 0.7

-~

* Horn (1971).

t Partition coefficient of 26-hydroxyecdysone is 0.39 in this system (Kaplanis e t al., 1973). phase of the first tube in the system chloroform-ethanol-water (1:1:1) is a convenient method for the removal of acidic impurities (Horn, 1971). At this stage of the procedure the available material is of the order of grammes and can be conveniently purified by adsorption chromatography. Karlson (195613) used alumina for this purpose, but later workers have preferred silica, especially for the isolation of ecdysterone. The recovery of small quantities of ecdysterone from alumina is generally poor (Horn et al., 1968; Miyazaki et al., 1973). Alumina prepared a considerable time before use, or which had formed “clumps” after deactivation was destructive to the ecdysones (Kaplanis et al., 1966b). Similar problems with silica have not been observed. Columns of silica gel or silicic acid with a sample-tosupport ratio of 1:ZO-30 (normaIly deactivated with 10 per cent water w/w) are eluted with a step-wise gradient of benzene-butanol, benzenemethanol, through pure methanol to aqueous methanol (Morgan et al., 1975a, b ; Bebbington 1975) or with chloroform-ethanol mixtures (Horn et al., 1968). The ecdysones are eluted with the benzene-methanol fractions. The use of Floridin earth (a grade of magnesium silicate from FIorida) instead of siIica gave little separation of ecdysones from the bulk material obtained by extraction of the desert locust (Woodbridge, 1971).

2.3

ISOLATION OF ECDYSONES FROM CONCENTRATE

An active fraction with a high score by bioassay is obtained at this stage. Further purification by adsorption chromatography is rarely successful as the ecdysones and associated contaminants have similar adsorption proper-

ESTIMATION OF ECDYSONES

27

ties. On occasions when a source rich in ecdysones has been extracted, simple recrystallization is all that is required by way of final purification. Separation of the active fraction by preparative layer chromatography (preparative TLC) gives higher resolution than is obtainable with columns and can give a useful further fractionation (Woodbridge, 1971; Morgan et al., 1975a, b). It has been reported that the losses of very small amounts of ecdysones on preparative layer plates can be substantial (Horn et al., 1968). The most efficient method for the further fractionation of the active fraction is reversed-phase column chromatography using cyclohexane-butanol (1:7) as stationary phase coated onto hydrophobic celite (Horn et al., 1968; Galbraith e t al., 1969b; Bebbington, 1975). Celite is the trade name of the Johns-Manville Co. for their grade of calcined diatomaceous earth. This phase allows a separation of ecdysterone and 2-deoxyecdysterone to be achieved. Horn et al. (1968) and Galbraith e t al. (1969b) used reversed-phase chromatography with further chromatography on a column of CM-Sephadex (Pharmacia AB) and final purification with silicic acid (a slightly acidic form of precipitated silica gel) in the isolation of ecdysterone from samples of Jasus hhndei and Calliphora vicina. Columns of CM-Sephadex are much superior to Sephadex G-25 for this purpose and enable the separation of inokosterone and ecdysterone to be achieved (Horn,1971). The fractionation of the active extracts by a countercurrent distribution of the material in cyclohexane-butanol-water(5:5: 10) using a Craig-Port fifty tube train enables the separation of ecdysone, ecdysterone and 20,26-diiydroxyecdysone from each other and associated contaminants (Kaplanis et al., 1966b; Thompson e t al., 1967). In this system, 26-hydroxyecdysone overlaps ecdysterone and separation of the two hormones was achieved by preparative layer chromatography on silica gel (Kaplaniqet al., 1973). The physical properties of arthropod ecdysones are summarized in Table 4. In the nuclear magnetic resonance spectra, there are significant differences in the chemical shifts of the methyl signals measured in pyridine and methanol solvents which has been used to advantage in structure elucidation (Galbraith et al., 1968a, 1973). The chemical shift of the C-19 methyl signals of ecdysones measured in pyridine are 0.3 ppm further upfield than the corresponding A/B trans fused isomer ( 5 4 , and this difference provides a useful means of determining the configuration of the A/B ring junction (Horn, 1971). Because of the large sample size (several mg) required for nmr spectroscopy, in many cases it has been impractical for arthropod ecdysones, but the Fourier transform technique, and accumulated spectra, increased instrument sensitivity and smaller sample tubes have already reduced sample size to pg for the most modern instruments, and still further reduction in sample size may be possible.

N

03

TABLE 4 Physical properties of arthropod ecdysones Ultraviolet absorp tiont Ecdysone

Optical rotation

nmr 60 MHz (perdeuteropyridine)

m.p.("C) * Emax

(EtOH)

c-18

c-19

c-2 1

C-26

C-27

C-28

[&I;' (MeOH)

r" Ecdysone Ecdysterone 3-Epi-ecdysterone 26-Hydroxyecdysone 2-Deoxyecdysterone Inokosterone Makisterone A 20,26-Dihydroxyecdysone

237-239 241-242.5 225-229 252-256 -

2 55 (decomp.) 263(decomp.) 149-153

242 242 245 245 243 243 243 245

12 400 12 400 10 800 11 600 -

12 100 12 400 10 400

0.70 1.20 1.20 0.74 1.21 1.19 1.21 1.22

1.05 1.07 1.08 1.08 1.04 1.07 1.09 1.08

1.25 1.56 1.58 1.2311.32 1.57 1.52 1.54 1.58

* Ecdysones lose water of crystallization with partial melting at lower temperatures, e.g. t Absorption maxima shifted +6 nm in water and -6 nm in hexane.

1.38 1.38 1.38 -

1.35 -

1.29 -

1.38 1.38 1.38 1.47 1.35 1.03 1.32 1.48

-

1.05 -

+67.8" +60.5' -

- . -

+74.3" -

0

2 z

%0

z

P0

., ecdysone at 160-165°C.

Pz rn

ESTIMATION OF ECDYSONES

29

The infrared spectra (in KBr discs) are characterized by a strong absorption at 1645 cm-' ( G O ) and 1612 cm-' (C=C) for the a,P-unsaturated ketone, and at 3330-3400 cm-' for the hydroxyl groups. There are characteristic bands at 1383 cm-' , 917 cm-* and 789-843 cm-' in ecdysterone that are not present in ecdysone. The optical rotatory dispersion spectra of ecdysones in dioxane have a positive Cotton effect with an amplitude of 60-80, which is characteristic of the A/B-cis fused rings (Horn, 1971; Nakanishi, 1971). The use of ord spectroscopy for ecdysones is discussed in detail by Nakanishi (1971). The mass spectra of ecdysones show weak molecular ions with consecutive losses of 1 8 mass units due t o elimination of water. Estimation of molecular weight from the mass spectrum must therefore be cautious, unless good, reproducible spectra are obtained. The molecular weight of ecdysterone was originally underestimated because of loss of water from the molecule in the spectrometer (Hoffmeister and Grutzmacher, 1966), and that of ecdysone was in doubt (Karlson et al., 1965). Conversion t o trimethylsilyl ether derivatives (sections 3.5.1 and 3.6) for mass spectrometry has advantages, but molecular ions are still weak. Pentafluorophenyl dimethylsilyl ethers of steroid alcohols give strong molecular ions and useful f r a p e n t a t i o n patterns (Morgan and Poole, 1975; Poole and Morgan, 197513). The method has not been applied t o ecdysones. It would have the disadvantage of producing compounds of very high molecular weight. Ecdysones with a 20,22-diol show a characteristic fragmentation of the 20-22 bond which aids in their identification (Horn, 1971; Galbraith and Horn, 1969). Further details of mass spectra are contained in the references t o Table 1 and in Hoffmeister and Grutzmacher (1966), Koreeda e t a l . (1969), Nakanishi (1971), and Zatsny et al. (1975).

3 Microdetermination of ecdysones 3.1

THIN-LAYER CHROMATOGRAPHY

In thin-layer chromatography, resolution is normally greater than that achieved with packed columns, and the separated components of a mixture can be identified simultaneously on the surface of the adsorbent by a variety of visualization techniques. Iodine vapour is a quick and simple technique. Using uv light, as little as 0.5 pg of ecdysones can be observed on silica gel containing a fluorescent additive absorbing at 254 nm (Poole, 1975). Ecdysones also give characteristically coloured spots when the plate is sprayed with vanillin-sulphuric acid followed b y heating at 110 *C for fiveminutes (Stahl, 1962; Galbraith et al., 196813; Horn, 1971). Other spray

30

E. DAVID MORGAN AND COLIN F. POOLE

rc'igents were found less useful, including 2,4-dinitrophenylhydrazine (Izevinson and Shaaya, 1966). The vanillin-sulphuric acid reagent is very useful for relatively pure samples of ecdysones but the colour reaction is e'isily masked by the presence of other material which limits its use for the analysis of crude samples. The quantitative determination of ecdysones by a densitometric method on fluorescent TLC plates has been described (S'irdini et al., 1974). The recovery of small amounts of ecdysones from silica gel plates with methanol is often poor and extracted samples are easily contaminated with p'irticles of silica and binder (Horn et al., 1968; Schooley and Nakanishi, 1973). Thin-layer chromatography has been used to separate mixtures of ec dysones, its precursors and metabolites, which were labelled with H or 14 C. The ecdysones were determined by either radiochromatography using a 'I'LC scanner (King and Siddall, 1969; Moriyama et al., 1970; Hoffmann ct al., 1974; King and Marks, 1974; Sannasi and Karlson, 1974; Galbraith rt al., 1975) or by removing discrete bands for scintillation counting (Nakanishi et al., 1972; Romer et al., 1974; Hikino et al., 1975). Ecdysones have also been eluted from TLC plates and determined by bioassay (Willig and Keller, 1973; Hoffmann et al., 1974; King et al., 1974; Koolman et al., 1975) and radioimmunoassay (Borst and Englemann, 1974). In these ex
31

ESTIMATION OF ECDYSONES TABLE 5

Rf values of Arthropod ecdysones on silica gel TLC eluted with CHCIJ -95 per cent EtOH(4: 1) Ecdysone Ecdysone

Ecdysterone

2-Deoxyecdysterone 26-Hy droxy ecdy sone 3-Epi-ecdy sterone Inokosterone Makisterone A 20,26-Dihydroxyecdysone

Rf values* 0.37 0.23 0.20 0.20 0.24 0.14 0.13 0.16 0.17 0.15 0.30 0.08 0.17 0.16 0.16 0.08

References Thompson et al. (1967) Galbraith et al. (1968a) Horn (1971) Woodbridge (1971) Thompson et al. (1967) Galbraith et al. (1968a) Horn (1971) Horn (1971) Woodbridge (1971) Kaplanis et al. (1973) Galbraith et al. (1968a), Horn (1971) Kaplanis et al. (1973) Thompson et al. (1974) Horn (1971) Horn (1971) Thompson et al. (1967)

* Rf values are approximate as different methods of deactivating the silica gel are used (e.g. equilibrated over saturated brine, equilibrated with the atmosphere or oven dried and stored in a dry box). the expense of time, and continuous development for many hours is required for good separation (Borst and O'Connor, 1974). In terms of efficiency (plates per minute) the continuous flow TLC technique performs poorly by comparison with high-pressure liquid-adsorption chromatography. The formation of derivatives has been used to reduce the polarity of the ecdysones and obtain a better separation on TLC. For this purpose acetates have been most used. The steric environment of an hydroxyl group governs its rate of acetylation. Equatorial secondary ring hydroxyl groups react most rapidly and side-chain hydroxyl groups slightly faster than ring axial groups (Galbraith and Horn, 1969). For ecdysterone at 20 OC in acetic anhydride-pyridine (1:2), the order of acetylation is 2 > 3 > 22 > 25 (Galbraith and Horn, 1969; Horn, 1971). The various acetates have been separated and fully characterized (Galbraith and Horn, 1969; Canonica et al., 1975) and the pseudo first-order reaction rate constants determined (Galbraith and Horn, 1969). Thin-layer chromatography of the acetate mixture from ecdysterone produces four spots which have been used as a

E. DAVID MORGAN AND COLIN F. POOLE

32

TABLE 6 "lie

R f values of ecdysone and ecdysterone in various solvent systems on silica gel TLC plates

Solvent sytem

CIIC13-95% EtOH ( 7 ~ 3 ) C:IICI3-95%EtOH (13:7)* C1 IC13-EtOH (3:2) CIIC13-MeOH (9:l) ClIC13-MeOH (5:l) CIICI3-MeOH (3 :2)

Ecdysone 0.39 -

0.10 -

-

Ecdysterone 0.34 0.31 0.50 0.07 0.23 0.40 0.55

CHC13-MeOH (1:l) C1lC13-n-propanol (9:5) (:HC13-EtOH-MezCO (6:2:1) (:IICl,-MeOH-Me2CO (6: 2: l ) t CHzC12-MezCO-MeOH( 2 : l : l )

0.21 0.69

0.12 0.48 0.33 0.62

Cl-IzClz-MezCO-EtOH (16:4:5)

0.32

0.10

CHz Clz -MeOH-C6H6 (25:5:3) CHz Clz-MezCO-H20 (15: 62.5: 10)

0.65

0.19

(:H2CIz-MeOH-H20 (79: 15:1)" (:6H6-MeOH(7: 3) t.tOAc-EtOH (4:1)$ LtOAc-EtOH (4:l)S 1 tOAc-EtOH (4: 1) CHzC12-MeOH-25%NH3-H20 (77: 20: 2: 1)

0.32 0.40 0.49

0.19 -

-

-

0.47q

-

0.60 0.46 0.32 0.40q

Reference Woodbridge (1971) Kaplanis et al. (1973) Hornet al. (1968) Siddall et al. (1966) Imai et al. (1967) Borst and O'Connor (1974) Borst and O'Connor (1974) King and Siddall (1969) Imai et al. (1967) Woodbridge (1971) Levinson and Shaaya (1966) Heinrich and Hoffmeister (1967) Imai et al. (1967) Heinrich and Hoffmeister (1967) Sardini et al. (1974) Karlson et al. (1965) Takemoto et aZ. (1967a, c) Sardini e t al. (1974) Woodbridge (1971) Romer et al. (1974)

* I n this system 20,26-dihydroxyecdysone has Rf 0.22. t In the same solvent system on aluminium oxide, ecdysterone has Rf 0.16. $ In this system inokosterone has Rf 0.60.

8 In this system makisterone A has Rf 0.50. 11 In this system makisterone A has Rf 0.25. f Estimated values.

"fingerprint" to confirm the presence of the hormone in an extract ('Takernoto et al., 1967a; Galbraith et al., 1968a, 1973; King and Siddall, 1969; King, 1972; Nakanishi et al., 1972; King and Marks, 1974; King et d.,1974; Hikino et al., 1975). Brief acylation at room temperature allows the monoacetate derivative to be obtained in high yield (Galbraith and LIorn, 1969; King and Sidall, 1969; Horn, 1971). The mono-acetate is often

ESTIMATION OF ECDYSONES

33

more amenable t o recrystallization from semi-pure samples than the ecdysone itself. With the vanillin-sulphuric acid spray reagent, the acyl esters of ecdysterone produce the characteristic olive green coloration of ecdysterone itself unless the C-22 hydroxyl group is acylated in which case the spot is a leaf-green colour (Galbraith and Horn, 1969). Formation of the 20-acetate and cleavage of the 20,22-glycol with 0.1 M sodium metaperiodate in 50 per cent aqueous methanol has been used to distinguish between ecdysone and ecdysterone (King and Siddall, 1969; King, 1972; King and Marks, 1974). The reaction can be performed o n 10 pg of material and the product, 2~-acetoxy-3~,14~-dihydroxy-5~-pregn-7-en-6,20-dione, recognized by its R f value on TLC. Other derivatives which have been used to characterize ecdysones by TLC include boronates (Galbraith et al., 1969a) and acetonides (Galbraith and Horn, 1969; Koreeda et al., 1969; King and Marks, 1974; Thompson et al., 1974). These reagents react selectively with the cis-diol functions in ecdysones. Under appropriate conditions it is possible t o prepare both the mono- and diacetonides of ecdysterone (Galbraith and Horn, 1969). The 20,22-monoacetonide is particularly resistant t o hydrolysis (Galbraith and Horn, 1969). The structural conformation of the hydroxyl groups in 3-epi-ecdysterone was decided with the help of the acetonide derivative, as the trans-2,3-diol does not form a cyclic derivative under the usual conditions (Thompson et al., 1974). 3.2

BIOASSAY

The establishment of a satisfactory bioassay is a prerequisite when seeking an unknown chemical substance which has biological activity. It indicates the biological potency of semi-pure material and is essential for monitoring fractionation and purification steps. It is possible t o use one species t o measure biological potency in another, but this presupposes that the chemical substance inducing the biological response is the same in both. Moreover the bioassay may not be specific for a single chemical substance and may give a positive response with chemical analogues. Other chemically related compounds or prohormones, of slightly different structural features, for example, bearing a 5a-hydrogen atom or a 22s-hydroxyl group, would not be detected by bioassay. The most commonly used bioassay for moulting hormone is the dipteran assay based on the work of Fraenkel (1935) and refined by Karlson (1956b), Karlson and Shaaya (1964) and others. The method has been reviewed recently by Thomson (1974); the intention here is only to assess its usefulness, in comparison with other methods in isolating and determining ecdysones. The method depends upon the induced sclerotization of the

34

E. DAVID MORGAN AND COLIN F. POOLE

larval cuticle of an abdomen which has been ligatured and the head removed. The most popular systems are summarized in Table 7. TABLE 7 Test species used in moulting bioassay Species

Common name

Test name

Reference

Calliphora erythrocephala

Blowfly

Calliphora

Calliphora stygia Musca dornestica Chilo suppressalis Sarcophaga peregrina

Blowfly Housefly Rice stem borer Fleshfly

Calliphora Musca Chilo Sarcophaga

Karlson (1956b), Kadson (1 963), Karlson and Shaaya (1964) Thomson et al. (1970) Kaplanis et al. (1966a) Sato et al. (1968) Ohtaki et al. (1967)

Desert locust

Locust abdomen

Morgan et al. (1975a), Morgan et al. (1975b)

Freshwater crayfish

Crayfish

Lowe and Horn (1967)

DIPTERA

ORTHOPTERA

Schistocerca gregarh CRUSTACEA

Cherax destructor*

* Strictly, this is a test for a different hormone which co-chromatographs with ecdysones. The nonspecialized larva-to-larva moult used in the locust abdomen test provides a rather different system (Morgan et al., 1975a, b). The method has the advantage of being able to distinguish between new cuticle formation and apolysis, but may suffer from the disadvantage of being less close to a complete living system. The crayfish test is really a test for dilation of the chromatophores and was used only because the chromatophore-stimulating hormone was found to accompany ecdysones through all the preliminary extractions of Jasus lulandei (Lowe and Horn, 1967). The routine use of a bioassay requires a large supply of test animals of similar age which must be ligated, injected with sample, incubated and visibly assessed for degree of sclerotization. The test abdomens are compared with a control group injected with solvent only but otherwise treated as the test group. For quantitative work a second control group injected with known amounts of hormone are required. This second group is required because of the variation in the physiological state of the test animals. The same ecdysone should be used in the control as is being

ESTIMATION OF ECDYSONES

35

assessed in the test. If it is an unknown ecdysone, ecdysterone should be used in the control since it appears to be the most common ecdysone and has good solubility properties in aqueous ethanol. The degree of sclerotization is assessed visually and expressed as a percentage of the surviving test abdomens to give a measure of biological potency (Karlson, 1956b; Kaplanis et al., 1966a). The sensitivity of the assay is of the order of 5-50 ng ecdysterone per abdomen in the Calliphora test (Thomson e t al., 1970) and 5-6ng ecdysterone per abdomen in the Musca test (Kaplanis e t al., 1966a). The sensitivity of the locust abdomen test is similar (Morgan e t al., 1975b). For simply deciding which fractions are active from a purification step, five abdomens per fraction is sufficient, but for quantitative work 20 t o 25 abdomens are required. Thus for quantitative work the least detectable amount of ecdysterone is of the order of 0.1 pg. Bioassays are expensive of material, laborious and unspecific in that they cannot distinguish between different chemical substances with the same physiological action, but are essential in a complet'ely new area of investigation. Other problems include poor solubility of test material in the injection solvent without having to use too large a volume of liquid injected and at times a high degree of toxicity of the partially purified material resulting in a high death rate in the test. Impure barnacle fractions proved to be very toxic in the locust abdomen assay (Morgan and Bebbington, unpublished). A variation on the dipteran assay, an in vitro test using a piece of integument from a ligatured and nonpupated larva of Chilo suppressalis has been reported by Agui (1973), with a sensitivity of approximately 15 ng of hormone per test.

3.3

OPTICAL SPECTROSCOPY

The ecdysones are characterized by a strong uv absorption (em a x 10 000 to 14 000; A,, 240 to 245 nm, see Table 5) in alcoholic solvents. This means a limit of detection of 0.5 pg with an instrument reading to 0.01 optical density units, but increasing instrument accuracy will lower this considerably. The decrease in uv absorption obtained by the addition of sodium borohydride to a biological extract has been used to determine the amount of ecdysones present (Takemoto et nl., 1968). It has been shown that ecdysones in a sulphuric acid-ethanol mixture fluoresce strongly when excited by light of a suitable wavelength (Takemoto et al., 1968; Gilgan and Zinck, 1972). Also, the fluorescence intensity of inokosterone in ammonia solution increased markedly by comparison with that of ecdysterone. This difference in relative fluor-

E. DAVID MORGAN AND COLIN F. POOLE

36

exence output in acidic and alkaline medium enabled the composition of mixtures of ecdysterone and inokosterone t o be determined (Takemoto et al., 1968). Gilgan and Zinck (1972) have investigated the conditions for mctximum fluorescence of ecdysterone in sulphuric acid-ethanol medium and claim a detection limit of 50 ng for crystalline ecdysterone. Several ec dysones were found t o fluoresce under the same conditions indicating the gcBnerality of the response. The application of the technique t o biological m'tterial was not shown but the limiting factor of sensitivity was the blmk value. In a study of sulphuric acid-ethanol induced fluorescence of the synthetic model compound 2/3,3/3,5a-trihydroxycholest-7-en-6-one, Bc bbington (1975) has shown that blank values were generally high due to iri [erference with the fluorescence emission by Rayleigh scattering and R'tman emission of the solvent. This limited the overall sensitivity t o concentrations higher than 0.1 pg ml-' . Attempts t o identify the model cc )mpound in biological extracts containing known additions of the steroid wrre unsuccessful due t o excessive interference from other fluorescing siibstances in the extract. 3.4

KADIOIMMUNOASSAY

The specific binding proteins used in radioimmunoassay are antibodies ohtained by active immunization with the substance t o be measured. Sr eroids themselves are not immunogenic but when covalently coupled t o piotein carriers they become so, acting as haptens. Borst and O'Connor (1 372, 1974) coupled ecdysterone as its oximino-acetic acid derivative t o bovine serum albumin (see also Borst, 1973). Lauer et al. (1974) formed a hi,misuccinate derivative of ecdysterone which was coupled to human srrum albumin (see also Nakanishi et aZ., 1973). Injection of white rabbits w Lth the hapten triggers an immune response, resulting in the formation of tibodies. Not all animals exhibit an immune response o n injection and the degree of response is variable. Antibodies obtained by bleeding the a 1 imal are titred with labelled ecdysterone and those showing a relatively hiqh titre are frozen until use. To perform the assay, a limited amount of specific antibody (Ab) is w..tcted with ecdysterone (E*) labelled with a radioisotope. Upon addition 0 1 an increasing amount of the unlabelled ecdysterone (E), a corresponding &,creasing fraction of total labelled ecdysterone (*E) is bound t o the .in tibody. After separation of bound labelled ecdysterone from free labelled a dysterone by ammonium sulphate precipitation, the amount of radioa Livity in the bound protein is determined by dissolution and scintillation counting. This information is used t o construct a standard curve against which the unknown samples are compared. Blanks and control samples are

37

ESTIMATION OF ECDYSONES

normally run with biological samples t o check on the performance of the assay. *E+Ab

r

*EAb

+ E

EAb The effectiveness of the antibodies as an analytical reagent is usually determined in terms of its specificity, affinity and sensitivity. Specificity refers t o the uniqueness of the induced antibody site when analogues of the primary antigen are presented to it. The specificity depends on the structure of the reacting hormone and similarly structured compounds may bond t o the antibodies, giving a nonspecific cross-reaction In the assay devised by Borst and O'Connor, most ecdysones compete effectively with ecdysterone for antibody binding sites and the assay has a low specificity which can be applied to determining total ecdysones (Borst and O'Connor, 1974). As ponasterone A and 22,25-dideoxy-5a-ecdysone do not react significantly with the antibody, it is likely that a side-chain terminal hydroxyl group is necessary for hapten-antibody interaction. The assay of Lauer et al. (1974) was shown t o be more specific for ecdysterone; ecdysones lacking a (2-20 hydroxyl substituent were either weakly competitive or noncompetitive in the assay. The affinity refers t o the vigour of binding displayed by an antibody and is usually expressed as a temperature-variable constant. The competitive binding phenomena is then partially controlled by this factor. Borst and O'Connor (1974) obtained a value of 2 x 10' 1 mol-' for their assay which by comparison with other steroid assay systems would be described as moderate and probably explains in part the low specificity achieved. The sensitivity refers t o the minimum amount of antigenic material that may be determined with a degree of confidence. The sensitivity * is dependent on the activity of the radiolabelled ecdysterone. Borst and O'Connor (1972) using ecdysterone of specific activity 6 Ci mmol-" could detect 200 pg of hormone, while with ecdysterone of 50 Ci mmol-', less than 100 pgcould be detected. Lauer et al. (1974) claim a detection limit of 80 pg in their assay. Some of the disadvantages of the radioimmunoassay technique are the long time required to develop the procedures for the production of good quality antisera, the rather large quantities of expensive ecdysterone needed to form the initial hapten complex and that tritiated ecdysterone of high

38

E. DAVID MORGAN AND COLIN F. POOLE

specific activity is required for measurement of the competitive binding process. The linear range of the calibration curve is short and the time per malysis is rather long. Its advantages are that it is a sensitive technique with some degree of \pecificity that can be applied to crude biological extracts. It determines molecules with a three-dimensional structure similar to ecdysterone. The method is potentially very useful if the antiserum could be made available commercially and is probably capable of still greater sensitivity. The radioimmunoassay of Borst and O'Connor has been used t o determine ecdysones in Drosophila melanogaster (Borst et al., 1974), Manducta sexta (Bollenbacher et al., 1975) and Leucophaea maderae (Borst and Engelmann, 1974). If the ecdysone molecule could be linked t o the protein through a less important part of its structure (e.g. the 2P-OH) a more specific mtiserum might be produced.

3.5

GAS-LIQUID CHROMATOGRAPHY

Gas-liquid chromatography (GLC) is widely used for the separation of organic compounds which are volatile and sufficiently thermally stable t o survive the processes of vaporization and passage through the chromatography column. The highly polar ecdysones do not meet these requirements unless converted t o less polar derivatives before analysis. The most useful form of nonpolar derivative of an hydroxyl group is the trimethylsilyl ether, made by methods which are discussed in section 3.5.1. y

ROH + CH3-Si-X

I

3

CH3

-

CH3 I RO-Si-CH3 + HX AH3 A trimethylsilyl ether

X is one of a variety of groups

The separation of the structurally similar ecdysone trimethylsilyl (TMS) cthers presents a problem in that relatively high column temperatures are necessary for their analysis and this limits considerably the number of stationary liquid phases which can be used. In essence, only the thermally stable silicone oil phases meet the requirements of low bleed at the (emperatures (260-300 "C) employed in the analysis. A comparison of the separation of a mixture of three ecdysone TMS ethers on three thermally stable silicone phases (OV-101, Dexsil 300 GC and OV-17) (Poole, 1975) is wmmarized in Table 8. Only the column of OV-101 gave a base line separation of ecdysone and ecdysterone TMS ethers. None of the liquid phases gave a base line separation of ecdysterone and inokosterone TMS

39

ESTIMATION OF ECDYSONES TABLE 8

The separation of ecdysone TMS ethers on 3-ft columns, 1 per cent loading on Gas ChromQ, nitrogen 60 ml min-' and temperature programmed 250-300 OC a t 6 O C min-' Ecdysone TM S ether

Retention time (min)

ov-101

A B C D

2.60 4.50 5.05 5.40

Dexsil300GC

A B C D

5.30 5.75 5.90

Stationary phase ~~

~

ov-17

A

B C D

3.30

2.90 4.65 4.90 5.35

A = 2~,3~,14&tris(trimethylsiloxy)-5~-cholest-7-en-6-one. B = 2~,3~,14~,22,25-pentakis(trimethylsiloxy)-5~-cholest-7-en-6-one (ecdysone penta-TMS* ether). C = 2~,3~,1~,20,22,25-hexakis(trimethylsiloxy)-5~-cholest-7-en-one (ecdysterone hexa-TMS ether). D = 2~,3~,1~,20,22,26-hexakis(trimethylsiloxy)-5~-cho1est-7-en-6-one (inokosterone hexa-TMS ether). * TMS = trimethylsilyl.

ethers, which differ only in the position of one side chain TMS group. The best resolution of this pair was again achieved on the OV-101 column. Recently, two thermally stable polar phases (Poly-S 179 and PZ-176) of potential use for steroid analysis have been introduced (Mathews, 1970, 1974). Their application to the separation of ecdysone TMS ethers has not been reported. Low phase loadings, typically 1-2 per cent, are used so as t o maintain good peak shape at moderate temperatures. This requires that only the best column support material should be used to avoid peak tailing and decomposition at active centres. The support should be of the diatomite type, acid washed and silylated by the manufacturer. Coating of the support with the stationary phase by the rotary evaporator technique followed by fluidized drying gives very efficient columns (Kruppa et ul., 1967). After coating and conditioning the support can be further deactiva-

E. DAVID MORGAN AND COLIN F. POOLE

40

Led by on-column silylation when necessary. The columns should be well onditioned before use with the electron capture detector. The useful life of tile column depends upon its treatment. With careful handling, columns I lave been used continually for 2-3 years without deterioration. The choice of GLC detector depends on the sensitivity required. The flame ionization detector (FID) will detect at maximum sensitivity 50 ng of liexaltis-TMS ecdysterone (50 pg ml-' ) and the electron capture detector (IXD), 5.0 pg (5.0 ng m1-l) of the same compound. The FID is robust and simple t o operate whereas the ECD requires more skill and gives poor I vsults when mishandled. The physiological levels of ecdysones in arthro1)oda normally require the greater sensitivity of the ECD for their t letermination. I

'i.5.1 Formation of trimethylsilyl ethers of ecdysones

'I'rimethylsilyl ethers can be prepared with one or more of the commerc ially available silylation reagents, some of which are shown below. CH3

I

0--Si( CH3 ) 3 CH3'C 'N-s~(cH,)

CH3-Si-Cl

I

CH3 (9) Trimethylchlorosilane

TMCS

(10) N,O-bis(trimethylsily1) acetamide BSA

/=N ( CH3) Si-NJ ( 1 1 ) Trimethylsilylimidazole TMSIm

With ecdysones, some or all of the hydroxy groups can be converted to \ilyl ethers t o make the molecule more volatile for gas chromatography, but I eproducible conditions are required. Katz and Lensky (1970) reported a I'MS ether of ecdysone by heating the steroid for one minute at 80 "C in V.0-bis( trimethylsi1yl)acetamide (BSA) (10) and pyridine. A single peak was obtained on gas chromatography but no evidence was given for the number o f hydroxyl groups reacted. Morgan and Woodbridge (1971) first converted (he ketone groups t o an 0-methyloxime (methoxime) and then by a Iirolonged treatment (70 h ) with BSA at room temperature converted the 2,3,22 and 25-hydroxyl groups t o silyl ethers in ecdysone and ecdysterone. I'he nature of the product was determined by mass spectrometry (Morgan xid Woodbridge, 19 74). The ecdysone derivatives were conveniently \ olatile and gave characteristic double peaks due t o incomplete separation

ESTIMATION OF ECDYSONES

41

of the syn and anti methoxime isomers. However, the two-stage derivatization procedure was inconveniently long and the double peaks could be troublesome in complex mixtures.

a

CH,ONH,HCI

m*'

+

, H

H O

OCH~

H N CH~O'

Methoxime isomers

Galbraith et al. (196813, 1969a) and Thompson et al. (1970) have shown that the l k - h y d r o x y groups in some ecdysone analogues were incompletely silylated b y BSA in dimethylformamide at 80 "C for up t o 18 h. Thus BSA can be used selectively t o protect some hydroxyl groups but lacks the reactivity required for preparing fully trimethylsilylated derivatives. Si(CH,),Cl

* Q

OSi(CH3)3 Formation of enol silyl ether

Certain advantages are t o be gained by the use of trimethylsilylimidazole (11) for the formation of ecdysone TMS ethers. It is the strongest trimethylsilylating reagent known and the by-product of the reaction is the weakly amphoteric imidazole which does not promote enolization of the ketone group in ecdysones. The use of acid catalysis (e.g. trimethylchlorosilane, TMCS) (9) to improve the silylation strength of the reagents commonly used to form TMS ethers of steroids has t o be avoided with ecdysones due to the ready enolization of the unsaturated ketone group. Ikekawa et al. (1972) claimed that all the hydroxyl groups in ecdysterone could be quantitatively trimethylsilylated with TMSIm at 100 "C for one hour. The C-20 hydroxyl group was found t o be the slowest t o react and the extent of trimethylsilyl ether formation was confirmed by combined gas chromatography and mass spectrometry (GC-MS). Other authors have reported the use of TMSIm for the formation of ecdysterone TMS ethers (see Miyazaki et al., 1973; Lafont et al., 1974; King et al., 1974-0.5 h, 9 6 "C; Borst and O'Connor, 1974-0.25 h, 100 "C). In an attempt to repeat the work of Ikekawa et al. (1972), Poole et al. (1975) found that heating ecdysterone in TMSIm at various temperatures resulted in the formation of different products easily

E. DAVID MORGAN AND COLIN F. POOLE

42

separated by GLC. Heating the reaction mixture for 4 h at 100 "C gave a quantitative yield of a TMS ether which after heating overnight ,it 140 "C was converted to a derivative of shorter retention time. It was shown that the 14a-hydroxyl group was not trimethylsilylated readily at LOO OC but could be converted t o the TMS ether at the higher temperature. The order of reactivity of the hydroxyl groups in ecdysterone towards N S I m was established as 2,3,22,25 > 20 >> 14. The order of reactivity was confirmed by the use of simpler model compounds, infrared spectroscopy aid mass spectrometry (Morgan and Poole, 1976). The relative retention times on gas chromatography and Rf values on TLC of some fully and p,irtially protected TMS ethers of ecdysones are summarized in Tables 9

v11tually

TABLE 9 Rc-tention time data for some TMS ethers of ecdysones on a 3 ft x 0.25 in 0.d. column of 1 per cent OV-101 on Gas ChromQ, a t 266 O C and nitrogen 60 ml min-' Retention time min

Ecdysone TMS ether 'I etrakis-TMS ecdysone

3.90 3.10 4.60 5.30 4.10 6.20 4.50

Pvntakis-TMS ecdysone 'I ctrakis-TMS ecdysterone Pc ntakis-TMS ecdysterone Hcxakis-TMS ecdysterone Prntakis-TZIS inokosterone H exakis-TMS inokosterone

TABLE 10 'TRIS ethers of ecdysones: Rf values on silica gel TLC plates deactivated by heating at 110 OC for 1 h before use

Solvent systems Steroid

Hydroxyl groups silylated ~~

Toluene-ethyl Acetate ( 9 : l ) ~

26, 30, 1k-Trihydroxy- 20, 30 -5fl-choIest-7-en-6-one 20, 30, 1 k l k tlysone I;( tiysterone

Tolune-ethyl Acetate (7:3)

~

0.39 0.51

-

-

0.69

20,30, 22,25 2/3,30, 1401,22,25

0.58

-

20, 30, 22, 25 20, 3$, 20, 22, 2 5 20, 30, 1401, 20, 22, 25

0.22 0.69

0.54 0.67 0.75

43

ESTIMATION OF ECDYSONES

and 10. It seems that 1kekawa.et al. failed t o recognize incomplete TMS ether formation with their reaction conditions. The conditions used in this laboratory for the formation of TMS ethers of ecdysterone are summarized in Table 11. The reactions are conveniently carried out in screw-capped reacti-vials. It should be noticed that pyridine when added t o the reaction medium leads t o an extension of the reaction time. The effect of various catalysts on the formation of TMS ethers of ecdysterone has been discussed by Morgan and Poole (1976). TABLE 11 Experimental conditions for the formation of TMS ethers of ecdysterone Reaction time with TMSIm

Hydroxyl goups

Room temperature 15 min 100 OC, 4 h 100 'C, 6 h 140 'C, 16 h

2P, 30, 22, 25 20

140 ___

Comments

~

~

___

No solvent Pyridine as solvent No solvent

___

* Time for complete reaction depends on the purity of the TMSIm reagent. Some of the confusion in reported silylation conditions is dependent upon the quality of the reagent. We have found that TMSIm freshly prepared in the laboratory is a more powerful reagent than the commercial material, but that it deteriorates considerably on contact with air. Whether or not the 14a-hydroxyl group is silylated depends upon the quality of the reagent as well as temperature and reaction time. Very unreactive hydroxyl groups can be silylated by adding trimethylchlorosilane (TMCS) t o the reaction mixture, but this can encourage partial formation of enol silyl ether. In the author's laboratory it was found that synthetic model ecdysones containing a 5a-hydroxyl group were thermally unstable unless the hydroxyl group was protected. To achieve this the ketone must first be converted t o its methoxime and then the 5a-hydroxyl group can be silylated with a mixture of BSA, TMSIm and TMCS (5:5:4). 3.5.2 Formation of trimethylsilyl heptafluorobutyrates Ikekawa et al. (1972) have described a method for formation of a mixed trimethylsilylheptafluorobutyryl (TMS-HFB) derivative of ecdysones. To the TMS ether in TMSIm was added heptafluorobutyrlimidazole (HFBIm) with a catalytic amount of heptafluorobutyric acid. The conversion was about 9 0 per cent of theoretical b y GLC after heating for 2 h at 50 'C. The reaction can be carried out with or without the 14a-hydroxyl group

44

E. DAVID MORGAN AND COLIN F. POOLE

protected as its TMS ether and the product can be detected at the picogram le\el with the electron capture detector (Ikekawaetul., 1972; Miyazaki e t al., 1973; Poole, 1975). Direct reaction of ecdysterone with HFBIm or hcptafluorobutyric anhydride gave a mixture of products. In the case of etdysterone it was shown that the exchange reaction allowed the introduction of one heptafluorobyrl group at the C-2 position (Miyazaki et al., 1973). The TMS ethers of ecdysones are sensitive to electron capture detection at the picogram level without resorting t o formation of the mixed II1.IS-HFB derivatives. The TMS-HFB derivatives are more volatile than the r h l S ethers on GLC and their formation is useful t o help confirm the dcntity of an ecdysone by providing further retention time data. Reactions o I this type are increasingly important in the ecdysone field due to the low IeLels of the hormone in arthropods which often precludes the collection of rn'tss spectral data. This approach was used in the confirmation of the piesence of ecdysterone in Balunus balanoides (Bebbington, 1975). Both the acetonide and n-butylboronic ester derivatives of adjacent diol groups In ecdysterone are cleaved by TMSIm, therefore acetonides or boronic esters cannot be used as alternative derivatives for confirming identity of structure. '3.5.3 The electrophore in ecdysones Steroids, as a class, do not show strong electron capturing properties, the rather poorly understood requirement for determining with the selective E(:D detector. The ecdysones are an exception in this respect in that they contain a conjugated electron capturing group which enables them to be dcterrnined at the picogram level. The centre of initial electron attachment in a molecule is described as the electrophore. The necessary structural

TMso O M T *S

TMS 0

Least detectable amount 1.0 ng R=OH 0.06ng R = OTMS 0.005 ng The electrophore of ecdysone TMS ethers

R =H

ESTIMATION OF ECDYSONES

45

elements of the electrophore in,ecdysones were identified as the 7-en-6-one (unsaturated ketone) group, the substituent at the C-14 position with a smaller contribution from the remote trimethylsiloxy groups at C-2 and C-3 (Poole and Morgan, 1975a). Trimethylsilylation of the l4a-hydroxyl group increases the sensitivity of the ecdysone t o the detector by a power of ten. However, this extra sensitivity is not always required in the determination of ecdysones in arthropoda and in these cases the extra time required t o trimethylsilylate the l k - h y d r o x y l group is not justified. A linear response over the range 60-700 x lo-'' g was obtained for ecdysone and ecdysterone with a free 14a-hydroxyl group and 5-700 x lo-'* g for the ecdysones with a 14ol-TMS group. The response of the ecdysone derivatives t o the electron capture detector is markedly temperature dependent (Poole and Morgan, 1975a). Maximum response was obtained at low detector oven temperatures. Moderate retention times for ecdysone TMS ethers require a column oven temperature of 265-280 OC with a slightly higher temperature for the detector oven, so that the best compromise is a detector oven temperature of 300 "C. 3.5.4 Sample preparation f o r ECD To determine the very low concentrations of arthropod ecdysones with the electron capture detector requires careful sample preparation, to avoid contamination with electron-capturing impurities. Above all, chlorinated solvents must be avoided. A very efficient procedure used in the authors' laboratory for ecdysone analysis in Schistocerca gregaria is shown in scheme 1 (see p. 46). It makes use of three highly efficient partition systems followed by a further purification of the extract by thin-layer chromatography (TLC) on silica gel of the trimethylsilyl ethers. An area from R f = 0.5-0.9 is scraped from the plate and the ecdysone TMS ethers eluted with sodium-dried diethyl ether. The ether is removed with a stream of nitrogen at 30 OC and injection of the residue in an appropriate volume of toluene is made €or gas chromatography. The recovery of ecdysterone added t o the methanol extract of adult desert locusts is shown in Table 12. Scheme 1 was designed €or the efficient separation of polar ecdysones from biological material and will not be as efficient for the determination of the more hydrophobic ecdysone metabolites with only a few hydroxyl groups. The analysis of isolated tissues may not require the use of the complete scheme for acceptable results. The quality of the chromatograms obtained is illustrated for the determinations of ecdysone (A), ecdysterone (B) and an as yet unidentified compound (C) in 4th instar nymphs, day 5 of Schistocerca gfegaria (Fig. 1).

46

E. DAVID MORGAN AND COLIN F. POOLE SCHEME 1

+

Ten insects homogenized in methanol

Insoluble residue

Methanol solution

1

(discard)

Residue, partition

4

4 Aqueous methanol

Hexane (discard)

4

Residue, partition

r-7 Butanol

Aqueous

&

(discard)

Residue, partition

4 Aqueous

Ethyl acetate (discard)

4

Residue

4 GLC-ECD C TLC t silylate

Borst et al. (1974) have described a sample “clean-up” procedure for the determination of ecdysones in Drosophila melanogaster by GLC-ECD, scheme 2. The ecdysones contained in the butanol are subjected t o TLC on silica gel in CHCl3 : 95 per cent EtOH (7:3). Some preliminary work in our laboratory indicated that the recovery of trace quantities of ecdysones by alcoholic elution from TLC plates gave poor recoveries and contributed t o the GLC detector solvent front by contaminating samples with impurities from the silica gel even though this had been thoroughly washed prior t o use. I t was for this reason that chromatography was confined t o TMS ethers in scheme 1 as they can be recovered more efficiently and with a weaker eluent, e.g. ether, which does not add any impurities t o the gas chromatogram. This also enabled us t o avoid the use of a water-hexane partition before GLC t o destroy excess silylating reagent. The authors also used a very high detector oven temperature (400 “C) for the final determination which, because of the temperature dependence of the detector response, would cause considerable loss in sensitivity.

47

ESTIMATION OF ECDYSONES TABLE 12 The recovery of ecdysterone added to the methanol extract of adult locusts

TMS ether formed

Pentakis-TMS ecdysterone

Hexakis-TMS ecdysterone

Amount of added ecdysterone I-lg

Range of recoveries % 80-90

10-1 1-0.1 0.01-0.001

75-85 70-85 90-100 80-90 70-85

10-1 0.1-0.01 0.001-0.0001

i

C

Time + Fig. 1. Gas chromatograph trace of ecdysones present on day 5 of the 4th instar of Schistocercu greguriu, using an electron capture detector. A, ecdysone; B, ecdysterone; C, unidentified polar ecdysone.

Morgan et al. (1975a) have described a simpler “clean-up” procedure which was adequate for determining methoxime-silyl ethers with the flame ionization detector. Miyazaki et al. (1973) have described a rather long

48

-

E. DAVID MORGAN AND COLIN F. POOLE

SCHEME 2

Larvae blend in methanol

Residue (discarded)

Methanol solution

4

Add water and CHC13

4 I

4

?

Lower phase

Upper phase

I

I

_______J

1

L

Residue

Re-extract

Rutanol

Water (discarded)

1 Silica Gel TLC

1

Elute with ethanol

1 1 Silylate

Residue

Water (discarded)

Hexane

1 GLC-ECD

extraction procedure for the analysis of ecdysones from pupae of Bombyx mori. The pupae were dried and ground with sea sand before soxhlet extraction with tetrahydrofuran for twenty-four hours. The tetrahydrofuran extract was concentrated, adsorbed onto silicic acid and lipids etc. removed by soxhlet extraction for one hour each with hexane, benzene and ether before extraction of the ecdysones with tetrahydro furan. This extract was purified by passage though a short column of silica gel followed by preparative layer TLC and the residue eluted from the plate used t o form the TMS ethers. Formation of the TMS-HFB derivative followed by preparative layer TLC was used for the final analysis by GLC-ECD.

ESTIMATION OF ECDYSONES

3.6

49

MASS FRAGMENTOGRAPHY

The ion collector of a modern mass spectrometer is so sensitive t o small amounts of material that the mass spectrometer can be used as a specific and quantitative gas chromatograph detector. Because the mass spectrometer has t o operate at high vacuum, the gas chromatograph is coupled to the mass spectrometer through an interface (a semi-permeable membrane or jet separator) to remove the carrier gas. For efficient operation of the technique, the conditions of the gas chromatograph, interface and mass spectrometer must all be optimized. The mass spectrometer does not scan the mass range but is set t o record ions of a specific mass-charge ratio (m/e). For single ion monitoring (SIM), a single mass is selected from the mass spectrum of the compounds of interest and this ion is monitored throughout the GLC run. Thus only peaks from the GLC containing the selected ion are recorded by the mass spectrometer and the output (mass chromatogram) is in the conventional form of Gaussian peaks on a time axis, like most GLC detectors. Selectivity can be improved by the use of multiple ion detection (MID) in which several ions are sequentially brought to focus at the detector by rapid switching of the accelerating voltage using an accelerating voltage alternator and the output accumulated in different channels. Each ion has its own channel and a multipen recorder is used to display the data in the form of mass chromatograms. Under optimum conditions, l o - ' g of an organic compound can be detected. Not all mass spectrometers are suitable for the mass fragmentographic analysis of ecdysones. For compounds of low volatility a doubly pumped mass spectrometer is essential (Poole, 1975). The MID unit is only available for mass spectrometers of recent origin. The very high cost of a mass spectrometer and the expertise needed in its operation will probably limit this technique for the analysis of ecdysones to a few specialist laboratories. The mass fragmentographic analysis of the TMS ethers of ecdysones was first performed by Miyazaki et al. (1973). The fragments m/e 561 (I) for ecdysterone and m/e 564 for ecdysone TMS ethers were used as the selected masses. The limit of detection was l o - ' g (100 pg) and both ecdysone and ecdysterone were measured from pupae of Bombyx mori. This method has been used by Lafont et al. (1974) and Chino et al. (1974). It is important when using the mass fragmentographic technique to be sure of the composition of the derivative formed. For ecdysterone both the hexakis (TMS) ether and pentakis (TMS) ether have the ion m/e 561 as their base peak. Their gas chromatographic retention times are very different, and if incomplete reaction at the 14a-hydroxyl group had occurred, failure t o appreciate this point could lead t o error in the interpretation of the mass chromatogram. As the same ion m/e 561 is

E. DAVID MORGAN AND COLIN F. POOLE

50

obtained with both derivatives, its structure is probabh. hettcr reprrsented Iiy (13) than (12). The situation with the TMS ethers of ecdysone is difterent. The mass spectra of tetrakis (TMS) ecdysone has a strong ion at m/e 564 but in pentakis (TMS) ecdysone, the ion m/e 564 is weak with a strong ion at mfe 567 (Morgan and Poole, 1976). Thus, although either derivative is juitable for mass fragmentography, it is essential that the derivative !ormation reaction is thoroughly investigated first as this controls the \election of the ion t o be monitored. Insufficient information is available to propose structures for the ions m/e 564 and 567.

:::I::; cP

TMSO

OTMS

TMSO

*=OTMS

y

3

-+

TMSO TMSO

H O

H O m/e 561 (13)

For quantitative analysis by either SIM or MID an internal standard is iequired to permit the analyst to make allowances for losses caused by \ ariation in injected sample volume, column adsorption effects and all those variations in ion current or voltage which influence the intensity, I ocusing o r measurement of the ion beam. The plant ecdysone, cyasterone, its trimethylsilyl ether derivative was used for this purpose by Miyazaki t t al. This compound is less volatile than the insect ecdysones and unless high temperatures are used, peak shapes are very broad. The ideal ecdysone Iiiternal standard would seem t o be an isotopically labelled insect hormone \I hich could be distinguished from the natural hormone by using multiple ) n detection of the similar fragment in labelled and unlabelled compound. I(

:i. 7 HIGH-PRESSURE LIQUID CHROMATOGRAPHY

'I he very polar nature of the ecdysone molecule and the initial difficulties cuperienced in forming suitable derivatives for GLC led several groups to

investigate the possibility of ecdysone analysis using high-pressure liquid c hromatography (HPLC). As derivative fprmation is not required for aiialysis, ecdysones might be determined directly and the effluent collected f o r subsequent analysis by other physical techniques or b y bioassay. I;urthermore, the ecdysones strongly absorb ultraviolet light at 254 nm

ESTIMATION OF ECDYSONES

51

(section 3.3) which is convenient for detection purposes. The ultraviolet detector is currently the most sensitive of all HPLC detectors available for those compounds which absorb radiation between 200 and 700 nm. A detection limit for the determination of ecdysones b y HPLC using uv detection has not been published, but theoretical calculation indicates that the detection of 10 ng of ecdysones should be possible. By the use of high pressures to force the mobile liquid phase through columns of incompressible micro-particulate stationary phase, resolution in a time compatible with that associated with GLC can be obtained. The separation of ecdysones by HPLC is conveniently considered in terms of the nature of the stationary phase employed. 3.7.1 Reversed-phase chromatography Reversed-phase chromatography has important advantages over other forms of adsorption chromatography for the analysis of water-soluble organic compounds. The separation process is based on the attraction of the hydrophobic stationary phase for hydrophobic compounds (Simpson, 1972). Hydrophilic solvents are strong eluents because they are capable of displacing the sorbed polar molecules. For ecdysones, the reversed-phase technique is specially attractive as the very polar impurities found in crude extracts (e.g. sugars and glucosides) are eluted from the column before the less polar ecdysones and the column material can be made ready for re-use by solvent recycle. Hori (1969) described the separation of ecdysones by reversed-phase chromatography using Amberlite XAD-2 with a wateralcohol eluting gradient. The Amberlite resin is not available in a particle size suitable for HPLC and had t o be ground and sieved before use. Since the resin is compressible, maximum pressures were limited, which set an upper limit on flow rates. Although the separation of structurally similar ecdysones was often quite good, the retention times were inconveniently long (ecdysterone 420 min, ecdysone 540 min). The method has been used for the separation of ecdysones from plants (Imai et al., 1968a, b; Hori, 1969) and insect material (Mnriyama et al., 1970; Schooley et al., 1972; Hikino et al., 1975). Separations on the preparative scale have been described for ecdysones from crude plant extracts (Schooley and Nakanishi, 1973). The authors caution that for this application the very polar ecdysones may not be sufficiently retained by the resin (low column capacity factor) to give an adequate separation from impurities. For this purpose, more polar Amberlite resins might be used to advantage (Simpson, 1972). The separation of ecdysones on the semi-rigid porous polymer, Poragel PN, has been described (Bombaugh, 1971; Schooley and Nakanishi, 1973). Poragel PN is a copolymer of divinylbenzene and ethylene glycol dimethacrylate with heavy cross-linking (40 per cent) compared with Amberlite XAD-2

52

E. D A V I D MORGAN AND COLIN F. POOLE

which is a lightly cross-linked polystyrene polymer. The ester groups of the I'oragel confer a degree of polarity t o the polymer which probably explains the greater retention volume of ecdysterone on Poragel compared with .\mberlite XAD-2. The separation of milligramme quantities of ecdysones was achieved but analytical separations (sub-microgramme level) were poor, due in part t o the inherently slow mass-transfer and limited capacity factor o f the polymer, particularly at high flow rates (Bombaugh, 1971; Schooley, 1973). The reversed-phase separation of ecdysones from crude plant extracts using polyamide has been described (Jizba and Herout, 1967). Pellicular "olyamide phases suitable for HPLC are available commercially (Pellidon, Reeve Angel) but their application t o the analysis of ecdysones has not been reported. King et al. (1974) have described the analysis of ecdysones 1 n tissue culture medium using Vydac reversed-phase (CIS hydrocarbon permanently bonded to a pellicular support) and eluting with a mobile phase of 25 per cent methanol in water. This method has been used in the Identification of ecdysones in Drosophila melunogaster (Borst et al., 1974) .ind Leucaphaea maderae (King and Marks, 1974). Using Corasil CIS, and a mobile phase of 35 per cent water in methanol ZP,3&14a-trihydroxy-5$holest-7-en-6-one had a retention time of 12.0 min (1.0 ml min-' , column I m x 2.1 mm). Under these conditions ecdysterone is poorly retained and rlutes with the injection solvent (Poole, 1975). The change in capacity I x t o r for the model steroid with solvent composition can be illustrated b y I he following data: (

0.5 ml min-' 29 per cent water in methanol: R , 5.75 min, 0.5 ml min-' 25 per cent water in methanol: R t 1.40 min. i n an attempt to increase the capacity factor by using a more polar

eversed-phase column (ETH Permaphase), ecdysterone was not retained when water-alcohol mixtures were used as the mobile phase.

1

.7 .2 Liquid -1iquid part it io n chro mu tography liquid partition chromatography of ecdysones is difficult due t o a lack of permanently bonded polar stationary phases. Those separations which have I)een achieved have used polar liquid phases mechanically held on a solid jupport. To prevent stripping of the stationary phase, the mobile phase has to be pre-saturated with stationary phase. Gradient elution is not practical m d high flow rates usually sheer the stationary phase from the support. l'he most successful analysis of ecdysones have been achieved with 1 per < ent P,P'-oxydipropionitrile (BOP), on Zipax using hexane mixed with letrahydrofuran as the mobile phase (Henry et al., 1971; Schmit, 1971). A method for increasing the percentage loading of BOP on Zipax has been 'k

ESTIMATION OF ECDYSONES

53

reported (Kirkland and Dilks, 19 73). Permanently bonded liquid phases such as Corasil CI8 and ETH Permaphase show no retention of ecdysones using hexane mixed with chlorinated hydrocarbons as the mobile phase. 3.7.3 Liquid-solid absorption chroma tograp hy From the early days of ecdysone analysis, the separation of the hormones by silica gel chromatography has been important. The extension o f this work t o HPLC using highly efficient small-particle silica1 columns was predictable. Chino et al. (1974) separated ecdysone from ecdysterone on Zorbax Sil (25 cm x 2.1 mm column, 0.3 ml min-' , dichloromethanemethanol 9: 1).Peak shapes were reasonably sharp and the separation quite good (ecdysone R , 23 min and ecdysterone R , 30 min). Nigg e t al. (1974) have described the separation of ecdysones on Corasil I1 (a high-capacity pellicular silica) using mixtures of chloroform-ethanol as the mobile phase. King and Marks (1974) have described the separation of ecdysone from tissue culture medium in which pairs of prothoracic glands of Leucophaea maderae were incubated, using Lichrosorb SI 60 (0.5 m x 2.2 mm column, 10 per cent methanol in dichloromethane, 0.4 ml min-' ). With Porasil A, an irregularly shaped porous silica (37-75 pm) and tetrahydrofuran or tetrahydrofuran mixed with methanol, ecdysterone is rapidly eluted. With 40 per cent tetrahydrofuran in dichloromethane, 2/3,3/3,14~-trihydroxy-5/3-cholest-7-en-6-one is eluted in 2.25 min (1.5 ml min-', 50 cm x 2.1 mm) but the retention time of ecdysterone is very long (Poole, 1975). The optimum mobile phase composition was tetrahydrofuran-methanol-dichloromethane (20: 1:29). The addition of a small amount of methanol as a polar phase-modifier reduced peak tailing substantially. With a flow rate of 1 ml min-', the retention time of ecdysterone was 11.O min and for 2~,3@,14a-trihydroxy-5/3-cholest-7en-6-one was 2.60 min. The still relatively broad peak for ecdysterone is probably a consequence of the effect of diffusion in the stagnant mobile phase in the pores of the support making an important contribution t o the mass transfer term. To overcome this problem the use of a more efficient silica gel adsorbent is required. High-pressure liquid chromatography is still in a state of rapid advance. With improved detection cells and new adsorbents and liquid phases we may expect t o learn of more efficient and more versatile separations for ecdysones.

4 Summary of analytical methods Seven different methods for the micro-determination of ecdysones have been described in the previous section. With the exception of the acid-

E. DAVID MORGAN AND COLIN F. POOLE

54

induced fluorescence method they can all be applied t o the analysis of c.cdysones in biological material. The remaining six methods are contrasted in terms of sensitivity, selectivity, ease of application to routine samples, degree of ‘skill required for their operation and relative cost per analysis. The least detectable amount of ecdysterone which can be determined by I he physical methods described in section 3 is given in Table 13. Thin-layer chromatography with uv detection, and bioassay methods are the least TABLE 1 3 The sensitivity of current analytical techniques for the analysis of ecdysterone ~

Method

Detection limit 10-~

Comments ~~

Thin -layer chromatography (IIV detection)

500

Bioassay (quantitative determination) Fluorescence spectroscopy

100

50

Radioimmunoassay

0.08

Gas-liquid chromatography (electron capture detection) Mass fragmentography High-pressure liquid chromatography (uv detection)

0.005

0.10 10

Nonspecific detection easily masked by biological material Approximately 25 for nonquantitative detection Not applicable for crude biological material Dependent upon availability of antiserum 0.06 with a free 1 k - O H group Expensive equipment Approximate value by calculation

sensitive. Thin-layer chromatography is an inexpensive technique, requiring llttle skill in its operation. Selectivity in terms of resolution of ecdysones is poor unless the time per analysis is extended as in continuous flow TLC. In 1 erms of efficiency it performs badly by comparison with high-pressure liquid chromatography but the capital cost of equipment is much less. The I)ioassay technique is simple to perform but requires large numbers of test ‘rbdomens, the upkeep of a stable insect colony and a degree of skill in handling, and scoring the test species. It should be remembered that the Iiioassay does not detect all compounds of ecdysone type, shows a variable I esponse between individual ecdysones and can give a positive response t o c.cdysone precursors. It responds to substances which induce moulting as this is the effect observed and not t o ecdysones in general. A preliminary purification step is often required before analysis and the resolution

ESTIMATION OF ECDYSONES

55

obtained by this step controls the specificity. When TLC is used at this stage, the specificity may be poor unless conditions are optimized for the separation. Radioimmunoassay is capable of detecting ecdysones in crude biological material at trace levels. A degree of specificity is obtained in that the immunization process yields antibodies to molecules of a certain threedimensional structure. Specificity between individual ecdysones is not normally large and cross-reaction occurs. To establish the assay requires considerable time and labour with a high degree of skill. The time per assay can be inconveniently long and if counting equipment is not available, then expensive to set up. The establishment and operation of the assay requires relatively large quantities of expensive ecdysones and radiolabelled ecdysones. Availability of the required antiserum could make the method much more attractive. Gas-liquid chromatography with electron capture detection of ecdysone TMS ethers is the most sensitive of all the assay techniques and is directly applicable to those ecdysones containing 7-en-6-one and l4a-hydroxyl groups, a combination found in all arthropod ecdysones so far identified. The combination of the high resolution of the gas chromatograph and sensitivity of the electron capture detector cannot be matched by another technique for the analysis of ecdysones. Application t o biological material requires a preliminary purification procedure which extends the time per analysis. The continuous operation of an electron capture detector requires some experience and skill but, if treated with care, analyses can be carried out routinely. Variability in the conditions necessary for silyl ether formation can cause difficulties. The gas chromatograph-electron capture detector combination is not prohibitively expensive t o purchase and running costs are very low. Mass fragmentography makes use of a mass spectrometer as a gas chromatographic detector. It is a sensitive technique with probably the highest degree of specificity of all the assays. The high resolution of the gas chromatograph is coupled to the mass discriminatory power of the mass spectrometer and thus even close t o the detection limit a high degree of confidence can be placed in both the identity of the compound and its concentration in the sample. The method ties up a very expensive piece of equipment that may be serving a variety of uses. The capital cost required to establish the technique is the highest of all the assays. If the instrument is also available for general use then allowance needs to be made for the time required to re-establish the optimum conditions and stabilize the instrument each time. Some preliminary purification is also required for biological material unless the mass spectrometer is operated under high resolution conditions at an overall lower sensitivity. High-pressure liquid chromatography is a relatively new technique with

56

E. DAVID MORGAN AND COLIN F. POOLE

,In enormous potential for the separation of mixtures into their components. The uv detector is at present the most sensitive of all HPLC cletectors. Crude biological material tends t o contain uv absorbing impuri1 ies which mask the detector and as a consequence a preliminary extraction 1)rocedure is necessary which adds t o the time per analysis. The initial cost o f equipment is high. It is relatively easy t o operate, if the operator has c,uperience in chromatography. Depending on the mobile phase used, ic~placementof solvent can considerably add t o the operating cost of the asay.

5 Conclusion ‘1 his review has attempted to bring together a body of information that is

s( attered through the chemical and biological literature. The study of ai thropod moulting and development has excited the interest of research

orkers in a wide range of disciplines. Our own interest arises from possible means of pest control. Thus far, discoveries have provided even fewer new ideas for insect control than has the area of juvenile hormone, but the assembling of the jig-saw puzzle has c,nly begun. As the picture emerges, one can expect fundamental discoveries that will stimulate entirely new strategies for insect control. 11

Acknowledgements

Work in the authors’ laboratory has been supported by the Overseas rkvelopment Administration, the Ministry of Overseas Development, and the Science Research Council, whose help we are glad t o acknowledge. References Agui, N. (1973). Quantitative bioassay of moulting hormone in vitro. Appl. Ent. ZOOL 9, 236-238. Urbbington, P. M. (1975). A chemical investigation into the moulting hormones of the barnacle Batanus balunoides. Ph.D. Thesis, Keele University. Bollenbacher, W. E., Vedeckis, W. V., Gilbert, L. I. and O’Connor, J. D. (1975). Ecdysone titres and prothoracic gland activity during the larval-pupal development of Manduca sexta. Devl Biol. 44, 46-53. Bombaugh, K. J. (1971). A comparison of separation mechanisms for liquid chromatography applications. In “Modern Practice of Liquid Chromatography” (Ed. J. J. Kirkland). pp. 360-374. WiIey-Interscience, New York. Borst, D. W. (1973). The analysis of ecdysones in biological tissue. Ph.D. Thesis, University of California (Dissertation Abs. No. 73 32052). Borst, D. W. and Engelmann, F. (1974).In vitro secretion of u-ecdysone by prothoracic glands of a hemimetabolous insect Leucophaea muderae (Blattaria). J. ex$ ZooL 189, 415-419.

ESTIMATION OF ECDYSONES

57

Borst, D. W. and O’Connor, J. D. (1972). Arthropod moulting hormone radioimmune assay. Science, 178, 418-419. , Borst, D. W. and O’Connor, J. D. (1974). Trace analysis of ecdysones by gas liquid chromatography, radioimmunoassay and bioassay. Steroids, 24, 637-656. Borst, D. W., Bollenbacher, W. E., O’Connor, J. D., King, D. S. and Fristrom, J. W. (1974). Ecdysone levels during metamorphosis of Drosophila melanogaster. Devt Biol. 39, 308-316. Burdette, W . J. (Ed.) (1974). “Invertebrate Endocrinology and Hormonal Heterophylly”. Springer, New York. Butenandt, A. and Karlson, P. (1954). h e r die Isolierung eines MetamorphoseHormons der Insekten in kristallisierten Form. 2. Naturf. 9b, 389-391. Canonica, L., Danieli, B., Ferrari, G., Krepinsky, J. and Weisz-Vincze, I. (1975). A novel method of isolation of phytoecdysones from Kaladana seeds. Phytochem. 14, 525-527. Carlisle, D. B. (1965). The effect of crustacean and locust ecdysones on moulting and proecdysis in juvenile shore crabs, Carcinus maenas. Gen. comp. Endocr. 5, 366-372. Chino, H., Sakurai, S., Ohtaki, T., lkekawa, N., Miyazaki, H., Ishibashi, M. and Abuki, H. (1974). Biosynthesis of a-ecdysone by prothoracic glands in vitro. Science, 183, 529-530. Clever, U., Clever, I., Storbeck, I. and Young, N. L. (1973). The apparent requirement of two hormones, (Y- and 0-ecdysone for moulting induction in insects. Devl Biol. 31, 47-60. Faux, A., Horn, D. H. S., Middleton, E. J., Fales, H. M. and Lowe, M. E. (1969). Moulting hormones of a crab during ecdysis. Chem. Commun. 175-176. Fraenkel, G. (1935). A hormone causing pupation in the blowfly Calliphora erythrocephala. Prof. R. SOC. B, 118, 1-12. Gagosian, R. B., Bourbonniere, R. A., Smith, W. B., Couch, E. F., Blanton, C. and Novak, W. (1974). Lobster moulting hormones: isolation and biosynthesis of ecdysones. Experientia, 30, 723-724. Galbraith, M. N. and Horn, D. H. S. (1969). Insect moulting hormones: crustecdysone (20-hydroxyecdysone) from Podocarpus elatus. Aust. J. Chem. 22, 1045-1057. Galbraith, M. N., Horn, D. H. S., Middleton, E. and Hackney, R. J. (1968a). Structure of deoxycrustecdysone, a second crustacean moulting hormone. Chem. Commun. 83-85. Galbraith, M. N., Horn, D. H. S., Middleton, E. J. and Hackney, R. J. (1968b). A silyl ether as a protecting group in the synthesis of an isomer of ecdysone, 2OR-hydroxy22-deoxyecdysone. Chem. Commun. 466-467. Galbraith, M. N., Horn, D. H. S., Middleton, E. J. and Hackney, R. J. (1969a). Moulting hormones of insects and crustaceans: the synthesis of 2-deoxy-3-epicrustecdysone. Aust. J. Chem. 2 2 , 1059-1067. Galbraith, M. N., Horn, D. H. S., Thomson, J. A., Neufeld, G. J. and Hackney, R. J. (1 969b). Insect moulting hormones: crustecdysone in Calliphora. J. Insect Physiol. 15, 1225-1233. Galbraith, M. N., Horn, D. H. S., Middleton, E. J., Kaplanis, J. N. and Thompson, M. J. (1973). Structure of podecdysone C, a steroid with moulting hormone activity from the bark of Podocarpus elatus R. Br. Experientia, 29, 782.

E. DAVID MORGAN AND COLIN F. POOLE

58

(;albl-aith, M. N., Horn, D. H. S., Middleton, E. J., Thomson, J. A. and Wilkie, J. S. (1975). Metabolism of 3~,14~-dihydr0xy-5~-(3a-~H)-chOlest-7-en-6-one in Calliphora s t y p a J. Insect Physiol. 21, 23-32. Gilbert, L. I. and King, D. S. (1973). Physiology of growth and development: Endocrine aspects. I n “The Physiology of Insecta (Ed. M. Rockstein), vol. 1, pp. 250-368. Academic Press, New York and London. Gilgan, M. W. and Zinck, M. E. (1972). Estimation of ecdysterone from sulphuric acid induced fluorescence. Steroids, 20, 95-104. IIampshire, F. and Horn, D. H. S. (1966). Structure of crustecdysone, a crustacean moulting hormone. Chem. Commun. 37-38. I leinrich, G. and Hoffmeister, H. (1967).Ecdyson als Begleitsubstanz des Ecdysterons in Polypodium vulgare L. Experienta, 23, 995. Ilenrv, R. A., Schmit, J. A. and Dieckman, J. F. (1971).The analysis of steroids and derivatized steroids by high speed liquid chromatography. J. Chromat. Sci. 9,

5 13-520. IIikino, H., Ohizumi, U. and Takemoto, T. (1975). Steroid metabolism in Bombyx mori, 1. Catabolism of ponasterone A and ecdysterone in Bombyx mori. Hopp‘peSeyler’s 2. physiol. Chem. 356,309-314. I locks, P. and Weichert, R. (1966). 2O-Hydroxy-ecdyson, isoliert aus Insekten. Tetrahedron Lett. No. 26, 2989-2993. :;offman, J. A., Koolman, J., Karlson, P. and Joly, P. (1974).Moulting hormone titre and metabolic fate of injected ecdysone during the fifth larval instar and in the adult of Locusta migratoria (Orthoptera). Gen. conp. Endocr. 2 2 , 90-97. I loffnieister, H. (1966). Isolierung eines neuen Insektenhormons aus Seidenspinnerpuppen. 2. Naturf. Zlb, 335-336. Iloffmeister, H. and Grutzmacher, H. F. (1966). Zur Chemie des Ecdysterones. Tetrahedron Lett. No. 33,4017-4023. 1 lori, M. (1969). Automatic column chromatographic method for insect moulting steroids. Steroids, 14,33-45. Ilorn, D. H. S. (1971). The ecdysones. In “Naturally Occurring Insecticides (Eds M. Jacobson and D. G . Crosby), pp. 333-459.Marcel Dekker, New York. Ilorn, D. H. S., Middleton, E. J., Wunderlich, J. A. andHampshire, F. (1966).Identity of the moulting hormones of insects and crustaceans. Chem. Commun. 339-341. Ilorn, D. H. S., Fabbri, S., Hampshire, F. and Lowe, M. E. (1968). Isolation of crustecdysone from a crayfish (Jasus lalandei). Biochem. J. 109,399-406. liorn, D. H. S., Wilkie, J. S. and Thomson, J. A. (1974). Isolation of 0-ecdysone (20-hydroxyecdysone) from the parasitic nematode Ascaris lumbricoides. Experisntia, 30, 1109-1110. Il>iao, T. H., Hsiao, C. and deWilde, J. (1975). Moulting hormone production in the isolated larval abdomen of the Colorado beetle. Nature, 255, 727-728. lIktber, R. and Hoppe, W. (1965). Zur Chemie des Ecdysons VII. Die Kristall-u. Molekiilstrukturanalyse des insektenverpuppungs-hormons Ecdyson mit der Automatisierten Faltmolekulmethode. Chem. Ber. 98, 2403-2424. ILrkawa, N., Hattori, F., Rubio-Lightbourn, J., Miyazaki, H., Ishibashi, M. and Mori, C. (1972).Gas chromatographic separation of phytoecdysones. J. Chronat. Sci. 10,

233-242.

ESTIMATION OF ECDYSONES

59

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

60

E. D A V I D MORGAN AND COLIN F. POOLE

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

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

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

ESTIMATION OF ECDYSONES

61

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

E. DAVID MORGAN AND COLIN F. POOLE

62

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

i.

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

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

.

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

.

.

.

.

.

.

.

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

64

HUGH FRASER ROWELL

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

NEUROSECRETORY CELLS

65

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

2 The anatomy of the insect neurosecretory system

2.1

RECOGNITION OF NEUROSECRETORY CELLS

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

66

HUGH FRASER ROWELL

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

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

N EUROSECR ETO RY CELLS

67

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

2.2

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

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

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

68

HUGH FRASER ROWELL

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

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

N E U ROSECR ETORY CELLS

69

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

Full name

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

70

HUGH FRASER ROWELL

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

2.3

GENERAL RESULTS OF MORPHOLOGICAL STUDIES OF NEUROSECRETORY CELLS

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

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

N EU ROSEC R ETO R Y CELLS

71

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

2.4

THE DISTRIBUTION OF NEUROSECRETORY CELLS IN INSECTS

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

BRAIN

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

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

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

HUGH FRASER ROWELL

72

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

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

In most of the stomatogastric ganglia examined, the neurosecretory axons present derive from NSCs located in the brain or the corpus cardiacum. Jwo NSCs are found in the frontal ganglion of sphingid and saturniid inoths (Bounhiol et at., 1953; Borg et at., 1973). The hypocerebral ganglion o f Aedes (Diptera, Culicidae) was reported t o contain a few NSCs (Clements, 1956) but this has not been confirmed (Burgess and Rempel, 1966). Secretory cells, possibly not neural, occur in the hypocerebral ganglion of Oncopeltus (Hemiptera, Lygeidae) according t o Unnithan et al. (1971).

ti.

SUBOESOPHAGEAL GANGLION, GANGLIA OF THE VENTRAL NERVE CORD

NSCs have been demonstrated in the SOG and VNC of all insects iiivestigated, since they were first described by Day (1940) and Scharrer

N EU ROSEC R ETO R Y CELLS

73

(1941) from sphingids and blattids respectively. Most of them remain without clear functional attribution, though in different individual animals humoral factors controlling diapause, diuresis and antidiuresis, chromatophores, and luminescent organs have been associated with the ganglia. Most of the NSCs of the VNC are associated with the medial neurohaemal organs (see below), and they show much segmental replication. In some groups the somata of the NSCs are found within the NHOs, rather than in the ganglia. e.

EXTRAGANGLIONIC NSCS

In Phasmida and Diptera (Finlayson and Osborne, 1968) NSCs are found outside the ganglia, associated with segmental abdominal nerves. Possibly they are derived from sensory neurones. Fourteen neurosecretory somata are also found in the thorax of M y z u s (Aphidae) and their axons are part of the nerves which radiate from the CC. The cell bodies are located on the aorta, and in the thoracic musculature and tracheal system (Bowers and Johnson, 1966). Their homologies are obscure, but by analogy with the structural variety associated with the median neurohaemal organs (Raabe et al., 1974) they may be part of the corpus cardiacum. Apart from the soma position, and often the fine structure of the vesicles, remarkably little anatomical information is available about these cells. With few exceptions (Adiyodi and Bern, 1968; Highnam and West, 1971; Mason, 1973; Schoonveld, 1974b) nothing is known of any dendritic structures. Transport of neurosecretory material down the axons has been demonstrated frequently; release appears to be by exocytosis, to be associated with depolarization of the membrane, and t o be dependent on Caz+ influx (e.g. Normann, 1973, 1974) in the same way as in vertebrate chrom affin cells or in the release of transmitter substance from normal neurones. In an aphid and a chrysomelid beetle (Johnson, 1963; Schoonveld, 1974b) individual NSCs give rise t o more than one major axon, with different destinations, and this has been suggested for several other insects too. Golgi or dye injection preparations would in general be required t o substantiate this sort of anatomical claim. After transection o f the axon, the amount of stainable material in the cell proximal t o the cut increases (either b y accumulation of secretion, or as a component of the injury response) and dendritic structures then sometimes become apparent which were not previously visible (Schoonveld, 1974b). The axons of NSCs terminate in a great variety of ways. In some cases they form well-defined neurohaemal organs (NHOs). Sometimes these are associated with the aorta, especially in the Hemiptera (Junqua, 1956; Seshan and Ittycheriah, 1966, Dogra, 1967a, b; Srivastavah and Dogra, 1969; Furtago, 1971; Unnithan et al., 1971, who also give a review) but

74

HUGH FRASER ROWELL

.ilso in other groups (e.g. Normann, 1965). More commonly, however, VHOs occur at the surface of a ganglion or nerve, where they are separated from the haemolymph only by the neural lamella. Such NHOs are reported from the. CC of many insects, the CA (Kind, 1965; Tombes and Smith, 1970), the NCA I1 of crickets (Huignard, 1964; Awasthi, 1968; Weber and Gaude, 1971), the brain of Orth‘optera (Geldiay, 1973; Geldiay and Edwards, 1973) and possibly of moths (Kobayashi, 1957), the frontal connectives (Ganagarajah, 1965), the medial nerves of all orders of insects (Ra‘ibe, 1965; Brady and Maddrell, 1967; recently reviewed by Thomas and Raabe, 1974, and by Raabe et al., 1974), the ganglia of the VNC (Grillot, 1970a) and the abdominal nerves of Hemiptera (Maddrell, 19.66a). In dddition to those associated with NHOs, some NSCs appear t o innervate their target directly, or at least t o end in a very restricted space. Direct neurosecretory innervation of the gut (Willey, 1962; Johnson and BoGers, 1963; Fletcher, 1969), heart (sources reviewed by Miller and Rees, 1973), epidermis (Maddrell, 1965), skeletal muscle (Willey, 1962; Johnson and Bowers, 1963; Osborne et al., 1971), luminescent organ (Smith, 1963), pro thoracic or homologous gland (Normann, 1965; Hintze-Podufal, 1970; Thomas and Raabe, 1974) and salivary gland (Bowers and Johnson, 1966) have been reported; the neurosecretory innervation of the CA may be another similar instance. Some of these examples may be unique to a particular group of insects, and in some, at least, the evidence for the innervation being neurosecretory is not only purely morphological, but is even contradicted by the available physiological evidence. For example, Miller and Usherwood (19 71) recorded normal postsynaptic potentials from heart muscles in response t o stimulation of allegedly neurosecretory axons. If these units cannot be shown to have any additional function, which is not merely the transient alteration of subsynaptic membrane permeability to ions (the functional definition of a transmitter substance) there would be a strong case for considering them simply as motor neurons with an unusual ultrastructural morphology. If this interpretation is general, it may be that many of the apparently neurosecretory units associated with the heart and gut in insects are actually motor neurons. Brown (personal communication and 1976) has described the isolation from cockroaches of a pentapeptide which causes the contraction of gut muscle when iontophoresed in pharmacological quantity onto synaptic drc,ts. A neuron secreting a peptide transmitter substance would presumably have a morphology like that of an orthodox NSC. Anwyl and Finlayson (1974) have proposed that the same neurons function alternately as motor neurons and as NSCs, utilizing at least two different secretory products. A further blurring of the division between neurosecretory cell and a cell secreting “neurohumors” in the sense of Scharrer (1969) is seen

NEUROSEC R ETO RY CELLS

75

in the case of the neurons which appear t o bring about the plasticization of the cuticle in Rhodnius (Maddrell, 1965). The active agent appears to be 5-hydroxtryptamine (Reynolds, 1974) which is normally secreted from neurons which lack the ultrastructural morphology of a NSC. Finally, there are neurosecretory axons which appear to end in the ganglionic neuropil without innervating peripheral effector organs (e.g. the NSCs which run from the brain to the terminal abdominal ganglion in culicids (Fuller, 1960; Burgess, 1973) or from the SOG to the thoracic ganglia in acridids (Delphin, 1975; Chalaye, 1967)). Further studies are required t o determine the function and release point of these units. They may affect directly the ganglionic neurons, and a number of neurotropic factors appear t o be involved in the hormonaIly controlled release of motor activity (Truman and Riddiford, 1974a).

2.5

DIVERSITY OF NEUROSECRETORY CELL COMPLEMENT IN INSECTS

2.5.1 How conservative are the insects? The universality of the major NSC groups (e.g. of the pars intercerebralis, the corpus cardiacum, etc.) shows that at least some neurosecretory features are the same throughout the insects. To assess conservation in evolution one obviously requires knowledge of physiology, as well as anatomy. In theory, homologous groups of NSCs in different animals could have totally different products, or different functions even if they elaborated chemically identical products. In most cases, unfortunately, this physiological knowledge is not yet available. One would expect that the basic endocrine mechanism would be fairly stable within the insects, but that there would also be wide variation in the use of hormonal mechanisms for specific adaptations. Panov (1964) reviewing the distribution of type A NSCs in the VNC of various Orthoptera, has already advanced the concepts of “principal” cells, found in most taxa, and “additional” cells, peculiar to a few; together these form a statement in anatomical terms of the same hypothesis. As insects are arguably the most diverse and ecologically wide-ranging of all organisms, and as the function of the neuroendocrine system is broadly to translate environmental signals into hormonal action, one might expect to see an unparalleled radiation of NSC function within the insects. In specific cases, where both anatomical and physiological information is available, examples of both these trends (conservation and adaptive radiation) can be cited. Thus, all known insects appear t o utilize prothoraco tropic hormone (PTTH) to initiate ecdysone synthesis during growth and moulting, and the PTTH of different insects are mutually effective, suggesting closely similar chemical structures. Conversely, some very indi-

76

HUGH FRASER ROWELL

vidual neurosecretory functions are known. Only the mosquito Aedes is known t o involve a protocerebral hormone i n ' t h e later stages of vitellogenesis, and in other insects juvenile hormone alone suffices. Similarly, only in lampyrids (Coleoptera) has a gonadotropic hormone of brain origin been demonstrated; in these animals, sex at the phenotypic level is determined by cerebral neurosecretion (Naisse, 1966b, 1969). Neurosecretory control of chromatophores has been shown in phasmids (Dupont-Raabe, 1957) and in an odonatan (Veron, 1973). In the majority of cases, however, one lacks physiological knowledge of this sort, and further comparison must at present be derived from comparative anatomical data. The most basic information is the number and groupings of NSCs in the different taxa of insects. This information is summarized in Tables 2-4, which also contain the references. Good numerical data have become common only recently, and there appears t o have been no previous general review of the quantitative studies. Despite this, a number of qualitative generalizations have become current. Two of the most pervasive, for example, are that the more primitive insects have larger numbers of smaller NSCs than the more evolved, and that larvae have fewer NSCs than do adult insects. In the text that follows, these generalizations and others are examined in the light of the available data. 2.5.2 Variation between taxa: the protocerebral medial and lateral NSCs Table 2 presents most of the available information about the numbers of somata in the most intensively researched groups of NSCs, the median and lateral protocerebral groups of adult insects. Much is known of the axonal projections of these cells in the brain and retrocerebral complex, and something of their functions, and the evidence available suggests that they are functionally homologous throughout the Pterygota. They should therefore provide appropriate material t o assess conservancy of structure, or t o display any major anatomical trends in evolution. The figures given must be assessed for probable completeness in the light of the staining procedure used and Table 1. The following points emerge from the Table: i. Palaeoptera Unfortunately, no quantitative data are available for either of the existing orders. It seems that they have fairly numerous medial and lateral cells, probably more than 2 x 50 medial cells and 2 x 15 lateral cells. The true figures could be much higher.

ii. Polyneoptera This group shows a huge range in numbers of median NSCs, over nearly 2 orders of magnitude. The earwig Anisolabis has remarkably few cells, fewer than most moths or bugs. The cockroach and the primitive grylloblattid have medium numbers of cells (2 x 50-70) whereas the Orthoptera (grasshoppers, crickets and katydids) have uniquely

TABLE 2 Estimates of numbers of median and lateral protocerebral NSCs in the adults of various insects Taxonomic division

Median NSCs

Lateral NSCs

Technique

Reference

n

n rn

1. Palaeoptera

Ephemeroptera Ephemera Ecdyonurus Odonata S y mpetrum, A esc hna Caloptery x, Agrion Aeschna 2. Polyneoptera

I

Isoptera Embioptera Zor apt era Grylloblattodea Schizodactylus Orthoptera (Grylloidea) Gryllotalpa Acheta

(Acridoidea) Locusta

4

“Numerous”

2 x >25*

2x7* 2 x 3*

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

Present

“Numerous”

Present

Az, CHP, PF

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

Present Az, Fuchsin, Methyl Green, CHP PSI

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

2 x 66

2x6

CHP, PF

Khattar (1968)

2 x 300+

Not seen 2 x 12-16 Present No information

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

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

Very numerous” 2 x 400 No information

2 x 8-12

per formic-acid-Schiff,

EM

5 < rn r r v)

U

a3

TABLE 2-conlinued Taxonomic division

Median NSCs

Lateral NSCs

Schistocerca

2 x c. 1000 2 x c. 400

2 x 10

2 x 604

Anacridium

“Numerous”

Melano pus

2 x 400+

2 x 12

Technique CHP, PF Cobalt Iontophoresis EM of NCC I

Reference Highnam (1961) Mason (1973)

PF, PAVB

Rowell and Mason (unpublished) Girardie and Granier

PAVB. CHP

Dogra and Ewen (1970)

PF, silver impregnation

Willey (1961)

(1973)

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

Phasmida Cliturnnus Carausius, Clitu mnus Bacillus, Eurycnema

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

Not seen Not seen

CHP, PF CHP, PF

I

C 2 x >40* “Numerous”

Present 2 X 5-6

Az, CHP, M3C

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

2 x >40*

CHP

“Un g a n d nombre” Plecoptera ISOPerla

Fiiller (1960) Pipa (1962 and personal communication)

2 x >30*

Present

CHP

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

t)

I n I]

Rrn

R R

0 r r

D errnaptera Forficu la, Lab idu la Anisolabis

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

i

z

“Numerous” 2 x 10-20

CHP, AF, M

Cazal (1948) Ozeki (1958)

m C

a

am 0

a rn

-I

0

< Present

Gabe (1966)

Present

0

rn r r v)

2 x 20* 2x65

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

Present Not positively identifiedperhaps 2 x 2

No information 2 x 19-20

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

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

2 x 12

2x3

PF, CHP, PAVB

Ewen (1962)

2x7 2 x 14-16

2x43 2x2

PF, CHP PF, PAVB, CHP, Az

Johansson (1957,1958) Furtado (1971)

2 x 16 2 x 9-10

2 x 3-4 2 X 4-6

CHP, PF PF, PAVB, PATh

Nayar (1955) Dogra (1967)

2 x 5+ 2 x 8-9+

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

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

03 0

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

(Aphoidea) Aphis Drepano sip hu m

Median NSCs

Lateral NSCs

2 x 8-10+

Not seen

PF, PAVB

Dogra (1969)

2 x 9-10+

2 x 3-4

PF, PAVB

Dopa (1967)

Present

Present, though not laterally situated

2 x 4-6 2 x 4-6

2x2

No information

Present

Technique

Reference

Cazal (1948)

Az, CHP, Pf

Johnson (1963)

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

Cazal (1948)

I C GI

r n

“Numerous” 2 x >14*

Present

2 x >20*

2 x >5*

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

Arvy (1956)

ID

50

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

c

2 x 6-8 2 x 38-47

2 x 2-3 2 x 12

Az, Iron haematoxylin

Rehm (1950,1951)

n

Az, CHP, PF

Delipine (1965)

0, m

2x8

2 x 9-10

CHF, PF, PTh

Panov and Kind (1963)

rn -I 0

2 x 10 (2x8

2x7 2 x 8-10

CHP, PF CHP, PF, PATh

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

0

0

n

n

<

m I-

i

Antherea Malacosoma, Eudia

v)

2x8

2 x 8-10

CHP, PF, PATh

Panov and Kind (1963)

2x5 2x 7

Not stated CHP, PF

Van der Kloot, (1961)

2x9 (2x8

2x5 2 x 8-10

CHP CHP, PF, PTh

Ichlkawa (1962) Panov and Kind (1963)

2x8

2 x 9-10

CHP, PF, PTh

Panov and Kind (1963)

PAVB,PF/HM

Burgess (1971, 1973) Meola and Lea (1972)

Hyalophora

(Noctuoidea) Dicranura, Phalaera Spiloso ma, Dasy c him, Acronycta, Dendrolimus, Lymantria, Orgya (Cossoidea)

.

Herman and Gilbert (1965)

cossus

(Papiliono idea) Vanessa, Pieris Diptera (Nematocera) Culiseta Aedes

__

__---_2 2x12

1 4 total

___________ 2x5

PF, EM

2

m

TABLE 2-continued

N

Taxonomic division

Median NSCs

Lateral NSCs

Tipula (Brachycera) Tubanus (Cyclorrapha)

2 x >15*

No information

NS

Cazal ( 1948)

2 x>5*

2 x >4*

NS

Cazal (1948)

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

RefIected Iight CHP, PF CHP CHP CHP PF, PAVB

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

2x6

PF

Gawande (1968)

Not seen

CHP

M. Thomsen (1954)

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

Andrenu ( 0 )

Coleoptera (Caraboidea) Nebrio

(i

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

Technique

Reference

N o information

2 x c. 100 2x

>loo

I

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

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

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

5 b

I -n II

D

v)

m II SD

0

2x8

2 X 2-4

AF, CHP,'AB/P silver inpregnation

Ganargarajah (1965)

5r... r

(Hydrophiloidea) Hydrous

3

2 x 20

Aulacophora (Curculionoidea) Slaps Hypera Sitophilus

(Cantharoidea) Lampyris

X

4-6

GHP, Qiemsa, Feullgen

De Lerma (1954)

n .. 3

1

(Dermestoidea) Dermestes (Chrysomeliodea) Galeruca Leptino tarsa

2

Ern

2x5

Ladduwahetty (1962) quoted by Siew (1965)

2

0

2 x 54-64 2 x 80

2 x 8-16 2 X 6-27

2 x 8-10

2x2

2

--

X

24-26

2 x 7-12

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

No information

2 x 11

2 x 12

Present

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

Siew (19 6 5) Schooneveld, (1970)

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

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

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

Naisse (1966a)

rn r

r cn

Saini (1967)

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

w

84

HUGH FRASER ROWELL

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

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

NEU ROSEC R ETO R Y CELLS

85

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

86

HUGH FRASER ROWELL

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

NEUROSECRETORY CELLS

87

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

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

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

Acheta

Schistocerca

Medial and lateral NSCs

144

830

c. 2000 1228

Technique

Author

PF, CHP

Khattar (1968)

PF, CHP, PAVB,EM

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

1

PF, CHP EM, PF/ PIC

1

SOG

NSCs

30

VNC NSCs

Technique Reference

no information

PF, CHP

Khattar (1968)

PTh

Panov (1964)

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

Rowell (unpublished)

Az, PF Delphin (1965) CHP, ABP M3C

Dictyoptera

Periplaneta

c. 120

120 optic

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

20

209

Az, CHP PF, RSR

de Besse (1965,1967) I

C

c, I n

Hemiptera Rhodnius

Oncopeltus

a 170 64 32

Az, CHP PF/HM

Baehr (1968) Steel and

CHP, PF

Harrnsen (1971) Johansson (1957, 1958)

34

None

106

18

A,CHP

CHP, PF

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

9 YJ YJ

0

%

Lepidoptera Galleria Adult

118

“Nymph”

38

“Preiiymph”

32

( Bombyx

Diptera L u c ilia Larva

Culiseta Larva, Pupa Coleoptera Leptino tarsa

36

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

[PTh

Delipine (1 965)

8

Del6pine (1965)

None

72

Delkpine (1965)

28

80

Kobayashi (1957)

80-100

129

>500t

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

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

Panov and Kind (1963)

42

CHP, PF, AB

Fraser (1959a)

None

50

CHP, PF

Fraser (1956b)

28

PF/HM

Burgess (1971, 1973)

None

32-48

PF/HM PAVB

Burgess (1971)

CHP, PF

Schooneveld (1 970)

CHP, P F

Schooneveld (1970)

214

2

No information

dD .(

2r

‘

(D

0

Taxonomic divison

Blaps Adult Larva

Medial and lateral NSCs

76 114

Technique

ABP, PF ABP, PF

Authoi

Fletcher (1969) Fletcher (1969)

SOG NSCs

c. 30 c. 30

VNC NSCs

239 171

Technique

ABP, PF ABP, PF

Reference

Fletcher (1969) Fletcher (1969)

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

I G) C

-n

30

$ rn sa

m

0

5r

r

91

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

cc

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

cc

1232 20 137 695
120 240

20

From Table 2 Raabe (1963a),Mason (1973), Brousse-Gaury (1967) From Table 3 From Table 3 18 intrinsic neurons of the storage lobe are not NSCs (Highnam and Galdsworthy, 1972; Cazal et al., 1971). The latter authors consider the intrinsic glandular cells to be nonneural

Table 2 From Beattie (1971) Raabe (1963a), Browse-Guary (1967) from Table 3

209


Guess

c. 700

Phasmida Carausius Medial and lateral NSCs

45 0

Deuto- and tritocerebral NSCs SOG + VNC NSCs

280

(Brady and Maddrell(l967) found 2 x 11+ NSC axons in a segmental medial neurohaemal organ, which extrapolates to a closely comparable figure) Peripheral NSCs

cc

loo+


Guess, based on qualitative comparison with Schistocerca Raabe (1963a) Raabe (1965) (Clitumnus, not Carausius)

Finlayson and Osborne (1968) Guess

92

HUGH FRASER ROWELL TABLE 4-continued

Hemip t era R hodinus Brain, total (Steele and Harmsen (1971) making the assumption that cell staining types were totally mutable, derived minimum estimates of 64 NSCs in the adult pars) SOG VNC

cc Stilbocoris Brain, total NSCs “A” cells of thoracic and abdominal ganglia Other NSCs of thorax and abdomen, by extrapolation

cc

170

34 106 <50 ___ <360, >254

Baehr (1968)

Baehr (1968) Baudry (1968) Guess

38

Furtado (1971)

24

Furtado (1971)

40 <40

Guess Guess

142 Homoptera aphid Brain, total NSCs VNC, other ganglia

cc Peripheral NSCs of thorax Lepidoptera Galleria Brain, total NSCs SOG VNCs VNC

cc

16 none 12

14 42

122 8 129 16

Drepanosiphum, Johnson (1963) CC intrinsic cells, M y z u s , Bowers and Johnson (1966) Bowers and Johnson (1966)

DelCpine (1965) Delipine (1965) Delipine (1965)

(Ephestiu, Rehm (1951))

275 Bombyx Medial and lateral cells Deuto- and tritocerebral NSCs

36 10

Frontal ganglion SOG

2 80

From Table 2 Larva of Manduca ( Sphingo idea), N ijhout (1975) Bounhiol et al. (1975) Kobayashi (1957)

93

NEUROSECRETORY CELLS VNC

cc

500-1000? 10-16

-Kobayashi (1957) 10 in larva of Mimas (Sphingoidea), Highnam (1958) 16 inxphestia (Pyraloidea), Rehm (1951) 1 4 in Ostrinia larva (Pyraloidea), McLeod and Beck (1963)

>644, <1144, Diptera Calliphorid Brain NSCs

SOG + VNC NSCs (Dogra and Tandan (1966) record 52 type “A” NSCs in the CNS of Sarcophaga) Additional type C cells of SOG and VNC Ocellar nerve NSCs Peripheral NSCs

CC and retrocerebral system Coleoptera Blaps Median + lateral NSCs Deuto- and tritocerebral NSCs SOG NSCs VNC NSCs

cc

40-50

50

50

40 in CaIliphora adult, Thomsen (1965) 42 in Lucilia larva, Fraser (1959a) 44 in Aedes (Culicoidea), Meola and Lee (1971) 46 in Drosophila, KGpf (1957) Lucilia larva, Fraser (1959b)

194

Guess Sarcophaga, Schlein (1972) Sarcophaga larva, Finlayson and Osborne (1968) Calliphora, Normann (1965); Thomsen (1969)

76 6+ 30 229 <50

Fletcher Fletcher Fletcher Fletcher Guess

20 2 16 -

(1969) (1969) (1969) (1969)

<390

not enough data t o make good estimates for either another homopteran or for a nonreduviid hemipteran; these are the two groups where really low totals might be expected; the figures available suggest that the two representatives chosen may have fewer than 50 and 150 NSCs respectively. The next lowest estimate is for a dipteran, probably less than 200 NSCs. A moth, a reduviid bug and a beetle all produce estimates of 300-400 cells.

94

HUGH FRASER ROWELL

The cockroaches and phasmids have prob,ably between 800 and 1000 MSCs, and by far the largest total once again comes from the Orthoptera, which seem t o have in excess of 2000 NSCs. No estimate can be made for the Hymenoptera, which are also expected t o have large numbers of cells. The aberrant figures for the silk moth Bombyx derive from Kobayashi (1957). His estimate of brain NSCs is in accord with other workers in the Lepidoptera. In the same paper he briefly refers t o 80-120 NSCs in all ganglia of the VNC, a statement which has been interpreted (e.g. by Delphin, 1965) t o mean up t o 1100 NSCs in addition t o the 36 he (Kobayashi) found in the brain. This puts a total estimate for this moth in the order of 100-1500 cells, a value approximately 5 times that obtained for the pyralid moth Galleria. It is noteworthy that Kobayashi was not using a specially wide spectrum of stains t o arrive at this large figure, and further examination of this species is needed. The larger figures for total numbers of NSCs are comparable t o similar estimates (of no greater accuracy!) which can be made for the total number of motor neurons innervating muscle in the insect. The interesting implication is that the CNS may devote as much of its integrative powers t o neurosecretory outputs as t o the more familiar motor neurons. 2.5.4 Vuriution during L$e history of the individual Table 5 compares various populations of NSCs which have been counted in comparable ways, preferably by the same worker, at different stages of the life history. For the obvious reason of the great amount of labour involved in counting and recounting a statistical sample, very few workers appear t o have assessed the variation inherent either in their own counting techniques or, more importantly, in individual animals. For this reason, Delphin’s (1965) counts of NCSs and in the VNC of Schistocercu at five-day intervals after the final moult are particularly important. The total number of cells recorded in any individual varied from 651 t o 832 cells, a fluctuation of some 30 per cent. This variation is comparable t o that obtained by GeIdiay (1959) in cockroaches. It is notable that some cell categories showed no variation at all, which provides a reassuring control as t o the accuracy of the counting technique Delphin employed, as does Chalaye’s (1967) close confirmation of his estimates in the related genus Locustu. If, however, one adopts the hypothesis that NSCs d o not change from one major staining category t o another (e.g. A celIs do not become B cells), but onIy become stainable or nonstainable, then the total figure is better derived from the sum of the maximum values obtained for each category. In Delphin’s case this would produce a total of no less than 1143 NSCs in the VNC, or 7 7 per cent more than the minimum number found in any individual at one time. This is a plausible hypothesis. The possibility. that cells change in major

2 61:

N d m w

0

NEUROSECRETORY CELLS

m

w 4

3

+ w

i

* N

0 03 0

m

W

2

v

3

; , 0 0 0N 0W 0I N

95

TABLE 5-curtlinued

(D

0,

Taxonomic division NSC population

Developmental stages Prehatch

(Sphingoidea) Manduca

Larva IV

LarvaV Pupa

Reference Adult * 6 cells (tritocerebral)

TotaI brain NS cells

shown only by cobalt, not by PF or CHP (a) Bassurmanova and Panov (1967) (b) Kobayashi (1958) (c) Panov and Kind

(Bombycoidea)

Bombyx

Median cells Lateral cells

(1963) Antherea

Median cells

Philosamia

Lateral cells Median cells

(d) Mitsuhashi (1963) (e) Ichikawa (1962) (f) Nijhout (1975)

Lateral cells (Noctuoidea) Lymantria Dendro limns

Diptera: Drosop hila Selected detail

Median cells Lateral cells

16

I C

(4 20 (4

(4

16

8 (4

t) X

26 (4 16 (4 28 (dl 20 (4 (Note: Panov and Kind (ref. c) did not describe the “posterior” cclls o f other authors, and their counts of “median” cells can therefore be increased by 4) Total brain NSCs Posterior cerebral cells

Larva (1-111) 16-20 0

Pupa 14 2

Adult 46 8

n VI

rn R

Ropf (1957%b )

Sarcophaga

Brain A cells (PAVB) Brain A cells (PF)

18 26

16 32

Dogra and Tandan (1966)

Calise ta

Hymenoptera (Apoidea) Apis (Formicoidea)

Neomyrma Leptothorax Campono tus Formica Coleoptera Blaps

larva 2x10 2x2 2x2 Larva

“Mediate” “Intermediate” “Lateral”

Median MSCs A cells of brain (PF) A cells of A cells of A cells of A cells of

brain brain brain brain

(PF) (PF) (PF) (PF)

Total NSCs Median cells Lateral cells Tritocerebral cells “Anterior” cells SOG T I, T I1 T 111 A I-A IV AV-A VII (larva) Last abdominal g. (adult)

c.

130

Larva 24 12 24 24

Pupa 2 x 10 2x2 2x2 Adult “About the same” Adult (queen) 109 60 150 136

Larva 54 60 6 5 30 15 15 18 18

Adult 52 24 6 0 30 18 27 26

340

311

z

m

Last instar Adult 2 x 10 2x2 2x2

rn 4

0 zu

<

Formigoni (1 956)

0

rn

r r v)

Gawande (1968)

Fletcher (1969)

32

Variation in NSC complement with age. The examples are selected for comparability; where possible the figures are derived by the same author, and where this is not possible variations in staining technique are noticed.

w

98

HUGH FRASER ROWELL

staining type with development or secretory ,cycle has been extensively debated, but the balance of recent opinion seems t o regard this possibility as unlikely, or at least rare (see e.g. Prent4, 1972). On the other hand, there is much evidence that cells are only stainable at certain times, and can appear and disappear in successive ages (p. 70), and have slightly different appearances at intermediate stages of this process. If cell types are so narrowly defined as t o differentiate between these intermediate stages, then it is obviously true that they change with time. Steel and Harmsen (1971) freely admitted the possibility of unrestricted transformation of cell type, adopted the most parsimonious estimates of cell number, and also used a single, though wide-range, staining technique. Their estimate of the NSC population was half that obtained b y more conventional methods (Table 2) but even so they were forced t o admit the probability that the population included two chemically distinct types of cell. The belief is frequently expressed that larval insects have fewer neurosecretory cells than do adults. Probably most workers would interpret this statement to mean “stainable” neurosecretory cells, for there is a total absence of evidence for the formation of new NSCs, presumably by cell division from undifferentiated neuroblasts at the final moult. There is clearly an increase in cell stainability which is associated with maturation of function. For example, only 9 NSCs were detected in a 42-day embryo of a cockroach, as opposed t o about 50 seen by the same workers in the adult (Khan and Fraser, 1962). This apart, the collected data do not support the generalization. It is apparently true of some animals, but not of others, and detailed analysis of subpopulations o f NSCs strongly suggests that some of them are more prominent in the larvae, some in the adult, and that the final total visualized is more o r less fortuitous and can show a change in either direction at metamorphosis. This makes intuitive sense: while adults need to reproduce, larvae need t o moult and sometimes to metamorphose; in addition, they often have different physiological and environmental requirements t o mediate. There is no good reason why one should require more or less NSCs than the other, but every reason why they should require different ones, or at least different functions for the same ones. The most dramatic increase in numbers of cells is reported among the A cells of cerebral NSCs of ants (Gawande, 1968) where larvae have only 24 and adult queens around 100 pairs. This is obviously an extreme case, for not only is the ant larva actively maintained in a very constant environment with relatively few neurosecretory demands, but also the queen could well be expected t o have unusually numerous neurosecretory requirements. Furthermore, only A cells were counted, and other cell types may well behave differently, as in other species. For comparison, the 5th instar larval brain of Munducu (Sphingidae, Lepidoptera) contains 42 NS cells (Nijhout,

NEUROSECR ETO RY CE L LS

99

1975). Panov and Kind (1963) found 36 cells in adults of the closely related general Sphinx and Herse, but their staining techniques would not be expected to reveal the 6 tritocerebral cells which stain only with azan or with cobalt via fills of the NCC IV. Correcting for this deficiency, the estimates for larva and adult are identical. Similarly, Burgess (1971) found regular changes in stain affinity as Culiseta (Diptera, Culicidae) passed from larva, through pupa, to adult, but the absolute number of bnainNSCs remained constant. 3 Implications of the identifiable cell concept for the insect neurosecretory system 3.1

ANATOMICALLY SIGNIFICANT DIFFERENCES BETWEEN NEUROSECRETORY CELLS

AND OTHER NEURONS

NCSs can be grouped along a continuum between two e,xtremes, the cell which releases its secretion at a NHO and utilizes the general blood circulation to transport its product to the target organ, and the cell which directly innervates its target and releases its product in very intimate proximity. (In animals with a highly vascularized circulatory system, such as crustacea, some molluscs, or vertebrates, there is also the case of the NSC which releases its product into a capillary which transmits it to a specific target directly, but this circumstance does not arise in the open haemocoel of insects.) In the latter case, the anatomically significant differences between the NSC and a regular neuron, say a motor neuron, are likely to be minimal. It is true that, at the fine structural level, the somatic elaboration and axonal transport of secretory product must differ from the mechanisms needed to produce and recycle transmitter substance, in which all the necessary synthetic and enzymatic machinery may be localized peripherally in the terminals; and there are likely to be small differences between the classical synapse and the point of release of the NSM. However, the NSC is not freed from any of the anatomical restraints that apply to regular neurons. The NSC that ends in a NHO, in contrast, differs from the regular neuron in two important ways. First, it loses the constraint of having to approach its target closely with an axon. Provided it is in contact with the circulation, the NHO can be located almost anywhere. It is still difficult to alter the position of the input to the cell-the premotor neurones still have to make the usual synaptic connections-but the output structures can evolve much more freely. This seems t o have happened in the evolution of the median NSCs of the protocerebrum. Their position in the brain is almost constant throughout the insects, but their axons terminate in a great variety of NHOs in different species (pp. 73-74).The second consequence of

100

HUGH FRASER ROWELL

not innervating the target organ directly is that the appropriate threshold titre of secretion has t o be reached using the whole blood volume, and this in turn implies that the NSC utilizing an NHO probably has t o produce much larger quantities of secretion than either a regular neuron or a directly innervating NSC, or, alternatively, that there must be duplication of cells. This point is returned t o below.

3.2 CONSTANCY, UNIQUENESS, AND REDUPLICATION

IN NEUROSECRETORY CELLS

If NSCs are similar in their organization to other neurons of the insect CNS, they could be expected t o be constant in number and position and individual morphology, and t o display a minimum of functional reduplication, other than that associated with segmentation. To what extent do these predictions seem t o be true? And what departures from this prediction could we anticipate due t o the genuine differences between NSCs and other neurons, considered in the previous section?

Constancy The work reviewed in section 3 indicates that at the gross level insect NSCs are constant among individuals in number and in grouping, and are species-specific in these respects. A similar situation exists in at least some other invertebrate groups, e.g. pulmonate molluscs (Wendelaar-Bonga, 1970) and leeches (Hagadorn et al., 1963). In some populations of insect cells no variation at all has been detected (e.g. the A cells of the orthopteran VNC: Panov, 1964; Delphin, 1965) and in others apparent variation can plausibly be associated with variation in stainability rather than in absolute number. Few dye injections into individual neurons have yet been made (section 4) which would test the constancy of the fine morphology of the NSC, but there is no reason t o suppose that (at least at their input end) NSCs will be any less constant than other neurons-the problems of making predetermined synaptic connections are the same in both. (NSCs associated with NHOs, as noted above, may well be more variable at the output end.) There is thus every reason to believe that NSCs can in principle be characterized as individuals, and recognized as such from animal to animal. IJniqueness Before considering uniqueness and duplication of function I should declare an underlying assumption. This is that any individual neuron will normally manufacture only one major secretory product. I t is well known the neurons do release more than one substance. For example, detectable amounts of proteins are released from motor nerves, in addition to TS, and these are sometimes postulated t o be the mediators of “trophic” influences of nerve on the subsynaptic tissue; and neurophysins are released from vertebrate neurohypophysial cells. In general, however, only one

N EU ROSECR ETO R Y CELLS

101

product is liberated in quantity (though there has been a recent suggestion that a neuron in a snail may change its TS from serotonin t o acetyl choline at different seasons of the year (Hanley et al., 1974). The extension of this common assumption t o the NSC implies that each would produce large amounts of only one characteristic secretory product, probably peptide or protein. If more than one function is t o be performed by this molecule in the body, the diversity must be obtained at the targets and it should be impossible to have more neurohormones than NSCs. If, on the other hand, the assumption proves t o be incorrect, and it transpires than an individual NSC can synthesize more than one molecular species of hormone, then this constraint does not apply. So far there is no evidence arguing against the assumption, and I will make it for the remainder of this article. It is hard to see how a cell which did produce two products of hormonal significance could liberate them other than in a fixed ratio in response to depolarization, which should limit the utility of such dualism, though Anwyl and Finlayson (1974) have proposed that insect neurons may function successively as NSCs and as motor neurons. The nearest approaches t o duplication of function in regular neurons in insects are in laterally replicated sensory arrays (e.g. the optic lobe and retina) and in segmental homologues. The first category does not seem to have any direct parallel in NSC organization, but the issue of segmental reduplication is a real one. It is noteworthy that those insects with the fewest NSCs are also those which have condensed the primitive segmentally replicated ganglionic chain down t o a few large compound ganglia. Perhaps condensation has allowed them t o reduce the number of segmentally duplicated NSCs. It is possibly significant that most of these orders are also relatively small, either absolutely (Hornoptera, Hemiptera, Diptera) or in relative blood volume (Lepidoptera), and so less demanding of the large quantities of hormones that reduplicated cells could provide. Segmental reduplication might be expected t o apply especially t o those NSCs which directly innervate their target, as those utilizing NHOs are liberated from the need to have a defined relationship with their target. There is some support for this, in that at least some candidate NSCs of this type (the dorsal unpaired median neurones, for example, treated in section 4.3) are arranged segmentally down the ganglionic chain; but on the other hand, the median NHOs of the thoracic and abdominal ganglia are also segmentally replicated and can sometimes survive in their original numbers in nervous systems where there is fusion of the main VNC ganglia (Thomas and Raabe, 1974). Assuming that they produce a similar spectrum of secretions in each ganglion (each contains a large variety as judged by EM appearances (Brady and Maddrell, 1967) and at least two staining types by LM (Thomas and Raabe, 1974)) one must suppose that the absolute number of NHOs is

1a2

HUGH FRASER ROWELL

required t o maintain a titre, and that they are,synchronized to work as a unit by interconnecting premotor neural connections. Support for the distributed synthesis of a particular hormone comes also from the common finding that many parts of the CNS have some specific pharmacological activity when assayed after homogenization. There are, however, a number of objections to this argument, and even if sustained it carries no evidence that the homologous source cells in the different ganglia ever function in concert. They might have quite different afferent connections. An NSC utilizing an NHO could be unique in either (a) its secretion-i.e. only that cell secretes that compound, or (b) its input connections-i.e. it is unique in the stimulus situation which causes it to release its secretion. (A directly innervating NSC is also unique in terms of its anatomical destination, as in regular neurons.) To what extent are NSCs likely to be unique in either of these ways? a. Uniqueness of secretion, or, one pair of cells to one hormone This is an exciting and stimulating possibility. The most obvious rough test of its validity is t o compare the expected number of neurosecretions with the experimentally ascertained number of NSCs. One immediately runs into two difficulties: To what extent does the diversity of neurohormonal function reflect the diversity of neurohormones? Can the same hormones be produced in different insects by NSCs of morphologically different areas? The first question is confused by the remarkable ability of insects to use the same molecule in different functional contexts. Bursicon has been shown t o induce tanning, but also postulated to end ecdysial motor outputs (Truman and Endo, 1974) and t o have diuretic function (Mills and Whitehead, 1970). To draw a further example from nonneural endocrine systems, the many functions of ecdysone and juvenile hormone are notorious in insect physiology. An example is the use of both in the control of growth, moulting and metamorphosis of the larval stages and then again in the induction and control of vitellogenesis in the adult. (It may be, however, that these different functions are to some extent partitioned between the different chemical forms of JH which are found within the individual (Judy et al., 1973, and personal communication)). Similarly, if it could be shown that the target systems responded in different ways to precise mixtures of synergistic hormones, it would be possible t o explain a large number of hormonal functions by using only a small range of neurohormones. A further way of controlling a diversity of process with one hormone is t o make the targets susceptible t o temporal patterning. Truman and Riddiford (1974b) have explored the temporal sequence of PTTH ecdysone release in pupation, and temporal factors are important in the hormonal determination of coloration in acridids (Rowell, 1971, for review). The second question is harder t o come t o grips with. Suppose one

NEU ROSECR ETO R Y CE LLS

103

compares two animals, each with the same total number of NSCs. One has only 10 pairs of cerebral NSCs, the other 30 pairs. Does this imply that the latter averages three times as many cells per brain hormone as the former? It clearly does not if the total hormonal synthetic requirement is spread over the whole complement of NSCs, but could one not expect some conservation in evolution as t o what part of the CNS produced what sort of hormones? If the cerebral cells have well-defined roles, not replicated by those of the VNC, then the hypothesis would be justifiable. Bursicon, however, appears t o be functionally produced from NSCs of both brain and abdominal ganglia (see Goldworthy and Mordue, 1974, for review). When one reviews the numbers of NSCs in insects (section 3) and compares them with the expected numbers of hormones, it seems likely that in some species at least the one hormone/one cell pair situation will exist, especially in small individuals (see p. 104) of those groups with condensed CNS and small numbers of cells such as the Homoptera and Diptera. For example, it is hard t o believe that the brain of any insect would produce much fewer than 7 different neurosecretions. Both the crustacean X organ and the vertebrate hypothalamus are known already t o produce approximately this number, and the life of the insect is certainly as complex. Wyatt (1972) lists 1 3 neurohormones of proven occurrence, including CC hormones, and mentions that more are known from individual taxa. Minimally, one would expect prothoracotrophic hormone, bursicon, ecdysis-releasing hormone, at least one hormone associated with control of the corpus allatum, diuretic hormone, and either or possibly both a hypo- and hyperglycaemic factor, to be of general occurrence in the brain. Special cerebral neurosecretory functions include the lampyrid gonadotropic hormone (Naisse, 196613, 1969) and dipteran pupation factor (Zdarek and Fraenkel, 1969). (Other common neurosecretory functions such as control of oviposition, diapause, diuresis, and chromatophores, seem to be confined t o the SOG, or VNC, and others (adipokinetic hormone, glycaemic factors, c?rdiac accelerating factor, melanin-inducing factor) t o be associated primarily with the CC.) Yet Aphis apparently has only 7 pairs of cerebral NSCs (Table 2). Following a previous argument (p. loo), it is unlikely that this brain can produce more than 7 distinct neurosecretions. This interpretation suggests that with one or two intracellular electrodes it ought t o be possible t o monitor, abolish or control an entire neurosecretory pathway in these insects. At the other extreme, the numbers of NSCs present in the Orthoptera make it effectively certain that the same hormone is produced by many cells. It is impossible t o imagine that a locust needs 100 times as many different cerebral hormones as an aphid, for example, or ten times as many neurohormones of all sorts as a fly even if one allows for the greater effect

104

HUGH FRASER ROWELL

o f segmental reduplication in the VNC of the locust. None the less, the second type of cellular uniqueness could still be obtained by having each NSC of a certain molecular species driven by a different integrative network. This would be especially appropriate where one hormone had to be produced in response to a variety of environmental situations, say at very different times in the life history, or where sustained production over a long period of fluctuating sensory input was required. By this argument, one might expect insects with a more complicated life history, environment, or life style to split up their hormone production among more numerous NSCs, even if they did not increase the actual number of products. The data are not yet sufficient to examine this possibility, though it is of interest t o see that the social Hymenoptera have more numerous and smaller NSCs than any other Oligoneopteran group. Perhaps the Coleoptera, which show large variation in NSC number among closely related forms, would be suitable material for such an investigation. A corrolary anatomical approach would be to investigate the morphology of comparable NSCs from animals with relatively few and relatively numerous cells. The argument would suggest that NSCs from the less numerous population would have larger and more elaborate dendritic trees or a higher density of input synapses, whereas the larger population of cells would have relatively simple input structures. It is, however, hard to imagine that all the 1000 or more pairs of NSCs of some insects are unique, even in subtle details of input. The great range in abosolute numbers of NSCs in the insects is in considerable contrast to the relative constancy of number of regular motor neurons. For example, Schistocercu (Orthoptera), Bombus (Hymenoptera), and Cuhphoru (Diptera) all have about the same absolute number of flight motor neurones, despite considerable differences in the morphology of the flight apparatus. There is one obvious reason why NSCs might be more prone t o reduplication than regular neurones, and that is their frequent utilization of the blood as a carrier, coupled with the dependence of the target on concentration of hormone. The larger the animal, the more reduplication of NSC will be necessary for each NHO, in order t o keep the effective titre the same. If this hypothesis is founded in fact, there ought t o be a correlation between the size of the animal and the total volume of NSCs serving its NHOs. Taking the median NSCs as the best known example of the latter, I have calculated the total volume of their somata for a variety of insects, using published figures for number and cell size. This calculation is even more approximate than the preceding ones, because cell dimension is almost always given as a linear measure, and hence all errors are cubed when converting to volume; further, I have assumed spherical somata, and most NSCs have pear-shaped or ellipsoid somata. The circulating blood volume

N EUROSECR ETORY CELLS

105

ought t o be the best correlate of NSC volume; unfortunately, this is a difficult figure t o obtain experimentally, and I have not been able t o find enough estimates t o make a useful comparison. None the less, Table 6 TABLE 6 Estimated total volumes of medial neurosecretory cell somata in a variety of insects, arranged in orde,rof magnitude Blood volume, where known (pl)

Taxonomic divison Drosophila (fruit fly) Nebria (ground beetle) Glossina (tse-tse fly) Oncopeltus (plant bug) Apis (honeybee, worker) Ranatra (water strider) Stilbocoris (plant bug) A niso labis (earwig) Periplaneta (cockroach) Bornbyx (silkmoth) Leptinotursa (potato beetle) Zphita (shield bug) Rhodnius (predatory bug) Schistocerca (locust) Belostoma (giant water bug) Biggest Saturniid moths (composite estimate)

7 000 13 000 17 000 18 000 27 000 30 000 56 000 72 000 84 000 105 000 112 000 168 000 170 000 2 2 5 0 000 1 300 000

160 (Wheeler, 1963)

202 (Lee, 1961)

2 61 7 000 maximum

shows that when the species are listed in order of calculated NSC volume, the ranking fits quite well with that expected from the size of the animal. This result suggests that the hypothesis o f reduplication of NSCs to maintain hormone titre in large blood volumes is true. It also suggests that there is some measure of equivalence between lots of small cells and a few large ones. It is a striking example of the cube law that 1000 cells of 15 pm diameter have the same volume as only 20 cells of 55 p m diameter, and this makes the differences between, say, the medial NSCs of Orthoptera and Lepidoptera much less than appears at first sight. Both groups, it may be noted, have 2 x 3 large tritocerebral cells which give rise to the NCC 111. The cube relation may also explain why one sees little difference between the NSC array of a small and large species of one taxon (e.g. the large locust Schistocerca and the small grasshopper Melanoplus, Table 2), as large changes in blood volume can be equated with small changes in cell lineur

106

HUGH FRASER ROWELL

dimensions. For example, a 30 per cent change, in blood volume could be balanced by only about 3 per cent change in NSC soma diameter. There is also some anecdotal support for the hypothesis. Gawande (1968) reported that of a group of formicid genera the one with the largest body size had the largest number of NSCs, and Dupont-Raabe (1951) noted that the giant phasmid Euryonema had a particularly “voluminous” NSC area in the pars. In larval Schzstocerca the diameter of the individual medial NSC is half what it is in the adult (Gawande, 1969). In a system with reduplicated NSCs, there must be provision for driving them synchronously. A massive reduplication of presynaptic input is perhaps improbable, and might be expected t o be visible in the neuroanatomy, which is not obviously the case. It seems more likely that the NSC population would be coupled laterally. This could be accomplished either by extensive electrotonic coupling (it is of interest t o note that this has been found between the numerous small bag cells of the large sea slug Aplysia, where again reduplication, relative t o a hypothetical small gastropod ancestor, seems a logical necessity) or alternatively by chemical synapses. It is possible that the synaptic junctions between the axons of cerebral NSCs reported from the CC of some insects (e.g. Normann, 1965) may function in this way. If synchronous release of secretion is the only aim, there is no reason why the neurone should not have its spikes initiated near the terminal, rather than at the beginning of the axon, as long as the terminal membrane is depolarized. The frequency of firing of recorded NSCs is so low that collision and occlusion of orthodromic and antidromic spikes would be of negligible probability. It is also possible that the local post-synaptic potential could suffice t o cause release of secretory product, without regenerative spiking.

4 Available techniques, research strategy, and some examples

4 . 1 FILLING OF NEURONS WITH DYE FROM SEVERED NERVE STUMPS Mulloney (1973) has reviewed his recent synthesis of techniques by which cells are filled with dye, either by diffusion or by axonal iontophoresis, starting with a cut nerve stump. This technique can be applied in either the retrograde or prograde directions, filling either soma or the axonal terminations. Depending on the anatomical configuration (e.g. what proportion o f axons within the nerve are those of NSCs) more or less additional

N EU ROSECRETORY CELLS

107

investigation is required to, ascertain whether the cells so revealed are NSCs or not.

4.2 FILLING OF

NEURONS WITH DYE THROUGH AN INTRACELLULAR MICROPIPETTE

ELECTRODE

This technique is closely allied with the above, and gave rise t o it. Dye is injected by pressure or by electrophoresis (see Kater and Nicholson, 1973, for review of techniques). The advantages are that only a single cell is filled (most of the dyes are unable to cross even electrical synapses) and that the electrode simultaneously records electrophysiological events which help to characterize the cell. NSCs are often easy to identify electrophysiologically, because of their relatively long duration and slow rise-time action potential when recorded intracellularly. Both these intracellular dye techniques supply incontrovertible positive evidence, but only weak or meaningless negative evidence. If the structure fills, it can be assumed that it is in cellular continuity with the injection site. If a structure does not fill, it may merely indicate a technical failure. Fills are most successful with relatively thick axons of short length-the longer and the thinner, the less the probability of a complete fill, although successful fills are attainable even with axons well below five microns in diameter.

4.3 INTENSIFICATION OF COBALT STAINING BY SILVER PRECIPITATION The utility of both of the above techniques is greatly enhanced when cobalt is utilized, because trace amounts of cobalt sulphide become visible in paraffin sections after silver intensification by Timm’s technique. for the detection of heavy metal sulphides (Tyrer and Bell, 1974). Using this method, the finest dendritic processes become visible, defining structures invisible in the original preparation. To my knowledge, the dye injection techniques described above have so far been applied to only three neurosecretory systems. The neuroanatomical results are briefly summarized below. i. Mason (1973) investigated the origins and terminations of the neurones contained in the major nerves of the retrocerebral complex of Schistocerca (Orthoptera, Acrididae). Retrograde filling of the NCC I and I1 with cobalt filled the expected medial and lateral NSCs, and in both cases also filled a number of other somata not previously identified as associated with these nerves, and which are not stained with “specific” stains. They are assumed to be the somata giving rise to at least some of the 200 axons devoid of neurosecretion seen in both LM and EM sections of these nerves (Strong,

108

HUGH FRASER ROWELL

1965; Rowel1 and Mason, unpublished), and which are probably concerned with cerebral control of release of materials from the intrinsic glandular (NSC?) cells of the CC and CA. Scharrer (1963, 1964) shows EMS of Leucophaea in which presynaptic terminals onto intrinsic CC and CA cells have the appearance of regular axons, not NSCs. Other authors have since published similar findings. The extra somata filled through the NCC I1 are closely associated with the lateral NSCs, but at least some of those whose axons run in NCC I are in contrast tritocerebral, and have extensive dendritic arborizations elsewhere in the brain. The somata filled via the NCC 111 appear to correspond to the tritocerebral type C NSCs described by Raabe (196313). Prograde filling of the same nerves indicates the areas innervated directly by the somata (both NSCs and regular neurones) described above, and in general confirmed the more tentative conclusions derived from physiological and histological investigations. In addition, however, it was found that the axons of the NCC 111project almost in their entirety to the CA, which was not previously suspected. Filling of the NCA I and I1 gave similar results, including a confirmation of the projection from the suboesophageal ganglion to the corpora allata of the cells described by Chalaye (1967) as type C NSCs in Locusta from azan staining. This innervation had not been previously demonstrated in the subfamily to which Schistocerca belongs, and not confirmed in Locusta. This raises a conflict with the work of Odhiambo (1966), who reported no NSC axon profiles after an EM study of the NCA I of Schistocerca. If Chalaye correctly interpreted his Azan stains, electron-lucent vesicles would be expected in EM (see Maddrell and Brady, 1968; Smalley, 1970) (see Note added in proof, p. 123). On the other hand, Staal (1961) and Strong (1965) were unable to show any effect on CA function by cutting the NCA 11. Perhaps these neurons transmit to the SOG, and not from it, which might explain the absence of effect on the corpus allatum after the nerve is cut. ii. Axonal iontophoresis of cobalt into the brain of the hawkmoth ~lfunduca(Lepidoptera, Sphingidae) via the retrocerebral nerves has produced results interestingly comparable with the above (Nijhout, 1975). In the moths the division of the brain NSCs into median and lateral groups is almost without anatomical meaning. The cobalt staining shows that many of the same functional connections are present, even though the distribution of somata is quite different. The moth NCA I also contains neurosecretory axons derived from brain NSCs, but these somata are located contralaterally, rather than ipsilaterally as in the orthopteran, and comprise both medial and lateral cells of the pars intercerebralis. The chiasma takes place within the brain. Some of these cells may also innervate the corpus cardiacum, which they traverse to reach the NCA I, but axons which end specifically in the corpus cardiacum derive from ipsilateral cells,

NEUROSEC R ETOR Y C E L LS

109

as opposed t o contralateral cells in the orthopteran. Van der Kloot (1961) noted that in the saturriiid Hyalophora the CC was innervated from both ipsi and contralateral NSC. A number of associated nonneurosecretory cells also fill in the same area as the NSCs, as in the locust. Stumm-Zollinger (1957) had previously demonstrated that the moth NCC contained axons without neurosecretory granules. The origin of the NCC I11 appears to be identical in the two insects, three pairs of cells staining neither with A- nor B-type stains in the tritocerebrum. The distal projections of these cells was not studied. iii. The dorsal unpaired medial (DUM)neurons which occur in the thoracic and abdominal ganglia of many insects have an interesting history in research which typifies those cells of the VNC which appear to be NSCs but are largely without physiological correlate t o date. They are cells of uncommon morphology, having their somata medially and dorsally in the VNC ganglia, and sending large axons symmetrically out of one or more roots on both sides of the ganglion t o the periphery. They were first figured by the early insect neuroanatomists, such as Zawarzin, and again by Seabrook (1967) and Plotnikova (1969) in their studies of the ganglia of locusts. In Locusta they were described as Type C NSCs from azanstained preparations by Chalaye (1967), and by Fletcher (1969) from the beetle Blaps. Fortuitous microelectrode penetrations of comparable cells in the cockroach attracted attention because of the unusual characteristics of the action potential, which is of longer duration than is usual in insect neurons and which invades the soma; iontophoresis of cobalt showed these cells t o be DUM neurons (Crossman et al., 1971a, b y 1972). Up t o this point none of these authors appear t o have been aware of the others. Hoyle et al. (1974) described 7 such cells from the metathoracic ganglion of Schistocerca, showed that the largest of them sent axons bilaterally to the fast extensor tibiae muscle of the jumping leg, and that the terminal showed an EM profile of large electron-dense vesicles typical of some NSCs, thus extending the observations of Chalaye (1967). The only function so far ascribable t o this neuron is that it inhibits a slow spontaneous rhythm of contraction and relaxation, itself of unknown function, which is present in the muscle (Hoyle and O’Shea, 1974; Hoyle, 1974). No intracellular muscle recordings are yet available which would illuminate the mode of action of the neural product, and perhaps allow a distinction t o be drawn between a polypeptide transmitter substance (in the sense given o n p. 74) and some other type of neurosecretion. Four cells of similar morphology have been cobalt-filled by iontophoresis through the spermathecal nerve of Schistocerca, and again present a neurosecretory facies in EM transverse section (Okelo, 1974). These appear t o be the cells first figured by Seabrook (1967).

HUGH FRASER ROWELL

110

4.4

ELECTROPHYSIOLOGICAL

RECORDING

AND

INTRACELLULAR

CURRENT

INJECTION

When the soma position is known, either by dye injection or orthodox histology, it is in principle possible to penetrate it with a recording microelectrode. Proof of identity requires a subsequent transelectrode dye injection, at least until the physiological characteristics of the cell are so well known that positive identification can be made on electrophysiological grounds alone. Once the entire cell morphology is known, regions other than the soma can be selected as electrode targets; though harder to hit initially, much more definite information is recordable from the integrating segment of a neuron. Obviously the best chance of identifying individual cells repeatedly will come from insects with small numbers of large NSCs. Once achieved, a microelectrode penetration can be used in two basic ways: passively, to record voltage fluctuations, ionic fluxes, and (with suitable electrodes) ionic concentrations; or, by actively injecting ions, to manipulate the membrane’s potential, ionic permeability, and, indirectly, the release of the secretory product. The former techniques can be expected to have the same consequences in NSCs as they have had in regular neurons. They allow one to ascertain the connectivity of the cell, and to decide whether its activity is a consequence of presynaptic driving or whether it is endogenous. In an insect medial NSC, for example, one could ascertain directly whether the cell was influenced by ocellar input, and with more difficulty one could ascertain whether the cell was a pacemaker or showed other endogenous activity t o suggest that it could release its product without presynaptic cues. Current injection is classically utilized to establish the function of a unit by either driving it or by inhibiting its activity. In the case of most NSCs, the difficulty is likely to be the long-term nature of the response to the neurohormone, often exceeding the expected life of a neurophysiological preparation. The situation, however, is by no means irremediable. The DUM neurons, described above, show how the technique may be used t o investigate short-term effects of putative NSCs. Some classes of NSC, however, seem obviously suited for this analysis. A number of cases have been described where a complex behaviour is the result of activity in an intrinsic neural mechanism which in turn is triggered or caused to function by release of a hormone(s) (reviewed by Truman and Riddiford, 1974). For example, Truman (1973 and personal communication) considers that the release of ecdysial motor patterns in the moth pupa is probably the consequence of hormonal release from a medial NSC, and their termination t o be due to the release of bursicon from the abdominal VNC. Such cells are obvious targets for neurophysiological techniques. This hypothesis could be directly tested via microelectrode penetration of the cell in question. Apart from ecdysis,

NEU ROSECR ETO R Y CE L LS

111

there appear to be a considerable number of behaviour patterns of this class, though the source of the hormone is unknown in most cases: assembling and other mating behaviour (Riddiford and Williams, 1971), various aspects of oviposition (Nayar, 1958; Okelo, 1971; Truman and Riddiford, 1971; Rowell, unpublished), cocoon spinning and other prepupal behaviour (Truman and Riddiford, 1974b). The combination of intracellular recording and current injection simultaneously allows a number of refinements and some new capabilities, such as voltagc clamping of a single cell, and direct measurement of the degree of electrical coupling between adjacent cells which are simultaneously penetrated. The hypothesis that orthopteran rpedial NSCs may be electrotonically coupled (p. 106) is for example susceptible t o direct investigation by this means. In principle, there seems no reason why any of the various hypotheses erected or discussed in this review should not be experimentally proven b y a combination of these techniques. Many of the controversies mentioned, such as the correlation of staining type and function, or the possible transformation of an individual cell from one staining type t o another, can only ultimately be resolved when cells are characterized as individuals of known function and morphology. In practice, extremely little work has so far been carried out. A number of modern microelectrode studies of NSCs have been made (Gosbee et al., 1968; Wilkens and Mote, 1970; Cook and Milligan, 1972; Normann, 1973), but none has utilized dye injection or made any other attempt t o characterize the recorded cells as individuals; most have been concerned with establishing that NSCs follow normal neuronal procedures in their ionic and cellular mechanism, such as release of secretory product. In addition, none of these studies has used insects with unusually few, easily characterized, NSCs. Though there are obvious technical difficulties, mostly related t o lack of immediate consequences of release of the neurosecretory product, and the frequent inaccessibility of the axon t o extracellular electrodes, these do not appear insurmountable. In my opinion major advances in neuroendocrinology will be made when a suitable preparation is found t o which the concepts and techniques of the single identifiable neuron can be applied, and this review has attempted t o indicate those insect groups in which success is most likely. Acknowledgements I am deeply grateful t o the many authors who discussed with me their published or unpublished work and allowed me t o quote from it, and also to my colleagues H. A. Bern, S. B. Kater, W. Loher and C. A. Mason, who read and commented on various drafts of this manuscript. The review was written during the tenure of a Research Professorship of the Miller Institute for Basic Research in Science.

I12

HUGH FRASER ROWELL

Abbreviations

CA, corpus allatum; CC, corpus cardiacum; CNS, central nervous system; EM, electron microscope; LM, light microscope; NCA, nervis corporis allati; NCC, nervis corporis cardiaci; NHO, neurohaemal organ; NSC, neuro5ecretory cell; SOG, suboesophageal ganglion; VNC, ventral nerve cord; PTTH, prothoracotropic hormone; JH, juvenile hormone. For abbreviations of staining methods see Table 1. References 4diyodi, K. G. and Bern, H. A. (1968). Neuronal appearance of neurosecretory cells in the pars intercerebralis of Periplaneta americana (L.). Gen. comp. Endocr. 11, 88-91. 4nwy1, R. and Finlayson, L. (1974). Peripherally and centrally generated action potentials in neurons with both a motor and a neurosecretory function in the insect Rhodnius prolixus. J. comp. Physiol. 91, 135-146. k v y , L. (1956). Le systeme pars intercerebralis-corpus cardiacum-allatum chez Euroleon nostras Foucroy (Niroptireide: Plannipenne). In “Bertil Hanstrom. Zoological Papers in Honour of his 65th Birthday” (Ed. K. G. Wingstrand), pp. 47-55. Zoologica Institute, Lund. Arvy, L. and Gabe, M. (1952). DonPes histophysiologiques sur les formations endocrines rttro-c6rCbrale.s de quelques Odonates. Annls Sci. nut. (Zool.) 14, 345-374. Arvy, L. and Gabe, M. (1953). Donnies histophysiologiques sur la neurosicrition chez quelques Ephimiroptkres. Cellule, 55, 201-222. Arvy, L. and Gabe, M. (1954). The intercerebralis-cardiacum-allatum system of some Plecoptera. Biol. Bull. mar. biol. Lab., Woods Hole, 106, 1-14. Awasthi, V. B. (1968). The functional significance of the nervi corporis allati 1 and nervi corporis allati 2 in Gryllodes sigillatus. J. Insect Physiol. 14, 301-304. Awasthi, V. B. (1969). Effect of temperature on the neurosecretory activity in Nerara viridula Linn. (Heteroptera: Pentatomidae). Experientia, 25, 1164-1166. Baehr, J.-C. (1968). Etude histologique de la neurosicrition du cerveau et du ganglion sous-oesophagien de Rhodnius prolixus St%l (Himiptkre). C.r. hebd. Skanc. Acad. Sci. 267, 2364-2367. Baehr, J.-C. (1973). ContrBle neuroendocrine du fonctionnement du corpus allatum chez Rhodnius prolixus. J. Insect Physiol. 19, 1041-1055. Bargmann, W. (1949). ‘Ilber di neurosekretorischer Verkniipfung von Hypothalamus und Neurohypophyse. 2. Zellforsch. mikrosk. Anat. 34, 610-634. Bassurmanova, 0. K. and Panov, A. A. (1967). Structure of the neurosecretory system in Lepidoptera. Light and electron microscopy of type A neurosecretory cells in the brain of normal and starved larvae of the silkworm Bombyx mori. Gen. comp. Endocr. 9, 245-262. Baudry, N. (1968). Etude histologique de la neuroskcrition dans la chaine nerveuse ventrale de Rhodnius prolixus St%l (HCmipttre). C.r. hebd. Sbanc. Acad. Sci. 267, 2 35 6-235 8. Baudry, N. and Baehr, J.4.(1970). Etude histochimique des cellules neurosicritrices de l’ensemble du systeme nerveux central de Rhodnius prolixus St%l (HCtCroptere, Reduviidae). C.r. hebd. Skanc. Acad. Sci. 2 7 0 , 174.

N EU ROSECRETORY CELLS

113

Beattie, T. M. (1971).Histplogy, histochemistry, and ultrastructure of neurosecretory cells in the optic lobe of the cockroach, Periplaneta americana. J. Insect Physiol. 17,

1843-1855. Bern, H. A. (1966). The hormonogenic properties of neurosecretory cells. Neurosecretion: IV. International Symposium on Neurosecretion, Strasbourg, 25-27Juillet 1966. (Ed. F. Stutinsky). Springer-Verlag, Heidelberg and New York. Bessk, N. de (1965). Recherches histophysiologiques sur la neuroskcrktion dans la chaine nerveuse ventral d’une blatte, Leucophaea maderae (F.). C.r. hebd. Skanc. Acad. Sci. 260, 7014-7015. BessC, N. de (1967).NeurosicrCtion dans la chaine nerveuse ventrale de deux Blattes, Leucophaea maderae (F.) et Periplaneta americana (L.). Bull. SOC.zool. Fr. 92,

73-86. Bloch, B., Thomsen, E. and Thomsen, M. (1966).The neurosecretory system of the adult Calliphora erythrocephala. 111. Electron microscopy of the medial neurosecretory cells of the brain and some adjacent cells. 2 . Zellforsch. mikrosk. Anat. 70,

185-208. Borg, R. M., Bell, R. A. and Picard, D. J. (1973).Ultrastructure of neurosecretory cells in the frontal ganglion of tobacco hornworm, Manduca sexta. Tissue and Cell, 5,

259-267. Boulton, P. S. and Rowell, C. H. F. (1969).Degeneration and regeneration in the insect central nervous system. 11. Z. Zellforsch. microsk. Anat. 107, 119-134. Bounhiol, J. J., Gabe, M. and Arvy, L. (1953). DonnCes histophysiologiques sur la neuroskcrktion chez Bombyx mori L., et sur ses rapports avec les glandes endocrines. Bull. biol. Fr. Belg. 87, 323-333. Bowers, B. and Johnson, B. (1966). An electron microscope study of the corpora cardiaca and secretory neurons in the Aphid Myzus persicae (Sulz.). Gen. comp. Endocr. 6,213-230. Brady, J. (1967).Histological observations on circadian changes in the neurosecretory cells of cockroach suboesophogeal ganglia. J. Insect Physiol. 13, 201-213. Brady, J. and Maddrell, S. H. P. (1967). Neurohaemal organs in the medial nervous system of insects. Z. Zellforsch. mikrosk. Anat. 76, 389-404. Browse-Gaury, P. (1967). GCnCralisation, i divers insectes, de l’innervation deutoctrkbrale des corpora cardiaca, et r61e neurosicretoire des nervi corporis cardiaci IV. C.r. hebd. Sianc. Acad. Sci. 265, 2043-2046. Brown, B. E. (1976).F’roctolin: a peptide transmitter candidate in insects. Life Sciences, 17, 1241-1252. Burgess, I,. (1971). Neurosecretory cells and their axon pathways in Culiseta inornata (Williston) (Diptera: Culicidae). Can. J. Zool. 49,889-901. Burgess, L. (1973). Axon pathways of the intermediate neurosecretory cells in Culex tarsalis Coquillet (Diptera: Culicidae). Can. 1 .Zool. 51,379-382. Burgess, L. and Rempel, J. G. (1966).The standard nervous system, the neurosecretory system, and the gland complex in Aides aegypti (L.) (Diptera: Culicidae). Can. /. Zool. 44,731-765. Cassier, P. and Fain-Maurel, M.-A. (1970). Contribution B 1’Ctude infrastructurale du systkme neurosCaCteur retrocCrCbra1 chez Locusta mipatoria migratoriodes (R. et F.) 11. Le transit des neuroskcrCtions. Z. Zellforsch. Microsk. Anat. 111,

483-492.

114

HUGH FRASER ROWELL

Cazal, P. (1 948). Les glandes endocrines retroctrtbrales, des Insectes. Etude morphologique. Bull. Biol. Fr. Belg., suppl. 32, 1-228. Cazal, M., Joly, L. and Porte, A. (1971). Etude ultrastructurale des corpora cardiaca et de quelqqes formations annexes chez Locusta migratoria L. 2. Zellforsch. mikrosk. .4nat. 114, 61-72. Chalaye, D. (1967). Neurostcrttion au nivea,u de la chaine nerveuse ventrale de Locusta migratoria migratorioides R. et F. (Orthoptkre acridien). Bull. SOC. zool. Fr, 92, 87-108. Chalaye, D. (1974a). Ultrastructure de la masse ganglionnaire metathoracique de Locusta migratoria migratorioides (R. and F.) (OrthoptPre). I. Les cellules neurostcrttrices et leurs prolongements dan le neuropile. Acrida, 3,19-34. Chalaye, D. (1974b). Ultrastructure de la masse ganglionnaire metathoracique de Locusta migratoria migratorioides (R. and F.) (OrthoptPre). 11. Les organes ptrisympathiques abdominaux et thoraciques. Acrida, 3, 35-46. Clements, A. N. (1956). Hormonal control of ovary development in mosquitoes. J. e i p . Riol. 33, 211-213. Cook, D. J. and Milligan, J. V. (1972). Electrophysiology and histology of the medial neurosecretory cells in adult male cockroaches, Periplaneta americana. J. Jnsect Physiol. 18, 1197-1214. Cymborowski, B. (1973). Les variations diurnes de l’activitb des cellules neurosbcrktices du cerveau du grillon (Acheta domesticus).Acrida, 2, 299-306. Day, M. F. (1940). Neurosecretory cells in the ganglia of Lepidoptera. Nature, Lond. 145,264. de Lerma, B. (1954). Osservazioni sulla neurosecrezione in Hydrous piceus (Coleotteri). Pubbl. Staz. 2001. Napoli 24 (suppl.), 56-58. Deltpine, Y. (1965). Recherches sur la neurostcrttion dans l’ensemble du systkme nerveux central d’un lepidopthre, Galleria meflonellu. Bull. SOC. 2001. Fr. 90, 525-540. Delphin, F. (1965). The histology and possible functions of neurosecretory cells in the ventral ganglia of Schistocerca gregaria Forskhl (Orthoptera; Acrididae). Trans. R. t x t . SOC. Lond. 117, 167-214. Dogra, G. S. (1967a). Studies on the neurosecretory system of the female mole cricket Gryllotalpa africana (Orthoptera: Gryllotalpidae). J. 2001.London, 152, 173-178. Dogra, G. S. (196713). Studies on the neurosecretory system and the functional significance of neurosecretory material in the aortal wall of the bug, Dysdercus koenigii. J. Insect Physiol. 13, 1895-1906. Dogra, G. S. ( 1 9 6 7 ~ ) .Studies on the neurosecretory system of Ranatra elongata Fabricius (Hemiptera: Nepidae) with reference to the distal fate of NCC I and 11. J. Morph. 121, 223-240. Dogra, G. S. (1969). Studies in situ on the neuroendocrine system of the giant water bug, Belostoma indica (Lep. and Serv.) (Heteroptera: Belostomatidae). Acta anat. 72, 429-445. Dogra, G. S. and Ewen, A. B. (1970). Histology of the neurosecretory system and the retrocerebral endocrine glands of the adult migratory grasshopper, Melanoplus sanguinipes (Fab.) (Orthoptera: Acrididae). J . Morph. 130,45 1-465.

NEUROSECRETORY CELLS

115

Dopa, G. S. and Tandan, B. K. (1966).An in situ study of cystine and cysteine-rich neurosecretory cells in the brain of Sarcophaga ruficornis (Fabricius 117941 ) (Diptera: Cyclorrhapha).Proc. R . ent. SOC.Lond. (A), 41,57-66. Dupont-Raabe, M. (1951). Etude morphologique et cytologique du cerveau de quelques Phasmides. Bull. SOC.2001. Fr. 76, 386-397. Dupont-Raabe, M. (1956). Quelques donnkes relatives aux phinomines de neurosecrttion chez les phasmides. Annls Sci. mat. ( Z o o l ) . 18(2),293. Dupont-Raabe, M. (1957). Les mtcanismes de l’adaptation chromatique chez les insectes. Archs 2001.exp. gen. 94,61-294. Engelmann, F. and Luscher, M. (1956). Die hemmende Wirkung des Gehirns auf die Corpora Allata bei Leucophaea maderae (Orth.). Verh. d . zool. Gesell., Hamburg 1956. 2001. Anz. Suppl. 20, 215-220. Ewen, A. B. (1962). Histophysiology of the neurosecretory system and retrocerebral endocrine glands of the alfalfa plant bug, Adelphocoris lineolatus (Goeze) (Hemiptera, Miridae).J. Morph. 111, 253-273. Finlayson, L. H. and Osborne, M. P. (1968). Peripheral neurosecretory cells in the stick insect (Caransius ntorosus) and the blowfly larva (Phorrnia terra-novae). J. Insect Physiol. 14,1793-1801. Fletcher, B. S. (1969).The diversity of cell types in the neurosecretory system of the beetle Blaps mucronata. J. Insect Physiol. 15, 119-134. Formigoni, A. (1956). Neuroskcrition et organes endocrines chez Apis mellifica L. Annls Sci. nut. ( Z o o l ) . 18,283-291. Fraser, A. (1959a).Neurosecretion in the brain of the larva of the sheep blowfly Lucilia caesar (Diptera). Q. Jl microsc. Sci. 100,377-384. Fraser, A. (1959b).Neurosecretory cells in the abdominal ganglia of larvae of Lucilia caeser L. Q. J . Microsc. Sci. 100,395-399. Freeman, R. (1970).“Classification of the Animal Kingdom”, English Universities Press, London. Fiiller, H. B. (1960). Morphologisches und experimentelle Untersuchungen iiber die neurosekretische Verhaltnisse im Zentralnervensystem von Blattiden und Culiciden. Zool. Jb. A b t Allg. Zool. 69,223-250. Furtado,A. F. (1971).Etude des cellules neuroskcrktrices, de leur site de dicharge et des corpora cardiaca chez une Punaise vivipare, Stilbocoris natalensis (HCtkroptires, Lygeidis). C.r. hebd. Sbanc. Acad. Sci., Paris, 272, 2364-2367. Gabe, M. (1966).“Neurosecretion”. Pergamon Press, Oxford. Gabe, M. (1972).Donntes histochemiques sur l’tvolution du produit de neurosecrttion protockphalique des insectes pterygotes au cours de son cheminement axonal. Acta histochem. 43, 168-183. Ganagarajah, M. (1965). The neuro-endocrine complex of adult Nebria breuicollis and its relation t o reproduction. J. Insect Physiol. 11, 1377-1387. Gaude, H. and Weber, W. (1966). Untersuchungen zur Neurosekretion bei Acheta domesticus L. Experientia, 22, 396. Gawande, R. B. (1968). A histological study of neurosecretion in ants (Formicoidea). Acta ent. bohemoslou. 65,349-363.

116

HUGH FRASER ROWELL

Gawande, R. €3. (1969). Neurosecretion in the Vth ,instar nymphs of Schistocerca gregaria Forsk. Acta ent. bohemoslou. 6 6 , 70-80. Geldiay, S. (1959). Neurosecretory cells in ganglia of the roach, Blaberus cranifer. Biol. Bull. mar. biol. Lab., Woods Hole, 117, 267-274. Geldiay, S. (1973). Histological, cytological and autoradiographical studies on the new neurosecretory centre in the brain of three Orthoptera species (Anacridium aegyptium L., Acheta domesticus L., Melanogryllus desertus Pall.) Scient. Rep. Fac. Sci. Ege Uniu. 190, (Biyoloji 126), 19 pp. Geldiay, S. and Edwards, J. S. (1973). The protocerebral neurosecretory system and associated cerebral neurohaernal area of Acheta domesticus. 2. Zellforsch. mikrosk. Anat. 145, 1-22. Girardie, A. (1970). Mise en Cvidence, dans le protockribron de Locusta migratoria migratorioides et de Schistocerca gregaria, de nouvelles cellules neurosicrhtrices controlant le mCtabolisme hydrique. C.r. heb. S h n c . Acad. Sci., Paris, 271, 504-497. Girardie, A. and Girardie, J. (1972). Les cellules neurosicrktrices de la zone sousocellaire de Locusta migratoria migratorioides, R. & F., Ctude histologique, cytologique, autoradiographique et Cxperimentale. Acrida, 1 , 205-222. Girardie, A. and Girardie, J. (1973). Structure et fonction des cellules neurosCcrCtrices latCrales de Locusta migratoria migratorioides (Insecte Orthoptire). Conf. Eur. comp. Endocrinologists, Abtrs. 7th, Budapest, 1973, 212. Girardie, A. and Granier, S. (1973). Systkme endocrine et physiologie de la diapause imaginale chez le criquet Cgyptien, Anacridium aegyptum. J . Insect Physiol. 19, 2341-2358. Girardie, J. (1973). Aspects histologique, histochimique et ultrastructural des perikaryones neurosicrtteurs latiraux du protocCrCbron de Locusts migratoria migratorioides (Insecta: Orthopttre). Z . Zellforsch. mikrosk. Anat. 141, 75-91. Goldsworthy, G. J. and Mordue, W. (1974). Neurosecretory hormones in insects. J. Endocr. 6 0 , 529-558. Golbard, G. A., Sauer, J. R. and Mills, R. R. (1971). Hormonal control of excretion in the American cockroach, 11. Preliminary purification of a diuretic and antidiuretic hormone. Cornp. Gen. Pharmac. 1, 82-86. Gosbee, J. L., Milligan, J. V. and Smallman, B. N. (1968). Neural properties of the protocerebral neurosecretory cells of the adult cockroach Periplaneta americana. J. Insect Physiol. 14, 1785-1792. Grillot, J.-P. (1970a). Recherches sur la localisation et la structure des organes neurohemaux mCtam6riques associbs a la chaine nerveuse ventrale chez ColCoptkres. C.r. hebd. Sianc. Acad. Sci., Paris, 270, 847-850. Grillot, J.-P. (1970b). Consid6rations sur la diversit6 des organes neurohemaux mitameriques associis a la chaine nerveuse ventrale de l’imago de Dytiscus marginalis L. (ColCopttre, Dytiscidae). C.r. hebd. Sianc. Acad. Sci., Paris, 270, 403-406. Hagadorn, I. R., Bern, H. A. and Nishioka, R. S. (1963). The fine structure of the supraoesophageal ganglion of the rhynchobdellid leech Theromyzon rude, with special reference to neurosecretion. Z . Zellforsch. mikrosk. Anat. 58, 714-758. Hanky, M., Cottrell, G . A., Emson, P. and Fonnum, F. (1974). Enzymatic synthesis of acetylcholine by a serotonin-containing neurone from Helix. Nature, Lond. 251, 631-633. Hanstrom, B. (1940). Inkretorische Organe, Sinnesorgane und Nervensystem des Kopfes einiger niederer Insektordnungen. K. suenska Vetensk Akad. Handl. 3 ser. 18 (8),266pp.

NEUROSECRETORY CELLS

117

Herlant-Meewis, H. and Paquet, L. (1956). Neurosicrition et mue chez Carausius morosus Brdt. Annls Sci. nut. (Zool.) 18, 163-169. Herman, W. S. and Gilbert, L. I. (1965). Multiplicity of neurosecretory cell types and groups in the brain of the saturniid moth Hyalophora cecropia (L.). Nature, Lond. 205, 926-927. Highnam, K. C . (1958). Activity in the brainlcorpora cardiaca system during pupal diapause “break” in Mimas tiliae (Lepidoptera). Q. J l microsc. Sci. 99, 73. Highnam, K. C. (1961). The histology of the neurosecretory system of the adult female desert locust, Schistocerca gregaria. Q. J l microsc. Sci. 102, 27-38. Highnam, K. C. and West, M. W. (1971). The neuropilar neurosecretory reservoir of Locusta migratoria migratorioides R. and F. Gen. comp. Endocr. 16, 574-585. Hintze-Podufal, C. (1970). The innervation of the prothoracic glands of Cerura vinula L. (Lepidoptera). Experientia, 26, 1269-1271. Hoyle, G. (1974). A function for neurons (DUM) neurosecretory on skeletal muscle of insects. J. Exp. Zool. 189, 401-406. Hoyle, G., and O’Shea, M. (1974). Intrinsic rhythmic contractions in insect skeletal muscle. J. Exp. 2001. 189, 407-412. Huignard, J. (1 964). Recherches histophysiologiques sur le contrble hormonal de l’ovogintse chez Gryllus domesticus L. C.r. hebd. Sianc. Acad. Sci., Paris, 259, 1557-1560. Ichikawa, M. (1962). Brain and metamorphosis of lepidoptera. In “Progress in Comparative Endocrinology” (Ed. K. Takewaki), Gen. comp. Endocr. Suppl. 1 , 331-336. Johansson, A. S. (1957). The nervous system of the milkweed bug Oncopeltus fasciatus (Heteroptera: Lygeidae). Trans. A m . ent. SOC.83, 119-183. Johansson, A. S. (1958). Relation of nutrition to endocrine-reproductive functions in the milkweed bug Oncepeltus fasciatus (Dallas) (Heteroptera: Lygaeidae). N y t . Mag. ZOO^. 7 , 1-132. Johnson, B. (1963). A histological study of neurosecretion in aphids. J. Insect Physiol. 9 , 727-739. Johnson, B. and Bowers, B. (1963). Transport of neurohormones from the corpora cardiaca in insects. Science, N . Y. 1 4 1 , 264-266. Judy, K. J., Schooley, D. A., Dunham, L. L., Hall, M. S., Bergot, B. J. and Siddall, J. B. (1973). Isolation, structure and absolute configuration of a new natural insect juvenile hormone from Manduca sexta. Proc. nut. Acad. Sci. U.S.A. 7 0 , 1509-1511. Junqua, C. ( 1 956). Etude morphologique et histophysiologique des organes endocrines de 1’Hydrocyrius columbae Spin. (Hemiptires belostomidis). Bull. biol. Fr. Belg. 90, 154-162. Khattar, N. (1968). The neurosecretory system of Schizodactylus monstrosvs (Drury) (Orthoptera). Bull. SOC. zool. Fr. 93, 225-232. Kahn, T. R. and Fraser, A. (1962). Neurosecretion in the embryo and later stages of cockroach (Periplaneta americana L.). Mem. SOC.Endocr. 12,349-369. Kind, T. V. (1965). Morphological study of the neurosecretory system of Spilosomo menthastri. Vestn. Lenigr. Univ., Ser. Biol. 3, 24-39. Kobayashi, M. (1957). Studies on the neurosecretion in the silkworm, Bombyx mori L. Bull. Seric. exptl. Sta., Tokyo, 15, 181-273. Kono, Y . (1975). Daily changes of neurosecretory type-11 cell structure of Pieris larvae entrained by short and long days. J. Insect Physiol. 21, 249-264. Kopf, H. (1957a). Zlber Neurosekretion bei Drosophila. I. Zur Topographie und

118

HUGH FRASER ROWELL

Morphologie neurosekretorischer Zentren bei der Imago von Drosophila. Biol. Zbl,

76,28-42. KSpf, H. (1957b). Beitrag zur Topographie und Histologie neurosekretorischer Zentren bei Drosophila. 11. Larven-und Puppenstadien. Verh. deutsch. zool. Ges., Graz. Zool. Anz. Suppl. 21, 439-443. Ladduwahetty, A. M. (1962).The reproductive cycle and neuroendocrine relations in Dermestes maculatus (Coleoptera: Dermestidae). University of London, Ph.D thesis. Langley, P. A. (1965).The neuroendocrine system and stomatogastric nervous system of the adult tsetse fly, Glossina morsitans. Proc. zool. SOC.Lond. 144,415-423. Lea, A. 0.and van Handel, E. (1970). Suppression of glycogen synthesis in the mosquito by a hormone from the medial neurosecretory cells. J. Insect Physiol. 16,

3 19-321. Lee, R. M. (1961). The variation of blood volume with age in the desert locust (Schistocerca gregaria Forsk.). J. Insect Physiol. 6 , 36-51. Maddrell, S. H. P. (1965).Neurosecretory supply to the epidermis of an insect. Science,

N.Y. 150,1033-1934. Maddrell, S. H. P. (1966a).The site of release of the diuretic hormone in Rhodnius-a new neurohaemal system in insects. J. exp. Biot. 45,499-508. Maddrell, S. H. P. (1966b). Nervous control of the mechanical properties of the abdominal wall at feeding in Rhodnius. J. exp. Biol. 44,59-68. Maddrell, S. H. P. (1967).Neurosecretion in insects. In “Insects and Physiology” (Eds J. W. L. Beament and J. E. Treherne), pp. 103-118. Maddrell, S. H. P. (1974). Neurosecretion. In “Insect Neurobiology” (Ed. J. E. Treherne), North-Holland, Amserdam, and Elsevier, New York. Mason, C. A. (1973). New features of the brain-retrocerebral complex of the locust, Schistocerca uaga (Scudder). Z. Zellforsch. mikrosk. Anat. 141,19-32. McLeod, D. G. R. and Beck, S. D. (1963).The anatomy of the neuroendocrine complex of the European corn borer, Ostrinia nubialis, and its relation to diapause. Ann. ent. SOC. A m . 56, 723-727. Meola, R. and Lea, A. 0. (1971).Independence of paraldehyde-fuchsin staining of the corpus cardiacum and the presence of the neurosecretory hormone required for egg development in the mosquito. Gen. comp. Endocr. 16,105-111. Meola, S. M. and Lea, A. 0. (1972). The ultrastructure of the corpus cardiacum of Aedes sollicitans and the histology of the cerebral neurosecretory system of mosquitoes. Gen. comp. Endocr. 18,210-234. Milburn, N. (personal communication). Production of neuroseaetory material by regenerating motor neurones in cockroach thoracic ganglia. Miller, T. and Rees, D. (1973).Excitatory transmission in insect neurornuscular systems. A m . Zool. 13, 299-314. Miller, T. and Usherwood, P. N. R. (1971). Studies of cardio-regulation in the cockroach, Periplaneta americana. J. exp. Biol. 54, 329-348. Mitsuhashi, J. (1963). Histological studies on the neurosecretory cells of the brain and on the corpus allatum during diapause in some lepidopterous insects. Bull. natn. Inst. agric. Sci., Ser. C, Tokyo, 16, 67-71. Mordue, W. and Goldsworthy, G. (1974). Some recent progress in acridid endocrinology. Acrida, 3, IX-XXXII.

NEU ROSECRETORY CELLS

119

Musko, I. B. and Novak, V. J. A. (1973). The structure and ultrastructure of the metameric neurohaemal organs in Gryllotalpa gryllotalpa L. In Conf. Eur. comp. Endocrinologists, Abtrs. 7th., Budapest, 1973, 223. Naisse, J. (1966a). ContrBle endocrinien de la differentiation sexuelle chez Lampyris noctiluca (Coltoptire Lampyride). 11. Phtnomines neurostcrktoires et endocrines au cours du developpement post embryonnaire chez le mile et la femelle. Gen. comp. Endocr. 7, 85-104. Naisse, J. (1966a). Controle endocrinien de la differenciation sexuelle chez Lampyris noctiluca (ColCoptire Lampyride) 111. Influence des hormones de la pars intercerebralis. Gen. comp. Endocr. 7, 105-110. Naisse, J. (1969). RBle des neurohormones dans la differenciation sexuelle de Lampyris noctiluca. J . Insect Physiol. 15, 877-892. Nayar, K. K. (1954). The neurosecretory system of the fruitfly Chaetodacus cucurbitae Cop. I. Distribution and description of the neurosecretory cells in the adult fly. Proc. Indian Acad. Sci. 40, 138-144. Nayar, K. K. (1955). Studies on the neurosecretory system of Iphita limbata Stil I. Distribution and structure of the neurosecretory cells of the nerve ring. Biol. Bull. mar. biol. Lab., Woods Hole, 108, 296-307. Nayar, K. K. (1958). Studies in neurosecretory system of Iphita limbata St%l V. Probable endocrine basis of oviposition in the female insect. Proc. Indian Acad. Sci. B, 47, 233-251. Nijhout, H. F. (1975). (Personal communication material in preparation for publication.) Cobalt backfills of retrocerebral nerves of Manduca sexta 5th instar larvae fill total of 42 cells in brain which appear to be NS, and a further 10-12 associated with “medial” cells which are probably not NS. Normann, T. C. (1965). The neuroseaetory system of the adult Calliphora erythrocephala. I. The fine structure of the corpus cardiacum, with some observations on adjacent organs. Z. Zellforsch. mikrosk. Anat. 67,461-501. Normann, T. C. (1973). Membrane potential of the corpus cardiacum neuroseaetory cells of the blowfly, Calliphora erythrocephala. J. Insect Physiol. 19, 303-318. Normann, T. C. (1974). Calcium-dependence of neurosecretion by exocytosis. J. exp. Bioi. 61,401-410. Normann, T. C. (1975). Neurosecretory cells in insect brain and production of hypoglycaemic hormone. Nature, Lond. 254, 259-261. Odhiambo, T. R. (1966b). The fine structure of the corpus allatum of the sexually mature male of the desert locust. J. Insect Physiol. 12, 819-828. Osborne, M. P., Finlayson, L. H. and Rice, M. J. (1971). Neurosecretory endings associated with striated muscles in three insects (Schistocerca, Carausius, and Phormia) and a frog (Rana).2. Zellforsch. mikrosk. Anat. 116, 391-404. Okelo, 0. (1971). Physiological control of oviposition in the female desert locust Schistocerca gregaria Forsk. Can. J. Zool. 49,969-974. Okelo, 0. (1974). Ph.D Thesis, University of California, Berkeley. Ozeki, K. (1958). Neurosecretion of the earwig, Anisolabis maritima. Sci. Pap. Coll. Gen. Ed. Univ. Tokyo, 8, 85-92. Panov, A. A. (1964). Neurosecretory cells of the abdominal ganglia of Orthoptera (Insecta). Entomol. Obozreniye, 43, 789-800.

120

HUGH FRASER ROWELL

Panov, A. A. (1965).The state of the protocerebral neurosecretory cells in the oak silkworm, Antheraea perneyi C u b . (Lepidoptera, Saturniidae) in the course of the larval development. Dokl. Akad. Nauk. S S S R (Biol.) 165,423-426. Panov, A. A. and Kind, P. V. (1963).The neurosecretory cell system in the lepidopteran brain (Lepid. Insecta). Dokl. Akad. Nauk S S S R . (Biol.)153, 1186-1189. Pflugfelder, 0.(1936). Vergleichendanatomische, experimentelle, und embryologische Untersuchungen iiber das Nervensystem und die Sinnesorganen der Rhynchoten. Zoologica, 34, 1-102. Pipa, R. L. (1961). Studies on the hexapod nervous system. IV. A cytological and cytochemical study of the neurones and their inclusion in the brain of a cockroach, Periplaneta americana L. Biol. Bull. mar. biol. Lab., Woods Hole, 121,521-534. Plotnikova, S. I. (1969).[Effector neurones with several axons in the ventral nerve cord of Locusta m i g a t o r i a ] . J. Euol. Biochem. Physiol. 5 , 339-341.(In Russian.) Prentd, P. (1972). Histochemistry of neurosecretion in the pars intercerebralis-corpus cardiacum system of the desert locust Chistocerca gregaria. Gen. comp. Endow. 18,

482-500. Raabe, M. (1963a). Mise en ividence, chez les insectes d’orders varics, d’iltments neurosicriteurs tritocCribraux. C.r. hebd. S h n c . Acad. Sci., Paris, 257, 1171-1173. Raabe, M. (1963b). Existence chez divers insectes d’une innervation tritociribrale des corpora cardiaca. C.r. hebd. Sianc. Acad. Sci., Paris, 257, 1552-1555. Raabe, M. (1965). Etude des phinomknes de neurosCcr6tion au niveau de la c h a h e nerveuse ventrals des Phasmides. Bull. SOC.2001. Fr. 90,631-654. Raabe, M. (1967). Recherches recentes sur la neurosicrition dans la chaine nerveuse ventrale des Insectes. Bull. SOC.2001. Fr. 92, 67-72. Raabe, M., Baudry, N., Grillot, J. P. and Provensal, A. (1974).The perisympathetic organs of insects. In “Neurosecretion-the Final Common Neuroendocrine Pathway” (Eds F. Knowles and L. Vollrath), pp. 60-71. VI International Symposium on Neurosecretion, London 19 73. Springer-Verlag. Heidelberg and New York. Raabe, M. and Monjo, D. (1970). Recherches histologiques et histochimiques sur la neurosiattion chez le Phasme, Clitumnus extradentatus: les neurosiaitions de type C. C.r. hebd. Sianc. Acad. Sci., Paris, 270, 2021-2024. Ramade, F. (1966). Sur l’ultrastructure de la pars intercerebralis chez Musca domestica L. C.r. hebd. Skanc. Acad. Sci., Paris, 263,271-274. Rehm, M. (1950). Sekretionsperioden neurosekretorischer Zellen im Gehirn von Ephestia kiihniella. Z. Naturf. 58, 167-169. Rehm, M. (1951). Die zeitliches Folge der Titigkeitsrythmen inkretorischer Organe von Ephestia kiihniella wahrend der Metamorphose und des Imaginallebens. Arch. EntwMech. Org. 145,205-248. Rehm, M. (1955). Morphologische und histochemische Untersuchungen an neurosekretionsche Zellen von Schmetterlingen. Z. Zellforsch. mikrosk. Anat. 42, 19-58. Riddifurd, L. M. and Williams, C. M. (1971). Role of the corpora cardiaca in the behavior of saturniiid moths. I. Release of sex pheromone. Biol. Bull. mar. biol. Lab., Woods Hole, 140,1-7. Rowell, C. H. F. (1971). The variable coloration of the acridoid grasshoppers. In “Advances in Insect Physiology” (Eds J. W. L. Beament, J. E. Treherne and V. B. Wigglesworth), vol. 8,pp. 146-198.Academic Press, London and New York.

NEUROSECRETORY CELLS

121

Saini, R. S. (1967). Neuroendocrine control of oocyte development in the beetle Aulacophora foveicollis Luc. J. hisect Physiol. 12, 1003-1008. Sandifer, J. B. and Tombes, A. S. (1972). Ultrastructure of the lateral neurosecretory cells during reproductive development of Sitophilus g-ranarius (L.). (Insecta: Coleoptera). Tissue and Cell, 4, 437-446. Scharrer, B. (1941). Neurosecretion. 11. Neurosecretory cells in the nervous system of cockroaches. J. comp. Neurol. 74, 93-108. Scharrer, B. (1963). Neurosecretion. XIII. The ultrastructure of the corpus cardiacum of the insect Leucophaea maderae. Z. Zellforsch. mikrosk. Anat. 60, 761-796. Scharrer, B. (1964). Histophysiological studies on the corpus allatum of Leucophaea maderae. IV. Ultrastructure during normal activity cycle. Z. Zellforsch. mikrosk. Anat. 62, 125-148. Scharrer, B. (1969). Neurohumors and neurohormones: definitions and terminology. J. Neuro- Viscer. Relat.lSupp1. IX. pp. 1-20. Schlein, Y. (1972). Post emergence growth in the fly Sarcophaga talculata initiated by neurosecretion from the ocellar nerve. Nature, New Biol. 236, 217-219. Schooneveld, H. (1970). Structural aspects of neurosecretory and corpus allatum activity in the adult colorado beetle, Leptinotarsa decemlineata Say, as a function of daylength. Netherlands J. 2001.20, 151-237. Schooneveld, H. (1974a). Ultrastructure of the neurosecretory system of the Colorado Potato Beetle, Leptinotarsa decemlineata (Say) 1. Characterisation of the protocerebral neurosecretory cells. Cell Tiss. Res. 154, 275-288. Schooneveld, H. (1 974b). Ultrastructure of the neurosecretory system of the colorado potato beetle, Leptinotarsa decemlineata (Say). 11. Pathways of axonal secretion transport and innervation of neurosecretory cells. Cell Tiss. Res. 154, 289-301. Schreiner, B. (1966). Histochemistry of the A cell neurosecretory material in the milkweed bug, Oncopeftusfasciatus Dallas (Heteroptera: Lygaeidae) with a discussion of the neurosecretory material/carrier problem. Gen. comp. Endocr. 6, 388-400. Seabrook, W. D. (1970). The structure of the terminal ganglionic mass of the locust Schistocerca gregaria (Forskll.) J . comp. Neurol. 138, 63-86. Seshan, K. R. and Ittycheriah, P. I. (1966). Iphita limbata St%l.: components of neurosecretory material. Science, N.Y . 153,427-428. Siew, Y. C. (1965). The endocrine control of adult reproductive diapause in the chrysomelid beetle Galeruca tanaceti (L.) I and 11.J. Insect Physiol. 11, 1-10,463-479. Smalley, K. N. (1970). Median nerve neurosecretory cells in the abdominal ganglia of the cockroach, Periplaneta americana. J. Insect Physiol. 16, 241 -250. Smith, D. S. (1963). The organisation and innervation of the luminescent organ in a firefly, Photuris pennsylvanica (Coleoptera).J. Cell. Biol. 16, 323-359. Srivastava, U. S. (1959). The prothoracic glands of some coleopteran larvae. Quart. J . micr. Sci. 100, 51-64. Srivastava, R. C. and Dogra, G. S. (1969). Studies in the neurosecretory system of the bug, Scutellera nobifis Fabr. (Heteropterd: Pentatomidae), with reference to the material in the aortal wall. Anat. Anz. 125, 525-534. Staal, G. B. (1961). Studies on the physiology of phase induction in Locusta migratoria migratorioides R and F. Publikatue Fonds Landbouw Export Bureau 1916-1918, nr. 40, 1-125.

122

HUGH FRASER ROWELL

Steel, G. G. H. and Harmsen, R. (1971). Dynamics of the neurosecretory system in the brain of an insect, Rhodnius prolixus, during growth and moulting. Gen. comp. Enclocr. 1 7 , 125-141. S ~ e r b a ,G . and Hoheisel, H. (1964). Darstellung des Neurosekretorischen Systems der Insekten mit Pseudoisocyanin. Z. mikrosk. anat. Forsch. 72, 31-48. Strong, L. (1965). The relationships between the brain, corpora allata, and oocyte growth in the central american locust, Schistocerca sp. 11. The innervation of the corporal allata, the lateral neurosecretory complex, and oocyte growth. J. Insect Physiol. 11, 271-280. S Iumm-Zollinger, E. (1957). Histological study of regenerative processes after transection of the nervi corporis cardiaci in transplanted brains of the cecropia silkworm (Platysamia cecropia L.). J . exp. Zool. 134, 315-326. Thomas, A. and Raabe, M. (1974). Les organes perisympathiques des Orthopttres. Bull. S O C . 2001. Fr. 99, 187-206. 1 homsen, E. (1952). Functional significance of the neurosecretory cells and the corpus cardiacum in the female blow-fly Calliphora erythrocephala Meig, J . exp. Biol. 29, 137-172. 1 liomsen, M. (1954). Neurosecretion in some hymenoptera. Danske Videnskaberns Selskab, Copenhagen: Biol. Skrifter. 7, 5 , l - 2 4 . rhomsen, M. (1965). The neurosecretory system of the adult Calliphora erythrocephata 11. Histology of the neurosecretory cells of the brain and some related structures. 2. ZellJorsch. mikrosk. Anat. 6 7 , 693-717. rhomsen, M. (1969). The neurosecretory system of the adult Calliphora erythrocephata. IV. A histological study of the corpus cardiacum and its connections with the nervous system. 2. Zellforsch. mikrosk. Anat. 94,205-219. rornbes, A. S. and Smith, D. S. (1970). Ultrastructural studies on the corpora cardiaca-allata complex of the adult alfalfa weevil, Hypera postica. J. Morph. 132, 137-148. ‘ri riman, J. W. (1973). How moths “turn on”; a study of the action of hormones o n the nervous system. A m . Sci. 61, 700-706. Triiman, .J. W. and Endo, P. T. (1974), Physiology of insect ecdysis: neural and hormonal factors involved in wing-spreading behaviour of moths. J. exp. Biol. 61, 47-5 6 . Truman, J. W. and Riddiford, L. M. (1971). Role of the corpora cardiaca in the behavior o f sdturniid moths. 11. Oviposition. Biol. Bull. mar. biol. Lab., Woods Hole, 1 4 0 , 5-14. ‘l’riiman, J. W. and Riddiford, L. M. (1974a). Hormonal mechanisms underlying insect I,ehaviour.In “Advances in Insect Physiology” (Eds J. E. Treherne, M. J. Berridge and V. B. Wigglesworth), vol. 10, pp. 297-352. Academic Press, London and New York. ‘l’riiman, J. W. and Riddiford, L. M. (197413). Physiology of insect rhythms. 111. The I emporal organisation of the endocrine events underlying pupation of the tobacco hornworm. J. exp. Biol. 60, 371-382. 17iiiiithan, G. C., Bern, H. A. and Nayar, K. K. (1971). Ultrastructural analysis of the Iieuroendocrine apparatus of Oncopeltus fasciatus (Heteroptera). Acta Zool. 52, 117-143.

N EU ROSECR ETORY CELLS

123

Van der Kloot, W. G. (1961). Insect metamorphosis and its endocrine control. Am Zool. 1, 3-9. Veron, J. E. N. (1973). Physiological control of the chromatophores of Austroleste annulosus (Odonata). J. Insect Physiol. 19, 1689-1703. Weber, W. and Gaude, H. (1971). Ultrastruktur des Neurohaemalorgans in Nervus corporis allati I1 von Acheta domesticus. Z. Zellforsch. mikrosk. Anat. 121,561-572. Wendelaar Bonga, S. E. (1970).Ultrastructure and histochemistry of neurosecretory cells and neurohaemal areas in the pond snail Lymnaea stagnalis (L.). Z. Zellforsch. mikrosk. Anat. 108, 190-224. Weyer, F. (1935). Uber driisenatige Nervenzellen in Gehirn der Honigbiene, Apis mellifica L. Zool. Anz. 112, 137-141. Wheeler, R. E. (1963).Studies on the total haemocyte count and haemolymph volume in Periplaneta americana with special reference to the last moulting cycle. J. Insect Physiol. 9,223-235. Wigglesworth, V. B. (1940). The determination of characters at metamorphosis in Rhodniusprolixus (Hemiptera).]. exp. Biol. 17, 201. Wilkens, J. L. and Mote, M. I. (1970).Neuronal properties of the neurosecretory cells in the fly Sarcophaga bullata. Experientia, 26, 275-276. Willey, R. B. (1961).The morphology of the stomodeal nervous system in Periplaneta americana (L.) and other Blattaria. J. Morph. 108,219-260. Williams, C. M. (1948).The endocrinology of diapause. Biol. Bull. mar. biol. Lab. Woods Hole, Suppl. 33, 52-56. Wyatt, G. R. (1972).Insect hormones. In “Biochemical Actions of Hormones” (Ed. G. Litwack), vol. 11, pp. 385-490.Academic h e s s , New York and London. Zdarek, J. and Fraenkel, G. (1969). Correlated effects of ecdysone and neurosecretion in puparium formation (pupariation) in flies. Proc. natn. Acad. Sci. U.S.A. 64,565.

Note added in proof Chalaye (1975)has reported work which specifically compares LM and EM profiles of a wide range of neurosecretory cells, including A, B, C types as defined on pp. 67-68. The most interesting result is to show that Type C cells, as defined with Azan, are a heterogeneous population under the EM (see p. 67). Also, EM confirms the presence in the NCAII of type C neurosecretory axons, deriving from the SOG (see p. 108). References Chalaye, D. (1975). Etude ultrastructurale des cellules neuroskcrktrices du ganglion sous-oesophagien de Locusta migratoria migratorioides (R. et F.) (Orthoptkres). Acrida, 4, 19-32.

This Page Intentionally Left Blank

Specification of the Basic Body Pattern in Insect Embryogenesis‘ Klaus Sander Biologisches lnstitut I (Zoologic), Freiburg, i, Br. Federal Republic of Germany

1 2 3 4

. . . . .

Introduction The basic body pattern-elements and origin Modes of early insect development: from short germ-type to long germ-type Specification of the longitudinal body pattern 4.1 Fundamental concepts illustrated by experiments on cricket eggs 4.2 Dragonfly eggs-the concepts of activation and differentiation centres 4.3 The short germ-type of development: pattern specification in a growing blastema 4.4 Leaf hopper embryogenesis: transplantation of a “centre” and the gradient concept. 4.5 Beetle eggs and the increasing involvement of anterior egg regions in pattern specification . 4.6 Hymenoptera and Lepidoptera: potency regions and early organ determination challenged 4.7 Lower dipterans-mirroring heads and abdomens 4.8 Higher dipterans: no special status 4.9 Generalizations concerning longitudinal pattern specification and some data not covered by these 5 Specification of the transverse bilateral body pattern 5.1 Patterns formed by longitudinal halves of the blastoderm or germ anlage 5.2 Differences between transverse and longitudinal pattern specification 5.3 The differentiation centre revisited . 6 Genetic tools in the study of pattern specification 6.1 Mutants transforming pattern elements 6.2 Mapping of developmental foci 6.3 Clonal analysis and developmental compartmentalization 7 Cytological and molecular aspects of embryonic pattern specification in insects 7.1 Cytoarchitecture of the oocyte 7.2 Mitotic waves during blastoderm formation 7.3 Functional differentiation of nuclei 7.4 Blastodermal cell boundaries

.

.

.

. .

. .

. . . . . . .

.

.

.

.

.

.

.

.

.

.

.

.

Dedicated in gratitude to Professor Gerhard Krause, Wiirzburg. 125

126 128 133 136 136 155 160 163 172 184 189 192 200 2Q8 208 212 212 216 216 219 219 220 220 221 222 223

KLAUS SANDER

126

7.5 Prospects for a molecular approach to pattern specification in the insect 223 embryo 8 Concluding remarks . 226 Acknowledgements . . 227 References 228

. .

. .

1 Introduction

The ways and means by which orders for different developmental pathways are handed out in proper spatial coordination to the parts of a developing system constitute one of the central problems of developmental biology. But despite many decades of research, most aspects of the problem must still be considered as unsolved. To quote Waddington (Counce and Waddington, 19 72): “But even though binocular microscopes, hair loops and glass needles have given way to ultracentrifuges, counters and fraction collectors, and the level of analysis is pushed down from the cellular to the molecular, the basic problem remains; what causal processes bring about the changes which we can see or otherwise detect as an egg develops into an ‘idult? The new methods of study have still to be applied to embryos if 1 hey are to provide answers to embryological problems.” In the field of pattern formation, optimal use can be made of these new methods of study only if they are applied with the proper questions in mind; starting off from the wrong hypothesis may mean years of detour. This consideration requires integrating old and new data from an animal &TOUP which, like the insects, holds much promise for the developmental biologist, especially from the genetical point of view. A special reason for writing this review was that recent evidence concerning the central problem outlined above has cast severe doubts on wme fundamental interpretations provided decades ago, and perpetuated since. It was long held that a differentiation centre associated with the 1 horacic region, and in some ways comparable to the amphibian organizer (see Haget, 1953, Seidel, 1961), conferred on the different regions of the prospective embryo specific orders for their future pathways of development. But from 1959 onwards evidence accumulated that it is the terminal rvgions of the pattern-forming system which play the ,key role in this process (Sander, 1959, 1960; Yajima, 1960). The possibility that these clifferent patterning mechanisms might be representative for different orders of insects was practically excluded by new data obtained since in a vxiety of species ranging from dragonfly to blowfly. This fact suggested reconsideration of the data on which the conflicting views are based. l‘herefore, the present review does not aim so much at providing an exhaustive source for references but rather should be considered as an

INSECT EMBRYOGENESIS

127

attempt to try and formulate a unifying conceptual framework from which to ask new (and hopefully, meaningful) questions. The author is well aware of the dangers involved in this type of approach and has therefore presented more original data than may be usual for a review-hoping thereby to counteract the bias necessarily involved in his line of argument. Some earlier interpretations which are doubted in the light of present knowledge, and some subsidiary aspects, are presented in smaller print in order to save space and t o enable the reader to by-pass these sections unless specially interested. Last but not least the author wishes t o apologize if he should have failed to quote correctly the source of one idea or the other. Considering the bulk of literature scanned and partly embodied (which includes some 25 research papers of between 50 and 120 pages each) it is nearly impossible to acknowledge the origins of every relevant thought. The present paper could not have been written in this style had not several recent reviews obviated the necessity for more exhaustive coverage. In this connection, particularly the two invaluable volumes of Counce and Waddington (1972) must be mentioned which tbe reader is urged to consult. Some reviews published since then will be quoted in the respective sections. With other literature, papers not included in these volumes have been quoted preferentially when there was a choice. The terminology employed requires some comment. Specification of developmental pathways is a term which has come into use rather recently (e.g. Wolpert, 1969; Bryant, 1974). Its use was prompted by the fact that previously used terms, such as determination, turned out to imply a variety of processes which should be treated (and therefore named) separately. The following processes involved in embryonic determination will be distinguished in this review:

1. Any embryonic cell or group of cells which later on will give rise t o a particular part of the body must be instructed by appropriate signals which specific pathway of development they should follow; this process will be called cellular instruction (see section 4.1.6.a). The term instruction does not imply transmission of an exhaustive set of prescriptions; it rather should be taken t o indicate signals which release appropriate programmes stored in the reacting cells.

2. Specification of a pattern occurs when different instructing signals 'are provided in a spatially coordinated manner. Pattern specification is considered t o represent the first step inpattern formation, a series of processes leading, via regionally different pathways of cell differentiation, to apparent differences between the different regions or pattern elements which together constitute the visible pattern. 3. In order to respond to a specific instructing signal by producing a

128

KLAUS SANDER

particular visible pattern element, an embryonic cell or group of cells must become committed to the developmental pathway indicated by that signal. The types of experiment reviewed here are not suitable for providing strict proof of the committed or determined state of cells or regions of the developing system; this can only be done by explantation (Gehring, 1972). Yet many experimental results described below have been interpreted to indicate a committed state, and others have possibly been misinterpreted because of failure t o distinguish between instruction and commitment as potentially separate steps in embryonic determination. Region-specific cellular commitment is thought to occur after pattern specification, and thus should fall outside the scope of this review. However, many experimental results can be interpreted to mean that specifying signals provided t o a given region of the system may change with time (e.g. Fig. 11 and Table 1); in this case the timing of commitment could decide which pathway a cell or group of cells is going to follow, and therefore cellular commitment cannot be excluded from the discussion of pattern specification. 4. Much evidence concerning pattern specification is derived from the study of egg fragments, i.e. parts of the system isolated functionally by removal (ligature, “pinching”, see section 4.6.1) or denaturation (cautery) of the remaining parts. If such fragments can produce visible pattern elements they are said to be capable of self-differentiation (independent differentiation). It is important to bear in mind that the capacity for selfdifferentiation may be lacking due t o other causes than the absence of instructing signals (section 4.4.3). The cells involved in the construction of a pattern element may be said to interpret the instructing signals which they received during pattern specification. Interpretation is yet another term proposed by Wolpert (1969) and it may be asked why 4;s stimulating concept of positional information has not been adopted, too, in this review. Actually, some results obtained in insect embryos fit in with this concept very well (Sander, 1975a), but I feel that t o some other results it could be applied only by stretching it beyond useful limits.

2 The basic body pattern-elements and origin The basic body pattern of pterygote insects, the central topic of this review, becomes visible at the late germ-band stage. This is the earliest stage where all segments and appendages of the insect body can be recognized. Incidentally, in many species it is also the last, because subsequent steps of development (e.g. head involution in higher dipterans, or specialization of

I NSECT EMB RYOG E N ESlS

129

mouth parts in hemipterans) tend to obscure the basic pattern. Stages prior to the germ band may also differ considerably (see sections 3 and 4). Therefore, the late germ band is the stage of maximum similarity between members of different insect orders and may thus be considered a hallmark of the whole taxon (KorpergrundgestaZt of Seidel, 1960). The insect germ band (Fig. l(c) and Fig. 5 ) initially is a two-layered structure corresponding to the ventral region of the larval body. The dorsal (= prospective inner) layer faces the yolk system while the ventral layer, . .

. . .

.

. .

(a 1 (b 1 (c1 (d1 Fig. 1. Some stages of embryonic development in the beetle Tenehio molitor L. (a) Fate map of the germ anlage stage as inferred by Anderson (1972) (slightly modified). The strictly embryonic territories (heavy outline) are surrounded by the prospective amnion (am). Longitudinal pattern: territories for procephalon (P), gnathocephalon (G), thorax (T) and abdomen (A). Transverse pattern: middle plate (hatched) and ectodermal material for fore-gxt and hind-gut (black), neurogenic ectoderm (outline dotted), purely epidermal ectoderm (lateral). The shape of the territories is conjectural (cf. shape of earlier germ anlage in Fig. 25(d)), but their relative positions are probably correct. (b) Invagination of the middle plate and circular constriction of the amniotic fold to cover the germ band. (c) The first segment borders appear in gnathocephalon and anterior thorax. (d) Germ band with appendage buds and f i s t signs of abdominal segmentation indicated. ((b)-(d) after Ullmann, 1964.)

which later on will furnish the whole body surface, is covered by the amnion. Yolk system and amnion are ensheathed by the serosa. This three-dimensional array of epithelia originates from a single cell layer, the blastoderm. By tracing cell movements back from germ band to blastoderm stage, a two-dimensional fate map can be constructed (Fig. l ( a ) ) which in the longitudinal as well as in the transverse direction may be considered to represent a linear series of different pattern elements. Both series are carried over t o the germ band, and in a formalized approach we may therefore view the basic body pattern as two linear patterns superimposed

130

KLAUS SANDER

on each other: a polarized antero-posterior sequence of pattern elements which will be termed the longitudinal body pattern, and the bilaterally symmetrical array of pattern elements oriented at right angles to this which will be distinguished as the transverse body pattern. These two components of the basic body pattern will be treated separately not only because of corresponding limitations in experimental approach, but also because apparently different formal principles are involved in their specification (Sander, 1971) (section 5.2). The elements of the longitudinal pattern fall into two classes: terminal structures and metameric segments. The anterior terminal structure, the procephalon, is very prominent; it consists of the head lobes including the anlagen of brain and eyes, and carries stomodeurn, labral, and antennal buds as visible landmarks. The posterior terminal structure is essentially limited to the region surrounding the proctodeum, but for simplicity the cerci in some species attached to the hindmost metameric segment (Fig. 5) will be considered terminal structures too. The metameric segments (metamers) include 3 gnathal segments (gnathocephalon), 3 thoracic segments, and between 8 and 11 abdominal segments. For identification of the metameric pattern elements, the following criteria are available: shape, position relative to other elements of the pattern, and specific appendages. With continued development, histological pecularities and specific cuticular structures may be added t o this list. Among the appendages, the different mouth parts as a rule can be identified rather early; in the abdomen, the pleuropodial glands of the first segment (considered t o be modified appendages) show early histological differentiation, while the cerci like the legs are useful mainly in connection with other landmarks. The transverse pattern as represented on the blastoderm fate map (Fig. l(a)) cannot be seen completely at the germ band stage (Fig. l(c)) because the most central pattern element, the anlage of mesoderm and mid-gut (also called middle plate), has sunk behind the lateral ectodermal parts (lateral plates) during gastrulation. The subsequent subdivision of this inner layer into different structures is probably under the control of the adjacent ectoderm (Bock, 1942; Haget, 1953; Louvet, 1964; see Krause, 1958b) and will not be treated here. The pattern elements which before gastrulation flank the middle plate laterally will later on give rise to epidermis and, from their dorsal surfaces, t o the ganglia of the ventral nerve cord. The pattern elements lateral to this neurogenic area turn into epidermis for body wall and appendage5. In the procephalic region, the middle plate fails t o form, and the ganglion-producing ectoderm covers a much greater area than in the metameric region; this is a speciality which will not be further considered. The blastodermal territories just considered are surrounded by the rather narrow anlage of the amnion, and by the rest of the blastoderm, which will

INSECT EMBRYOGENESIS

131

turn into the serosa (Fig. l(a)). The latter area is also referred to as the extra-embry o nic blast oderm. Development prior to the blastoderm stage is characterized by mitotic muItipIication of the original zygote nucleus. Each daughter nucleus becomes surrounded by a yolk-free island of cytoplasm. Such an aggregate is referred t o as an energid. It differs from a cleavage cell by the absence of a limiting membrane or plasmalemma. Therefore, during cleavage the egg cell instead of becoming subdivided into daughter cells turns into a multinucleate cell, here referred t o as egg plasmodium (in accordance with the terminology used in Physarum and the protozoan genus Plasmodium). Towards the end of cleavage, most energids become arranged at the surface of the egg plasmodium (plasmodial blastoderm stage). The oocyte plasmalemma thereafter extends and cuts in between the energids, and separates these from each other and finally from the yolky core of the egg plasmodium. The latter is then called yolk plasmodium (Krause and Sander, 1962), and the cells covering its surface constitute the blastoderm. It should be noted that the delay in subdivision of the ooplasm by cell borders provides physiological conditions quite different from those existing in the aggregate of blastomeres resulting from cleavage in most other animals; these conditions could permit or even require unusual modes of pattern specification during the plasmodial stages. The transition from blastoderm to germ band is initiated by changes in shape (and frequently also location) of some or most blastoderm cells. These changes lead t o subdivision of the blastoderm into germ anlage and extraembryonic region (Fig. 4). The next steps of development occur in a peculiar spatio-temporal order, starting from a focal region. This fact was first pointed out by Seidel (1924) who called the focal region differentiation centre (oifferenzierungstentrum). It is there that the processes of gastrulation and germ band segmentation are f i s t visible, and are first completed. This region according to Seidel is located somewhere in the prospective anterior thoracic region; but in other cases differentiation was observed to proceed in strict antero-posterior sequence (see Anderson, 1972). The lead taken by the differentiation centre is very conspicuous in some species (e.g. Apis) and less so in others; it is usually still manifest in later developmental processes such as histological differentiation. It should be noted that the differentiation centre was established on purely descriptive evidence and therefore represents the type of centre termed Initialbereich (initial region) by Krause and Krause (1957). The course of development just outlined must be programmed in the zygote genome and in the cytoplasm of the oocyte. The latter plays a decisive role for which the stage must be set during oogenesis. But as Mahowald (1972) put it: “The greatest lacuna in our knowledge of

KLAUS SANDER

132

oogenesis whether in insects or elsewhere is in the nature of the organization of the egg which produces normal development." So we must be content to draw conclusions concerning ooplasmic organization from observations and experiments during early embryogenesis. These indicate that considerable differences in oocyte organization must exist (see next section).

'""1

Fig. 2. The reference system used to convey topographical data. Total length of egg or embryo is set equal t o 100 per cent EL (egg length), with 0 per cent EL at the posterior egg pole (pp) (oriented towards bottom in all figures) and 100 per cent EL at the mterior egg pole (ap). Egg fragments are classified as posterior (left) or anterior (right); their size is characterized by determining the level of fragmentation on the per cent EL scale (for fragments shown, see values at the left).

(c)

(d)

( e l ( f ) (9) ( h )

( i )

Fig. 3. Various germ band patterns, and symbols used for classes of patterns in subsequent Figures. Patterns are classified as complete (f), posterior partial ((c)-(e)) or anterior partial germ bands ((g)-(i)). The individual germ bands shown represent the largest partial pattern classified under the respective symbol. The smallest partial pattern in the class would comprise one pattern element (segment) more than the neighbouring ~mallergerm band shown. With classes (c) and (i), the minimum partial pattern comprises only the terminal structures of abdomen or head. The absence of any element of the body pattern (extraembryonic development) is marked by symbol (a), patterns which could not be identified are represented by symbol (b). Rounded symbols (e.g. in Fig. 6) refer to results from uv irradiation.

133

INSECT EMBRYOGENESIS

The following conventions are used in this review. Developmental timing refers t o hours or days after egg deposition, unless stated otherwise. The egg side on which the germ anlage forms is considered the ventral egg side since the outer face of the germ anlage corresponds t o the ventral surface of the prospective body. With reference t o the longitudinal egg axis, locations are marked by their distance from the posterior egg pole, expressed in percentage of total egg length (per cent EL). The sire of egg fragments produced b y ligaturing etc. is characterized by indicating in per cent EL the location (level) where they were separated from the remainder of the egg; consequently, a large (long) posterior egg fragment will be characterized by a h k h value, and a large (long) anterior fragment by a low value of per cent EL (Fig. 2). Partial patterns resulting from operations will be symbolized as shown in Fig. 3 unless stated otherwise.

3 Modes of early insect development: from short germ-type to long germ-type Insect eggs display considerable variation with respect to overall size and to area of blastoderm representing the germ anlage proper (Fig. 4).However, the criterion best suited for their classification is representation of different body regions in the germ anlage (Krause, 1939). In one extreme, the short germ-type, the germ anlage represents essentially the procephalon and a budding zone which subsequent to the germ anlage stage produces the Hemimelobolo

(a1

lbl

lc)

7 Holometabo

Id1

(e)

(fl

lh) ( 1 1

lkl

IIl

Fig. 4. Some insect eggs drawn to scale, left-hand side views. Area covered by germ anlage shaded. Orthoptera: (a) Oecanthus pellucens (Scop.) (Sander, 1976); (b) Acheta domesticus L. (Seidel, 1966). Odonata: (c) Platycnemis pennipes (Pallas) (Krause, 1939). Homoptera: (d) Euscelis plebejus Fall. (Sander, 1959). Coleoptera: (e) Atrachya m a e t r i e s i Falderm. (Miya, 1965): (f) Leptinotarsa decemlineata Say ( W . Schnetter, 1965): (8) Bruchidius obtectus Say (Jung, 1966a). Diptera: (h) Smittia sp. (Kalthoff and Sander, 1968): (i) Drosophila melanogaster Meig. (Bier, 1970): (k) Calliphora erythrocephula Meig. (Alleaume, 1971). Hymenoptera: (1) A p k mellifica L. (Krause, 1939). References are to papers on which diagrams are based.

134

KLAUS SANDER

whole metameric part of the body (e.g. Tachycines, Fig. 15). In the other cxtreme, the long germ-type, the different body regions in the germ anlage ‘ire represented proportionally t o their relative dimensions in the germ band, and no differential growth occurs (e.g. Apis, Fig. 27). Between these cxtremes,’ intermediate forms exist where anterior metamers are repre\ented in proportion t o the procephalon while the remaining (posterior) inetamers originate from a budding zone (e.g. Tenebrio, Fig. 1);this mode I S referred to as the semilong germ, or intermediate type of early development. Compared to egg size, the germ anlage is usually small in the short germ-type while in the long germ-type it may reach from pole to pole; I)ut this relative length of germ anlage is not the essential criterion. Roughly correlated with these types is the extent of the peculiar movements of the embryonic blastema known as blastokinesis (Sander, 1976). In the short germ-type, the germ anlage soon after its appearance migrates around the posterior egg pole into the yolk system or to the dorsal cgg side, with head lobes trailing behind. The original antero-posterior orientation thereby becomes reverted. It is restored by another movement ,ifter visible segmentation and shortly before the flanks of the germ band ATOW out to close the body dorsally (Fig. 5 ) . In the long germ-type, a 1emporary stretching of the embryonic blastema occurs between germ anlage m d segmented germ band stages whereby the posterior tip is carried far up the dorsal side and back to the posterior pole. Although possibly a phylogenetic relic left from the movements observed in the short and intermediate germ types, this temporary elongation of the germ band must have some essential function which preserved it to this day. It could he required for gastrulation (Anderson, 1972, pp. 190, 198), or because the initial short germ anlage may not permit certain physiological differences 1 0 be set up within the blastema (Sander, 1975b). The special value of establishing these different types of development derives from their correlation with “determinateness” or cytoplasmic predetermination of early development. It had been noted by Seidel (1924) that insects on a descriptive basis may be grouped on a scale ranging from I ather “indeterminate” to most “determinate” modes of embryonic development, as exemplified by mode and timing of germ cell segregation. Krause (1939, 1961) reviewed further data relevant to this scale. He stressed the increase in cytoplasm, and particularly periplasm, as compared to yolk and lipid droplets in the oocyte, and the tendency towards nonproliferative embryogenesis with the more determinate forms. Bier (1970) traced these differences back into oogenesis and discussed their evolutionary implications (see also section 4). These may be summarized by stating that the evolutionary trend goes towards an ever-increasing degree of “prefabrication” of the embryo during oogenesis, the selective advantage

135

INSECT EMBRYOGENESIS

probably being an increase in number of generations per unit time, combined with reduction of the immobile stages between oviposition and hatching. Progress in this direction becomes evident when the one or two

6.5 10.5

3d

28

4d

42

50

54

62

h 70

10 d

Fig. 5. Embryogenesis in the cricket Acheta domesticus (L). (a) Cleavage and formation of the germ anlage at 23 "C. Stages: 6.5-h onset of cleavage (dots on ventral egg side (left) represent maIe pronuclei, H. W. Sauer, 1966); 10.5-h egg prior to 5th cleavage mitosis; 28-h uniform blastoderm; 42-h dorsolateral assembly of prospective germ anlage cells; 50-h germ anlage cells shifted ventrally; 54-h single-layered germ anlage; 62-h germ anlage during gastrulation; 70-h beginning of anatrepsis (see (b)) and subdivision of yolk plasmodium into large yolk cells ("yolk cleavage") (from Heinig 1967;courtesy of Dr S. Heinig, Marburg). (b) Anatrepsis and Katatrepsis. The posterior tip of germ anlage moves through the posterior egg pole and then in an anterior direction along the serosa. The remainder of the germ anlage follows suit and thereby gets immersed in the yolk system in reverted position (age 3-4 days at 23 "C). After segmentation and limb bud formation the germ band is being pulled back into its original position, mainly by contraction of the serosa (S) (age c. 10 days at 23 "C) (modified from Mahr; see Sander, 1976). A, amnion. (c) Germ bands after 44 and 61 days of development a t 23OC (drawings of G. Denger, courtesy of Prof. F. Seidel, Marburg). A, abdomen; G, gnathocephalon; H, head; P, procephalon; T, thorax.

KLAUS SANDER

136

months required until hatching in crickets, stick insects and termites are compared t o the 1-2 days required in higher dipterans (Anderson, 1972). Yet despite their high degree of prefabrication during oogenesis, even the most determinate egg types do not, with respect t o pattern specification, display the strict mosaicism that has traditionally been ascribed t o them (section 4.9).

4 Specification of the longitudinal body pattern 4.1

FUNDAMENTAL CONCEPTS ILLUSTRATED BY EXPERIMENTS ON CRICKET EGGS

Embryonic pattern formation has been studied in the cricket, Acheta domesticus (L.), by more types of experiment than in any other species or even family of insects. For this reason, and because it represents the intermediate type of development (section 3), the cricket data will be used to explain and discuss some basic concepts. These will be extended in subsequent chapters so as t o cover the short and long germ-types of early development. Some stages of normal development at 23 OC, the temperature used in all experiments reviewed here, are shown in Fig. 5. Cleavage leads to an uniform array of nuclei at the egg surface (28 h). Thereafter, streaming movements of the egg cytoplasm (H. W. Sauer, 1966; Vollmar, 1972) and mitotic divisions increase the number of nuclei per unit area in the region of the future germ anlage, so that by 42 h a large patch of closely adjacent nuclei can be observed in the as yet plasmodia1 “blastema” on each egg flank (bipartite germ anlage). These patches then merge ventrally to form the germ anlage proper (56 h) while their nuclei decrease considerably in volume (H. W. Sauer, 1966; Vollmar 1974). It must be during these changes that the prospective germ anlage nuclei become separated from each other by cell membranes, a process not clearly recognized in the cricket (Schwalm, 1965; H. W. Sauer, 1966); for practical reasons, the energids of the prospective germ anlage will here be called “cells” once they have started to assemble. Subsequently, the germ anlage becomes thicker and more clearly delineated, and shifts slowly towards the posterior pole and from there up the dorsal side. During this process, the head lobes and the anterior regions sink into the yolk; the germ anlage also stretches considerably and thereby acquires the shape of the germ band. The first segment borders make their appearance by 4 days. Their location indicates considerable growth of the prospective abdomen following the germ anlage stage (Fig. 5(c)).

INSECT EMBRYOGENESIS

137

4.1.1 Longitudinal patterns formed b y anterior egg fragments Anterior fragments of cricket eggs have been produced by ligature (Mahr, 1960), chemical denaturation of the posterior egg region (Mahr, 1960), and by pinching the egg transversely with a blunted razor blade (Vollmar, 1971; for technique see Sander, 1971). The following discussion will concentrate on the results of Vollmar (1971) since they were obtained with a superior fragmentation levels above curve A-A in Fig. 6(b), more than 50 per cent of germ band pattern, anterior fragments must not fall short of a certain critical length (9-13 per cent EL) irrespective of stage of separation. With fragmentation levels above curve v-v in Fig. 6(b), more than 50 per cent of the fragments fail to yield the complete pattern. Fragmentation levels above curve V-v in Fig. 6(b) in the majority of cases lead t o failure to form any germ band parts (extraembryonic development); this curve ascends rather steadily with age at fragmentation, starting from around 9 per cent EL and reaching about 30 per cent EL some time after germ anlage formation. With fragmentation levels between the two curves, incomplete germ bands are the predominant result from 45 h onward. All incomplete germ bands are anterior partial germ bands (see Fig. 3). When grouped according to the sets of pattern elements they produced, these fragments reveal three noteworthy facts: 1. For fragmentation from 47 h onward, the mean values of fragmentation level are inversely correlated to the range of pattern elements formed, i.e. long fragments form many pattern elements and shorter fragments fewer. 2. The mean values of fragmentation level for the smallest partial patterns increase steadily from 27 h t o 60 h (Fig. 6(b));a slight tendency to do so is indicated for larger partial patterns (0, 0) between 47 h and 60 h. 3. The spectrum of partial germ bands is stage dependent: the average number of pattern elements contained in incomplete germ bands increases with advance in age at which fragmentation is performed (Table 1).

(u)

4.1.2 Longitudinal patterns formed b y posterior egg fragments Posterior egg fragments have also been studied by Mahr (1960) and by Vollmar (1971) (Sander et al., 1970; Vollmar and Sander, unpublished). The results obtained clearly demonstrate that fragmentation by pinching off part of the egg is tolerated much better than fragmentation by ligature. Using the latter technique, Mahr (1960) was unable to obtain development in posterior fragments shorter than 30 per cent EL; with the pinching technique, a posterior per cent fragment as short as 16 per cent EL was

KLAUS SANDER

138

uv\......

. ..

lo0 40

t

30 J

w 20

8,

10 I

0

40

I

I

I

I

L

ic 1

3c 20 IC 7.32

51

51

50

31 I

h Fig. 6. Results from fragmentation of cricket eggs with the pinching technique (Vollmar, 1971) (diagram courtesy of H. Vollmar, Freiburg). The abscissa represents the time after oviposition when the fragmentation was performed. The point of fragmentation was varied and the ordinate indicates distance between site of fragmentation (fragmentation level) and posterior egg pole in percentage of egg length (per cent EL) (see arrows at inset). At hatching the larvae were examined and the nature of the pattern in the posterior (a) and anterior (b) fragments were examined. The various curves summarize the kind of patterns produced by fragmenting eggs at different levels. n, number of cases per age group. (a) Posterior egg fragments. When the egg was fragmented below the level represented by curve A-A, more than 50 per cent of fragments failed to produce recognizable patterns. With fragmentation above the level

139

INSECT EMBRYOGENESIS

TABLE 1 Frequencies of different results obtained from anterior egg fragments (6-12 per cent EL, see Fig. 5) of the cricket Acheta domesticus (L.) (Data of Vollmar, 1971, Fig. 49) Age at fragmentation Germ band terminating in 6h Head Thorax Abdomen Germ band complete

17h

28h

45-47 h

60h

16 1 0

8 1 0

7 2 2

1 4 5

0 2 6

3

11

7

11

2

found to produce a complete germ band (Sander et al., 1971). Discussion will therefore again be restricted t o results obtained by pinching off part of the egg (see Fig. 6(a)). Development of posterior egg fragments can take place only after some cleavage nuclei have reached this region. A fragment may then form either a complete germ band, a posterior partial germ band (see Fig. 3), or fail to form any recognizable germ band parts at all. In the last case, irregular aggregation of small nuclei, comparable to germ anlage nuclei, may occur (Vollmar, 1971). Failure to form any recognizable germ band parts is frequent throughout all stages tested (Table 2). The frequency of recognizable partial germ bands varies with stage. A few, lacking only parts of the head region, appear with the earliest fragmentation stage. The next two Fig. 6-continued represented by curve A-A, more than 50 per cent of fragments produced the complete pattern. Rectangular symbols indicate mean values of fragmentation level for fragments which produced the respective partial patterns (see Fig. 3). Positions of rounded symbols and dotted curves are transformed from Fig. 7(c) to comply with coordinates used here. (b) Anterior egg fragments. When fragmented a t levels below curve v-v , more than 50 per cent of the fragments produced complete patterns. Above curve v--0 there were more than 50 per cent cases of extraembryonic development. Rectangular symbols indicate mean fragmentation levels for partial patterns. For easier understanding of this frequently used type of diagram (see also Figs 7, 17, 25 and 30), the “profile” of results obtained a t 47 h will be described here. Fragments which produced nearly complete embryos lacking only parts of the abdomen were ligatured on the average at 9 per cent EL (mean value of all such fragments). Fragments producing the head and 1-3 thoracic segments averaged 13 per cent EL, and those which contained only head structures averaged 18 per cent EL. Positions of rounded symbols and dotted curves transformed from Fig. 7(b). (c) The above curves superimposed. Groups of circles indicate region of closest aggregation of nuclei (Schwalm, 1965). H, T, A, extent of the territories for head, thorax and abdomen in fate map established with small uv lesions by Kanellis (1952).

140

KLAUS SANDER

stages are charicterized by near-complete absence of partial germ bands (Table 2); therefore curves A-A and A-A in Fig. 6(a) run very close to each other between 28 and 47 h. Thereafter, the latter curve continues to rise while the former drops sharply; concomitantly, a sizeable percentage of partial germ bands appear among the results (Table 2). As in anterior fragments, the number of pattern elements produced by these posterior fragments is apparently correlated to fragment length (Fig. 6(a)). TABLE 2 Frequencies of different results obtained from posterior egg fragments (11-30 per cent EL) of the cricket Acheta domesticus (L.) (Data of Vollmar, 1971, Fig. 50) Age at fragmentation Result

17h

28 h

45-47 h

60 h

11

10 1

7 0

2 15

16

14

20

~~~~~~~

Complete germ band Posterior partial germ band N o germ band (extraembryonic development)

6*

18

* Only small defects in head lobes. 4.1.3 Patterns formed in anterior and posterior fragments of the same egg With early fragmentations including the stage 45-47 h, at best only one fiagment of a pinched egg produced pattern elements of the germ band. For stage 60 h, the situation is different. In about 30 per cent of cases an egg which formed a partial pattern in one fragment produced a recognizable partial pattern in the other fragment too. When considered together,
INSECT EMBRYOGENESIS

141

predominant wavelength 288 nm. If applied some time before germ anlage formation, irradiation with doses effective on later stages did not yield any germ band defects, nor did irradiation of larger areas. Even when the germ anlage had appeared, no defects could be produced by irradiating the anterior 315 of the head lobes or part of the prospective abdomen (hatched in Fig. 7(a)). However, irradiation in the intervening region frequently did yield defects, and these displayed a peculiar pattern. When defects were restricted to the larval thorax, only one segment was affected in 7 5 per cent of cases. The single segment defects dropped to roughly 50 per cent for irradiations which affected head parts, and to about 35 per cent when the damage extended into the abdomen. The other defects comprised mostly 2 segments if limited to the thorax; but defects located mainly or completely outside the thorax involved up to 4 (head) or even 7 segments (abdomen). These multiple-segment defects tended to appear in “polarized” fashion. When the uv strip was placed close to the anlage of the metathorax (as established by single-segment defects) the resulting damage as a rule was found to extend posteriorly so as to include some abdominal segments, while irradiations in the prospective gnathocephalon sometimes affected large areas of the procephalon, i.e. extended in anterior direction. Kanellis observed yet another and very interesting tendency among his results (Kanellis, 1952, Fig. 12): The correlation between irradiated region and pattern element(s) affected appears to be better when the region was determined with reference to the yolk plasmodium (distance from the posterior pole) rather than with reference to either anterior or posterior limit of the germ anlage, i.e. defects in a given pattern element appear to be due to irradiation of the cells located on a certain stretch of the yolk plasmodium rather than to irradiation of a certain stretch of germ anlage. For instance, in eggs with a particularly short germ anlage the antenna maps inside the head lobes, while in the remaining eggs it maps clearly behind the head lobes (loc. cit., pp. 453-454). If statistically significant, this difference might be of great value in recognizing the components of the developing system which carry specifying signals (see section 4.1.6.b). Using uv radiation of wavelengths around 264 nm (Baeckstroem filter), G. Sauer irradiated areas across the germ anlage which included the anterior or the posterior border and extended from there in posterior-anterior direction to various degrees (G. Sauer 1961b; Seidel, 1964) (see Fig. 7(b), (c)). With the doses used, irradiation proved ineffective with respect to the final germ band pattern if carried out before 40 h after oviposition, even if the whole prospective germ anlage was irradiated; this is not due to shielding of nuclei because these reach the egg surface before the 20th hour of development. From 40-42 h onward, a rapid change of reaction occurred. Tolerance for anterior as well as posterior irradiation became

~

KLAUS SANDER

142

00 I-

46- 50 h

80

---

a 60 0

$ 40

55- 62 h

-0------3 ---

20

0 I00

80

a 60 c3

$ 40 20

0

40

45

50

55

60

65

h

Fig. 7.(n) Shifting of germ anlage blastema and per cent GA scale with reference to per cent E L during formation of the cricket germ anlage (based on data of G. Sauer, 1961b). Small uv lesions in the hatched areas fail to produce any defects in the larval body pattern (Kanellis, 1952). (b) Results from uv irradiation of posterior regions of the germ anlage blastema (see inset). Ordinate refers t o distance indicated by arrow in inset. Irradiation up to level of unbroken curve fails in more than 50 per cent of cases to cause any pattern defects, while irradiation beyond level of broken cqrve results in extraembryonic development in more than 50 per cent of cases. Symbols indicate mean values of irradiation level for the respective partial patterns and age groups (based on data of G. Sauer, published in Seidel, 1964, Fig. 3). (c) Results from corresponding anterior uv irradiations (based on data of G. Sauer, published in Seidel, 1964, Fig. 4). (Calculdtions and diagrams courtesy of Dr H. Vollmar, Freiburg.)

strongly reduced so that less than 10 h later only the posterior 1/5 or the anterior 1/5 of the germ anlage could be irradiated without impairment to t h e final pattern (see unbroken curves in Fig. 7(b), ( c ) ) , Irradiation beyond

INSECT EMBRYOGENESIS

143

the limits indicated by these curves was followed by complete suppression of the germ band if carried out early. By contrast, later irradiations transgressing these curves yielded incomplete germ bands as the predominant result; with these irradiations, complete suppression of the germ band was obtained only if the irradiated area exceeded the limit indicated by broken curves in Fig. 7(b), (c). With anterior irradiations, the partial germ bands were of the posterior type, and vice versa; they can be classified according to the sets of pattern elements they represent (for symbols see Fig. 3). The mean values of length of irradiated area for these classes, as calculated for two age groups (Vollmar and Sander, unpublished), are set out in Table 3. They do not reveal any significant differences between earlier and later stages for either type of irradiation. Moreover, with the nonterminal classes (8 and @), no difference exists between corresponding mean values from anterior and posterior irradiations (Table 3). TABLE 3 Average limits of uv-irradiated area for production of different classes of partial germ bands in Acheta domesticus (L.). Mean values and standard deviations calculated from data of G . Sauer as published in Seidel, 1964, Figs 3 and 4 (For symbols see Fig. 3, for % GA see Fig. 7(a)) Posterior limit of anterior irradiation Anterior limit of posterior irradiation (see Fig. 7(c)) (see Fig. 7(b))

43-54 h

0

%GA

%EL* % GA

0

% GA

%EL

55-62 h

* 7.0

61.1 f 10.7 31

62.0

40.4 f 15.0 25

36.9 f. 8.8 17

38.6 13.5 25

24.0 f 10.0 14

% GA

23 % GA

* 41.1 * 10.8 50.7

8.7 28

53.8 f 5.6 21

25

36.5 ? 9.5 17

* % EL calculated for position of germ anlage at 50 and 60 h respectively t Based on less than 10 cases.

55-62 h

46-54 h

26.6t (14) (see Fig. 7a).

4.1.5 Irradiation of cricket eggs with X-rays The effects of X-rays on cricket eggs have been studied with a view to pattern formation by Schwalm (1965) and Heinig (1967). Schwalm (1965) was able t o show that in a germ anlage irradiated with 500 rad most of the nuclei are destroyed but the germ anlage is reconstructed by the remaining nuclei and by energids which migrate there from more anterior egg regions (Fig. 8). When the germ anlage region alone was irradiated with 1000 rad,

144

KLAUS SANDER

49h

+I0

4-16

+24

4-45 h

g .......I ::.......

.......

(a)

(b)

(cl

(d)

(el

Fig. 8. Substitution of the germ anlage in the cricket after X-irradiation of the whole egg (after Schwalm, 1965). The irradiated germ anlage (outline dotted) decays while viable cells assemble at its site to form a new germ anlage ((c)-(e)).

47h

+20

1 +26

4-42

+76h

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

Fig. 9. Substitution of the cricket germ anlage after uv irradiation of the germ anlage region so that only a narrow dorsolateral strip of viable cells remains (shown black in inserted cross section) (after G. Sauer, 1962). A new germ anlage forms while the irradiated germ anlage decays (outline dotted).

all germ anlage nuclei were destroyed. Yet a new germ anlage was formed with energids which immigrated from the (shielded) anterior egg half. These had ceased to divide several hours before, as a first step in differentiating t o

INSECT EMBRYOGENESIS

145

form serosa cells. On arriving at the site of the germ anlage they resumed mitotic divisions, to reform the germ anlage within some 70 h after irradiation. This new germ anlage was capable of producing the complete body pattern in roughly 75 per cent of eggs irradiated between 47 and 48 h, and in about 20 per cent of eggs irradiated between 49 and 50 h. Schwalm also showed that irradiation of the posterior half of the germ anlage with a very high dose (6000 rad) at 65 h can be compensated for in some eggs. He did not study this process in detail but apparently the germ anlage retains its original position for an abnormally extended period of time (Fig. 29 in Schwalm, 1965); this might enable any cells reconstructing the posterior half t o pick u p instructions located there in the yolk plasmodium. The results which Heinig (1967) obtained by irradiation of the whole egg are even more interesting with respect to longitudinal pattern specification. He describes a variety of differential responses to LDS0 irradiations during various stages from early cleavage to the segmented germ band. Four phases characterized by different responses are of interest in our context: 1. Irradiation between 10.5 and 22 h after oviposition does not impair germ anlage formation or development, but causes defects in the serosa and in the yolk cells which subdivide the yolk system after completion of the germ anlage, i.e. about 50 h after irradiation.

2. Irradiations between 28 and 50 h produce grossly abnormal germ bands defective over many segments except in the anterior head region and in the abdomen. These germ bands cease development rather early. 3. Irradiations from 42 h onwards produce increasing percentages of germ bands defective in a single segment or at most a few segments, and capable of reaching the hatching stage. The defects depend on irradiation age in a clearcut spatio-temporal pattern (Table 4). Defects induced between 48 and 54 h are restricted to the maxillary, labial, and thoracic segments. From 58 h onwards, mandible segment and head lobes including antennae are frequently affected. Susceptibility for these defects vanishes in the time sequence of their appearance, beginning with the mesothoracic defects which cannot be induced any more after the 66th hour of development. A peculiar feature also observed by Kanellis (1952) after local uv irradiation is the occurence of duplicated appendages in the thorax with irradiations around 54 h (occasionally up t o 66 h). During the last phase, which begins around 74 h (with higher doses 70 h), the abdomen becomes involved. From the onset, localized defects may be found in any of its segments (Table 4). However, a subsequent decrease in X-ray sensitivity followed clearly an anteroposterior course, with the cerci being the last structure which still can be affected (as late as

KLAUS SANDER

146

TABLE 4 Frequencies of locally restricted pattern defects after irradiation of whole cricket eggs with X-rays (LDso) (modified from Heinig, 1967,~. 476). For stages see Fig. 5. Bold type: Median value of frequency distribution per deficient pattern element (lines), and neighbouring age class when median value fell next to it

48

Age (h)

50

54

58

62

66

70

Procephalon Eye Antenna

74

80

86

29 12 5

12 1

6 -

12 12 9 14

-

-

5 14 5 5

3 10 20 21 8

98

112

-

2

Metamers Mandibular 2 Maxillar 1 Labial 4 Thorax I 1 Thorax I1 Thorax 111 Abdomen 1-3

-

1 4 2 2

1 3 8 9 5 1

4-6 7-9 Cerci

4 1 5 6 1 -

2 - 4 3 13 6 7 2 3 1 5 2 1

7 3 2 3 1

2

-

4 29 13

8 29

112 h). With higher doses than L D S 0 ,a further class of results is obtained from irradiation between 70 and 80 h. This is characterized b y complete or nearly complete suppression of the abdomen. Near-complete suppression has a very peculiar aspect: if all but 2 abdominal segments are lacking, one of these is marked as the first abdominal segment by bulging pleuropodial glands, and the other must at least in part correspond to the last abdominal segment, because it carries a well-developed pair of cerci. With any higher number of abdominal segments up t o the normal, these 2 pairs of modified appendages are also always present. In the period immediately preceding phase (4), i.e. between 66 and 70 h, supernumerary abdomens attached t o the rhorax are occasionally obtained with LDs irradiations. 4.1.6 Some basic concepts The experimental results described in the preceding sections can be used t o illustrate some basic concepts which will be useful for subsequent discussion of data from other insect species. These concepts will first be outlined under separate headings and then summarized t o provide a model of pattern specification in cricket embryogenesis. It goes without saying that this model represents an extreme simplification of the events actually taking place. Yet the heuristic value of such a model is thought t o justify

INSECT E M B R Y O G E N E S I S

147

the attempt. The main merit of the model is seen in the clear distinction between two aspects of “determination”: the aspect of signalling t o a cell or blastema of the developing system a certain pathway of development (instruction) and the aspect of restricting the cell or blastema to that pathway so it can no longer enter another pathway (commitment; see section 1). a. Shifting of specifying instructions Anterior egg parts separated from the hindmost egg region at various stages and levels of fragmentation produce different spectra of pattern elements. Fragments too short to yield complete germ bands (mean fragmentation levels above curve v - v in Fig. 6(b)) at best produce small sets of anterior pattern elements (head parts) if separated early; if separated later in development, they yield increasing percentages of larger sets of pattern elements (Table 1). The capacity to produce the additional pattern elements which appear only after later separation (segments of gnathocephalon, thorax and abdomen) must be conferred on that particular egg region during the intervening period of development, and the influence conferring this capacity must in some form or the other come from the posterior egg region. Fragments deprived early of their connection with the posterior egg pole form only head parts or no embryonic pattern elements at all, while fragments with longer contact with the posterior pole region produce on the average considerably more pattern elements. When a region near the posterior pole (below v-v in Fig. 6(b)) is included in an anterior fragment, this will form the complete germ band pattern irrespective of the stage of separation; accordingly, the region near the posterior pole must be able t o trigger crucial changes leading t o formation of the body pattern. The data indicate that the posterior pole region does not, or not only, provide some general stimulus required for germ anlage formation, but rather appears t o be involved in providing and/or moving anteriorly prerequisites for different pattern elements to be formed. These prerequisites may be considered as instructing signals which move anteriorly and reach the prospective site of the germ anlage shortly before the germ anlage becomes visible. Their spatial distribution there would be compatible with the fate map established from small uv lesions (see Fig. 6(c)). Anterior egg fragments separated early from the posterior pole region would then fail to produce thoracic or abdominal segments because their cells were never instructed to do so. This interpretation is strongly supported b y results obtained after shifting material forward from the posterior pol? region in a leaf hopper (see section 4.4.2). b. Egg components carrying these instructions Previous reviewers have noted that in the cricket the yolk plasmodium must be crucially involved in pattern specification (e.g. Seidel, 1964; Counce, 1972). Considering the

148

KLAUS SANDER

(ontlitions under which a new, fully functional germ. adage forms after destiwtitm of the original germ anlage (section 4.1.4 and 5) this conclusion is haid t o avoid. The observations of Kanellis (1952) that small uv defects ,Lppe,tr t o map more consistently with reference t o position on the yolk plasmodium than with reference to position in the germ anlage blastema (5ectlon 4.1.4) would also favour the former as a carrier of instructing sig~i~ils. Yet the question remains open whether the apparent movements of 5pecifying instructions as described above are due to some progressive chanqe within the plasmodial part of the system, or to signalling processes between the ensuing blastoderm cells. Traditionally, there is a strong feeling that the plasmodial parts, that is the yolk system, play the essential role in piding germ anlage formation (Seidel, 1935, 1961) and in the gradual establishment of pattern (Sander, 1959, 1960). This feeling is based on the iole the yolk plasmodium possibly plays in the accumulation of the prmpectlve germ anlage cells (see section 4.2.3), and on the fact that during germ anlage formation the nuclei and/or cellular components of the system mow opposite t o the specifying instructions and therefore cannot directly transport these (Sander, 1959, 1960). However, as pointed out by Lawrence (personal communication), the apparent shifting of instructions might be due to signals spreading within the moving blastema opposite to the main direction of its movement. This possibility is in keeping with prcscnt-day beliefs on pattern specification in cell layers but would require a d d i ~ i o n dand as yet otherwise unsupported assumptions in order t o x c o u n t for some experimental results (see e.g. section 4.5.4). Therefore, for thc time being we shall assume that it is changes in the plasmodial part of thc 5ystem (egg plasmodium followed by yolk plasmodium) which become manifest as the apparent shifting of specifying instructions. c . 1)iactivation of nuclei or cells prior to instruction Until a few hours before germ anlage formation, large areas of the egg surface can be irradiated with uv light so as to eliminate all nuclei there, yet ultimately and after considerable migration of blastodermal energids a complete germ band pattern is formed in all cases; the irradiated area may be as large as one lateral half, or the posterior half, of the whole blastoderm (sections 4.1.3. and 5.1). This remarkable capacity for functional replacement of irradiated blastoderm nuclei and/or cells becomes progressively restricted fro11142 h onwards (Fig. 7 ) . Subsequent anterior or posterior uv irradiations lead to partial or complete suppression of the germ band if the irradiated area exceeds certain limits. This is somewhat surprising since elimination of the whole germ anlage by X-rays 5-6 h later, i.e. at 47-48 h, is followed in more than 50 per cent of cases by a complete germ band as the ultimate result of development (section 4.1.5). Different reactions of

INSECT EMB R YOG E N ESIS

149

cytoplasm and/or yolk to uv light and X-rays respectively, have been invoked to explain this difference (Seidel, 1964; Counce, 1972). But this possibility can be excluded because complete compensation may occur with uv irradiations up to the 52nd hour of development provided the treatment eliminates the germ anlage almost completely (Fig. 9, section 5.1). So there must be other causes for this difference, and these are not difficult to see. After X-irradiation at 47-48 h and after the type of uv irradiation just described, the decaying irradiated “blastema” is quickly overgrown by viable energids or cells from neighbouring areas (Schwalm, 42

54

42 h

1i (a 1

(b)

( C )

Fig. 10. Shifting of irradiated cells following bilateral uv irradiation of the areas indicated (data of G. Sauer, 1961b). Stippled areas mark location of defective cells and/or cell debris still integrated in the migrating germ anlage blastema (see Fig. 7(a)). Scale in 5 4 h egg indicates normal position of germ anlage. The experiments shown here are followed by formation of a complete germ band pattern, but later irradiations likely to cause accumulation of defect cells or cell debris somewhere between the two stippled areas yield defective patterns.

1965; G . Sauer, 1962); the result is an intact cell sheet located in the germ anlage region from about 65 h onward for quite some time (see Fig. 9). With anterior and posterior uv irradiations, the situation is different (G. Sauer, 1961b): the irradiated cells are not overgrown by unirradiated cells. Rather, they are being pushed along within the blastema over the yolk plasmodium of the germ anlage region (Fig. 10). After irradiation of appropriate areas, these inactivated cells will be located somewhere in the germ anlage region during the period when their intact neighbours receive (assumedly from the underlying yolk plasmodium) the signals indicating their future pathways of development. The irradiated cells cannot pick up

150

KLAUS SANDER

and/or carry out the instructions provided t o them, and this failure will cause the absence of anterior or posterior pattern elements observed after irradiation. Additionally or alternatively, the irradiated cells might constitute some kind of barrier for developmental stimuli spreading subsequently within the blastema (see sections 4.1.6.d and 4.1.6.e). The essential point in this interpretation is that cells damaged by an irradiation which causes a pattern defect need at the time of irradiation neither be determined nor even instructed t o produce the absent pattern elements, a fact first realized in principle by Seidel (1935) (cf. G . Sauer, 1961b, p. 210). This permits reconciliation of apparently conflicting results from posterior uv irradiation and from anterior egg fragments. In both cases, the same types of partial embryos may result, but it is only with experiments carried out between 55 and 62 h that the border of the irradiated area corresponds approximately, to the pinching level (cf. symbols 0 and fl in Fig. 6(b)). With the earlier stages (42-54h), the border of the irradiated area for a given class of partial germ bands is located much more anteriorly than the pinching level representative for the same class, and this applies to both anterior and posterior irradiations. This discrepancy can be understood on the basis that the irradiated cells subsequently move backwards (Fig. 7(a)) while the instructing influences move anteriorly from behind during the same period (Fig. 6(b)). Identical partial patterns would then be obtained in experiments at or before 50 h for two different reasons: uv irradiation would inactivate the as yet uncommitted energids or cells required later on for certain pattern elements, while the pinching operation would exclude from the anterior fragment the instructions needed for specification Qf these pattern elements. Finally it should be pointed out that the uv irradiations (and the pinching experiments) fail to support the notion that instructing influences spread from a differentiation centre within the germ anlage blastema (section 4.2.3): no significant differences were demonstrated between early and late irradiations with respect to defect location within the moving germ anlage blastema (Table 3). This result, on the other hand, is compatible with the interpretation proposed above: the prospective but as yet uninstructed and uncommitted blastoderm cells for various pattern elements apparently maintain their relative positions within the moving blastema throughout the period under investigation, even when irradiated.

d. Acquisition of the capacity for independent differentiation Some results obtained from posterior egg fragments require a further assumption in addition t o those made in the previous paragraphs. Apparently some regions of the prospective germ anlage initially need physical connection with others in order t o produce visible pattern elements. This will be appreciated by studying curves A-A and A-A in Fig. 6(a), which indicate an

INSECT EMBRYOGENESIS

151

all-or-nothing type reacti,on of posterior fragments up till at least 47 h of development. With the next stage tested (60 h), much smaller fragments than before prove capable of producing germ bands (albeit partial ones) (Table 2). The failure of comparable fragments separated earlier to yield any germ band parts cannot-under the assumptions made so far-be due to lack of instruction, for it is just these regions which become instructed very early (Fig. 6(b)). The conclusion must then be that these regions require some other stimulus in order t o translate their instructions intc visible pattern. They acquire this capacity from 47 h onwards-apparently not by receiving instructions which pattern elements to form, but by some other change. This change may be the inclusion of greater numbers of nuclei or cells in fragments separated once the germ anlage has started to form, but there might also be a specific stimulus required for cell differentiation which spreads from more anterior regions of the germ anlage subsequent to 47 h of development. This assumption is based on the suggestions of Heinig (1967) concerning determination and self-differentiation in the germ anlage (see next paragraph). The spreading of such a stimulus could also explain the “polarized” multiple segment defects observed by Kanellis (section 4.1.4). These could result from failure of the irradiated cells to pass on the stimulus required for further differentiation. The cells “leeward” to this functional barrier would then lack the stimulus, and therefore fail to carry out their instructions. Farther distally, the stimulus might spread from the unirradiated half over the mid-line and enable the cells on the other side to differentiate. One .possible type or effect of such a stimulus might be the separation of blastoderm nuclei by cell membranes.

e. Differential timing of commitment to segment-specific pathways of development At some time between germ anlage formation and the appearance of visible differences between body segments, the cells in different prospective segments must become committed to carry out different instructions, i.e. reach a status of determination at least corresponding to that revealed by in uivo culture of imaginal discs (Gehring, 1972). No direct evidence bearing on this process is available, but the period of differential X-ray sensitivity observed during germ anlage and early germ band stages (Table 4) has been considered as linked to it (Heinig, 1967). By superimposing on each other the curves shown in Fig. 7(b) and (c), G. Sauer (1961b) and Seidel (1964) concluded that from 46 h onward a process reducing the capacity for regulation spreads in both anterior and posterior directions from a “region of constitutive factors for differentiation” represented by the central 1/3 of the germ anlage. The reasoning on which this conclusion is based may be questioned, as will be pointed out on discussing the results of Alleaume (1971) (see section 4.8.2). Yet if

KLAUS SANDER

152

inferred correctly, the spatio-tempora1,aspects of this process would closely resemble those observed by Heinig with respect t o increased X-ray sensitivity (Fig. 11). The “constitutive factors for differentiation” could then trigger regional .commitment and/or confer the capacity for self differentiation on the more terminal germ anlage regions (Seidel, 1964, p. 131).

I

0

I

50

h

I

I

I00

132

Fig. 11. Diagram to illustrate pattern specification in the cricket as inferred from experimental results (see text, Figs 5 and 6-10, and Table 4). The movements of the blastema relative to the egg axes (see Fig. 5(b)) have been neglected. In the as yet plasmodia1 system, instructing signals (hatched) appear t o spread anteriorly from the posterior pole region; these are capable of releasing region-specific morphogenetic reactions in the ensuing germ anlage cells (see Fig. 6). Towards the end of this period, the blastema which meanwhile has formed loses the capacity to compensate for uv damage (dotted curves) (see Fig. 7). In the prospective embryonic region of the superficial blastema (stippled), segment-specific periods of differential X-ray sensitivity (hatched bars, see Table 4) may indicate commitment of blastema cells t o segment specific tasks originally indicated to them by plasmodial signals. In the prospective abdomen, differential X-ray sensitivity (black crescents, see l a b l e 4) appears to be linked to increased mitotic activity; it is apparently during this period that the pattern of abdominal segments becomes specified, because no segmentally restricted defects can be induced earlier in the abdomen (Table 4; Kanellis, 1952; Sauer, 1961b). Germ band drawing of G. Denger (courtesy of Prof. F. Seidel, Marburg).

Heinig ( 1967) considered the process causing increased X-ray sensitivity also to be involved in qualifying the segment anlagen for independent differentiation (section 4.1.6.d). The regions traversed by this process would then have acquired a status of developmental mosaicism at least down t o the level of segment specifity. This notion is compatible with the appearance, after fragmentation at 60 h, of the first cases where the partial

INSECT EMBRYOGENESIS

153

patterns from anterior v d posterior fragments of an individual egg add up to nearly the complete germ band pattern (Vollmar, 1971), and with the appearance of duplicated appendages after irradiation with X-rays and uv light (section 4.1.5). f. Pattern specification during proliferative growth According t o the uv fate map (Fig. 6(c)) the abdomen occupies proportionally less space in the germ anlage than in the segmented germ band (Fig. 5). It must therefore grow more intensely than the other prospective body regions after the germ anlage stage. This special status of the abdomen is reflected by several experimental results, particularly the fact that it always retains-or subsequently gains-perfect bilateral symmetry when more anterior body regions develop unilaterally after lateral irradiation of the germ anlage (section 5.1). The growth process, which is also indicated by strong mitotic activity after the 70th hour of development (Fig. 15 in Seidel, 1964), is accompanied by a temporary increase of X-ray sensitivity. This appears to affect simultaneously all abdominal segments from 70 h onwards, but subsides in a clear antero-posterior sequence (Table 4). The terminal structures, especially the cerci, frequently form even when a series of more anterior abdominal segments were completely eliminated by the X-rays. Heinig (1967) ascribed these results t o interference with a subterminal budding zone. The specifying processes going on there might be related to the model which Summerbell et al. (1973) have developed to interpret proximo-distal pattern specification in the chick wing. The prolonged capacity to compensate for uv damage, which was observed in the abdominal region of the germ anlage, is also found in the head lobes after anterior or locally restricted irradiations (section 4.1.4).The head lobes and the terminal structures of the abdomen' were also the only typical parts in the aberrant germ bands observed by Sander et al. (1970),and in those obtained from X-irradiation in phase 2 by Heinig (section 4.1.5). This might suggest a similar mode of pattern specification in both anterior and posterior extremities of the germ band, But the procephalon is much more resistant to several types of insult than the abdomen, as documented by formation of isolated heads or head parts following irradiation of the germ anlage (G. Sauer, 1962; Schwalm, 1965);also no phases of increased sensitivity to X-rays were observed there so far. It remains to be seen how far this special status of the most anterior germ anlage region is connected with the special possibilities for cell replacement existing there (see Fig. 10)and/or the bulk of some structures deriving from it (brain!).

4.1.7 A comprehensive model of longitudinal body pattern formation in the cricket The concepts discussed in the preceding paragraphs have been summarized in a comprehensive diagram (Fig. 11). It distinguishes between processes occurring at two different levels of structural organization, the level of

KLAUS SANDER

154

ool)lasmic (plasmodial) and the level of intrablastemic reactions (Krause, 1 9 bXb). During cleavage and subsequent plasmodia1 stages, gradual changes m ' c Iir (hatched pattern in Fig. 11) which confer on different egg regions the cap,icity to signal different courses of development t o the blastoderm cells wh1c.h move there during germ anlage formation. For reasons set out above (sec.rion 4.1.6.b)these changes are thought t o occur at the ooplasmic level. 'I'he events at the blastemic level are represented in a manner which disi c,gards the movements of the blastema relative t o the yolk plasmodium (set. Figs 4 and 7(a)). The first change probably occurring at the blastemic ievc~lis the progressive loss of the faculty to compensate for uv damage (see Fig. 7(b); (c)). This effect may be linked t o restrictions concerning mowments (and perhaps proliferation) of energids or cells while these apptoach the site where the germ anlage will form. In the germ anlage, the cells become committed to the tasks indicated t o them by instructing si,i'n.ils (assumedly from the underlying yolk plasmodium) and may qualify con urrently for independent differentiation at the level of individual segments or body regions. These processes are thought t o be linked t o a temi)orary state of increased X-ray sensitivity (hatched bars in Fig. 11) which sprc.'ids in a characteristic spatio-temporal order from the prothoracic regii )n through gnathocephalon and posterior thorax. This state of inci cased radiation sensitivity requires about 4 h t o spread from one segment t o the next, and lasts roughly 15-20 h per segment (Heinig, 1967). 'The germ anlage then begins t o move posteriorly and passes around the pos~erioregg pole to the dorsal side (see Fig. 4); during these movements its ovet ~ l llength increases, mainly because of proliferative growth of the prohpective abdomen. Abdominal growth is accompanied b y a particular pat1 cm of temporarily increased X-ray sensitivity, which may indicate pro( esses of both cellular instruction and commitment. This pattern (black bars in Fig. 11) is characterized by simultaneous onset, but sequential termination of increased X-ray sensitivity in different regions of the abdi )men-a type of reaction possibly related t o successive pattern specification by a progress zone (section 4.1.6). In the end, the basic body pattern becomes visible when the segment borders appear in a spatio-temporal ordrr rcminiscent of that observed for the decrease of specific X-ray sensitivity (cf. Fig. 5 and Table 4). l'his model can account for most data presented here. The most nott worthy exception is the observation that with increasing fragmentation age posterior fragments have t o be increasingly longer in order t o produce the complete pattern (see curve A-A in Fig. 6(a)). This is a type of result raw in longitudinal pattern formation in insects, yet commonly expected from fragments of "regulating" embryonic systems: the more time available (

INSECT EMBRYOGENESIS

155

for regulation, the better they should do. Perhaps this result is to be seen in connection with the forward spreading of instructing influences (Fig. 6(b) and (c)) but even then it would require some additional assumptions.

4.2 DRAGONFLY EGGS-

CONCEPTS OF ACTIVATION AND DIFFERENTIATION CENTRES

The extensive studies of Seidel (1926-1936, 1961, 1966) on the egg of the damsel fly Platycnemis pennipes (Pallas) have provided a conceptual framework which many authors have used to interpret their results. Yet the original data, however carefully .collected and interpreted, have meanwhile been surpassed in several respects by those established for the cricket and some other insects, notably leaf hoppers and beetles. In the light of new concepts derived from these I shall try to reassess the interpretations Seidel offered for his data. Time will doubtless restore both original merit and present criticism t o their deserved proportions. 4.2.1 Longitudinal patterns formed b y fragments of dragonfly eggs Anterior fragments of dragonfly eggs produced by ligature, cautery or pinching have been studied with respect t o pattern formation by Seidel (1926-1936) and by Vollmar and Sander (unpublished data); the studies of Schanz (1965) and Bergmann-Schanz (1967) were only marginally concerned with the segment pattern a germ anlage could produce. The pinching experiments performed by Vollmar and Sander (unpublished) on the egg of Aeschna cyanea L. yielded results compatible with those from the cricket shown in Fig. 6(c). Whether the spectrum of partial germ bands also varies with stage as shown in Table 1 for the cricket remains at present open, because only a single type of partial pattern (isolated head parts) has been obtained so far. Anterior egg fragments produced by cauterization of the posterior egg pole in Platycnemis display a similar range of reactions. Figure 1 2 summarizes the relevant data published by Seidel (1929a). The overall resemblance to the data obtained by pinching cricket eggs (Fig. 6(b)) is obvious. Complete germ band patterns result in Platycnemis (at 20.3 "C) independent of stage only if the level of cauterization does not exceed values between 10 and 12 per cent EL (cricket: 9-13 per cent EL). Partial germ bands are not observed at all with the earlier stages, but from 25 h onward they appear in ever shorter fragments, as in the cricket. The resulting shift from extraembryonic development to partial germ bands as the predominant result is also observed with other temperatures (see top of Fig. 12).

KLAUS SANDER

156

0 OR1

I

31 80%

20 = 5Q0/o

I = 2%

-

9 = 22%

7 = 18%

I I = 28%

9 = 2 I O/O

27 = 6l%

8 = 18%

1

0

t

I

I

I

I

17

20

30

38

h Fig. 12. Longitudinal patterns obtained after cautery of the posterior egg pole in the damsel-fly Platycnemis pennipes (data of Seidel, 1929a). The results in the diagram were obtained from eggs raised at 20.3 OC. The figures at the top represent pooled data (number of cases and percentage) from experiments at 20.3 OC, 25 OC and 17.9 OC corrected for differences in speed of development (Figs 6 4 6 6 in Seidel, 1929a). The upper curve illustrates Seidel’s “activation” effect, the lower curve (added here) marks the limit to which the egg apparently can be cauterized without causing defects to the final pattern. U = units of eye piece grid used by Seidel.

4.2.2 The activation centre concept The results summarized in Fig. 12 were originally ascribed by Seidel to influences spreading from a centre located near the posterior egg pole. A dual effect was thought t o emanate from there: the centre was not only supposed to activate the egg t o form a germ anlage, but it was also thought to influence the specific courses of development which the different parts of the system were to take. Vom Bildungszentrum der Keimanlage geht nicht nur ein aktivierender Einflufi aus, sondern es ist auch an der Bestimmung der Teile fur ihre Aufgaben beteilzgt. (Seidel, 1929a, p. 433). Accordingly, it was considered to represent a “determination centre” (Seidel, 1926, pp. 340-341). However, a few years later this centre was stripped of its influence on pattern specification and reduced to the role of activating anterior egg regions, and particularly the differentiation centre, for formation of a germ anlage blastema (e.g. Seidel, 1961, p. 127). Only recently a return to the original interpretation was indicated by Schanz (1965, p. 89).

INSECT EMBRYOGENESIS

157

Before deserting the idea of a determination centre, Seidel(1929a, p. 437) proposed a crucial test to demonstrate the influence of the posterior centre on pattern specification: to permit the centre to exert its influence on other egg regions than normal. Without knowledge of this proposal, such an experiment was performed almost 30 years later in the leaf hopper Euscelis and yielded the proof Seidel appears to have expected when proposing the test in 1929 (see section 4.4.2).

4.2.3 The dzjferentiation centre concept With the decline of the posterior egg region from determination centre (Seidel, 1926) to activation centre, the differentiation centre (see section 2) became invested with a physiological function-or, more probably, increasing evidence in favour of a physiological role for the differentiation centre sent the Determinierungszentrum-and the relevant data!-into oblivion. The first definite physiological role attached t o the differentiation centre was to cause, in a rather mechanical way, the blastoderm nuclei or cells to assemble so as to form the germ anlage (Seidel, 1934); in the end, the centre was invested with the function of assigning t o different regions of the germ anlage their developmental tasks or potencies (e.g. Seidel, 1961, pp. 136 and 140). How firm is the evidence in favour of this idea, which is at variance with both Seidel’s earlier interpretation and many results obtained since? As for the mechanical (“dynamic”) activities of the differentiation centre stressed by Seidel (1934), investigations on related species have failed to support his notion that local contraction of the yolk system is as such sufficient to cause formation of a germ anlage. In the damsel fly Zschnura elegans, Schanz (1965, p. 89) found that partial suppression of the activation centre by cautery did not prevent the retraction of yolk plasmodium from egg shell, yet no part of the germ band was formed. Similarly, Vollmar (1974) observed that after fragmentation of the cricket egg at 30 per cent EL during cleavage, blastoderm nuclei assemble in the anterior fragment where the head lobes should form, but fail to turn into (small) germ anlage nuclei, and no part of the germ band is produced. Apparently, in both cases an additional (probably instructing) influence from the posterior egg region is needed t o cause these cells to form parts of the embryo. The notion of the differentiation centre as a region from which 66 potencies’’ for the different elements of the germ band pattern spread in anterior and posterior directions was most clearly stated by M. Schnetter (1934a) on the basis of fragmentations of honey bee eggs (see section 4.6). Schnetter’s idea was adopted by Seidel for Platycnemis (Seidel, 1961, p. 140). The experimental evidence in the latter species has not been published but only reviewed (Seidel, 1961). It is illustrated by Fig. 13. The solid transverse lines on the egg indicate a fate map established for the late

158 Instr. BI.

KLAUS SANDER

Def.

uv+

Fig. 13. Mapping wit. regional uv-defects in Phtycnemis. Right: E-stodermal anlagen (continuous border lines) and “potency regions” (stippled) as inferred from defects due to small uv lesions inflicted at c. 49 and 43 h, respectively, after oviposition (approximately 21 “C) (after Seidel, 1961). The differences between the two maps are here interpreted as due to progressive loss of the capability to compensate uv damage. Note also the restriction of the early defects to the ventral (= median) region; this is compatible with medio-lateral progress of cellular commitment and/or loss of capability to compensate for uv damage as demonstratedby other experiments (see section 5 ) . Left and centre: Model to explain the topographic discrepancies between “anlagen” and “potency regions”. Left: Assume that a sample of small lesions (Def. +) evenly distributed over the prospective territory for a certain pattern element (D) were produced by inflicting one lesion per egg. The meanvalue (a) of location of these lesions for all eggs which developed a defect in that pattern element will then be Iocated half-way between territory borders (top figure). If part of the territory at the time of irradiation could still compensate uv damage (part not hatched in bottom figure), irradiations located in this part would produce no visible pattern defects (Def. -) and the mean value for irradiations which visibly affect the pattern element would shift accordingly (see X in bottom figure). Note that the energids or cells in the irradiated blastema need not yet be instructed t o produce that particular pattern element (see section 4.1.6.b). Centre: Assuming that the capacity to compensate uv damage is lost progressively over a series of territories (A-E) starting from a central region (C), a sample comprising the stages marked 1, 2, and 3 will yield “shifted” or nonrepresentative mean values for all but the most central pattern elements (see ‘‘X1--3’’). The degree of deviation from the mean values for segment anlagen established by “late” irradiations (at right) would increase with distance from the centre (C)-as found by Seidel. The same effect could also be obtained with several slightly different assumptions based on the same principle.

blastoderm stage by defect mapping with small uv lesions (Seidel, 1935). The shaded areas represent a map of “Potenzbereiche” for individual segments (not for body regions as defined by Schnetter) derived by the same rationale from experiments carried out during late cleavage (c. 34 h at 20.9 “C), i.e. c. 6 h earlier. Comparing both maps, a striking longitudinal

INSECT EMBRYOGENESIS

159

aspect is revealed: with increasing distance from the middle region of the blastoderm fate map (i.e. from Th, segment, see arrow), the shaded areas shift more and more towards the proximal border of the corresponding anlagen, but do not transgress these borders. Since the shaded areas were inferred statistically, this kind of arrangement suggests increasingly skewed distributions, with concomitant shifting of mean values. Considering the

‘J‘

t

.. <’,

?

-I

w

s -

i I

f-

Fig. 14. (a) Diagram to illustrate Seidel’s concepts of activation centre (AC) and differentiation centre (DC) in Platycnemis. Once the AC has been triggered by a cleavage energid (not shown) it activates progressively more anterior egg regions (ascending curve) so that they may autonomously produce a germ anlage. Instructions specifying the body pattern spread then (arrows) from the DC where they were assembled as “potency regions” (see Fig. 13, egg at right). Under these assumptions, anterior egg fragments endowed with an activated DC should either produce complete germ bands, or partial germ bands deficient due to lack of space for realization of the potencies present; in this case the horizontal lines in the diagram might indicate the spatial restrictions imposed on the expression of the potency t o form the respective pattern elements. With early fragmentations, when fragments have t o be long in order to include part of the already activated region, lack of space would affect only the most posterior pattern elements. Therefore the earliest partial germ bands to be obtained (arrow a t left) should comprise large sets of pattern elements. This could also be expected with mosaic-type preformation of determinants for the various pattern elements; in this case the horizontal lines would reflect the spatial arrangement of these determinants. (b) Coordinated forward spreading of instruction required for various pattern elements, as suggested by results obtained in the cricket (Figs 6(b) and 11).With this mode of pattern specification, the earliest partial germ bands to be obtained (arrow) should comprise only very few pattern elements. t , time axis. Fragments terminating in the dotted regions should contain the potency to form a complete pattern.

well-established fact that uv defects can still be compensated for in more terminal regions when this is no longer possible in between (Seidel, 1961, p. 139: Figs 7 and 2 5 ) , one may speculate how a skewed distribution of recognizable defects could result from uv lesions on a basically unaltered pattern of segmental instructions, as shown in Fig. 13. However, it must be born in mind that higher uv doses were used with the earlier stage (Seidel, personal communication), a fact which may also have influenced the results.

160

KLAUSSANDER

It may be useful to close this discussion with an attempt to grasp diagrammatically the essential differences between Seidel’s final interpretation of his Platycnemis results, and the interpretation derived above (section 4.1.6.a) from pinching experiments in the cricket. The diagrams in Fig. 1 4 permit some predictions. With an “instructing” differentiation centre, anterior fragments activated for germ anlage formation by the spreading activation centre effect (dotted in Fig. 14(a)),when isolated during early stages, must either all yield complete germ bands, or complete germ bands and partial germ bands comprising large sets of pattern elements (0, left arrow in Fig. 14(a)). With the spreading of instructing influences from the posterior pole region, on the other hand, the earliest partial germ bands to be obtained can merely comprise small sets of pattern elements (fl, arrow in Fig. 14(b)), and larger sets may only be obtained later on. The cricket results clearly fulfil the latter expectations (Table l ) , and OUT Aeschna results are compatible with it because the only type of partial germ band obtained so far comprised exclusively head parts (section 4.2.1). Seidel’s Platycnemis data (Fig. 12) appear quantitatively insufficient for any decision on this issue; the two early cases of large sets of pattern elements (0) would favour an interpretation as in Fig. 14(a), but the remaining data are also compatible with an interpretation as in Fig. 14(b\. Certainly, further results are needed t o show whether Platycnemis really differs from Aeschna and the cricket with respect to the mode of pattern specification.

4.3

THE SHORT GERM-TYPE OF DEVELOPMENT: PATTERN SPECIFICATION IN A GROWING BLASTEMA

In the examples treated so far, the germ anlage was of the intermediate type (see section 2). In a variety of forms belonging to different insect orders, the germ anlage corresponds mainly to the head lobes (short germ type); in these cases, transformation of the germ anlage into the germ band is accompanied by considerable growth not only of the prospective abdomen but also of the thoracic and gnathal regions. With some of these species, specification of the segment pattern appears to occur more or less independently of specific instructing cues from the yolk plasmodium. This is indicated by several observations. In Curuusius morosus Br. a fully functional “replacement germ anlage” may form when a well-advanced germ band, including probably the adjacent yolk cells, is caused by mechanical injury to sink into the yolk system and degenerate there (Vigneau, 1967; Cavallin, 1971). In Schistocercu greguriu ForskP1, an ectopic germ anlage may form when its prospective region had been cauterized after early cleavage (Moloo, 1971). In Kalotermes fluuicollis Fabr. the position of the germ anlage may vary in wide limits (Truckenbrodt, 1964). In the beetle Atruchya menetriesi Falderm., a single egg when chilled may form up to 4 functional germ anlagen located in different egg regions, and eggs ligatured at 35 per cent EL during blastoderm stages are capable of forming two complete germ bands, one in each fragment (Miya and Kobayashi, 1975) (see section 4.5).

INSECT EMBRYOGENESIS

161

In these instances the role of the yolk system can at best,be to induce cell aggregations and perhaps to convey polarity on these. Instruction with respect t o the different body segments must essentially occur within the germ anlage blastema. Since this grows considerably before turning into the germ band it is not unreasonable to assume that the processes involved in pattern specification resemble those inferred for the cricket abdomen (section 4.1.7). Very extensive investigations have been carried out by G . and J. Krause on the camel cricket Tachycines usynamorus (Adelung) (for reviews see Krause, 1958a, b). The results of cutting with a glass needle the germ anlage in various directions and at different stages are summarized and interpreted in Fig. 15. Damage t o the central, dotted iegion of the early germ anlage 6

9

12

18

21

27 h

(a 1 (b) (C) (d 1 (e 1 (f) Fig. 15. Developmental tendencies during germ band formation in the camel cricket Tachycines (after Krause, 1953). Zero time is the heart-shaped germ anlage. Fine stippling in (b)-(d) indicates gastrulation. P, procephalon; G, gnathocephalon; T, thorax. Small circles in (d) and (e) represent segment primordia thought to establish the capacity for development of the respective structures in the lateral areas (arrows).

prevents formation of any germ band parts; this region will later on invaginate to form the mesoderm. Head lobes isolated somewhat later prove to be capable of turning into procephalic parts. For the regions behind the head lobes, successive appearance of tendencies t o form gnathocephalon, thorax and abdomen is inferred. The individual segment anlagen lose the capacity for defect compensation in a spatio-temporal pattern comparable to that of the decrease of X-ray sensitivity in the cricket (see Table 4). This effect may be ascribed t o a prothoracic differentiation centre as discussed below (section 4.9.3). But an instructing role, as postulated for this centre in Platycnemis (section 4.2.3), is unlikely because in duplications of the cruciata type all metameric pattern elements, starting from right behind the head lobes, may be specified in the prospective amnion (Fig. 16). If the longitudinal anterior cut which effected duplication had reached into the prospective prothorax of the germ anlage blastema, the crossing point of the

KLAUS SANDER

162

duplication should be located in that segment: if ,the cut did not reach that far, it is difficult to see how it should have caused a prothoracic differentiation centre t o become established in the prospective amnion. Krause (1962) discussed 2 models to account for this type of duplication. One is based on the streaming of specifying agents from a differentation centre; this was considered less probable. The other model, which was not described in detail, apparently foreshadows that of Summerbell et al. (1973) (section 4.1.7) in so far as it envisages translation of a temporal sequence of functional states . andert into spatial pattern. In dem kunstlich erzeugten kreuzformigen Blastemfeld sich also der Funktionszustand aller Zellen schrittweise, d.h. nach einem zeitlichen Muster so, dap ein raumliches Muster der Segmentanlagen ,fur kreuzweise verdoppelte

..

v

a ,---*-

Fig. 16. Cruciata type duplication in Tachycines (from Krause, 1962, courtesy of Prof. G. Krause, Wiirzburg). (a) Germ anlage cut anteriorly with a glass needle. The structure shown consists actually of two layers (see cross-section in (b)) continuous at the margin:

the amnion (stippled) and the germ anlage proper. Both layers fuse along the edges of the median cut, and amniotic material is caused to turn into two additional body halves ((c) and (e); see also Fig. 36). In the uncut posterior region, the median stretch of amnion becomes induced to form a supernumerary germ band ((b), (d) and (f)). The transition from one mode of duplication to the other may occur in the body region, even immediately behind the head lobes (g). E m b y o n e n entsteht. (Krause, 1962, p. 380). As in the cricket, further investigations will have to show how far pattern specification during budding growth can be explained on this basis.

The germ band defects which Moloo (1971) obtained by ligaturing eggs of Schzitocerca appear to be due t o toxic effects of substances liberated from the yolk plasmodium under mechanical stress, and therefore may have no bearing on longitudinal pattern specification. Of greater interest are the results from centrifugation experiments (Moloo, 1971). They display an age-dependent spectrum of defects (Table 5 ) which again indicates a leading role for the gnatho-thoracic region, this time apparently in a process which enables prospective germ anlage regions to continue development after centrifugation. This process might involve an increase in cell adhesion: areas

163

INSECT EMBRYOGENESIS

of the prospective germ anlage where cell adhesion is strong would stay intact during centrifugation while weak cell contacts might lead t o dispersal or improper mixing of cells during or after centrifugation. However, centrifugation could of course also interfere with the progress of some other process or signal essential for germ band segments to form, particularly since the results obtained during gastrulation (see Table 5 ) do not readily fit in with the above speculation. However, if it is assumed that gastrulation temporarily weakens cell adhesion, the head lobes would not be touched by this weakness and might therefore persist intact through centrifugation (Table 5 ) . TABLE 5 Pattern elements formed (+) after centrifugation of eggs of Schistocerca gregaria (Data of Moloo, 1971, p. 291) Stage at centrifugation

Moloo’s type of defect

Blastoderm to germ anlage

Germ anlage Gastrulation

Pattern elements formed Procephalon

Gnathocephalon Thorax Abdomen

+ +

+ + + +

(+)

-

-

-

+

-

-

-

-

+

+ + +

+

-

(+) Abdomen partially present.

4.4 LEAF HOPPER EMBRYOGENESIS: TRANSPLANTATION OF A “CENTRE” AND THE GRADIENT CONCEPT

The leaf hopper Euscelis plebejus Fall. is comparable to the cricket with respect t o most descriptive aspects of early development and thus belongs to the intermediate germ type (section 2). Experimental results reveal considerable similarities in pattern specification too. But in the leaf hopper, the role of the posterior pole region in this process can be demonstrated positively by “intracellular transplantation” of posterior pole material. The results indicate that the qualitatively differing characters which distinguish different pattern elements may possibly derive from quantitative differences concerning some kind of morphogen (morphogenetic gradients) within the plasmodia1 system. A significant difference to the cricket results is obvious in the development of posterior egg fragments. Their capacity for

KLAUS SANDER

164

pattern formation points towards additional influences exerted by more anterior egg regions on pattern specification. These influences will be demonstrated more clearly in some beetles (section 4.5.1). 4.4.1 The influence of posterior pole material on pattern specification Results obtained from anterior egg fragments in Euscelis resemble those from the cricket, with the exception that instructions for the most anterior pattern elements appear to be located rather anteriorly to begin with, and

36

0

7 12

3E

h Fig. 1 7 . Results from ligaturing eggs of the leaf hopper Euscelis plebejus. Representation based on frequencies per size class of Fragments (Sander, 1959). (a) Changes in the pattern-forming capacity of anterior egg fragments between oviposition and early germ anlage stage (condensed from Fig. 41(a) in Sander, 1959). With fragmentation levels above a given curve, the pattern elements anterior t o the body region indicated at the curve will on average be formed in the anterior egg fragment. P, procephalon; G, gnathocephalon; T, thorax; A, abdomen. The broken curves separate the individual segments of the thorax. The dotted region cannot be analysed with the technique used. (b) Changes in the pattern-forming capacity of posterior egg fragments (condensed from Fig. 25(a) in Sander, 1963). With fragmentation levels below a given curve, the pattern elements posterior to the body region indicated at the curve will on average be formed in the posterior egg fragment. 0, extraembryonic development. Other signs as above. Fragments comprising only the dotted zone are incapable of independent differtntiation (see section 4.1.6.d).

do not shift much during subsequent development (top curve in Fig. 17(a)). The more posterior pattern elements frequently fail to show up in partial germ bands from earlier stages (Fig. 17(a)).These observations suggest that instructions for at least the posterior 2/3 of the body pattern are being generated and/or released from the posterior pole region subsequent to egg deposition. This notion was shown to be correct by transposition of components from the posterior egg pole into more anterior egg regions (see also section 7.5).

INSECT EMBRYOGENESIS

165

The posterior egg pole in Euscelis (as in most leaf hoppers) contains a globular mass of microbial symbionts which, shortly before formation of the egg shell, assemble in a cup-shaped depression at the posterior pole of the oocyte. The symbiont mass is thus originally located in the extracellular compartment of the egg, but it becomes almost completely engulfed by the egg cell. On pushing this symbiont mass and the adherent material anteriorly through the egg cell and depositing it somewhere near the egg periphery during cleavage, some most drastic effects on pattern formation can be obtained (Fig. 18) (Sander, 1960-1962). We shall consider only four results.

(a)

(b)-

(C

1

(d1

(e 1

(f,)

(f*)

Fig. 18. Diagram combining types of operation and typical results for a variety of operations performed on Euscelis eggs (after Sander, 1975a). Black disc indicates posterior pole material, horizontal bar separation by ligature. Pattern elements: X, serosa; procephalon; B, gnathocephalon; C, thorax; D, anterior region of abdomen; E, posterior region of abdomen. The symbols at the upper and lower ends of each series indicate the approximate locations of the blastodermal anlagen for the respective pattern elements; the remaining symbols have been distributed evenly in between. Body regions next to the site of fragmentation, or t o the plane of mirroring symmetry ((d) and (fz)), may lack some segments. (a) Control pattern produced by the untreated egg. (b) See Fig. 19(a), (c) See Fig. 19(b) andTable 7. (d) and (e) See Figs 20 and 21. (f) See Sander, 1962, Fig. 2d-f. Operations (b)-(fl) were performed during late cleavage, (fz) at early germ anlage stage (21 h later at 22 "C).

4,

1. Anterior fragments so short as t o form only head parts-fragmentation levels between curves R and G in Fig. 17(a)-will in 70 per cent of cases produce the complete germ band pattern when supplied with the pole material (Figs 18 and 19) (Sander, 1960). 2. By depositing the pole material in the anterior region of a posterior fragment, a partial pattern of inverted antero-posterior polarity can be produced (Figs 18, 20 and 21), again with very high yield (Sander, 1961a, b).

166

KLAUSSANDER

Fig. 19. Effects of transposition of posterior pole material in Euscelis (data of Sander, 1959, 1960). (a) Control fragmentation. a l , Egg ligatured during cleavage, with pole material (black disc) left in place. az, Typical result: synophthalmic procephalon in anterior fragment, partial germ band consisting o f metathorax and abdomen in posterior fragment; abdomen carries globular “mycetome” harbouring symbionts. (b) Fragmentation after transposition of pole material. bl, Pole material included in anterior fragment. bz, Typical result: complete and viable embryo in the anterior fragment, partial germ band consisting of two thoracic segments and the abdomen (lacking the syrnbionts) in posterior fragment.

(0) (b) (C) (d 1 (e) (f) Fig. 20. Reversal of polarity in Euscelis after transposition of posterior pole material to the anterior region of a posterior egg fragment during cleavage (see Fig. 18(e)) (from Sander, 1961b). (a) Control fragment with posterior partial germ band consisting of two gnathal segments, thorax and abdomen. (b) and (c) Reverted germ bands consisting of abdomen and two or one thoracic segment(s) respectively. Note that number of pattern elements is smaller than in control fragment although the fragments are larger. This statistically significant difference is typical; it is predicted by the two-gradient hypothesis (see Fig. 23(f). (d)-(f) Mirror-type longitudinal duplications, with transverse plane of symmetry in metathorax (d) or abdomen ((e) and (f)).

3. If the egg is not ligatured immediately after transposition of the pole material, but shortly before the germ anlage stage (21 h later at 22 “C),the anterior fragment may form a complete germ band while the posterior

INSECT EMBRYOGENESIS

167

fragment may produce an,inverted partial germ band (Fig. 18(f)).The germ band in the anterior fragment will be complete more frequently when the pole material (as marked by the symbiont mass) is included in this fragment, but it may als6 be complete when the symbiont mass is excluded (see Fig. 18(f 2 ), left pair of eggs).

4. With results (2) and (3) the inverted posterior partial germ band may be joined in mirror symmetry fashion to an identical part with normal polarity; the latter may lack the terminal segments of the abdomen (Figs 18, 20, and 21).

Fig. 21. Thionine-stained and mounted posterior fragments corresponding to results shown in Fig. 20(a), 20(b) and 20(d) (from Sander, 1961a).

4.4.2 The concept of pattern specification b y a morphogenetic gradient The results described in the preceding section demonstrate positively that the posterior region of the egg plasmodium plays a direct role in specifying pattern. They also provide some clues concerning the formal mechanisms involved. The simplest model to account for the results assumes some kind of morphogenetic gradient to which nuclei or cells react differently according to local level. Two results in particular suggest such a model. Firstly, the apparently perfect reversal of a large sequence of pattern elements when the pole material is located anteriorly instead of posteriorly to a certain region (Figs 18, 20 and 21). Secondly, in those cases where an anterior fragment supplied with pole material fails t o yield the complete pattern, the pole material then does not evoke formation of the hindmost abdominal segments which normally develop in its vicinity, but rather enhances the anterior partial pattern by those segments which are missing immediately adjacent to i t (Table 7, cf. Fig. 18(c) left egg, cf. Fig. 18(b)). This result shows that the pole material is not a specific “inductor” for posterior abdominal pattern elements but rather exerts some more general influence

#’

168

KLAUS SANDER

on pattern specification. How this could be accomplished by building up a morphogenetic gradient is set out in Fig. 22.

Fig. 22. Gradient interpretation of the curves in Fig. 17(a). As in other figures, the vertical axis of the diagram represents the longitudinal egg axis. In the lateral blocks, the horizontal axis refers to gradient level or t o morphogen concentration (assuming a gradient in the concentration of some morphogen). Gradient levels within the range marked p are assumed t o trigger in germ anlage cells the programmes for procephalon formation, levels in the range marked g the programmes for gnathocephalon, and so on. It is also assumed that the change in gradient shape occurring between oviposition (left box) and early germ anlage (right box) is caused by the posteriormost region of the egg (at bottom, marked by black disc) and that a stretch of gradient separated from this region by ligature does not subsequently change its shape. The model is insufficient to explain other data, e.g. those shown in Fig. 17(b). It is solely intended t o illustrate the principle of transformation of quantitative differences (e.g. in the concentration of a morphogen) into qualitative differences (as exist between different elements of the body pattern), and to demonstrate some consequences of a change in gradient shape. (b) Interpretation of the result shown in Fig. 18(fz) (right egg) in terms of a morphogenetic gradient. Posterior pole material split up (half discs).

4.4.3 Anterior instructing influences in pattern specification The influence of more anterior egg regions on pattern formation in Euscelis becomes evident when the partial patterns produced by posterior egg fragments are studied (Sander, 1959, 1963). In contrast to the cricket results (Table 2, Fig. 6(a)), posterior partial germ bands are frequent in Euscelis right from the beginning, and the minimum length required for complete germ bands decreases considerably with increasing age at fragmentation. The latter is also true for the average fragment lengths at which partial embryos representing certain sets of segments were obtained (Fig. 17(b)). From these and other results it was concluded that the formation of most pattern elements in the leaf hopper is dependent not only on posterior but also on anterior instructing influences or prerequisites, and that these shift backward by 10-15 per cent of the egg’s length (i.e. c. 113 of germ anlage length) between cleavage and germ anlage formation (Sander, 1959).

169

INSECT EMBRYOGENESIS

This interpretatioo so far cannot be supported with an experiment as persuasive as the transposition of posterior pole material (see above). However, it can be shown that something more than simply lack of space is involved (Sander, 1975a). Fragments isolated at 40-55 per cent EL during cleavage fail t o produce complete germ bands; the head lobes at least are always lacking (Fig. 17(b)). When, however, the eggs before being ligatured were centrifuged mildly (5 min at c. 600 g) in radial position, a high percentage of comparable posterior fragments became capable of producing complete gem bands (Table 6 ) (Sander, 1963). The centrifugation procedure, therefore, must induce or speed up some change in the system comparable t o the change occurring normally during the subsequent 24 h (see top curve in Fig. 17(b)). Whatever that change might be, it appears t o be linked to the cytoplasm and not t o the major fractions of yolk particles or lipid droplets which partly separate in the centrifuge, because this centrifugation effect can be obtained with both centrifugal and centripetal orientation of the posterior egg pole. TABLE 6 Effects of centrifugal force (600 g, 5 min) applied parallel t o the longitudinal egg axis before or after fragmentation. Leaf hopper Euscelis plebejus; data of Sander, 1965, Fig. 22(a-c). Posterior fragments of 40-55 per cent EL; for symbols see Fig. 3 Type of pattern produced Centrifugation

Cleavage stage _

_

I

~

U

0 ~~

6 19

0 12

0

7

12

0 0

7 38

12 40

Before fragmentation

Early Middle + late

18 18

After fragmentation

Middle + late

None

Early Middle + late

TABLE 7 Frequencies of different patterns produced by anterior egg fragments (21-40 per cent EL) of the leaf hopper Euscelis plebejus in the presence or absence of material from the posterior egg pole (see Fig. 18; after Sander, 1975a) Pattern formed* Posterior pole material present Posterior pole material absent

X...E

X...D

23 0

4 0

X...C 12 0

X...B

0 10

X+A

X

1 21

0 6

* Letters represent pattern elements (see Fig. 18). 4.4.4 A two-gradient hypothesis for pattern specification in Euscelis In the experiment shown in Fig. 18(c), the abdominal and thoracic pattern elements were frequently formed twice within the same egg, one set in the

170

KLAUSSANDER

Q O0o O /0 ,

0

0 0

0

0 0

0 0

iA

I I((’ I Doooooo .*@

O O 0

... *. * .

0

.

.

Fig. 23. Double gradient hypothesis for formal interpretation of the results obtained in Euscelis by egg fragpentation and by transposition of posterior pole material (after Sander, 1960, 196lb). (a)-(.) Normal development from oviposition to the germ anlage stage. The posterior gradient corresponds to that shown in Fig. 22 (increasing towards the left; see arrow at bottom). An opposite anterior gradient (upper left; increasing towards the right; see arrow at top), is thought to build up under the influence of the egg region in front of 60-65 per cent EL (open circle). The relation of anterior to posterior gradient levels ( a / p ratio), which varies continuously along the egg axis (see assumed values for this ratio in (c)), is assumed to trigger at the germ anlage stage different pathways of development in cells located in different regions. (d)-(f)Status of gradients at the germ anlage stage in different experiments; the area covered by each (disfigured) gradient is equal to that covered in (c) (= same quantity of morphogen). (d) Fragmentation during cleavage. Subsequent accumulation of “anterior” substance in the anterior, and “posterior” substance in the posterior egg fragment causes a discontinuity in the alp ratio at the level of fragmentation (transverse bar). Consequently, the alp ratios which trigger formation of some pattern elements become established neither in the anterior nor in the posterior fragment (“gap phenomenon”, see section 4.4.4, Table 8 and Fig. 18(b)). ( e ) Transposition of posterior pole material prior to fragmentation. For result see Fig. 19(b). The gradient shape in the anterior fragment results from the assumption that the region in front of 60 per cent EL somehow inactivates the effects of the posterior pole material, an assumption suggested by some experimental results (Sander, 1960, Fig. 7). (f) Transposition of posterior pole material to the anterior region of a posterior fragment. For results see Fig. 20(b) and (c). Note that the ratio alp changes here more gradually along the egg axis because both gradients slope in the same direction (albeit with differences in steepness); this would explain the formation of fewer but longer pattern elements as compared to control fragments (cf. Fig. 20(b)).

INSECT EMBRYOGENESIS

171

anterior and the qther in the posterior fragment (see right egg in Fig. 18(c)). In some instances, even the gnathal segments were duplicated this way. With specification coming exclusively via a posterior gradient, this result could be understood rather easily: However, Fig. 17(b) indicates that specification of at least the gnathal and thoracic elements of the pattern may also be subject t o anterior influences spreading backwards during early development. When assuming a specific anterior prerequisite for each of these pattern elements, it is almost impossible to understand how these anterior prerequisites could be split, by translocation of posterior pole material, into two sets arranged one behind the other (as required to interpret the result shown at the right in Fig. 18(c). The dilemma can be resolved by assuming that the anterior influences on pattern specification inferred from Fig. 17(b) were also due to a gradient built up before the germ anlage stage. A two-gradient hypothesis elaborated on this basis (Sander, 1960) was adequate to account on a quantitative basis for the essential results from all experiments described here (Sander, 1960, 1961) (Fig. 23). By assuming more dynamic interactions between components of the system, and by testing these with a computer simulation (Meinhard and Gierer, 1974), it should now be possible to construct models for pattern specification in Euscelis which are more compatible with general knowledge on cell physiology than were the rather static and independent gradients proposed earlier. However, the principle of translating quantitative differences into qualitatively differing characters will probably have to be embodied in more advanced models too.

4.4.5 The ‘kap phenomenon ”-hallmark of antero-posterior interaction One further aspect of the leaf hopper results should be pointed out because it will be encountered with most holometabolan species investigated by egg fragmentation (see the following sections). On superimposing the curves from Fig. 17(a) on those in Fig. 17(b) it becomes apparent that the partial sets of pattern elements produced by anterior and posterior fragments of an individual-egg should not add up to the complete pattern if the egg was fragmented early (Sander, 1959) (e.g. Fig. 18(b); for illustration of the argument, see Fig. 31). This expectation was borne out by experiments performed to test it (Table 8). At first sight, the ensuing “gap” in the body pattern might appear to be due to some unspecific sensitivity of the early stages to the constriction experiment. This interpretation, however, is ruled out by the results obtained when posterior pole material was transposed prior to constriction (Fig. 18(c)). These show that the “gap” phenomenon must be due to prevention of further interaction between the separated anterior and posterior egg regions. Temporary separation of egg parts leads to the same conclusion: egg parts reunited immediately or c. 5 h after

I

KLAUS SANDER

172

separation will form the complete pattern in more than 90 per cent of cases (Fig. 24). Apparently, normal pattern specification can be resumed if anterior and posterior fragments are permitted to interact again.

n = 37

32

18

36

6

S

I

Im

E

100,

0’ Stage

D

Fig. 24. Effects of temporary separation of egg parts on pattern specification in Euscelis (data of Armbruster and Sander, from Sander, 1975a). The pinching technique employed (Sander, 197 1) permits complete separation and subsequent reunion of egg parts within the intact egg shell. Egg parts separated at approximately 35 per cent EL during late cleavage were allowed to reunite immediately (D), or after having reached one of several subsequent morphological stages (S-E); stage E corresponds to early germ anlage, a stage reached 16 h after late cleavage during normal development. Results: 1, complete germ band pattern; 2, “subcomplete” pattern, single germ band lacking some gnathal or thoracic segments; 3, two partial germ bands separated physically at the gap in segment pattern; n, number of cases.

4.5

BEETLE EGGS AND THE INCREASING INVOLVEMENT OF ANTERIOR EGG REGIONS IN PATTERN SPECIFICATION

Beetle eggs cover an unusually wide range on Krause’s (1939) scale of developmental types (section 3). They comprise the short, intermediate and (moderate) long germ-types. In this order, these types appear t o reflect an increasing role for anterior egg regions in pattern specification, as summarized below (section 4.5.7). It may be worth noting that leaf hoppers, where these anterior influences were first encountered (section 4.4.3),and beetles

INSECT EMBRYOGENESIS

173

both differ with respect to oogenesis from the species discussed earlier. In Euscelis and in all species treated subsequently, the ovarioles are of the meroistic type, i.e. contain nutritive chambers located anterior to the growing oocyte(s) (Telfer, 1975). If not fortuitous, the coincidence between appearance of nutritive cells and of anterior influences on pattern specification might have several causes. The polarized influx of products from the nutritive cells through the anterior pole of the oocyte (Winter, 1974) could provide cues utilized for pattern specification, especially in the anterior egg region (Engels, 1973). More indirectly, additional cues for pattern specification might be a prerequisite for full utilization of the main benefit provided by meroistic oogenesis, i.e. the capacity for rapid embryonic development (Bier, 1970). Pattern specification via proliferative growth is likely to be more time-consuming than instruction of a sheet of blastoderm cells via the yolk plasmodium (or by some other means). However, if large sets of pattern elements are t o be specified by the latter method, instruction by influences from only one terminal region (e.g. the posterior as in the cricket) might not be the optimal solution. In this case, introduction of additional specifying influences, that is from anterior egg regions, could have provided some evolutionary advantage. A closer study of oogenesis in beetle species representing different types of embryonic pattern specification might be rewarding from this point of view.

4.5.1 Egg fragments of Bruchidius: antagonistic terminal influences Extensive series of experiments have been performed by Jung (1960, 1971) on the long germ-type bruchid beetle Bruchidius obtectus Say. Some results obtained by ligaturing eggs at different stages and levels are summarized in Fig. 25(a). The upper, descending curves correspond to those from Euscelis shown in Fig. 17(b). They indicate that the capacity to produce a given set of pattern elements is found in ever shorter posterior fragments with increasing age at separation. Fragments from early stages yield much smaller sets of pattern elements than would be expected from a fate map of the germ anlage stage (see below). By the argument used in discussing the leaf hopper results (section 4.4), it would appear that a given posterior egg region requires contact with more anterior regions during early development in order to produce its normal share of the segment pattern. Attempts of Jung and Krause (1967) t o shift material responsible for this “anterior” influence towards more posterior egg regions, and demonstrate its efficiency there, gave no convincing results. This leaves room for alternative explanations but so far none have come forward. The study of patterns formed by anterior egg fragments of Bruchidius

174

KLAUS SANDER

0

10

20

0

I 0

20

30

40

30

40

h

Fig. 25. Synoptic review of data obtained by cautery, ligaturing or uv irradiation in beetle eggs. Stages: I, nuclei reach oocyte surface; 11, formation of radial cell boundaries between blastoderm nuclei; Ill, germ anlage stage before germ band extension. (a) Eruchidius obtectus, patterns produced by anterior and posterior fragments of ligatured eggs (based on data from Jung, 1966, Figs 22 and 23) (rearing temperature 20 "C). Upper curves: mean fragment lengths required for formation of posterior partial germ bands beginning with first (I), second and third thoracic segments, and first abdominal segment (A1 ) respectively. Lower curves: mean fragment lengths required for anterior partial germ bands terminating with labial (L), first, second and third thoracic (111) segments respectively. The differences between earliest and latest stage are highly significant, as are for most curves the differences between neighbouring stages. The comparable curves for segments of head and abdomen established by Jung are omitted

INSECT EMBRYOGENESIS

1

175

(Jung, 1966b) yielded,curves ascending as in the sample sho,wn in the lower part of Fig. 25(a). In their trends these curves are comparable to those published for the leaf hopper (see Fig. 17(a)), yet they transgress a much larger part of the egg. Attempts to shift anteriorly, during early stages of development, the posterior material probably responsible for these changes (Jung and Krause, 1967) have been somewhat more successful and demonstrate positively the influence of the posterior egg region on pattern specification. The similarity between these results and those obtained in the leaf hopper (section 4.4.1.3) is underlined by the fact that the “gap phenomenon” (section 4.4.5) is very evident in Bruchidius (Table 8), as would be expected from the large distances transgressed by the upper and lower curves in Fig. 25(a). These curves may serve to underline yet another aspect realized first on the basis of the less dramatic changes occurring in Euscelis (Fig. 17(a) and (b)). The influences exerted by opposite terminal egg regions on the body pattern may be called antagonistic (Sander, 1959). They counteract each other with respect to location of pattern elements. The thoracic segments for instance, which normally form from blastodermal cells located between approximately 40 and 60 per cent EL (see stage 40 h Fig. 25-continued here. G, T, A, territories for gnathocephalon, thorax and abdomen in the early germ anlage stage, as evidenced by these experiments and by uv-induced defects (see below). (b) Bruchidius obtectus, pattern defects resulting from small uv lesions (mostly unilateral, see inset at left) (data from Jung, 1971, Table 3). The curves delimit the (calculated) target areas for damaging individual segments in gnathocephalon and thorax, and abdominal segments nos 1-4; dotted curves are subject to error due to the compensation of uv damage (see Fig. 13). No pattern defects were obtained after irradiation of the hatched regions a t the respective stages. Irradiation of the whole ) is followed by formation of prospective germ anlage soon after oviposition complete germ band pattern while unilateral irradiation of the early germ anlage results in contralateral half-embryos (see section 5 ) . (c) Dermestes frischii, bilateral uv irradiation denaturing the yolk plasmodium right to the centre (data of Kiithe, 1966, rearing temperature 22 “C). Denaturation of anterior or posterior egg half (hatched) prior to blastoderm formation does not cause pattern defects. Thereafter, anterior or posterior irradiations transgressing levels of curves cause defects t o the body regions indicated between curves. For individual segments, such curves can be established only from 7 h onward. H, head. Denaturation of one longitudinal egg half before 6.5 h is followed by formation of bilateral germ band, later irradiation yields half-embryos (see section 5 ) . (d) Tenebrio molitor, cautery of the posterior egg pole (data of A. Ewest, 1937, rearing temperature 22.5 “C). With denaturation beyond level W-V, more than 50 per cent of eggs fail to produce the complete pattern; with denaturation beyond level V - V, the majority of eggs do not produce any pattern elements. With denaturations restricted to hatched area a t bottom, all eggs produce complete patterns. Hatched region at top was shown by ligature to be dispensable for pattern specification. Dotted: germ anlage. For germ bands inserted see text.

(0

(0)

(0) (a)

KLAUS SANDER

176

TABLE 8 Average numbers of body segments which fail to be formed after fragmentation at various stages (“gap phenomenon”) Stage at fragmentation Species and reference

Plasmodia1 Cleavage blastoderm

Acheta (Vollmar, 1971)

-*

Cellular blastoderm

-

Germ anlage

-

3

2-3

2

Germ band

Numbers represent

Median value

Euscelis (Sander,

1959, 1963)

(9) (5-6) (3-4)

Necsobia (Trendelenburg, 1971)

5

6

8

0

Medianvalue

2

Median value

3

Mean

Bruchidius fJung, 1966;see Herth and,Sander ,

1971)

7-8

(8)

Apis (M. Schnetter, 1934)

5

Greater

Smaller

Estimate

Srnittia (Sander,

1975a) Pro tophormin (Herth and Sander, 1971)

4

7

6

4

2

1

1

Median value

0

Mean

* Only one fragment of the egg will form a pattern. ( ), Figures based on a few cases, because in most eggs only one fragment will develop to a stage permitting analysis of the pattern formed.

in Fig. 25(a)) will be formed from the blastoderm around 10-30 per cent EL in the absence of the posterior, and from the bIastoderm around 70-90 per cent EL in the absence of the anterior influence (see results from fragmentation at 0-2 h in Fig. 25(a)). Obviously in either case the influence eliminated by ligature would have been required t o push the signal for thorax formation towards its proper location. 4.5.2 Egg fragments and fate maps Fate maps are intended to show where, during some earlier stage of development, the material is located which later on gives rise t o certain structures of the body. The partial patterns produced by egg fragments as described above (Figs 1 7 and 25(a)) invite attempts to construct such maps.

INSECT EMBRYOGENESIS

177

Thus, a fate map for the pre-cleavage stage based on posterior fragments of Bruchidius eggs would locate the thorax segments between roughly 70 and 90 per cent EL. However, a glimpse on the results from anterior fragments immediately indicates a fallacy: with these fragments, the material for the thorax should be located between roughly 10 and 30 per cent EL! This shows clearly that the method employed, when applied to only one type of fragment (anterior or posterior) will lead to false conclusions with an egg type reacting like Bruchidius. What these fragments show is not the prospective fate of an egg region in normal development, but at best its fate under the conditions of the experiment. It follows that no fate map or anlagen plan as defined above can be constructed by studying patterns formed in egg fragments, unless the results from both anterior and posterior fragments agree-which in Bruchidius is not the case until shortly before the germ anlage stage. We shall return to these considerations in section 4.6.

4.5.3 The capacity of egg fragments f o r independent differentiation Bruchidius egg fragments below a certain length are unable to produce any recognizable pattern elements. Whatever its reason, this failure to form germ band parts may be described as lack of capacity for independent differentiation (see section 4.1.6.c); the cells in these egg regions can differentiate quite well when these are connected with neighbouring regions, but not without this connection. The critical fragment length for self-differentiation is stage dependent (Fig. 26(a)). It should be noted that failure to differentiate recognizable germ band parts may have many causes, with lack of proper instruction being only one possibility. On the other hand, the presence in an egg fragment of instructions concerning pattern elements to be formed will not be revealed when other causes do not permit development up t o the segmented germ band stage. These considerations have a bearing in the interpretation of results which Haget (1953) obtained by cutting eggs of the Colorado beetle Leptinotarsa decemlineata Say. Figure 26(b) represents an attempt to produce from his data a coherent picture concerning the capability for self-differentiation in Leptinotarsa. It shows that generally speaking the fragment length required for independent differentiation of germ band parts decreases with increasing egg age at cutting. It should be noted that the right part of this figure resembles Fig. 26(a). In both cases, at first only fragments comprising 1/2 of the egg or slightly less can form germ band parts, but later on increasingly shorter fragments become capable of doing so. Haget (1953) explained the result presented in the left part of Fig. 26(b) as due to disturbances concerning a basophilic cortical gradient or quantitative relations between nuclei and cytoplasm, while the results from 1 8 h onward were ascribed to effects spreading from a centre diffkenciateur (see sections 4.5.5 and 5.3).

178

KLAUS SANDER I00

.G

h

I00

50

a9 0 0

20

10

27

h

Fig. 26. Stage-dependent limitations of the capacity for self-differentiation in beetle eggs. (a) Bruchidius obtectus (after Jung, 1966, Fig. 20). Fragments as shown are incapable of producing typical germ band parts while slightly larger fragments are capable of doing so. Added are the upper three curves from Fig. 25(b). (b) Leptinotarsa decemlineata, “limits of tolerance” for germ band formation in transversely cut eggs (data of Haget, 1963, Figs 1 4 and 21). Hatched egg parts are incapable of reaching the germ band stage when isolated at the stage indicated. Slightly longer fragments could reach germ band stage, but, unless longer than indicated by stippling, would fail to acquire the larval level of differentiation. The blank region between hatched parts on the right was termed “indispensable” by Haget (1953); actually it is dispensable excepting its very margins. MW 1-5: Timing of mitotic waves according t o Bergerard and Maisonhaute (1967). I, 11,111, stages as in Fig. 25.

4.5.4 Regional uu irradiation of Bruchidius eggs Jung (1971) irradiated restricted transverse areas (longitudinal extent 67 or 125 pm) of Bruchidius eggs at different stages with uv light of 270-330 nm. Some of his results are summarized in Fig. 25(b). Doses which with later stages produce germ band defects do not inflict permanent defects in the majority of eggs if applied before the nuclei reach the surface of the egg cell. Shortly afterwards, a change of reaction occurs. Irradiations within, or extending into, an equatorial belt then usually lead to defects in the germ band, while more distal irradiations (in regions indicated by hatching in Fig. 25(b)) are still without lasting effects. This lead of the more central regions in losing the capacity to compensate uv damage is reminiscent of the cricket data (section 4.1.4). But in Bruchidius the central region can be

lNSECT EMBRYOGENESIS

179

permanently damaged some.time before formation of radial cell boundaries (see marks in Fig. 25(a)) while in the cricket both events may be linked (H. W. Sauer, 1966). Mapping of the uv defects (curves in Fig. 25(b)) reveals a rather time-invariant spatial pattern in Bruchidius (as in the cricket with respect t o the germ anlage blastema; Table 3). At first sight, the data obtained by regional uv irradiation in Bruchidius appear t o be incompatible with those derived from egg fragments (cf. Fig. 25(a) and (b)). However, both sets of data coincide fairly well with respect to the latest stage studied (right margin of diagrams). Shortly before the germ anlage stage, the uv defects map in rather close agreement with the fragmentation fate map (section 4.5.2). This fact and the histological data provided by Jung (1 97 1) suggest the following interpretation. As in the cricket, the blastoderm nuclei and/or cells before germ anlage formation are not yet committed, and probably not even instructed, t o follow specific pathways of development within the germ band pattern; uv irradiation under these circumstances may impair their regional instruction during subsequent stages. This view is compatible with cytological observations of Jung (1971).According to these, early irradiations (which do not cause pattern defects) delay development somewhat but fail t o cause damage recognizable with histological methods. This is different with later irradiations leading to pattern defects. With these, the nuclei decay. The irradiated region eventually becomes repopulated by nuclei from surrounding regions, but the cytoplasm between these nuclei and the yolk plasmodium remains abnormal up to the latest stages studied histologically. Thus the abnormal cytoplasm in the irradiated region could cause failure of the cells formed there to react to instructions provided later on by the yolk plasmodium in this region. W. Schnetter (1965)observed that in Leptinotarsa failure of cell walls to form in previously cauterized egg regions is linked to development of defective embryos.

On this basis, the data from uv irradiation and constriction experiments are compatible. As in the cricket (section 4.1.6.b), ligature in Bruchidius would interfere with the generation andfor proper distribution of instruc; tions (presumedly in egg plasmodium and yolk plasmodium) while uv damage could locally prevent the blastoderm cells from carrying out these instructions. The minor mapping differences between uv results from different stages-most segments suffer an apparent shift amounting t o less than 5 per cent (see Table 3 in Jung, 1971)-might be caused by some sort of cortical deplacement or contraction during these stages, which could carry along areas damaged earlier. Jung (1971) also irradiated complete belts of 67-125 pm width situated at right angles t o the longitudinal egg axis. The results were compatible with those from unilateral irradiation as described above; Fig. 25(b) is indeed based on both types of experiment. However, these belt irradiations

180

KLAUS SANDER

yield some very valuable information when .compared to ligaturing experiments. The “gap” caused in the segment pattern by belt irradiation does not differ for different stages where defects can be induced by uv (Jung, 1971) while the gap caused by fragmentation decreases with advancing fragmentation age (Table 8 ) . This could mean that the terminal influences on pattern specification spread despite regional inactivation or destruction of the blastoderm-which would single out the yolk plasmodium as their substrate (see section 4.1.2). Yet further experiments with narrower uv belts will be required t o affirm this interpretation.

4.5.5 Other long germ-type beetles-data and interpretations Among the data collected over several decades from other beetle species which also represent the long germ-type of early development, some are consistent with the interpretations offered for Bruchidius in the preceding sections while others, at least as interpreted by their authors, are not. The cutting experiments performed by Haget (1953) on eggs of Leptinotarsa produced an impressive amount of information, but the data are presented in a rather biased way which makes independent evaluation difficult. As pointed out above (section 4.5.3), Haget maintains that in Leptinotarsa a differentiation centre located in the prospective prothorax serves to instruct (via intrablastemic reactions) adjacent egg regions about their specific pathways of development (see also section 5.3). This view is conflicting not only with the Bruchidius data, but also with evidence from other species and from other types of experiment performed in Leptinotarsa . W. Schnetter (1965)cauterized different regions of Leptinotarsa eggs before and during cleavage and found that damage to terminal egg regions always led to defective patterns whereas damage to equatorial egg regions (including the prospective prothorax) did so in less than 50 per cent of cases. Schnetter therefore favoured for Leptiaotarsa, too, a mode of pattern specification involving terminal influences. In another bruchid beetle, Callosobruchus maculatus Fabr. (= Bruchus quadrimaculatus), such influences have recently been established with the pinching technique by van der Meer and Miyamoto (personal communication). For this species, Brauer and Taylor (1936) had already stated: “. . . establishment of prospective values appears to be due to a wave of organization emanating from the posterior extremity of the egg, immediately after the (p. egg is laid, and spreading gradually forward toward the anterior extremity.. 147.) Apparently the data of these authors were insufficient to recognize the additional anterior influence on pattern specification, but in hindsight some cases clearly indicate this, because very large posterior parts from eggs cauterized early in development failed to form anything but abdominal segments. Such results were also obtained by Jura (1957)in Melasoma populi L. All these observations are compatible with the Bruchidius data and with the concept of pattern specification from opposite terminal egg regions. With respect to Haget’s interpretation (see above) it should be pointed out that

. .”

INSECT EMBRYOGENESIS

181

Bmchidius and Callosobruchus aiso display phenomena apparently spreading from the middle towards more terminal regions of pattern or egg, and that these concern the capability for self-differentiation in Bruchidius (Fig. 26(a)) and the transition from “labile” t o “definite” determination of bilaterality (i.e. commitment, see section 1) in Cdlosobruchus (Brauer, 1938, p. 261) (see section 5.1). This justifies the proposition that Haget’s differentiation centre effect, as established by transverse cutting, might be related somehow t o acquisition of the capacity for self-differentiation and/or t o cellular commitment, but not to pattern specification. This proposition will be backed by demonstrating that Haget’s main evidence for an “instructing” differentiation centre is not compelling (section 5.3).

4.5.6 Intermediate and short germ-type beetles The clerid beetle Necrobiu rufipes Deg. may be considered to represent the intermediate germ-type of early development. The results obtained by Trendelenburg (1971) in this species with the pinching technique resemble in some respects the Euscelk/Bruchidius results, while in other respects they are comparable to results obtained in short germ-type beetles (see below). The “gap phenomenon” which indicates pattern specification by opposite terminal influences is quite evident in Necrobiu (Table 8 ) , and TABLE 9 Frequencies of different classes of patterns produced by anterior egg fragments (45-50 per cent EL) of the beetle Necrobia rufipes. (Data of Trendelenburg, 1971, Figs 4-8) Anterior partial germ band terminating in Procephalon Gnathocephalon Thorax Abdomen

Stage at fragmentation* ~

~~

I

I1

I11

IV

V

18 4 0 0

12 0 0 0

9 3 0 0

2 8 2

0 3

0

7

2

* I, early cleavage; V, germ anlage. fragments of a given size may form increasing numbers of pattern elements with advance in age at separation (Table 9). The latter effect, however, may in part be due t o movements of the late blastoderm which in this species are exceptionally strong. Two types of result link Necrobiu to short germ-type development. Firstly, the egg region anterior to 65 per cent EL is dispensable for pattern specification throughout early development. Secondly, a sizeable posterior egg region, (up to 30 per cent EL) including part of the germ anlage, can be removed at the germ anlage stage without impairment of the final germ band pattern (see Tenebrio below).

182

KLAUS SANDER

In the short germ-type beetles studied, Atruchyu menetriesi Falderm. (chrysomelid) and Tenebrio molitor L. (tenebrionid), the complete germ band pattern can be formed in the absence of large anterior stretches of the original egg cell. In Atruchyu, the germ band pattern is also independent of the posterior 30 per cent of the egg. Ligaturing experiments performed by Miya and Kobayashi (1974) during cleavage have shown that in this species the complete germ band pattern may be produced by the anterior or the posterior egg fragment provided it includes the region between 30 per cent EL and 47 per cent EL. If the ligature is applied within this region during cleavage, no germ anlage will form at all (Table 10). The state of the system apparently changes with arrival of cleavage nuclei in the cortex of this region. Thereafter, eggs ligatured between 33 per cent EL and 37 per cent EL are capable of producing 2 complete germ bands, one in each fragment (Table 10). The interpretation offered for these results by Miya and TABLE 10 Results from ligatured eggs of the beetle Atrachya nenetriesi. (Data of Miya and Kobayashi, 1975) Stage at ligaturing

Fragment

Cleavage

Anterior Posterior

Blastoderm

Anterior Posterior

Fragmentation level

< 30% EL

3047% EL

-

-

< 33% EL

33-37% EL

+

+

-

i

+

> 47% EL -

i

> 37%

+

EL

+, Complete germ band; -, extraembryonic development.

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

INSECT EMBRYOGENESIS

183

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

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

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

184

KLAUS SANDER

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

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

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

4.6

HYMENOPTERA AND LEPIDOPTERA: POTENCY REGIONS AND EARLY ORGAN DETERMINATION CHALLENGED

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

INSECT EMBRYOGENESIS

185

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

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

KLAUS SANDER

186

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

(01)

(a2)

(bl)

(b,)

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

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

INSECT EMBRYOGENESIS

187

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

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

KLAUS SANDER

188

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

11 6

Gnathocephalon Thorax 19

9

2

7

Abdomen

3 6

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

INSECT EMBRYOGENESIS

189

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

4.7

LOWER DIPTERANS-MIRRORING HEADS AND ABDOMENS

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

KLAUS SANDER

190

a& r

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

~

& I

@-Q

..... .....

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

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

INSECT EMBRYOGENESIS

191

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

92

KLAUS SANDER

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

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

!.8 HIGHER DIPTERANS: NO SPECIAL STATUS

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

INSECT EMBRYOGENESIS

193

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

i ~

i

’

194

KLAUSSANDER

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

INSECT EMBRYOGENESIS

195

70

70 .-•

-J

w

s 20

20 0

I

3

2

0

I

h

2

3

h

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

I00

4

8

50

5

6 35 O/O

q;; 7 53 ‘10 EL

10

0

OP

opm

yy

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

KLAUS SANDER

196

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

Number of segments (+1?) Stage at fragmentation

Lleavage Plasmodia1blastoderm

9

10

11

12

13

14

15

16

0 6

2 9

1 13

2 17

0 5

0

1

0

C.ellular blastoderm (;astrulation Relative frequencies

w

A I

3 7%

48 %

Relative frequencies

0

3

4

25

4 3

15%

0

0

0

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

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

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

INSECT EMBRYOGENESIS

197

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

2

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

I

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

198

KLAUS SANDER

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

INSECT EMBRYOGENESIS

199

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

E M

L

-/+

-

A + -

~

~ 12

-/+

-/+

-la

-

I

2

-I+

-

~

I

~

I1

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

4

21

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

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

200

KLAUS SANDER

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

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

k.9

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

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

INSECT EMBRYOGENESIS

201

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

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

202

KLAUS SANDER

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

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

INSECT EMBRYOGENESIS

203

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

LOCATION

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

t t lnstruc t ng signal (s)

Cells or cell groups

Cellulor commitment i

Anterior Poster tor Instructing influences (prerequisi tesl

J

Plosmodial system

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

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

204

KLAUS SANDER

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

INSECT EMB RYOG EN ESlS

205

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

206

KLAUS SANDER

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

INSE CT EMB R Y OG ENES IS

207

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

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

21 1

Not visible

22 32

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

KLAUS SANDER

208

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

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

5.1

PATTERNS FORMED BY LONGITUDINAL HALVES OF THE BLASTODERM OR GERM ANLAGE

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

INSECT EMBRYOGENESIS

209

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

210

KLAUS SANDER

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

I 24

m

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

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

INSECT EM6 RYOG EN ESlS

21 1

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

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

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

212

KLAUS SANDER

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

5.2

DIFFERENCES BETWEEN TRANSVERSE AND LONGITUDINAL PATTERN SPECIFICATION

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

5.3

THE DIFFERENTIATION CENTRE REVISITED

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

INSECT E MB RYOG ENESI S

213

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

G 2 G 3 T

I

T I I

T

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

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

214

KLAUS SANDER

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

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

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

INSECT EMBRYOGENESIS

21 5

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

KLAUS SANDER

21 6

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

MUTANTS TRANSFORMING PATTERN ELEMENTS

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

INSECT EMBRYOGENESIS

21 7

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

218

KLAUS SANDER

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

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

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

I NSECT EMI3 R Y OG ENESI S

219

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

CLONAL ANALYSIS AND DEVELOPMENTAL COMPARTMENTALIZATION

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

KLALJSSANDER

220

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

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

7.1

CYTOARCHITECTUREOF THE OOCYTE

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

INSECT EMBRYOGENESIS

22 1

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

MITOTIC WAVES DURING BLASTODERM FORMATION

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

222

KLAUS SANDER

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

7.3

FUNCTIONAL DIFFERENTIATION OF NUCLEI

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

INSECT EMBRYOGENESIS

223

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

7.5 PROSPECTS

FOR A MOLECULAR APPROACH TO PATTERN SPECIFICATION IN THE

INSECT EMBRYO

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

224

KLAUS SANDER

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

I NSECT EMB R Y OG EN ESI S

225

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

KLAUS SANDER

226

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

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

ed t ly

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

8 Concluding remarks

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

INSECT EMBRYOGENESIS

227

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

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

228

KLAUS SANDER

grants from the Deutsche Forschungsgemeinschaft, SFB 46. This review was submitted for publication in September, 1975. References Achtelig, M. and Krause, G. (1971). Experimente am ungefurchten Ei von Pimpla turionellae L. (Hymenoptera) zur Funktionsanalyse des Oosombereichs. Wilhelm Roux Arch. EntwMech. Org. 167, 164-182. Agrell, I. (1963). Mitotic gradients in the early insect embryo.Ark. 2001. 15, 143-148. Alleaume, N. (1971). Contribution A l’analyse Cxperimentale des facteurs de la dtterrnination et de la diffirentiation des ibauches dans le germ des DiptPres supkrieurs (Calliphora erythrocephala. Meig. et Drosophila melanogaster Meig.) ThPse, Universiti de Bordeaux I. Anderson, D. T. (1961). A differentiation centre in the embryo of Dacus tryoni. Nature, Lond. 190, 560-561. Anderson, D. T. (1972a). The development of hemimetabolous insects. In “Developmental Systems: Insects” (Eds s. J. Counce and C. H. Waddington), vol. I, pp. 95-1 63. Academic Press, London and New York. Anderson, D. T. (1972b). The development of holometabolous insects. In “Developmental Systems: Insects” (Eds S. J. Counce and C. H. Waddington). vol. I, pp. 165-242. Academic Press, London and New York. Bakken, A. H. (1973). A cytological and genetic study of oogenesis in Drosophila melanogaster. Devl Biol. 3 3 , 100-122. Bergerard, J. and Maisonhaute, C. (1967). ActivitC mitotique des premiers stades ernbryonnaires du Doryphore Leptinotarsa decemlineata Say (ColCoptPre Chrysomalidae.) C.r. hebd. S h n c . Acad. Sci. Paris 2 6 4 0 , 2134-2137. Bergmann-Schanz, G. (1970). Embryonalentwicklung der Libelle Ischnura elegans. Begleitveroffentlichung Filme D 928 & D 929. Inst. Wiss. Film, Gottingen. Bier, K. H. (1970). Oogenesetypen bei Insekten und Vertebraten, ihre Bedeutung fur die Embryogenese und Phylogenese. Verh. d t . rool. Ges. 6 3 , 7-29. (Zool. Anr. Suppl. 33). Bock, E. (1941). Wechselbeziehungen zwischen den Keimblattern bei der Organbildung von Chrysopa perla (L.) 1: Die Entwicklung des Ektoderms in mesodermdefekten Keimteilen. Wilhelm Roux Arch. EntwMech. Org. 141, 159-247. Bownes, M. ( 1973). Determination in Drosophila Embryos. Thesis, University of Sussex. Bownes, M. and Kalthoff, K. (1974). Embryonic defects in Drosophila eggs after partial uv irradiation at different wavelengths. J. Embryol. exp. Morph. 31, 329-345. Bownes, M. and Sang, j. H. (1974a). Experimental manipulations of early Drosophila embryos. I. Adult and embryonic defects resulting from microcautery at nuclear multiplication and blastoderm stages. J. Embryol. exp. Morph. 32,253-272. Bownes, M. and Sang, J. H. (1974b). Experimental manipulations of early Drosophila embryos. 11. Adult and embryonic defects resulting from the removal of blastoderm cells by pricking. J. Embryol. exp. Morph. 32, 273-285. Brahmachary, R. L. (1973). Molecular embryology of invertebrates. Adu. Morphogenesis, 10, 115-173.

INSECT EMBRYOGENESIS

229

Brauer, A. (1938). Modifications of development in relation t o differential susceptibility of the bruchid (Coleoptera) egg t o KCN during different metabolic phases. Physiol. Zool. 1 1 , 249-266. Brauer, A. and Taylor, A. C. (1936). Experiments t o determine the time and method of organization in bruchid (Coleoptera) eggs. J. exp. Zool. 73, 127-151. Bruhns, E. (1974). Analyse der Ooplasmastromungen und ihrer strukturellen Grundlagen wahrend der Furchung bei Pimpla turionellae L. (Hymenoptera). I. Lichtmikroskopisch-anatomische Veranderungen in der Eiarchite ktur koinzident mit Zeitrafferfilmbefunden. Wilhelm Roux Arch. EntwMech. Org. 174, 55-89. Bryant, P. J. (1974). Determination and pattern formation in the imaginal discs of Drosophila. Curr. Top. Devl. Biol. 8 , 41-80. Bryant, P. J. and Zornetzer, M. (1973). Mosaic analysis of lethal mutation in Drosophila. Genetics, 75, 623-627. Bull, A. L. (1966). Bicaudal, a genetic factor which affects the polarity of the embryo in Drosophila melanogaster. J. exp. Zool. 161, 221-241. Capdevila, M. P. and Garcia-Bellido, A. (1974). Development and genetic analysis of bithorax phenocopies in Drosophila. Nature, Lond. 250, 500-502. Chan, L-N. and Gehring, W. (1971). Determination of blastoderm cells in Drosophila melanogaster. Proc. natl. Acad. Sci. U.S.A. 68, 2217-2221. Counce, S. J. (1972). The causal analysis of insect embryogenesis. In “Developmental Systems: Insects” (Eds S. J. Counce and C. H. Waddington), vol. 11, pp. 1-156. Academic Press, London and New York. Counce, S. J. and Waddington, C. H. (Eds) (1972). “Developmental Systems: Insects”. 2 vols. Academic Press, London and New York. Davis, C. W. C. (1967). A comparative study of larval embryogenesis in the mosquito Culex fatigans Wiedemann (Diptera: Culicidae) and the sheep-fly Lucilia sericata Meigen (Diptera: Calliphoridae). I. Description of embryonic development. Aust. J. Zool. 15, 547-579. Duspiva, F. (1969). Molekularbiologische Aspekte der Entwicklungsphysiologie. Naturwks. Rundsch. 22, 191-202. Ede, D. A. (1964). An inherited abnormality affecting the development of the yolk plasmodium and endoderm in Dermestes maculatus (Coleoptera). J . Embryol. exp. Morph. 12, 551-562. Engels, W. (1973). Das zeitliche und raumliche Muster der Dottereinlagerung in die Oocyte von Apis mellifica. Z. Zellforsch. Mikrosk. Anat. 142, 409-430. Ernst, G., Sauer, H. W. and Wegener, G. (1975). Unterschiedliche Proteinmuster in embryonalen und extraembryonalen Eihalften des Keimstreifstadiums der Grille. Verh. d t . 2001. Ges. 67, 192-196. Ewest, A. (1937). Struktur und erste Differenzierung im Ei des Mehlkafers Tenebrio molitor. Wilhelm Roux Arch. EntwMech. Org. 135, 689-752. Fausto-Sterling, A. (1971). On the timing and place of action during embryogenesis of the female-sterile mutants fused and rudimentary of Drosophih melanogaster. Devl Biol. 26,452-463. Fullilove, S . L. and Jacobson, A. G. (1971). Nuclear elongation and cytokinesis in Drosophila montana. Devl Biol. 26,560-577.

230

KLAUS SANDER

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

INSECT E M B RYOGEN ESlS

231

Howland, R. B. and Sonnenblick, B. P. (1936). Experimental studies on development in Drosophila melanogaster. 11. Regulation in the early egg. J. exp. 2001. 73, 109-125. Idris, B. E. M. (1960). Die Entwicklung im geschniirten Ei von Culex pipiens L. (Diptera). Wilhelm Roux Arch. EntwMech. Org. 152, 230-262. Illmensee, K. (1972). Developmental potencies of nuclei from cleavage, preblastoderm, and syncytial blastoderm transplanted into unfertilized eggs of Drosophila melanogaster. Wilhelm Roux Arch. EntwMech. Org. 170, 267-298. Illmensee, K. (1973). The potentialities of transplanted early gastrula nuclei of Drosophila melanogaster. Production of their imago descendants by germ-line transplantation. Wilhelm Roux Arch. EntwMech. Org. 171, 331-343. Illmensee, K. and Mahowald, A. (1974). Transplantation of posterior polar plasm, in Drosophila. Induction of germ cells at the anterior pole of the egg. Proc. natl. Acad. Sci. U.S.A. 71, 1016-1020. Ivanova-Kasas, 0. M. (1972). Polyembryony in insects. In “Developmental Systems: Insects” (Eds S. J. Counce and C. H. Waddington), vol. I, pp. 243-271. Academic Press, London and New York. Jung, E. (1966a). Untersuchungen am Ei des Speisebohnenbfers Bruchidius obtectus SAY. (Coleoptera). I. Entwicklungsgeschichtliche Ergebnisse zur Kennziechnung des Eitypus. Z. Morph. Okol. Tiere, 56, 444-480. Jung, E. (1966b). Untersuchungen am Ei des Speisebohnenbfers Bmchidius obtectus SAY (Coleoptera). 11. Entwicklungsphysiologische Ergebnisse der Schniirungsexperimente. Wilhelm Roux Arch. EntwMech. Org. 157, 320-392. Jung, E. (1971). Die Entwicklungsf&igkeit des Eies von Bruchidius obtectus SAY nach partieller UV-Licht-Bestrahlung (Coleoptera). Wilhelm Roux Arch. EntwMech. Org. 167, 299-324. Jung, E. and Krause, G. (1967). Experimente mit Verlagerung polnahen Eimaterials zur Analyse der Bedingungen fiir die metamere Gliederung des Embryos von Bruchidius (Coleoptera). Wilhelm Roux Arch. EntwMech. Org. 159, 89-126. Jura, C. (1957). Experimental studies on the embryonic development of the Melasoma populi L. (Chrysomelidae, Coleoptera). Zoologica Pol. 8, 177-199. Kalthoff, K. (1970). Reversionsmoglichkeiten der Entwicklung zur Missbildung “Doppelabdomen” im UV-bestrahlten Ei von Smittk spec. (Diptera, Chironomidae). Verh. dt. 2001. Ges. 64, 61-65. Kalthoff, K. (1971). Temperature effects on embryogenesis in Smittia spec. (Diptera, Chironomidae): Q1,-,-values of normal development and frequency of “double abdomens” after UV irradiation. Wilhelm Roux Arch. EntwMech. Org. 168, 85-96. Kalthoff, K. (1971). Position of targets and period of competence for UV-induction of the malformation “double abdomen” in the egg of Smittiu spec. (Diptera, Chironomidae). Wilhelm Roux Arch. EntwMech. Org. 168, 63-84. Kalthoff, K. (1973). Action spectra for UV induction and photoreversal of a switch in the developmental program of an insect (Smittia). Photochem. Photobiol. 18, 355-364. Kalthoff, K. and Sander, K. (1968). Der Entwicklungsgang der Missbildung “Doppelabdomen” im partiell UV-bestrahlten Ei von Smittk parthenogenetica (Dipt. Chironomidae). Wilhelm Roux Arch. EntwMech. Org. 101, 129-146.

232

KLAUS SANDER

Kanellis, A. (1952). Anlagenplan und Regul@ionserscheinungen in der Keimanlage des Eies von Gryllus domesticus. Wilhelm Roux Arch. EntwMech. Org. 145,417-461. Kaufman, S . A. (1973). Control circuits for determination and transdetermination. Science, N . Y . 181, 310-318. Kiger, J. A. (1973). The bithorax complex-a model for cell determination in Drosophila. J. theor. Biol. 40,455-467. Krause, G. (1934). Analyse erster Differenzierungsprozesse im Keim der Gewachshausheuschrecke durch kiinstlich erzeugte Zwillings- und Doppel-Mehrfachbildungen. Wilhelm Roux Arch. EntwMech. Org. 132, 115-205. Krause, G. (1939). Die Eitypen der Insekten. Biol. Zbl. 59, 495-536. Krause, G. (1952). Schnittoperation im Insekten-Ei zum Nachweis komplementfer Induktion bei Zwillingsbildung. Naturwissenschaften. 39, 356. Krause, G. (1953). Die Aktionsfolge zur Gestaltung des Keimstreifs von Tachycines (Saltatoria), insbesondere das morphogenetische Konstruktionsbild bei Duplicitas parallela. Wilhelm Roux Arch. EntwMech. Org. 146, 275-370. Krause, G. (1958a). Neue Beitrage zur Entwicklungsphysiologie des Insektenkeimes. Verh. dt. 2001. Ges. 51, 396-424. (Zool. Any. Suppl. 21). Krause, G. (195813). Induktionssysteme in der Embryonalentwicklung von Insekten. Ergebn. Biol. 20, 159-198. Krause, G. (1961). Preformed ooplasmic reaction system in insect eggs. In “Symposium on the Germ Cells and Earliest Stages of Development”, Pallanza, pp. 302-337. Institut International d’Embryologie. Krause, G. (1962). Die Entwicklungsphysiologie kreuzweise verdoppelter Embryonen. Embryofogia, 6, 355-386. Krause, G. and Krause, J. (1957). Die Regulation der Embryonalanlage von Tachycines (Saltatoria) im Schnittversuch. 2001.Jb. (Anat.) 75, 481-550. Krause, G. and Krause, J. (1965). Uber das Vermogen median durchschnittener Keimanlagen von Bornbyx mori L. sich in ovo und sich ohne Dottersystem in vitro zwillingsartig zu entwickeln. Z. Naturf. 2Ob, 334-339. Krause, G. and Sander, K. (1962). Ooplasmic reaction systems in insect embryogenesis. Adv. Morphogenesis, 2,259-303. Kiithe, H. W. (1966). Das Differenzierungszentrum als selbstregulierendes Faktorensystem fur den Aufbau der Keimanlage im Ei von Dermestes frischi (Coleoptera). Wilhelm Roux Arch. EntwMech. Org. 157, 212-302. Kiithe, H. W. (1972). Veranderungen im Proteinmuster wahrend Oogenese und Friihentwicklung von Dermestesfrischi (Coleoptera).J. Insect Physiol. 18,241 1-2423. Kiithe, H. W. (1973). Synthese- und Einbaumuster von Eiproteinen wahrend der Rlastodermentwicklung in der friihen Embryogenese von Dnmestes frische’ (Coleoptera). Wilhelm Roux Arch. EntwMech. Org. 171, 301-311. Lawrence, P. A. (1973). A clonal analysis of segment development in Oncopeltus (Hemiptera). J. Embryol. exp. Morph. 30, 681-699. Lewis, E. B. (1963). Genes and developmental pathways. A m . 2001. 3, 33-56. Louvet, J. P. (1964). Repartition des ribonuclkoproteines et r81e directeur de l’ectoderme dans la segmentation de la bandelette embryonnaire, chez le Phasme carausius morosus Rr. C.r. hebd. Sianc. Acad. Sci., Paris, 259D, 4819-4821.

INSECT EMBRYOGENESIS

233

Liischer, M. (1944).Experimentelle Untersuchungen iiber die larvale und die imaginale Determination im Ei der Kleidermotte (Tineola biselliella Hum.) Revue suisse 2001.

51,531-627. Mahowald, A. P. (1963). Ultrastructural differentiations during formation of the blastoderm in the Drosophila melanogaster embryo. Deul Biol. 8 , 186-204. Mahowald, A. P. (1972). Oogenesis. In “Developmental Systems: Insects.” (Eds S. J. Counce and C. H. Waddington), vol. I, pp. 1-47.Academic Press, London and New York. Mahr, E. (1960).Struktur und Entwicklungsfunktion des Dotter-Entoplasmasystems im Ei des Heimchens (Gryllus domesticus). Wilhelm Roux Arch. EntwMech. Org. 152,

263-302. Maisonhaute, C. (1970). Observations cytologiques et autoradiographiques sur les premiers stades embryonnaires du doryphore (Leptinotarsa decemlineata Say.). C.r. hebd. Sianc. Acad. Sci., Paris, 272D, 1398-1401. Maschlanka, H. (1938).Physiologische Untersuchungen am Ei der Mehlmotte Ephestia Kuehniella Wilhelm Roux Arch. EntwMech. Org. 137, 714-772. Maul, V. (1970).h e r Schniirungsexperimente an Eiern zweier verschiedener Inzuchtstamme der Honigbiene. Verh. dt. 2001. Ges. 63,53-58.(Zool. A n r . Suppl. 33.) Meinhardt, H. and Gierer. A. (1974). Applications of a theory of biological pattern formation based on lateral inhibition. J. Cell Sci. 15,321-346. Meng, C. (1968). Strukturwandel und histochemische Befunde insbesondere am Oosom wahrend der Oogenese und nach der Ablage des Eies von Pimpla turionellae L. (Hymenoptera, Icheumonidae). Wilhelm Roux Arch. EntwMech. Org. 161, 162-208. Wya, K. (1956). Influence of centrifugal forces upon the embryonic development of the silkworm Bombyx mori L. I. The germ-band in the eggs centrifuged at the early stages. J . Fac. Agric. Iwate Univ. 2, 393-400. Mya, K. (1965). The embryonic development of a chrysomelid beetle, Atrachya menetriesi Faldermann (Coleoptera, Chrysomelidae). I. The stages of development and changes of external form. J. Fac. Agric. Zwate Uniu., Morioka, 7, 155-166. Wya, K. and Kobayashi, Y. (1974). The embryonic development of Atrachya menetriesi Faldermann (Coleoptera, Chrysomelidae). 11. Analyses of early development by ligation and low temperature treatment. j . Fuc. Agric. Zwate Uniu. 12,

39-55. Moloo, S. K. (1971).The degree of determination of the early embryo of Schistocerca gregaria (ForskL1) (Orthoptera: Acrididae). J. Embryol. exp. Morph. 25, 277-299. Moser, J. G., Bode, H. J., Niinemann, H., Collatz, S., Feldhege, A. and Herzfeld, A. (1970). Differenzierung des Aktomyosin-systems w a r e n d der Morphogenese der Hausgrille,Acheta domesticus L. Verh. d t . zool. Ges. 64,56-60. Nitschmann, J. (1959). Segmentverluste beim geschniirten Calliphorakeim. Verh. dt. m o l . Ges. 52,370-377.(2001.Anr. Suppl. 22.) Nothiger, R. and Strub, S. (1972).Imaginal defects after UV-microbeam irradiation of early cleavage stages of Drosophila melanogaster. Revue suisse Zool. 79 (Suppl.),

267-279. Oelhafen, F. (1961). Zur Embryogenese von Culex pipiens: Markierungen und Extirpationen mit UV-Strahlenstich. Wilhelm Roux Arch. EntwMech. Org. 153,120-157.

234

KLAUS SANDER

Okada, M., Kleinman, I. A. and Schneiderman, H. A. (1974q). Restoration of fertility in sterilized Drosophila eggs by transplantation of polar cytoplasm. Devl Biol. 37, 43-54. Okada, M., Kleinman, I. A. and Schneiderman, H. A. (1974b). Repair of ageneticallycaused defect in oogenesis in Drosophila melanogaster by transplantation of cytoplasm from wild-type eggs and b y injection of pyrimidine nucleosides. Dezd Biol. 37, 55-62. . Drosophila Okada, M., Kleinman, I. A. and Schneiderman, H. A. ( 1 9 7 4 ~ ) Chimeric adults produced by transplantation of nuclei into specific regions of fertilized eggs. Dcvl Biol. 39, 286-294. Overton, J. and Raab, M. (1967). The development and fine structure of centrifuged eggs of Chironomus thummi. DevlBiol. 15, 271-287. I’ietruschka, F . and Bier, K. (1972). Autoradiographische Untersuchungen zur RNS-und Protein-Synthese in der fruhen Embryogenese von Musca do mestica. Wilhelm Roux Arch. EntwMech. Org. 169, 56-69. I’ostlethwait, J. H. and Schneiderman, H. A. (1973a). Pattern formation in imaginal discs of Drosophila melanogaster after irradiation of embryos and young larvae. Devl Biol. 32,345-360. I’ostlethwait, J. H. and Schneiderman, H. A. (1973b). Developmental genetics of Drosophila imaginal discs. A . Rev. Genetics, 7, 381-433. I’rice. R. D. (1958). Observations on a unique monster embryo of Wyeomyia smithii (Coquillett) (Diptera: Culicidae). Ann. ent. Sac. A m . 51, 600-604. Reith, F. (1931). Versuche iiber die Determination der Keimesanlage bei Camponotus ligniperda. Z. wiss. Zool. 139, 664-734. Rice. T. B. and Garen, A. (1975). Localized defects in maternal effect mutants of Drosophila. Devl Biol. 43, 277-286. %poll, P. (1972). The embryonic organization of the imaginal wing disc of Drosophila melanagaster. Wilhelm Roux Arch. EntwMech. Org. 169, 200-215. Sander, K. (1959). Analyse des ooplasmatischen Reaktionssystems von Euscelis plebejus Fall. (Cicadina) durch Isolieren und Kombinieren von Keimteilen. I. Die Differenzierungsleistungen vorderer und hinterer Eiteile. Wilhelm Roux Arch. EntwMech. Org. 151, 430-497. Sander, K. (1960). Analyse des ooplasmatischen Reaktionssystems von Euscelis plebejus Fall. (Cicadina) durch Isolieren und Kombinieren von Keimteilen. 11. Die Differenzierungsleistungen nach Verlagern von Hinterpolmaterial. Wilhelm Roux Arch. EntwMech. org. 151, 660-707. Sander, K. (1961a). Umkehr der Keimstreifpolaritat in Eifragmenten von Euscelis (Cicadina). Experientia, 17, 179-180. Sander, K. (1961b). New experiments concerning the ooplasmic reaction system of Euscelis plebejus (Cicadina). In “Symposium on the Germ Cells and Earliest Stages of Development” Pallanza, pp. 338-353. Institut International d‘Embryologie. Sander, K. (1962). Uber den Einfluss von verlagertem Hinterpolmaterial auf das metamere Organisationsmuster im Zikaden-Ei. Verh. d t . zool. Ges. 55, 315-322. (Zool. Anz. Suppl. 25.) Sander, K. (1963). Untersuchungen iiber die Wirkung der Zentrifugalkraft auf die Eiarchitektur und das ooplasmatische Reaktionssystem von Euscelis plebejus F. (Cicadina). Habilitationsschrift, Universitat Wiirzburg.

INSECT EMBRYOGENESIS

235

Sander, K. (1968). Entwi+lungsphysiologische Untersuchungen am embryonalen Mycetom von Euscelis plebejus F. (Homoptera, Cicadina). Devl Biol. 17, 1638. Sander, K. (1971). Pattern formation in longitudinal halves of leaf hopper eggs (Homoptera) and some remarks on the definition of “embryonic regulation”. Wilhelm Roux Arch. EntwMech. Org. 167,336-352. Sander, K. (1974). Beeinflussen symbiontische Bakterien die Embryonalentwicklung von Insekten? Umschuu, 74, 619. Sander, K. (1975a). Pattern specification in the insect embryo. In “Cell Patterning”. Ciba Foundation Symp. 29 (new series), 241-263. Sander, K. (197513). Bildung und Kontrolle raumlicher Muster bei Metazoen. Verh. d t . 2001. Ges. 67, 58-70. Sander, K. (1976). Morphogenetic movements in insect embryogenesis. In “Insect Development” (Ed. P. Lawrence). Symp. R . ent. SOC. 8 . In press. Sander, K., Herth, W. and Vollmar, H. (1970). Abwandlungen des metameren Organisationsmusters in fragmentierten und in abnormen Insekteneiern. Verh. dt. rool. Ges. 63, 46-52. (ZooL Anz. Suppl. 33.) Sauer, G. (1961a). Ordnungsvorgange und ihre Regulationsmoglichkeit bei der Bildung der Keimanlage auf der Eioberfiche von Gryllus domesticus. Verh. d t . 2001. Ges. 54, 113-121. (Zool. Anr. Suppl. 24.) Sauer, G. (1961b). Die Regulationsbefahigung des Keimanlagenblastoderms von Gryllus domesticus beim Beginn der Wirkung des Differenzierungszentrums. Zool. Jb. (Anat.) 79,149-222. Sauer, G. (1962). Beziehungen zwischen der Bewegungsmoglichkeit und dem Regulationsvermogen von Blastodermbereichen im Grillenei. Verh. d t . zool. Ges. 55, 323-329. (Zool. Anr. Suppl. 25.) Sauer, H. W. (1966). Zeitraffer-Mikro-Film-Analyse embryonder Differenzierungsphasen von Gryllus domesticus. 2. Morph Okol. Tiere, 56, 143-251. Sauer-Loecher, E. (1954). Keimblatterbildung und Differenzierungsleistungen in isolierten Eiteilen der Honigbiene. Wilhelm Roux Arch. EntwMech. Org. 147, 302-354. Schanz, G. (1965). Entwicklungsvorgange im Ei der Libelle Ischnura elegans und Experimente zur Frage ihrer Aktivierung. Eine Mikro-Zeitraffer-Film-Analyse. Dips. Marburg/Lahn. Schmidt, O., Zissler, D., Sander, K. and Kalthoff, K. (1975). Switch in pattern formation after puncturing the anterior pole of Smittia eggs (Chironomidae, Diptera). Devl Biol. 46, 216-221. Schneider, R. (1975). Zeitliche und raumliche Variationen des Proteinmusters in Eiern der Hausgrille (Acheta domestica). Verh. d t . zool. Ges. 67, 187-191. Schnetter, M. (1934). Physiologische Untersuchungen uber das Differenzierungszentrum in der Embryonalentwicklung der Honigbiene. Wilhelm Roux Arch. EntwMech. Org. 131, 285-323. Schnetter, M. (1936). Die Entwicklung von Zwerglarven in geschniirten Bieneneiern. Verh. dt. zool. Ges. 38, 82-88. (Zool.A m . Suppl. 9.) Schnetter, W. (1 965). Experimente zur Analyse der morphogenetischen Funktion der Ooplasmabestandteile in der Embryonalentwicklung des Kartoffelkafers (Leptinotarsa decemlineata Say.) Wilhelm Roux Arch. EntwMech. Org. 155, 637-692.

236

KLAUS SANDER

Schnetter, W. (1967). Transplantation von Furchungs- und Blastodermkernen in entkernte Eier bei Leptinotarsa decemlineata (Coleoptera). Verh. d t . rool. Ges. 60, 494-499. (Zool. A m . Suppl. 30.) Schnetter, W . (1975). Zur Proteinsynthese in der friihen Embryonalentwicklung von Leptinotarsa decemlineata (Coleoptera): Rolle der mutterlichen und der embryoeigenen Messenger-RNS. Verh. d t . rool. Ges. 67, 197-201. Schwalm, F. E. (1965). Zell- und Mitosenmuster der normalen und nach Rontgenbestrahlung regulierenden Keimanlage von Gryllus domesticus. Z. Morph Okol. Tiere, 55, 915-1023. Schwalm, F. E. and Bender, H. A. (1973). Early development of the kelp fly, Coelopn frigida (Diptera). 11. Morphology of cleavage and blastoderm formation. 1. Morph. 141, 235-256. Seidel, F. (1924). Die Geschlechtsorgane in der embryonalen Entwicklung von Pyrrhocoris apterus L. 2. Morph Okol. Tiere, 1 , 429-506. Seidel, F. (1926). Die Determinierung der Keimanlage bei Insekten. I. Biol. ZbZ. 46, 321-343. Seidel, F. (1928). Die Determinierung der Keimanlage bei Insekten. 11. Biol. Zbl. 48, 230-25 1. Seidel, F. (1929a). Untersuchungen iiber das Bildungsprinzip der Keimanlage im Ei der Libelle Platycnemis pennipes, I-V. Wilhelm Roux Arch. EntwMech. Org. 119, 322-440. Seidel, F. (1929b). Die Determinierung der Keimanlage bei Insekten. 111. Biol. Zbl. 49, 57 7-60 7. Seidel, F. (1932). Die Potenzen der Furchungskerne im Libelleinei und ihre Rolle bei der Aktivierung des Bildungszentrums. Wilhelm Roux Arch. EntwMech. Org. 126, 21 3-276. Seidel, F. (1934). Das Differenzierungszentrum im Libellenei. I. Die dynamischen Voraussetzungen der Determination und Regulation. Wilhelm Roux Arch. EntwMech. Org. 131, 135-187. Seidel, F. (1935). Der Anlagenplan im Libellenei, zugleich eine Untersuchung iiber die allgemeinen Bedingungen fur die defekte Entwicklung und Regulation bei dotterreichen Eiern. Wilhelm Roux Arch. EntwMech. Org. 132, 671-751. Seidel, F. (1936). Entwicklungsphysiologie des Insekten-Keims. Verh. dt. zool. Ges. 38, 291-336. (Zool. Anz. Suppl. 9.) Seidel, F. (1960). Korpergrundgestalt und Keimstruktur. Eine Erorterung iiber die Grundlagen der vergleichenden und experimentellen Embryologie und deren Giiltigkeit bei phylogenetischen aerlegungen. Zool. Anr. 164, 245-305. Seidel, F. (1961). Entwicklungsphysiologische Zentren im Eisystem der Insekten. Verh. dt. zool. Ges. 54, 121-142. (2001.Anz. Suppl. 24.) Seidel, F. (1964). Analyse des Differenzierungsverlaufs im Insektenei (Gryllus) mittels UV- und Rontgenbestrahlungen. Verh. d t . zool. Ges. 57, 121-143. (2001.Anr. Suppl. 27.) Seidel, F. (1966). Das Eisystem der Insekten und die Dynamik seiner Aktivierung. Verh. dt. zool. Ges. 59, 166-188. (Zool. Anz. Suppl. 29.) Sokoloff, A. (1966). “The Genetics of Tribolium and Related Species”. Academic Press, London and New York.

INSECT EMBRYOGENESIS

237

Sokoloff, A. (1972). “Biqlogy of Tribolium with Special Emphasis on Genetic Aspects”. Clarendon Press, Oxford. Spemann, H. (1938). “Embryonic Development and Induction”. Yale University Press, New Haven. Stanley, M. S. M. and Grundmann, A. W. (1970). The embryonic development of Tribolium confusum. Ann. ent. SOC.A m . 63, 1248-1256. Summerbell, D., Lewis, J. S. and Wolpert, L. (1973). Positional information in chick limb morphogenesis. Nature, Lond. 244, 492-496. Tazima, Y. (1964). “The Genetics of the Silkworm”. Logos Press, London. Telfer, W. (1975). Development and physiology of the oocyte-nurse cell syncytium. In “Advances in Insect Physiology” (Eds J. E. Treherne, M. J. Berridge and V. B. Wigglesworth), vol. 11, pp. 223-319. Academic Press, London and New York. Trendelenburg, M. (197 1). Experimentelle Analyse des ooplasmatischen Reaktionssystems im Ei von Necro bin rufipes (Coleoptera, Cleridae.) Diplomarbeit Universitat Freiburg i. Br. Truckenbrodt, W. (1964). Zytologische und entwicklungsphysiologische Untersuchungen am besamten und am parthenogenetischen Ei von Kalotermes flavicollis Fabr. Reifung, Furchungsablauf und Bildung der Keimanlage. Zool. 16. (Anat.) 81, 359-434. Truckenbrodt, W. (1965). Sensibilitat undifferenzierter und differenzierter Kerne nach Rontgenbestrahlung in der fruhen Eientwicklung der Termite Kalotermes flavicollis Fabr. Wilhelm Roux Arch. EntwMech. Org. 156, 101-126. Truckenbrodt, W. (1971). Untersuchungen am Ei-Chorion der Termite Kalotermes flauicollis Fabr. unter normalen Bedingungen und nach Behandlung des Weibchens mit Colcemid (Insecta, Isoptera). Z . Morph. Okol. Tiere, 69,48-81. Ullman, S. L. (1964). The origin and structure of the mesoderm and the formation of the coelomic sacs in Tenebrio molitor L. (Insecta, Coleoptera). Phil. Trans. R . SOC. B. 248,245-277. Ulrich, H. (1952). Biophysikaliseh-entwicklungsphysiologischeBestrahlungsversuche an Drosophila-Eiern. Verh. dt. 2001. Ges. 45, 87-96. (Zool. Anz. Suppl. 16.) Verdier, M. (1961). Sur le gradient antho-posttrieur et la mitamirisation des insectes. Bull. SOC.tool. Fr. 86, 302-304. Vignau, J. (1967). Le remplacement rigulier des embryons abortifs par des Cbauches embryonnaires nouvelles dans les oeufs du phasme Carausius morosus Br.: Observations histologiques sur cette “polyembryonie substitutive”. C r . hebd. Se‘anc. Acad. Sci. 265D, 1404-1407. Vollmar, H. (1971). Musterbildungsprozesse und friihembryonale Gestaltungsbewegungen im Ei von Acheta domesticus nach Fragmentierung, Vitalfsbung und Zeitraffer-Filmaufnahmen. Dissertation, Universitat Freiburg. i. Br. Vollmar, H. (1972). Friihembryonale Gestaltungsbewegungen im vitalgefarbten DotterEntoplasmasystem intakter und fragmentierter Eier von Acheta domesticus L. (Orthopteroidea). Wilhelm Roux Arch. EntwMech. Org. 171, 228-243. Vollmar, H. (1974). Verteilung und Grosse der Zellkerne wahrend der ersten Differenzierungsschritte in Eifragmenten von Acheta domesticus L. (Orthopteroidea). Wilhelm Roux Arch. EntwMech. Org. 174, 160-171. Went, D. F. and Krause, G. (1974). Alteration of egg architecture and egg activation in

238

KLAUS SANDER

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

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

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

.

1

. .

.

.

.

.

. . .

.

239

.

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

.

. .

-

i

-

.

. . . . . . .

240

J. E. STEELE

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

HORMONAL CONTROL OF METABOLISM IN INSECTS

24 1

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

2 The hormones 2.1

MOULTING HORMONE (MH)

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

242

J. E. STEELE

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

HO

HO a-Ecdysone

QH OH :

0-Ecdy sone Fig. 1.

The structure of (Y- and 0-ecdysone.

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

HORMONAL CONTROL OF METABOLISM IN INSECTS

243

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

244

J.

E. STEELE

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

CllJH or JH I

C1,JH or JH I1

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

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

2. 3

BRAIN HORMONE (BH)

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

HORMONAL CONTROL OF METABOLISM IN INSECTS

245

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

2. 4

DIAPAUSE HORMONE (DH)

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

246

2. 5

J. E. STEELE BURSICON

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

HYPERCLYCAEMIC HORMONE (HGH)

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

ADIPOKINETIC (AKH) AND HYPOLIPAEMIC FACTORS

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

HORMONAL CONTROL OF METABOLISM IN INSECTS

247

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

BIOGENIC AMINES

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

3. 1 MOULTING

HORMONE

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

248

J. E. STEELE

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

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

249

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

3.2

JUVENILE HORMONE

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

N

cn

0

TABLE 1 Control of glycogen metabolism by juvenile hormone Species

Stage

Operation

Effect

Reference

Carausius morosus

Nymphs and adults

Allatectomy

Increase in whole body glycogen

L’Helias (1953)

Pytrhocoris apterus

Adult 9

Allatectomy

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

Janda and S l h a (1965)

Calliphora ery thro cephala

Adult 9

Allatectomy

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

Thomson (1952)

Phormia regina

Adult 9

Allatec tomy

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

Orr (1964)

Musca domestica

Adult 9

AIIatec tomy

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

Liu (1974)

Aedes taeniorhyncus

Adult 9

Allatec tomy

No effect

Van Handel and

Drosophila melanogaster

Adult d

Implantation of CA + CC

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

Butterworth and Bodenstein (1969)

Lea (I 9 70)

c rn

Yni m r m

I 0

n I

TABLE 2 Control of protein synthesis by juvenile hormone

0

z Species

Sex and stage

Operation

Effect

Reference

B

I-

0

Leucophaea maderae

Nauphoeta cinetea

Peripkzneta americana

Periplaneta americana

Adult 0

Adult 0

Adult 0

Adult ?

Allatectomized. CA implanted into experimental insects

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

Engelmann (19 7 1)

Decapitated. CA implanted into experimental insects

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

Luscher (1968)

Allatectomized 2-4 days prior t o experiment

Incorporation of labelled amino acids into haemolymph protein decreased 33%

Thomas and Nation

Allatectomy

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

Thomas and Nation

Locusta migratoria

Adult ?

Allatectomy

L o custa m igrato rM

5th instar nymph

Allatectomy

Increase in total body protein depressed in operated insect.

0

f 5

0

I-

% 3 rn

(1966a)

k 3

z

(1966b)

5

m

n

d

Minks (1967)

Goltzene-Bentz et al. (1972)

N

2

TABLE 2-continued Species ~~~

Sex and stage

Operation

Effect

Reference

~

Schistocerca gregaria

Adult

Oncopeltus fasciatus Caliiphora erthrocephala

0

Allatectomy

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

Hill (1965)

5th instar nymph

Injection of JH (2.5 P d

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

Bassi and Feir (1971)

3rd instar larva

Ligature behind CA

Normal decline of body protein prior to pupation accelerated

Price (1968)

HORMONAL CONTROL OF METABOLISM IN INSECTS

253

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

J. E. STEELE

254

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

DIAPAUSE HORMONE

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

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

HORMONAL CONTROL OF METABOLISM IN INSECTS

255

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

Pupal age (days)

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

256

J. E. STEELE

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

HORMONAL CONTROL OF METABOLISM IN INSECTS

257

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

\ \

\

0

H (controi) H ( A ) Days after oviposition

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

J. E. STEELE

258

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

, Sorbitol NADPH Sorbitol-6-P

f

NADH 1 I

2ADP 2ATP

!

I

I I

FAT

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

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

HORMONAL CONTROL OF METABOLISM IN INSECTS

259

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

HYPERGLYCAEMIC HORMONE

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

I

Glycogen

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

Glucose

-

G-6-P

? UDP

ATp

Trehalose - 6 - P

J.

Trehalose Fig. 6. The biosynthesis of trehalose.

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

260

J. E. STEELE

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

261

HORMONAL CONTROL OF METABOLISM IN INSECTS

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

,

<.It-;.

0

4

8

12

I

16

,

I

20

,

, 24

I

+ 48

28 ' 4 4

Hours after injection of hormone extract Fig. 7. Concomitant changes in blood trehalose and fat body glycogen in Periplaneta americana after injection of corpus cardiacum extract. (After Kilby, 1965.)

3.4.3 In vitro effect o f HGH on f a t body Few studies have been carried out on the effect of HGH on fat body in uitro which is surprising in view of the ease with which the technique can be performed and much useful kinetic data obtained. Phormia fat body incubated in vitro with a combined CC + CA extract (CA were shown to be inactive) released 225.1 mg trehalose per mg fat body per hour t o the medium as compared with 173.5 mg in the control tissues, clearly a rapid rate which is easily monitored (Friedman, 1967). Using a similar method Wiens and Gilbert (1967) measured trehalose release from Leucophaea fat body with time and demonstrated that the rate o f release was faster in the presence of CC extract for the first 75 minutes (at which time the rate of release was about 70 per cent faster in the treated tissue) and then gradually decreased towards the control level.

J. E. S T E E L E

262

3.4.4 Effect of HGH on glucose oxidation The profound effect which CC extract has on glycogen and trehalose levels is also reflected in the oxidation and metabolism of glucose as revealed by radiotracer experiments. Wiens and Gilbert (1969) have determined the effect of CC extract on the oxidation of U-’4C-glucose and its incorporation into glycogen and lipid of Leucophaea fat body in vitro. The extract reduced the oxidation of glucose to COz by 31 per cent and incorporation into glycogen by 83 per cent. The strong shift towards the synthesis of ‘trehalose readily explains the reduction in glycogen synthesis but the reduction in oxidation of glucose was not predictable. The latter observation may be taken as evidence for a reduced flow of carbohydrate through the glycolytic pathway. 3.4.5 Activation of phosphorylase The events associated with the activation of phosphorylase by epinephrine and glucagon in mammalian liver are so well known that there is little need to repeat them in detail here. A schematic diagram showing each of the steps in the activation process is presented in Fig. 8. The interaction of the Inactive Phosphorylase b kinase

Cel I rnernbmn,

Ad rena Iin glucagon HGH ?

f‘

Cyclic AMP

+ ATP

Fig. 8. The activation of phosphorylase in liver and proposed mechanism in insect fat body.

hormone with a membrane-bound adenyl cyclase leads to an increase in the concentration of cyclic AMP within the cell. Cyclic AMP activates a protein kinase which in turn catalyses a phosphorylation of the inactive form of phosphorylase kinase to yield the catalytically active form. Phosphorylase, like phosphorylase kinase, can exist in two forms one of which (the “a” form) is active in the absence of 5‘-AMP and a second (the “b” form) showing only partial activity in the absence of 5’-AMP. The conversion of phosphorylase “b” t o “a” is catalysed by the active form of phosphorylase

HORMONAL CONTROL OF METABOLISM IN INSECTS

263

kinase. Among other things, it i s the relative proportions of the “a” and “b” forms which determine the rate at which glucosyl units are removed from the parent glycogen and, in the insect, might influence the rate at which trehalose is synthesized. Unfortunately, our knowledge of phosphorylases in insect fat body is exceedingly scanty but what exists suggests that the nature of the enzyme is basically similar to that found in vertebrates. The study by Stevenson and Wyatt (1964) on pupal fat body of Surnia Cynthia was the first kinetic study of insect phosphorylase and clearly showed the activation conferred by 5‘-AMP. Wiens and Gilbert (196713) were able t o obtain evidence that the conversion of inactive phosphorylase t o the active form in Hyalophora cecropia was mediated by a kinase and the reverse change by a phosphatase. A recent study by Applebaum and Schlesinger (1973) on locust fat body has shown that glucose-6-phosphate is competitive (Ki= 1 mM) with the phosphorylase substrate glucose-1-phosphate. This mechanism may constitute an important regulatory mechanism in the synthesis of trehalose from glycogen since glucose-6-phosphate is a primary substrate for the synthesis of trehalose. In view of the tissue specificity of HGH and its hyperglycaemic effect it is hardly surprising that this hormone has been compared with glucagon and epinephrine in the vertebrates. Attempts t o strengthen the analogy have centered on the possible activation of phosphorylase by HGH (Fig. 8). Steele (1963) demonstrated an increase of 140 per cent in total phosphorylase activity when cockroach fat body is incubated with HGH in vitro; similar results were obtained by Wiens and Gilbert (1967). In Locusta Mordue and Goldsworthy (1969) and Goldsworthy (1970) have shown that HGH will activate phosphorylase both in vitro and in vivo. The activation of phosphorylase is affected by the concentrations of Na+ in the cell although it is not possible at this time to attach any particular significance to this effect (Steele, 1969). Manipulations of the extracellular ionic environment that resulted in a three-fold increase in tissue Na+ enabled the hormone to increase phosphorylase activity by over 400 per cent when compared with controls. However, it is not clear whether the Na+ acts directly on the phosphorylase mechanism or induces a change in the intracellular concentration of another ion e.g. K + or Ca2+ which is responsible for the increased sensitivity of phosphorylase t o the hormone. The results do suggest that the maintenance of the proper ionic balance within the cell is an integral part of the mechanism controlling the degradation of glycogen. There is, however, an important exception to the generalization that fat body phosphorylase is activated by HGH. Wiens and Gilbert (1967b) have shown that although the phosphorylase system in the silkmoth appears similar t o that in other insects it was unresponsive not only to extracts of H. cecropia CC but also to those prepared from

264

J. E. STEELE

Periplaneta CC as well. Furthermore,, silkmoth CC were without a phosphorylase activating effect in the cockroach Leucophaea. Thus it appears that H. cecropia does not produce the phosphorylase-activating factor and therefore it is not surprising that the fat body lacks the ability to respond t o extracts known to be active. 3.4.6 Role of cyclic AMP In order to complete the analogy between the hormone-mediated phosphorylase system in insects and mammals it is essential t o know if cyclic AMP acts as a “second messenger” between the initial interaction of the hormone with its target and the ultimate activation of phosphorylase. Phosphorylase activity in isolated cockroach fat body is increased 279 per cent by 0.1 mM cyclic AMP; 1 mM caffeine will increase the activity by 68 per cent (Steele, 1964). The activation by caffeine is attributed t o the inhibition of a phosphodiesterase (PDE) which hydrolyses the ester bond of cyclic AMP yielding 5’-AMP. N o attempts t o measure the accumulation of cyclic AMP in the presence of HGH have yet been reported; indeed there is no evidence to suggest that it does accumulate. Efforts to show an activation of adenyl cyclase by HGH have so far been unsuccessful (G. R. Wyatt-personal communication). It should be noted that an accumulation of cyclic AMP in the presence of HGH would not conclusively prove that the primary mode of action of the hormone is through an effect on phosphorylase. A definitive proof would require a demonstration that inactive phosphorylase kinase is activated by cyclic AMP or cyclic AMPdependent protein kinase. It is also possible that HGH stimulation of cyclic AMP synthesis might drive another mechanism whose demand for energy is coupled to glycogen degradation through phosphorylase, the activity of which is governed by factors other than cyclic AMP. 3.4.7 Control o f trehalose synthesis in fat body The normal level of trehalose in the haemolymph may be regulated by feedback control of the enzyme trehalose-6-phosphate synthetase (Murphy and Wyatt, 1965). Silkmoth trehalose synthetase is inhibited u p t o 70 per cent by 8 mM trehalose which is well below the concentration present in the haemolymph (Murphy and Wyatt, 1965); thus trehalose is an inhibitor of potential physiological significance. In agreement with these suggestions are the observations by Friedman (1967) showing that output of trehalose by Phormia fat body is effectively halted if the tissue is bathed in a medium containing one-tenth as much trehalose as that normally present in haemolymph. It is suggested that in tissue treated with HGH the inhibition of trehalose-6-phosphate synthetase by trehalose is overcome by an increase in the intracellular concentration of glucose-6-phosphate arising from the

HORMONAL CONTROL OF METABOLISM I N INSECTS

265

degradation of glycogen. This idea is supported by the fact that trehalose inhibition of trehalose-6-phosphate synthesis is partially depressed in the presence of glucose-6-phosphate (Murphy and Wyatt, 1965). The finding in our laboratory that HGH doubles the concentration of glucose-6-phosphate in Perzplaneta fat body provides additional supporting evidence (Coulthart and Steele, unpublished observations). Of interest but uncertain importance is the finding that Phormia fat body contains a glucose-6-phosphatase subject t o inhibition by trehalose (Friedman, 1968). Friedman has suggested that, in Phormia at least, trehalose itself may be an important regulator of trehalose synthesis by virtue of its ability to alter the rate at which glucose-6-phosphate is made available t o the trehalose-6-phosphate synthetase. 3.4.8 Control of glucose levels in haemolymph The presence of glucose-6-phosphatase in fat body (Friedman, 1968) implies that free glucose is also present. The accumulation of free glucose is difficult to explain since utilization of glucose within a cell usually requires prior conversion t o the phosphorylated derivative (G-6-P). It is possible that this may be a mechanism for regulating the level of glucose in the haemolymph. Steele (1961) was unable t o show an effect of CC extract on blood glucose in Periplaneta; however, the techniqe used to measure glucose lacked specificity as well as the sensitivity required t o measure small changes at low concentrations. Bowers and Friedman (1963) using a sensitive glucose oxidase method clearly showed a rise from 2.0 mg mI- t o 7.6 mg ml-' in haemolymph glucose in Phormia after injection of CC extract. If a function of CC is t o maintain a certain level of glucose in the haemolymph it would be expected that cardiacectomy would lead to a decrease in haemolymph glucose. Removal of the glandular lobe of the CC in Locusta causes blood glucose t o fall from 2.61 mg ml-' to 1.51 mg ml-' (Cazal, 1971) and removal of the CC + CA in Schistocerca induces a similar effect (Lafon-Cazal and Roussel, 1971). Thus HGH may be viewed as a potential regulator of haemolymph glucose by virtue of trehalose activation of glucose-6-phosphatase.

'

3.4.9 Post-hormonal synthesis of glycogen Within 3 t o 4 hours after injection of CC extract the cockroach fat body is resynthesizing glycogen and continues to do so for about 2 days (Fig. 7). This return of trehalose t o storage as glycogen would be inefficient if the activity of phosphorylase could not be reduced. An interesting observation in this regard is the low level of phosphorylase activity(50 per cent of control) that persists for not iess than two days after injection of CC extract (Steele, 1963). This result cannot be explained by assuming

266

J. E. STEELE

glucose-6-phosphate inhibition of phosphorylase as proposed by Applebaum and Schlesinger (1973) since inhibition is due t o an increase in the relative avaunt of the “b” form of the enzyme. We are reinvestigating this interesting problem in enzyme regulation and have been able t o show that the decrease in the “a” form of phosphorylase from 85 per cent of the total enzyme to 25 per cent occurs between 1 and 5 hours after hormone injection (White and Steele, unpublished observations). This may result from a decrease in phosphorylase kinase activity or an increase in phosphorylase phosphatase activity or both. The dramatic change in phosphorylase activity of fat body during treatment with HGH and again during the post-hormonal state suggests that it may be an ideal system in which t o study glycogen metabolism and its regulation. The decrease in phosphorylase activity of Locusta fat body following treatment with HGH (Goldsworthy, 1970) does not exhibit the pronounced shift towards the inactive form of the enzyme as it does in the cockroach. The physiological significance of the difference between the two species is not clear but it suggests that the cockroach has a more efficient mechanism for the synthesis of glycogen and may explain why the locust is glycogen deficient under certain conditions and often gives a negative hyperglycaemic response (Goldsworthy, 1969). The idea that HGH favours the active form of phosphorylase is also suggested by cardiacectomy experiments in Locusta in which a progressive decrease in the “a” form of the enzyme was noted during the two days following the operation. The decrease in phosphorylase activity under these conditions also explains the lowered trehalose levels observed after cardiacectomy (Cazal, 197 1; Lafon-Cazal and Roussel, 1971). 3.4.10 Effect on nerve cord glycogenolysis Injection of CC extract into Periplaneta causes the glycogen level in nerve cord to fall from 9.86 mg g-’ t o 1.21 mg g-’ (Steele, 1963). This is the only tissue other than fat body known to respond t o the extract by increasing the degradation of glycogen. It seems likely that the factor having this effect is identical with that acting on fat body. Highly purified HGH is active o n both tissues (Hart and Steele, 1973) and activity in each system is lost if the extract is incubated with chymotrypsin (Brown, 1965; Robertson and Steele, 1973b). The glycogenolytic effect of the factor in nerve cord can only be demonstrated in vivo for the reason that nerve cords undergo spontaneous glycogenolysis in vitro (Steele, unpublished observations). Under these circumstances glycogen is converted into trehalose (Hart and Steele, 1974) but phosphorolysis of glycogen is not dependent on the conversion of phosphorylase “b” to “a”. The most reasonable explanation for these results is that phosphorylase “b” appears active

HORMONAL CONTROL OF METABOLISM I N INSECTS

267

because of an accumulation of 5’-AMP induced by the anaerobic state of the tissue. Although a glycogenolytic effect of the hormone cannot be demonstrated in nitro, the phosphorylase system responds t o CC extract, cyclic AMP and caffeine as indicated by the conversion of phosphorylase “b” to “a”. Although the evidence linking cyclic AMP t o phosphorylase activation is circumstantial it is premature t o conclude that it occurs as a result of phosphorylase kinase activation mediated by a cyclic AMPdependent protein kinase. The nerve cord is less sensitive t o the hormone than fat body by a factor of 10 (Hart and Steele, 1973) and therefore threshold concentrations for nerve cord may never occur if the primary site of action is the fat body. The physiological significance, if any, of the HGH stimulated conversion of glycogen t o trehalose in nerve cord is not apparent.

3.4.1 1 Control of hyperglycaemic hormone release Although nothing is known about normal levels of HGH in haemolymph the decrease in trehalose following cardiacectomy strongly suggests that the hormone is part of a normal regulatory mechanism. Removal of the CC in Locusta caused trehalose levels t o fall from 13.95 mg ml-’ to 9.08 mg ml-’ and in those insects from which only the glandular lobe of the CC had been removed the change was 14.48 mg ml-’ t o 8.06 mg ml-’ (Cazal, 1971) indicating that the glandular lobe was the site of hormone storage. A similar effect was noted in Schistocerca after removal of the CC + CA (Lafon-Cazal and Roussel, 1971). Vejbjerg and Norman (1974) have provided good evidence that the release of HGH in Calliphora is probably under nervous control and linked t o physical activity in the fly. If the brain and CC are intact the fly will maintain flight for at least 45 minutes during which period the trehalose level in haemolymph is kept constant at 23 g 1-’ , closely approximating that found in the resting insect. If the CC have been removed or denervated the trehalose level falls and flight ceases within 45 minutes. While at rest the trehalose concentration slowly returns t o the resting level, apparently without the intervention of the hormone, but this process can be accelerated if the CC, which have previously been denervated, are squeezed and caused t o release their content of hormone. It appears that normal secretion of HGH from the CC is under nervous control since release can be achieved by electrical means if the nervous pathway from the brain t o the CC is intact (Normann and Duve, 1969). Similar results have been obtained by depolarizing the tissue with high K + concentrations. The mechanism of release is by Ca2+dependent exocytosis (Normann, 1974). The finding that section of the nervus corporis cardiaci externus (N.C.C. 11) in Periplaneta induces a 50 per

J. E. STEELE

268

cent decrease in haemolymph trehalose ,4 days after the operation (Gersch, 1974) corroborates these findings.

3. 5

MEDIAL NEUROSECRETORY CELL (MNC) HORMONE

If mosquitos are starved the reserves of glycogen and fat fall to very low levels (Van Handel and Lum, 1961). On subsequent feeding with a mixture of glucose and fructose glycogen synthesis proceeds rapidly for about 6 hours when the rate declines and surplus sugar is converted into lipid (Van Handel and Lea, 1965). The decrease in glycogen synthesis 6 hours after the initiation of feeding seems to be controlled by a hormone from the MNC. Removal of these cells in Aedes taeniorhynchus, Aedes solicitans and Aedes aegypti prevents the decrease in the rate of glycogen synthesis and glycogen accumulates at the expense of lipid (Van Handel and Lea, 1970; Lea and Van Handel, 1970). If MNC are reimplanted into mosquitos lacking these cells glycogen synthesis is reduced t o the normal level but lipid synthesis is not elevated (Lea and Van Handel, 1970). The change in the rate of lipid and glycogen synthesis following MNC removal is not seen if the mosquitos have been fed blood rather than sugar (Van Handel and Lea, 1970) suggesting that a component of the normal diet may play an important role in determining the flux of substrates between the various pathways associated with the synthesis of lipid and glycogen. Removal of the MNC in Call$hora also results in an accumulation of fat body glycogen but differs from the mosquito in that lipid continues to accumulate and at a faster rate (Thomsen, 1952). The glycogenic effect of MNC removal is not restricted to the Diptera since ablation of these cells in Locusta also causes glycogen to accumulate in the fat body (Goldsworthy, 1971). Six days after the operation it increased by 82 per cent and by twelve days reached 184 per cent. During this period the control level of glycogen did not change nor did the concentration of carbohydrate in the haemolymph. Since the removal of the MNC, at least in mosquitos, does not impair the utilization of glycogen (Van Handel and Lea, 1970) one must conclude that a factor produced in the MNC acts directly t o suppress glycogen synthesis or indirectly to stimulate other biosynthetic reactions having a requirement for carbohydrate. The large increase in fat body glycogen following removal of the MNC in locusts would be expected to produce a much enhanced response to HGH. Surprisingly, there is no effect at all (Goldsworthy, 1971), nor can it be explained by any change in phosphorylase activity. Although Goldsworthy has implied that the lack of response to HGH is caused by the removal of the MNC it is important t o note that the hyperglycaemic

HORMONAL CONTROL OF METABOLISM IN INSECTS

269

response in Locusta is stage specific and not shown in mature locusts (Goldsworthy, 1969) as used by Goldsworthy (1971). 3. 6

OCTOPAMINE

3.6.1 Glycogen levels in nerve cord Because octopamine occurs in relatively high concentrations in the nerve cord of Periplaneta (Robertson and Steele, 1973a) the finding that it will deplete this tissue of much of its glycogen (Robertson and Steele, 1972) can no longer be considered a pharmacological curiosity. Nerve cord loses as much as 35 per cent of its glycogen on injection of 25 nmol of octopamine into the intact insect (Robertson and Steele, 1972, 1973b). This effect can only be seen in vivo since glycogen is rapidly degraded under in vitro conditions, even in the absence of active factors (Hart and Steele, 1973). The concentration of glycogen in both nerve cord and fat body of the cockroach is approximately 1 0 mg g-' ; however, the nerve cord because of its much smaller size contains considerably less total glycogen than does fat body (Steele, 1963). For this reason the nerve cord is not an important contributor t o the blood trehalose pool. The question then concerns the role of this glycogen, situated mainly in the perineurium cells of the ganglia and t o a lesser extent in the glial cells (Wigglesworth, 1960). Wigglesworth has suggested that it serves a trophic function in relation to the axons, although this has not yet been proved. The perineurium of the ganglionic sheath is thought t o represent an important permeability barrier between the axons and haemolymph. The observation by Maddrell and Treherne (1967) that this tissue is ultrastructurally similar to various fluid-secreting epithelia suggests that it may regulate transport between the haemolymph and the extra-axonal space. The glycogen present may represent a source of energy for this purpose. 3.6.2 Cyclic AMP and phosphorylase activation Consistent with the glycogenolytic effect of octopamine is the finding that 0.1 mM octopamine will activate phosphorylase by as much as 48 per cent (Robertson and Steele, 1972). The lower limit for activation when nerve cords are incubated in vitro is in the range of 0.5 PM . Since phosphorylase activity is augmented 22 per cent by 1 0 /LM cyclic AMP (Hart and Steele, 1973) there is a tendency t o ascribe this effect t o a direct activation by the nucleotide, as in mammalian liver. Indeed the finding that octopamine and caffeine, when used together in subthreshold concentrations, also increase phosphorylase activity by as much as 25 per cent supports the idea that cyclic AMP mediates the activation of phosphorylase by octopamine but cannot be used as evidence for its locus of action. Conclusive evidence that octopamine induces the synthesis of cyclic AMP has been presented by

270

J. E. STEELE

Nathanson and Greengard (1973) who have shown that 1.5 j l ~(= K , ) L-octopamine will increase the synthesis of cyclic AMP in homogenates by 250 per cent. In intact thoracic ganglia a 5-fold increase in enzyme activity was obtained with 0 . 2 5 - m ~octopamine. The same authors have shown that the amine acts specifically on an adenyl cyclase specific for octopamine which is distinct from those responding to dopamine and 5-HT. Since a large part of the glycogen present in the nerve cord is present in the perineurium cells it follows that these cells are probably the site of action of the hormone, although the immediate processes set in motion by cyclic AMP are unknown.

D

3. 7 5-HYDROXYTRYPTAMINE (5-HT) Ever since 5-HT was first demonstrated in insect nervous systems (Welsh and Moorhead, 1960; Gersch et al., 1961; Colhoun, 1963) much interest has been shown in this indole derivative. Although synthesis of 5-HT has been demonstrated in brain, nerve cord, and CC its physiological role in those tissues where it occurs is unknown. The only metabolic effect attributed to 5-HT in insects is an inhibition of phosphorylase in Periplaneta nerve cord (Hart and Steele, 1969, 1973). The reduction in activity which amounts to 27 per cent with 0.1 mM 5-HT can be accounted for by a shift in equilibrium towards the inactive form of the enzyme rather than an allosteric effect on the “a” form. This interpretation is favoured because 1 mM 5-HT not only blocks the activation of phosphorylase by HGH (normally about 75 per cent with one pair of glands per ml) but reduces the active form of the enzyme from 58 per cent t o 21 per cent (Hart and Steele, 1973). The effect of 5-HT on phosphorylase activity can be explained either by a decrease in the activity of phosphorylase kinase or an increase in that of phosphorylase phosphatase. It is possible that 5-HT may act by altering the intracellular concentration of CaZ+.CaZ+is known to be a potent activator of phosphorylase kinase (Brostrom et al., 1971) and has been shown to markedly increase the uptake of CaZ+by the salivary gland cells of Calliphora (Prince et al., 1972). The inhibition of phosphorylase activity by 5-HT (Hart and Steele, 1969, 1973) is in direct contrast t o the finding of Nathanson and Greengard (1973) that it increases the synthesis of cyclic AMP, a known activator of phosphorylase (Hart and Steele, 1973). The most plausible explanation for this apparent contradiction is that separate receptors for 5-HT may be present on different cell types within the nerve cord. 4 Control of lipid metabolism The first report relating endocrine activity to metabolism in an insect is that of Pfeiffer (1945) in which it was shown that allatectomy of female

HORMONAL CONTROL OF METABOLISM IN INSECTS

271

Melanoplus differentialis produced excessive accumulation of lipid in the fat body. Through these studies the concept’ of a “metabolic” hormone first came into being. This work is also of historical significance because it was the first t o identify a target organ for JH in the adult insect. The conclusion was drawn that the CA were responsible €or mobilization of fat reserves destined for the developing ova. This concept has not changed greatly in the intervening period, although the mechanism by which JH triggers the transfer of lipid t o the ovary and its role in fat body metabolism is poorly understood. Further complications have arisen out of the recent discovery that J H is also required €or the synthesis of a fat body protein or vitellogenin necessary for egg deveJopment (Engelmann, 1969). The purpose of the discussion which follows is t o investigate the relationship that exists between JH-stimulated protein synthesis and lipid metabolism in fat body.

4. 1 JUVENILE HORMONE

4.1.1 Lipid synthesis in fat body a. Effect o f allatectomy on fat body lipid Numerous studies have confirmed the abnormal accumulation of lipid in fat body following allatectomy. This effect has been demonstrated in Carausius (L’Helias, 1953), Periplaneta (Bodenstein, 1953), Locusta (Minks, 1967; Strong, 1968a, b), Schistocerca (Odhiambo, 1966; Hill and Izatt, 1974), Sphodoptera (El-Ibrashy and Boctor, 1970); Drosophila (Vogt, 1949), and Calliphora (Thomsen, 1952). There is, however, an exception t o the general rule. Van Handel and Lea (1970) have shown that allatectomy has no effect on synthesis or utilization of lipid in Aedes taeniorhynchus, in sugar-fed or blood-fed mosquitos. The reason for the lack of effect is unknown. Implantation of female CA/CC complexes into male Drosophila increased the lipid content of fat body cells from an average of 7.6 per cent to 13 per cent (Butterworth and Bodenstein, 1969). A similar effect was obtained with CA alone and synthetic JH (t,t,t C-17 methyl ester), the latter increasing the lipid content from 7 per cent to 18 per cent. These results are contrary to that which might have been expected. As the authors suggest, the reason may lie in a complex relationship between the implanted gland and the host’s own CA which had not been removed. This may be the reason since allatectomy alone produces the anticipated increase in fat body lipid in Drosophila (Vogt, 1949). That allatectomy and not general injury t o the nervous system is responsible for the biochemical lesion affecting lipid metabolism has been confirmed by implanting CA into previously allatectomized insects. In Schistocerca (Hill and Jzatt, 1974) and Locusta (Beenakkers, 1969) the

272

J. E. STEELE

increase in lipid content of the fat body of allatectomized female adults, which can be as much as three times that' of controls, can be prevented by the implantation of CA. Females which had not been allatectomized but had received CA implants synthesized less fat body lipid than did normal females, suggesting that the effect of the hormone is concentration dependent. In allatectomized Schistocerca adults total lipid increases from 258 mg g-' to 409 mg g-' but returns t o 259 mg g-' following implantation of CA (Odhiambo, 1966), indicating the effect of allatectomy is not restricted to females. The surgery associated with allatectomy causes some damage t o the nervous system; thus there is the possibility that normal feeding behaviour is affected. Walker and Bailey (1971b) have maintained that the primary effect of JH is on the regulation of growth and feeding. They show that allatectomy of male Schistocerca shortly after the adult moult induces a period of intense feeding activity which they suggest reflects an alteration in the growth pattern due t o the absence of JH. The extra lipid deposited in the fat body is believed to arise from carbohydrate of dietary origin. This view has been challenged by Hill and Izatt (1974) who provide evidence t o the contrary. They have shown that allatectomized Schistocerca females do not consume significantly more food than do control insects. Their period of somatic growth was not prolonged by the operation nor did they grow more than did the controls. Furthermore, the DNA content of the fat body was unaltered by allatectomy, indicating that the accumulation of lipid in the fat body is not a consequence of increased food intake. Allatectomy is also without an effect on feeding behaviour in Locusta (Strong, 1968a). b. Fatty acid saturation and lipid synthesis Stephen and Gilbert (1970) have presented an interesting hypothesis t o explain the inhibitory effect of JH on lipid synthesis in Hyalophora cercropia. It was observed that synthesis of lipid as determined by the incorporation of labelled acetate was relatively low during the last larval instar and again on the eighteenth to twentieth day of pharate adult life. These are periods when the titre of JH is known to be high. In contrast t o these results the rate of lipid synthesis was high in four-day-old pharate adults that have a very low titre of JH. Whereas the relationship between lipid synthesis and JH concentration is an inverse one there is a direct correlation between JH concentration and the concentration of saturated fatty acids in total body lipids. These observations led the authors t o suggest that JH acts t o suppress desaturation of fatty acids thus accounting for the increased level of saturated fatty acids coincident with the high titre of JH. The increase in concentration of saturated fatty acids is thought t o be augmented by the degradation of polyenoic fatty acids t o acetyl CoA which is subsequently

HORMONAL CONTROL OF METABOLISM IN INSECTS

273

reconverted into saturated fatty acids. The increase in concentration of saturated fatty acids is viewed as favouring the synthesis of diglyceride, whose release into haemolymph is also increased, thus opposing the accumulation of lipid in the fat body. With decreasing titres of JH it is suggested that desaturation of palmitic and stearic acids increases, thus lowering the concentration of saturated fatty acids inside the cell and limiting the synthesis of diglyceride so that lipid accumulates in the tissue. The hypothesis is interesting but has not been subjected t o rigorous analysis. The nub of the hypothesis concerns the postulated role of JH in preventing desaturation of the fatty acids for which experimental evidence is lacking. 4.1.2 JH-related protein synthesis in f a t body a. Quantitative aspects of protein synthesis The data presented in Table 2 show that the CA promote the synthesis of protein in adult insects. Allatectomy is consistent in lowering the rate of protein synthesis whereas treatment with J H invariably is accompanied by an increase. The fat body is the source of a number of proteins present in the haemolymph. This was first shown in fifth instar larvae of Bombyx mori (Shigematsu, 1960). At the same time the similarity between haemolymph proteins of Hyalophora cecropia and Philosamia Cynthia was noted by Laufer (1960). Numerous workers have attempted t o relate haemolymph protein composition to JH titre but the effect of allatectomy is ill defined. However, an increase in haemolymph protein is the predominant response to allatectomy (Table 3). This would seem inconsistent with the observation that a decreasing titre of JH is associated with a decreased rate of protein synthesis (Table 2). Since the CA also have a gonadotrophic effect on the ovary facilitating the uptake of vitellogenin (Bell, 1969; Bell and Barth, 1971), the accumulation of protein in haemolymph indicates that uptake b y the ovary, and possibly other tissues, ceases shortly after withdrawal of J H while syn;hesis continues for some time. The relatively long-lived mRNA ( 3 days or more) obtained from Leucophaea fat body after JH treatment (Engelmann, 1971) is in agreement with this idea. The most significant information arising out of data presented in Table 3 is the large increase in the absolute amount of protein present in haemolymph after allatectomy. These results are all the more remarkable if one considers that in the absence of JH, the synthesis of at least some of the proteins have been turned off and only by virtue of the long-lived nature of the mRNA does synthesis continue. Even greater differences would probably be seen if it were possible t o inhibit the gonadotrophic effect of JH without interfering with the protein biosynthetic mechanism. A large part of the increase in total haemolymph protein after alla-

TABLE 3 Control of blood protein levels by corpora allata ~

Species

Sex

Operation

Change in protein concentration

Reference

Leucophaea maderae

Allatectomy

Decrease: 125-91 mg ml-' 3 weeks after operation

Engelmann and Penney (1966)

Periplaneta americana

Allatectomy

Decrease: 17.1-10.9 mg ml-' 35 days after operation

Thomas and Nation (1966a)

Periplaneta americana

Allatectomy

Increase: 63-87 mg ml-'

Mills et al. (1966)

Periplaneta americana

Allatectomy and cardiacectomy

Increase: Change not given

Adiyodi and Nayar (1967)

Periplaneta americana

Allatectomy

Increase: 57-76 mg ml-' 25 days after operation

Bell (1969)

Lo custa migratoria

Allatectomy

Decrease: 20%. Not apparent until 3 weeks after operation

Minks (1967)

Schistocerca gregaria

Allatectomy

Increase: 58-113 mg ml-' 5 days after operation

Highnam et al. (1963)

Rhodnius prolixus

Allatectomy

Decrease: 4.79-2.7 mg m l '

Pratt and Davey (1972)

Pyrrhocork apterus

Allatectomy

Increase: 75-275 mg ml-'

Slima (1 964a)

Leptinotarsa decemlineata

Q

Allatectomy

Increase: 30-120 mg ml-'

DeLoof and DeWilde (1970)

Leptinotarsa decemlineata

d Pd

Allatectomy Allatectomy

Increase: 30-90 mg ml-'

DeLoof and DeWilde (1970)

+

Increase: 200%

Orr (1964)

I" cn

Phormia regina

2

m r rn

HORMONAL CONTROL OF METABOLISM I N INSECTS

275

tectomy seems to be due t o . t h e accumulation of vitellogenin, the only identifiable protein species for which we have information on the relationship between rate of synthesis and JH. A number of studies have demonstrated the dependence of vitellogenin synthesis on JH. The species involved include Leucophaea (Englemann and Penney, 1966; Brooks, 1969; Engelmann, 1969, 1971; Scheurer, 1969), Periplaneta (Menonm, 1965; Thomas and Nation, 1966; Adiyodi and Nayar, 1967; Bell, 1969), Locusta (Minks, 1967; Bentz and Girardie, 1969; Bentz et al., 1970) and Danaus (Pan and Wyatt, 1971). Although vitellogenin is not normally present in high concentration in the haemolymph it is concentrated by the ovary where it may consitute up to 80 per cent or more of the yolk protein (Dejmal and Brookes, 1968; Bell, 1970). The synthesis of vitellogenin places severe demands on the energy reserves of the insect. There is little information on the role of JH in the synthesis of the nonsex-specific proteins yet the rate of synthesis of these proteins, like that of vitellogenin, is enhanced by JH although not t o the same extent (Engelmann, 1971). In a similar vein, Minks (1967) has shown that allatectomy of Locusta causes a smaller decrease in the rate of protein synthesis in male fat body than in female fat body. Unlike adult females in which there is complete disappearance of vitellogenin from the haemolymph following allatectomy (Engelmann and Penny, 1966; Bell, 1969; Scheurer, 1969) there is no evidence to show the complete disappearance from the haemolymph of any of the nonsex-specific proteins. This suggests that JH may be responsible for de nouo synthesis of vitellogenin and modulates that of the nonsexspecific proteins. Although JH may affect the nonsex-specific proteins in both sexes in a manner which could significantly affect metabolism it seems certain that changes in vitellogenin synthesis will have a much greater effect. b. Relationship between lipid and protein Analyses of a number of vitellogenins have shown them to be lipoproteins. This has been shown qualitatively for Periplaneta (Adiyodi and Nayar, 1967), Leucophaea (Dejmal and Brookes, 1968), and in Leptinotarsa (DeLoof and De Wilde, 1970), while Pan (in Wyatt, 1972) has estimated that Cecropia vitellogenin contains 10 per cent lipid. Thus the link between lipid and vitellogenic protein as the carrier of lipid between fat body and ovary is well established. In Periplaneta lipid is conjugated with a number of proteins present in haemolymph (Siakotos, 1960). The haemolymph lipoproteins have been the subject of detailed investigation in recent years and a number of elegant studies have originated in the laboratories of Chino and Gilbert. These proteins are considered to be the vehicle by which lipid is transported from the storage site t o the site of utilization. Lipid contained in fat body is

276

J. E. STEELE

released to the haemolymph as diglyceride conjugated with a protein (Chino and Gilbert, 1965a). Subsequent studies by Chino et al. (1969) and Thomas and Gilbert (1968, 1969) have shown that diglyceride in the haemolymph is associated with two lipoproteins designated LP-I and LP-I1 containing approximately 45 per cent and 10 per cent diglyceride respectively. Both diglyceride-transporting proteins are synthesized in the fat body (Thomas, 1972; Peled and Tietz, 1973). Thus the relationship between haemolymph proteins of fat body origin and fat body lipid is an intimate one. Should JH be found to influence the synthesis of these diglyceride-transporting proteins an important facet of JH control of lipid metabolism will have been uncovered. 4.1.3 The cause of &id accumulation The numerous studies showing that allatectomy causes a decrease in the rate of protein synthesis by fat body and an accumulation of lipid in the same tissue leads to the inescapable conclusion that these effects cannot be divorced from each other. The idea is supported by the observations that both the effect of JH on vitellogenin synthesis (Adiyodi and Nayar, 1967; Bell, 1969) as well as the increase in lipid following allatectomy (Odhiambo, 1966) occurs over a period of days. It is possible that each effect may be independent but it is difficult to visualize how a single hormone could have two independent primary effects on the same tissue. The theme of the discussion which follows is that only the protein moiety of the vitellogenin or other lipoprotein fails to be synthesized in the a h e c t o m i z e d state. It is proposed that the lipid moiety which normally is conjugated with the protein is therefore without an acceptor and accumulates within the fat body cell. Thus the effect of allatectomy in stimulating lipid accumulation may be viewed as a failure in protein synthesis rather than a release of lipid synthesis from a state of inhibition. However, this simple concept cannot explain adequately why lipid accumulates in the fat body when the CA are removed, since it would be equally valid to argue that reduction in lipid turnover might also lead to a reduction in lipid synthesis through feedback control. The fact that it does not may be cited as evidence for an indirect effect of JH on lipid metabolism. The lack of suitable protein as a vehicle for the transport of lipid out of the fat body may account for only part of the lipid accumulating in the fat body. The accumulation of more total body fat in allatectomized insects (Vroman et al., 1965; Odhiambo, 1966) suggests an increased conversion of nonlipid as well as lipid precursors into fat. In addition to dietary lipid, carbohydrate, which under normal circumstances would provide much of the ATP required for protein synthesis, and unutilized amino acids would also be important substrates for lipid synthesis.

HORMONAL CONTROL OF METABOLISM I N INSECTS

277

a. Rate of lipid synthesis The increase in fat body lipid following allatectomy suggests either that utilization of lipid has been impeded or the rate of synthesis has been increased or both have occurred. A reduction in lipid utilization seems unlikely since it has been shown the U-' C-palmitate oxidation by Schistocerca fat body is unaffected by the operation (Walker and Bailey, 1971a) and haemolymph lipid levels appear normal (Walker and Bailey, 1971b). Doane (1961) also concluded that absence of JH did not interfere with the utilization of lipid stored in the fat body. The most conclusive evidence showing that JH inhibits lipid synthesis has been obtained by measuring the rate of incorporation of labelled fatty acids into fat body lipid. Vroman et al. (1965) have shown that l-14C-acetate injected into female Periplaneta had a higher rate of incorporation into total body triglyceride when the insect had been allatectomized. They suggested that the removal of the CA reduced the turnover of fatty acids although the data would seem to suggest that it is the rate of synthesis o f fatty acids which is affected. Using in uitro techniques Gilbert (1967) has shown unequivocally that CA depress the rate of neutral lipid synthesis in fat body from preformed fatty acids when l-14C- palmitate is the marker. Thus the hormone affects lipid synthesis rather than mobilization. More recently, the inhibitory effect of JH on lipid synthesis in the fat body has been confirmed in Schistocerca (Hill and Izatt, 1974). The incorporation of labelled acetate into fat body lipid after incubation of the tissue in uitro was many times greater for tissues taken from allatectomized donors but only after the period of somatic growth had been completed. Labelled acetate is readily converted into fat by insects (Robbins et al., 1960; Louloudes et al., 1961; Sridhara and Bhat, 1964; and Bade, 1964) while Tietz (1961) has shown that it is largely incorporated into triglycerides and phospholipids. Studies on the biosynthesis of fatty acids in mammalian tissues have shown that the mechanism is a reductive synthesis with an absolute requirement for NADPH. The biosynthesis of fatty acids in homogenates and subcellular fractions prepared from fat body has been described (Clements, 1959; Zebe and McShan, 1959; and Tietz, 1961). Tietz (1961) has shown that omission of NADPH from the incubation medium decreased the incorporation of acetate into lipid b y one-half. In insects, as in mammals, the source of much of this reduced NADP is the hexosemonophosphate shunt (pentose cycle). Thus fatty acid synthesis is intimately related t o the activity of the pentose cycle. In this context it is important t o note that following allatectomy of the male desert locust there was an increase in the activity of glucose-6-phosphate dehydrogenase in fat body coincident with an increase in fat body lipid (Walker and Bailey, 197 lc). This increase in enzyme activity signifies enhanced utiliz-

278

J.

E. STEELE

ation of the pentose cycle and is consistent with the observed elevation of lipid synthesis. b. Lipogenic effect of farnesyl methyl ether Although a large number of studies attest to the increase in fat body lipid following allatectomy there are a few reports in which the opposite effect has been described. The study by Butterworth and Bodenstein (1969) has already been cited. In a study which demonstrates how juvenile hormone analogues can disrupt normal metabolism, Mansingh (1972) has investigated the effects of farnesyl methyl ether (FME) on the conversion of fatty acids and glucose to lipid in the pharate adult of Malacosoma pluviale. Nine-day-old pharate adult females which had been treated topically with FME 7 days previously incorporated 100 per cent more glucose into lipid than did untreated controls. Similarly, when 1-’4C-palmitate was used as the marker incorporation into total body lipid was about 25 per cent greater. Incorporation of labelled glucose into glycogen was reduced by 50 per cent. These results may be cited as evidence opposing the idea that JH acts to suppress lipid synthesis, but a more detailed examination of the data reveals that the circumstances are unique. In treated insects the level of total lipid remains high but declines in untreated controls. This effect is reminiscent of the effect which allatectomy has in other species. FME has an anti-gonadotrophic effect in this species (Wellington and Maelzer, 1967) possibly related to the fact that CA or JH, as in most Lepidoptera, are not essential for ovarian development (Engelmann, 1970). Mansingh (1972) has suggested that FME may have a direct effect on protein synthesis, an explanation previously made by Blumenfeld and Schneiderman (1968) to account for the inhibitory effect which JH has on vitellogenin synthesis in fat body of Antheraea polyphemus. c. Conversion of carbohydrate to lipid The synthesis of lipid from carbohydrate is a widely occurring phenomenon taking place by means of well understood pathways. Glycerophosphate arising in the a-glycerophosphate cycle serves as the “backbone” of the lipid molecule while other carbohydrates, converted t o acetyl-CoA, serve as the starting point for the synthesis of fatty acids subsequently conjugated with it. Strong (1963) has demonstrated the conversion of U-’ C-glucose to lipid in aphids while Chino and Gilbert (1965b) obtained similar results with H. cecropia. Horie et al. (1968) using specifically labelled glucose were able t o show that incorporation of glucose into lipid in Bombyx occurred via acetyl-CoA. The possibility that JH decreases the conversion of carbohydrate t o lipid has not received much attention although this would be expected since the incorporation of glucose into lipid is increased by allatectomy (Morohoshi and Fugo, 1972). Bombyx pupae allatectomized just prior to spinning

HORMONAL CONTROL OF METABOLISM I N INSECTS

279

incorporate more U-' C-glucose into fat body lipid than do control insects suggesting that metabolic intermediates are redistributed in the absence of

JH. d. Change in glycolytic and Kreb's cycle enzymes There have been very few attempts to correlate changes in metabolic end products with enzyme activity. Walker and Bailey ( 1 9 7 1 ~have ) measured the activity of phosphofructokinase, pyruvate kinase, malic dehydrogenase, glucose-6-phosphate dehydrogenase, isocitric dehydrogenase, ATP citrate liyase and fructose 1,6-diphosphatase in fat body of allatectomized Schistocerca and found the first two enzymes t o be lower in activity than in controls whereas the remainder were higher in activity. The authors conclude that the change in activity is consistent with the observed increase in lipid synthesis; however, it is difficult t o visualize how a lower phosphofructokinase activity can account for an increased conversion of glucose t o lipid. It is unfortunate that data on the concentration of glycolytic intermediates were not obtained (e.g. glucose-6-P04, fructose-6-P04 and fructose 1,6-diP04) t o provide more information on the rate of flux of glucose through the glycolytic pathway. e. Synthesis of lipid from amino acids The reduction in protein synthesis by fat body (Table 2) following dlatectomy means that amino acids destined for the synthesis of proteins are diverted into alternative pathways. It is likely that many of these amino acids would be deaminated or transaminated and the carbon skeleton converted into lipid, thus increasing the accumulation of lipid in the fat body. Decapitation of adult female Rhodnius (which removes the CA among other things!) caused a doubling of the haemolymph amino nitrogen level when compared with normal females (Coles, 1965). This enlarged pool of amino acids in the haemolymph may well prove to be a major source of the extra lipid synthesized under these circumstances. The conversion of glycine and leucine into lipid has been demonstrated in the locust fat body by Clements (1959). The synthesis of lipid from amino acids suggests transaminase activity may be altered under conditions associated with a change in lipid metabolism. However, Nohel and Slima (1972) have shown that the JH analogue 3,7,1l-trimethyl-7,1l-dichloro-2 dodocenic acid methyl ester has no effect on glutamate-pyruvate transaminase in Pyrrhocoris apterus. Although the specific activity of the enzyme did not change it cannot be assumed that the rate of the reaction within the cell was unaltered since this could have been modified as a result o f change in substrate concentration. f. JH control of lipid synthesis: summary and conclusions We have seen that, in the absence of JH, the decrease in protein synthesis is accompanied by an increase in lipid and glycogen levels in fat body. It is suggested that

280

J. E. STEELE

amino acids and Carbohydrate, which otherwise would have been consumed in the process of protein synthesis, are converted to lipid. At the same time lipid reserves in the fat body are no longer depleted to provide the lipid moiety for conjugation with the apoprotein. The suggested flow of metabolites in the presence and absence of JH is depicted in Fig. 9. When JH is present protein synthesis will occur at a high rate, particularly in the femde, and wiIl require considerable ATP. Much of this is derived from the oxidation of carbohydrate, especially glycogen in the fat body, and to a lesser extent lipid in the same tissue. At the same time the synthesis of free fatty acids from acetyl-CoA requires NADPH generated by the pentose cycle, thus the utilization of glucose-6-P04 (for NADPH synthesis) and Glycogen

Glycogen

I

I

c

G~UCOS+G-6-P

Glucose *G-6-P

F-6-P

NADPH

F-6-P

J JH H

LiDids Lipids

I

'

cycle cycle (a) J H present

transport transport system system

1i

Protein

w

system system

(b) J H absent

Fig. 9. Scheme showing the postulated flow of metabolites in fat body in the presence and absence of JH.

acetyl-CoA (as well as a-glycerophosphate) for lipid synthesis is in direct competition with the generation of ATP for protein synthesis. Furthermore, the contribution by amino acids to oxidative metabolism and lipid synthesis is only minimal when the level of protein synthesis is high. Following allactectomy the situation is reversed. The rate of protein synthesis is reduced and amino acids present in excess of that required for protein synthesis will be deaminated or transaminated and the carbon skeleton made available for lipid and glycogen synthesis. The need for ATP is much reduced and therefore carbohydrate from which it originates is also available for conversion into lipid. The reduction in protein synthesis will mean less lipid is required for conjugation with protein resulting in greater accumulation of lipid due to lessened turnover. The strongest argument for a direct effect of JH on lipid synthesis is the observation that CA depress the rate of incorporation of preformed fatty

HORMONAL CONTROL OF METABOLISM IN INSECTS

28 1

acids into fat body lipid (Gilbert, 1967). The use of preformed fatty acids suggests that inhibition must occur after the fatty acids have been synthesized and would therefore be largely independent of changes in oxidative metabolism. It seems equally likely that the rate of incorporation of preformed fatty acids into lipid is a function of the fatty acid pool size and turnover rate, which, in turn, will be influenced by the rate of oxidation of acetyl-CoA. 4.1.4 Control of ovarian metabolism Although J H is a well-known gonadotrophin there is little evidence t o show that it directly affects metabolism in this tissue. Discounting the influence of the CA on vitellogenin uptake (Bell and Barth, 1971) as a metabolic effect, the demonstration that ovaries of H. cecropia incubated in vitro take up more palmitic acid if CA are present is probably the only study in which a direct relationship between the hormone and ovarian metabolism seems likely (Gilbert, 1967). An enhanced synthesis of phospholipid and glyceride was demonstrated but it is not clear whether JH stimulates lipid synthesis or facilitates the entry of fatty acids into the cell. It may be the latter since entry of vitellogenin, which is under JH control, is by way of the intercellular spaces in the follicular epithelium.

4. 2

DIAPAUSE HORMONE

It is not surprising that lipid metabolism, because of its relationship t o carbohydrate metabolism, also undergoes certain modifications in the diapause egg of the silkworm. These eggs are characterized by a higher lipid content than nondiapsuse eggs (Hasegawa and Yamashita, 1967). Removal of the SOG from pupae expected t o produce diapause eggs leads t o a reduction in lipid content of the eggs from 96.2 mgg-' t o 87.3 m g g - ' . Conversely, the injection of DH extracts into pupae destined t o produce nondiapause eggs causes the egg lipid to rise from 75.7 mgg-' to 81.5 mg g-'. The fat body is the source of lipid deposited in the pupal ovary, the process being either accelerated or extended over a longer period in the presence of DH (Morohoshi and Fugo, 1971). When the SOG is removed from pupae containing developing diapause eggs there is a decline in ovary lipid and a corresponding increase in that of fat body. If SOG are implanted into pupae containing developing nondiapause eggs there is an enhancement of lipid deposition in the ovary and a concomitant fall in the level of fat body lipid (Morohoshi and Fugo, 1971). As with carbohydrate, it is the ovary and not the fat body which appears to be the target for the hormone. Although DH may stimulate the uptake of intact lipids and longchain fatty acids by the ovary the increase in lipid may also result from an

282

J. E. STEELE

activation of fatty acid synthesis. The ,more rapid conversion of 1-' 4Cglucose into ovary lipid in the presence of DH (Morohoshi and Fugi, 1971) supports this suggestion. There is, however, an increase in trehalase activity at this time (Yamashita et al., 1972). This could increase the glucose pool within the ovary and as a result of m q s action accentuate the conversion of glucose into lipid. Thus the effect of DH on lipid synthesis may be secondary t o its effect on carbohydrate metabolism.

4. 3

HYPERGLYCAEMIC HORMONE

It is generally recognized that the pathways of carbohydrate and lipid metabolism are so intimately interwoven that it is impossible to affect one without affecting the other. In addition to its well-known effect on carbohydrate metabolism (section 3.4) HGH is thought t o influence. the oxidation of lipid in fat body. This was first shown by Wiens and Gilbert (1965) who investigated the effects of CC extracts on respiration in Periplanata fat body and noted a decrease in R.Q. from 0.944 t o 0.836. The extract acts to switch energy metabolism t o the oxidation of lipid in preference to carbohydrate. This was confirmed b y measuring the evolution of 4 C 0 2 from either 1-' 4C-acetate of 1-' 4C-palmitate (Wiens and Gilbert, 1967a). With either substrate the increase in I4CO2 in the presence of CC extract was approximately 30 per cent. Complementary studies on the metabolism of U-' C-glucose showed that the oxidation of glucose fell by 46 per cent, yet its incorporation into glycogen was also reduced by 83 per cent. This latter fact is not surprising since much of the glycogen is converted to trehalose under these conditions. Lipid synthesis from glucose is reduced by almost 40 per cent. We have investigated the same problem in our laboratory b u t have used an in vivo approach utilizing a respirometric gas analysis technique. In this method respiratory COz resulting from the oxidation of labelled glucose is monitored continuosuly in a flow-through system connected t o an ionization chamber and an associated electometer. The results are in good agreement with those obtained by Wiens and Gilbert (1965, 1967a) but also show that the enhancement of lipid oxidation is most rapid at the time of hormone injection and disappears 2-3 hours later (Coulthart and Steele, unpublished observations). Recently it has been reported that CC extracts COz from 1-'4C-acetate in intact cockroaches reduce the release of (Hoffman and Downer, 1974), an effect contrary t o those reported by Wiens and Gilbert (1967a). There is no explanation for the apparent contradiction. Because of the lipolytic action of HGH the synthesis and accumulation of ketone bodies in the fat body o f the locust is of considerable importance

'

'

283

HORMONAL CONTROL OF METABOLISM I N INSECTS

(Hill et al., 1972). The origin of the ketone bodies, acetoacetic acid and (3-hydroxybutyric acid, is depicted below. Lipids

Acetyl-CoA

lipase

Lynen cycle

Free fatty acids

Acetoacetic acid

0-oxidation

b

Acetyl-CoA

0-hydroxybutyrate DH

b

6-Hydro xybutyric acid

Desert locusts injected with CC extract increase the concentration of acetoacetic acid in fat body by 317 per cent over the normal level of 0.067 mol g-' The concentration of 0-hydroxybutyric acid declines 1 7 per cent from 0.142 pm mol g-' tissue. Hill et al. (1972) have attempted to relate this effect of the CC t o the adipokinetic hormone which is present but an effect by this factor seems questionable since its function, at least in locusts, seems to be the facilitation of diglyceride release into haemolymph (Mayer and Candy, 1969). Consequently little free fatty acid for ketone body formation would be expected to accumulate. The clear demonstration that HGH favours fatty acid oxidation in fat body (Wiens and Gilbert, 1967a), an effect distinct from that of the hypolipaemic factor (Downer and Steele, 1972) suggests HGH is the hormone favouring ketone synthesis. Its rapid effect on lipid oxidation noted above may indicate that this hormone is important in providing fuel for flight in the early stages as ketone bodies and later as trehalose.

.

4. 4

ADIPOKINETIC HORMONE

Almost from the time it was first recognized that insects capable of maintaining prolonged flight were largely dependent on lipid as a source of energy (Weis-Fogh, 1952) there has been much interest in the possibility that mobilization of fat is endocrine controlled. Diglyceride complexed with haemolymph protein is the main form in which lipid is transported from the fat body t o the flight muscle (Chino and Gilbert, 1965a). Many insects have been shown to have a high level of diglyceride in the haemolymph. The increase from 4.0 mg ml-' t o 16.9 mg ml-' in total fatty acid content of haemolymph in locusts during flight suggests that mobilization of lipid was subject to a high degree of regulation (Beenakkers, 1965). It is now known that this increase in total fatty acids during flight can be accounted for by an increase in the haemolymph diglyceride fraction (Mayer and Candy, 1967; Beenakkers, 1973).

284

J. E. STEELE

4.4.1 The hyperlipaemic response The demonstration of an adipokinetic factor in the CC of locusts is most interesting in view of the importance of lipid as a substrate for flight. Injected gland extract will raise the lipid level of haemolymph 3-4 fold (Mayer and Candy, 1969; Downer, 1972; and Goldsworthy et al., 1972a). The increase is primarily in the diglyceride fraction; the diglyceride is transported t o the site of utilization (Chino and Gilbert, 1965a) and therefore most likely to be subject to hormonal control. The factor obtained from Schistocerca is not species specific since it will cause a hyperlipaemic response in Tenebrio (Goldsworthy et al., 1972a). Tenebrio CC are similarly effective in Schistocerca (Goldsworthy et al., 1972a). It seems that diglyceride released t o the haemolymph does not necessarily arise from preformed triglyceride stored in the fat body but may arise de novo from free fatty acids in the same tissue or even haemolymph (Beenakkers, 1969). Labelled palmitate injected into the haemolymph simultaneously with CC extract is more rapidly incorporated into the newly synthesized diglyceride of hormone-treated locusts than in control locusts (Beenakkers, 1969). This suggests that the hormone activates synthesis of diglyceride as well as partial degradation of pre-existing triglyceride. a. Flight behaviour The flight pattern in locusts is thought t o reflect certain changes in the utilization of oxidizable substrates (Weis-Fogh, 1952). During the first 5-10 minutes of flight the speed is high but gradually decreases over a period of about 25 minutes before attaining a constant or “cruising” speed. During the initial high-speed flight carbohydrate is the preferred fuel whereas lipid is utilized during the low-speed or cruising period (Weis-Fogh, 1952). It has been claimed that the mobilization of lipid is mediated by AKH (Mayer and Candy, 1969; Goldsworthy et al., 1972a, b). Locusts which have had the glandular lobes (containing AKH) removed from the CC have a lower flight speed than do control insects or those in which the neurosecretory cells have been removed (Goldsworthy et al., 1973). When locusts lacking glandular lobes are injected with 0.01 pair of CC at the time flight is initiated there is a period of about 1 5 minutes in which flight speed decreases at the same rate as in control locusts from which the glandular lobes have also been removed. Over a subsequent period of 15 minutes the flight speed of locusts receiving extract returns to that of intact control locusts. When the injection of extract is made 30 minutes prior t o the initiation of flight the pattern is indistinguishable from that of intact controls (Goldsworthy et al., 1973). However, if trehdose is injected along with CC extract into cardiacectomized locusts on the initiation of flight, normal flight speed occurs during the first 30 minutes, presumably because trehalose is

HORMONAL CONTROL OF METABOLISM I N INSECTS

285

available for oxidation. In the period following, flight speed is also normal but not because of trehalose (which has by this time been oxidized) but because of the extract which has presumably mobilized the lipid. This effect of the extract in the post 30-minute period is not observed with starved locusts which are known to be refractory to AKH (Goldsworthy and Coupland, 1974). Injection of CC extract into cardiacectomized locusts raises the haemolymph lipid level t o that found in unoperated controls (Goldsworthy et al., 1972b). If normal locusts are injected with extract at the beginning of flight the initial flight speed is lower than usual and characteristic of that when lipid is the principal fuel. Thus the hormone is viewed as inducing an earlier utilization of lipid (Goldsworthy et al., 1973). The inhibition of the high initial flight speed by the extract has been interpreted as an inhibition of carbohydrate utilization by the hormone and suggests that after initiation of flight there is a lag before secretion of AKH begins. The assumption that reduction in initial flight speed caused by cardiaca extract can be attributed t o an inhibition of carbohydrate metabolism (Goldsworthy et al., 1973) is strengthened by the observation that 0 2 consumption of locust metathoracic muscle is reduced by the hormone when trehalose is the substrate but increased with dipalmitin (Robinson and Goldsworthy, 1974). These are important observations because they suggest the hormone acts directly on muscle as well as fat body. The initial flight speed is decreased by injection of dipalmitin into the haemolymph but it can be maintained at the normal level if CC extract is introduced at the same time, suggesting that utilization of dipalmitin by flight muscle requires the presence of the hormone (Robinson and Goldsworthy, 1974). The studies b y Goldsworthy’s group on the relationship between AKH and flight behaviour pose a number of interesting problems. The change from high initial flight speed to lower cruising speed is coincident with the change-over from carbohydrate t o lipid metabolism. The reason €or the reduction in flight speed is not apparent but possibly indicates that there is a rate-limiting point in the oxidation of fat with consequent reduction in the supply of ATP. b. Adipokinetic effect of epinephrine Bhaktan and Gilbert (1968) tested a number of hormones known to have adipokinetic effects in vertebrates. Cockroach fat body incubated with epinephrine released a significant amount of free fatty acids t o the medium b u t was without effect on mono-, di-, or triglycerides. This effect has been confirmed by a cytological procedure in which fat body of Hyalophora cecropia and Leucophaea maderae was incubated with epinephrine t o determine possible effects on lipid and glycogen stored in the tissue (Bhaktan and Gilbert, 1971). Cecropia fat body contains an appreciable number of lipid droplets but no

286

J. E. STEELE

discernible glycogen or protein bodies. Epinephrine produced a gradual depletion of the lipid with time. In contrast to Cecropia fat body that of Leucophaea contains glycogen and protein as well as lipid. In this tissue only glycogen was reduced. Since insect fat body in viuo releases fatty acids in the form of diglyceride rather than the free acids the physiological significance of these observations remains obscure. 4.4.2 The hypolzibaemic response ‘The adipokinetic effect elicited b y CC in locusts does not occur in cockroaches and is replaced in that species b y a hypolipaemic response (Downer and Steele, 1969, 1972; Goldsworthy et al., 1972a), which if not pronounced is statistically significant. The extract causes a decline in the level of haemolymph triglycendes and diglycerides, the effect on the former being greater (Downer and Steele, 1972). More recent in vitro studies have confirmed the original observations. Female CC have a more potent effect on fat body uptake of triglyceride from the haemolymph than male glands (Steele and Sholdice, unpublished observations). The factor may be physiologically important in directing dietary lipid into reserve storage in the fat body (Downer and Steele, 1972). Recent studies by Downer (1972) and Goldsworthy et al. (1972a) suggest that the hypolipaemic factor may be identical with AKH. Treatment of locusts with extract prepared from the corpora cardiaca of Penplaneta produces the typical hyperlipaemic response in the locust, yet it has a hypolipaemic effect in the cockroach. If the extract is prepared from locust corpora cardiaca the response obtained when tested in both species is determined by the host. It appears that the hormone may be identical. Gilbert and Chino (1974) have suggested that in the course of evolution the structure of the hormone did not change but rather the response as the principal metabolic pathways associated with the metabolism of lipid diverged.

5 Control of amino acid metabolism There are only three situations in which the metabolism of a specific amino acid in insects is known t o be directly controlled by a hormone; in each instance the amino acid is tyrosine whose function, following conversion to a quinone, is t o tan or sclerotize a protein. The role of quinones in the tanning of insect proteins was first described by Pryor (1940) and more recently the chemistry of the reactions has been discussed by Brunet (1965).

HORMONAL CONTROL OF METABOLISM IN INSECTS

5. 1 MOULTINGHORMONE 5.1.1 Synthesis of N-acetyldopamine-quinone Mature larvae of cyclorrhaphous diptera pupate within the larval cuticle that is retained and referred t o as a puparium. The change from larval cuticle t o puparium is characterized by a hardening and darkening of the structure due t o the sclerotization of the cuticular proteins. During this period of development metabolism of tyrosine is profoundly influenced by MH. Tyrosine metabolism is predominantly by way of transamination and degradative reactions prior t o pupation but o n release of MH is emphatically shifted towards the synthesis of N-acetyldopamine. The biosynthesis of the tanning agent N-acetyldopamine-quinoneas elucidated in Calliphora is by means of the pathway tyrosine + dopa + dopamine + N-acetyldopamine (Sekeris and Karlson, 1966). The quinone configuration is derived from N-acetyldopamine by the action of a phenol oxidase. Little is known of this important enzyme but it has been shown t o require an activator enzyme (Karlson and Schweiger, 1961). The activator is synthesized in response t o MH since removal of the ring gland causes its disappearance and a fall in phenol oxidase activity. Injection of ecdysone into Calliphora larvae lacking ring glands reinitiates synthesis of the activator and reappearance of phenol oxidase (Karlson and Schweiger, 1961). Although this enzyme undoubtedly affects the rate of tyrosine metabolism via dopa it is not responsible for diverting it from other catabolic routes t o that through dopa. That shift is made possible by the appearance of dopa decarboxylase in response t o MH. Decarboxylase activity is thought t o increase because of induction of new enzyme protein by the hormone (Karlson and Sekeris, 1962a) but this has not been proved conclusively. The importance of MH in redirecting the metabolism of tyrosine is shown by the fact that 80 per cent of U-I4C-tyrosine injected into late last-stage Calliphora larvae is incorporated into puparial cuticle, having traversed the route described above (Karlson, 1960).

5.1.2 The source of tyrosine The large amount of tyrosine utilized in the process of sclerotizing the cuticle places a severe demand on the reserves present in haemolymph and tissue. A pre-ecdysial increase in the titre of tyrosine has been demonstrated in blowflies (Fraenkel and Rudall, 1947; Hackman, 1956; Levenbook and Dinamarca, 1966), in mealworms (Patterson, 1957) and silkworms (Duchiteau-Bosson et al., 1962). The changing tyrosine concentration in the haemolymph of Pieris brassicae during the transition from the fourth to fifth instar has been described by Post and DeJong (1973). An

J. E. STEELE

288

increase in tyrosine, detectable just prior, t o apolysis, suggests that it is triggered by MH. Sarcophaga has been shown to contain at least two distinct tyrosine reserves (Seligman et al., 1969). Prior t o the adult moult there is approximately 140 nmol p l - ’ of tyrosine-0-phosphate in haemolymph, falling t o about 75 nmol1.11- 5 hours after the moult. During this period the concentration of tyrosine remains more o r less constant at 60 nmol PI-’. Since tyrosine phosphate can be converted t o free tyrosine the decrease in tyrosine phosphate is thought t o represent the replacement of the tyrosine utilized in the cuticle. High levels of tyrosine-0-phosphate have also been demonstrated in Drosophila (Mitchell and Lunan, 1964; Lunan and Mitchell, 1969). The kinetics of tyrosine phosphate turnover indicate there is insufficient t o account for the tyrosine being consumed, and an additional source of tyrosine is suggested. In Sarcophaga it is apparently the unique dipeptide P-alanyl-L-tyrosine (“sarcophagine”) (Bodnaryk and Levenbook, 1969). Injection of 0-ecdysone into young larvae produced precocious tanning of the larval cuticle and concomitant hydrolysis of the dipeptide (Bodharyk, 1971). The evidence suggests that release of tyrosine from the dipeptide is dependent on the appearance of a specific dipeptidase whose synthesis may be directed by MH but a direct demonstration of this is lacking. The distribution of the dipeptide may be restricted to a few species in which case the pre-ecdysial accumulation of tyrosine in other species would have another origin. The timing of MH release and tyrosine appearance suggests that an investigation of possible effects of ecdysone on this aspect of tyrosine metabolism would be profitable.

’

5.1.3 Tyrosine metabolism in adults It is not surprising that hardening and darkening of mosquito eggs is associated with the presence of dopa-decarboxylase. What is unique is that either the activation or synthesis of the ovarian enzyme in the adult is under the control of MH released from an unknown source following a blood meal (Schlaeger and Fuchs, 1974; Schlaeger et al., 1974). The finding that the effect of the hormone is mediated by cyclic AMP (Fuchs and Schlaeger, 1973) is of particular interest and may be important in the elucidation of ecdysone action.

5. 2 JUVENILE HORMONE 5.2.1 Glucoside synthesis in colleterial glands The cockroach ootheca consists of a tanned protein produced by the combined secretions of the right and left colleterial glands (Pryor, 1940).

HORMONAL CONTROL OF METABOLISM I N INSECTS

289

Unlike cuticle it contains no chitin. The left gland secretes a structural protein forming the bulk of the egg capsule and protocatechuic acid-4-0, 0-glucoside (Brunet and Kent, 1955). Cleavage of the glucoside by a glucosidase produced in the right gland frees the protocatechuic acid (3,4-dihydroxybenzoic acid) which, o n oxidation t o the quinone, tans the protein (Whitehead et al., 1960). There is no doubt that the quinone obtained from protocatechuic acid is the tanning agent since label derived from U-14C-tyrosine can be detected in the acid after injection (Brunet, 1963; Shaaya and Sekeris, 1970) but the intermediate steps in its synthesis are unknown. Decapitation of cockroaches prevents glucoside accumulation in the left colleterial gland while implantation of CA reinitiates the process (Willis and Brunet, 1966; and Takahashi, 1972). Essentially similar results have been obtained by Shaaya and Bodenstein (1969) who not only confirmed the requirement for the CA but also showed that another factor, present only in females, was necessary for glucoside accumulation in the gland. They conclude that synthesis of the glucoside in the gland is dependent on JH while Shaaya and Sekeris (1970) suggest, in the absence of direct evidence, that the hormone acts at some point in the biosynthetic pathway between tyrosine and protocatechuic acid prior t o the formation of the glucoside. The left colleterial gland is not the sole site of glucoside synthesis since Shaaya and Sekeris (1970) have demonstrated that fat body and integument are also able t o perform the synthesis. Although the enzyme system of the colleterial gland has the highest specific activity the total activity in either integument o r fat body is higher so that these sites may b e quantitatively more important. That JH does not directly affect the glucoside-synthesizing mechanism seems certain since allatectomy does not alter the rate of glucoside synthesis in the colleterial gland or fat body (Takahashi, 1972). Where then does the hormone act? The important clues may be the following observations.

1. When colleterial glands are transplanted t o male hosts in the presence of JH there is no accumulation of the glucoside in the gland (Shaaya and Bodenstein, 1969) even though male fat body and integument can synthesize the glucoside (Shaaya and Sekeris, 1970). An apparent difference between the male and female milieu is the absence from the former of the female specific protein o r vitellogenin.

2. Injection of actinomycin D will inhibit the accumulation of glucoside (Takahashi, 1972) and simultaneously inhibit the synthesis of vitellogenin by the fat body (Engelmann, 1971). 3. Allatectomy causes an accumulation of glucoside in the blood (Takahashi, 1972) suggesting that synthesis or activity of a transport mechanism has been impaired.

290

J. E. STEELE

As a working hypothesis it is suggested that vitellogenin functions in the transport of glucoside from its site of synthesis in fat body and integument to the colleterial gland. The effect of J H on glucoside synthesis would thrrefore be mediated through an effect on vitellogenin synthesis. This idea is attractive because it permits synchronization between egg and ootheca production. During these periods when vitellogenin is absent the tendency for glucoside to accumulate in haemolymph would cause its synthesis to be shut off b y feedback control. 5.2.2 Regulation of uric acid production The role of JH appears paradoxical t o the point where a generalized scheme of control, applicable even to a single species of insect, is impractical. According to Bodenstein (1953) and Bruchhaus (1972) allatectomy of Penplaneta does not alter the level of uric acid in fat body, nor its accumulation with time, if the operation is performed more than 3 br 4 weeks after the imaginal moult. However, whole body (less digestive tract) uric acid in Penplaneta remains constant at 45 mg g-' following allatectomy within 2 days of the final moult but that of sham-operated controls continues t o increase (Thomas and Nation, 1966b). Two weeks after the operation the controls contained twice as much uric acid as did allatectomized cockroaches. Why then does allatectomy of young cockroaches, but not older ones, lead to a reduction in uric acid accumulation? Parallel determinations on incorporation of labelled glycine, leucine and histidine into fat body and ovary protein showed that allatectomy reduced the rate of protein synthesis by as much as 60 per cent (Thomas and Nation, 1966b). A reduction in protein synthesis (Table 2) suggests that the additional amino acids available for transamination and deamination would increase the synthesis of uric acid. Indeed removal of the CA may lead t o large increases in the haemolymph concentration of amino acids as suggested by decapitation experiments on Rhodnius (Coles, 1965). The recent finding that allatectomy of male locusts causes uric acid excretion to increase from 86.7 mg t o 111.5 mg during the 9 days following the operation (Mordue and Goldsworthy, 1973) supports the idea that more amino nitrogen is being converted t o uric acid. A curious fact t o emerge from these studies is that the CA apparently favour storage of the uric acid within the insect rather than excretion although the CA, in the absence of the CC, appear t o reduce the accumulation of uric acid in the fat body. This effect of the CA is difficult t o explain because the CC also increase uric acid synthesis. 5.2.3 Transaminase activity The effect of J H and its analogues on transaminase activity is also difficult to interpret. Allatcctomy of Periplaneta led to a decrease in muscle

HORMONAL CONTROL OF METABOLISM I N INSECTS

29 1

glutamate-oxalacetate transaminase (GOT) activity during periods of 50-90 days after the operation (Wang and Dixon, 1960). Removal of the CA in Locusta was without effect on GOT both in flight muscle and fat body but glutamate-pyruvate transaminase (GPT) showed a significant increase in both tissues (Mordue and Goldsworthy, 1973). Injection of farnesyl methyl ether into Tenebrio pupae at the beginning of pupation increased GOT activity to 55 nmol min-’ mg-’ protein from 35 nmoI min-’ mg-’ protein (Emmerich et al., 1965) although Nohel and Slima (1972) found that the analogue trans, 3,7,11-trimethyl-7,1l-dichloro-2-dodocenic acid methyl ester was without effect on GOT activity in last instar nymphs of Pyrrhocoris apterus. The shortest period that must elapse before an effect of hormone on transaminase activity can be detected appears to be about 2 days (Emmerich et al., 1965). This suggests that the CA do not activate transaminase directly, the increase observed probably resulting from de nouo enzyme synthesis. The lack of effect of CA on GOT and GPT activity in fat body and flight muscle of Locusta during the 3-hour post-injection period (Mordue and Goldsworthy, 1973) supports this conclusion. Much controversy has surrounded the change in transaminase activity and its relationship to protein synthesis and degradation. It seems likely that both processes will affect transaminase activity since the need to convert dietary amino acids to a profile characteristic of the species may be just as important as the need to degrade unwanted amino acids. Whether enzyme activity increases or decreases will probably be determined by the quantity of amino acids being “processed”. 5. 3 BURSICON 5.3.1 Mechanism of cuticular tanning Tanning of the cuticle in newly moulted insects of many species is controlled by a hormone produced and released from the central nervous system. The occurrence of this factor was first demonstrated in blowflies by CottreIl (1962a, b) and Fraenkel and Hsiao (1962, 1963) and has been given the name “bursicon” (Fraenkel and Hsiao, 1965). Bursicon action can be distinguished from that of MH because the sclerotization which it induces is not affected by actinomycin or puromycin (Fogal and Fraenkel, 1969) suggesting it does not initiate de nouo synthesis of an enzyme. Cuticular tanning under the influence of bursicon is not altogether different from that occurring in the puparium. It is believed that the corresponding quinone of N-acetyldopamine is the primary tanning agent. The presence of N-acetyldopamine during tanning of the cuticle after ecdysis has been demonstrated in Tenebrio (Karlson and Sekeris, 1962b) and its synthesis from tyrosine shown in the blowfly (Sekeris, 1964), locust (Karlson and Herrlich, 1965) and cockroach (Mills et al., 1967). Thus the hormone

292

J. E. STEELE

appears to induce tanning by increasing the activity of one or more enzyme-catalysed steps in the synthesis of the quinone from tyrosine. 5.3.2 Site of dopamine synthesis Haemocytes are the primary site of dopamine and N-acetyldopamine synthesis associated with cuticular tanning in the cockroach (Mills and Whitehead, 1970). The synthesis of these compounds is much enhanced in the presence of bursicon. When U-' 4C-tyrosine is incubated with pre- and post-moult blood of Periplaneta the uptake of label into those haemocytes present in the post-moult haemolymph is almost doubled. Blood taken from this stage is known to contain bursicon. The pre-ecdysial build-up of tyrosine in haemolymph has already been referred t o (section 5.1.2). Following the moult there is a rapid decline in haemolymph tyrosine coinciding with the appearance of bursicon (Post and DeJong, 1973). In Sarcophaga the decline in tyrosine is not so apparent because reserves in the form of tyrosine phosphate (Seligman et al., 1969) and 0-alanyl-L-tyrosine (Bodnaryk and Levenbook, 1969) are present. Kinetic studies in which bursicon release was controlled by ligatures showed that consumption of tyrosine was much greater in the presence of bursicon (Seligman et al., 1969). Increasing the permeability of the haemocytes t o tyrosine facilitates the synthesis of its derivatives as indicated by the fact that the rate of dopamine formation is ten times greater in blood taken 3 0 minutes after eclosion than in blood taken immediately after (Whitehead, 1970). The presence of dopa-decarboxylase in the haemocytes of Sarcophaga dictates that tyrosine be converted t o dopamine via dopa. The biosynthetic pathway in the haemocytes of Periplaneta follows a different route and labelled tyrosine is converted t o tyramine by means of a tyrosine decarboxylase before being converted to dopamine. When a partially purified extract of the terminal abdominal ganglion was added t o the incubation medium the synthesis of tyramine increased four-fold and dopamine eight-fold (Whitehead, 1969), so that the path of synthesis appears t o be tyrosine + tyramine + dopamine. It is possible that dopamine is further converted t o N-acetyldopamine before it is exported from the haemocyte. 5 . 3.3 The effect of bursicon o n haemocytes The mechanism by which bursicon changes the permeability of the haemocyte is important because it appears to be the limiting step in the series of events leading up to the tanning of the cuticle. The cockroach h.iemocyte preparation used by Mills and Whitehead (1970) also exhibited a high rate of tyrosine uptake in the absence of hormone although showing a significant increase in permeability to tyrosine in the presence of bursicon. As Post (1972) points out this is an unlikely situation in the

HORMONAL CONTROL OF METABOLISM IN INSECTS

293

intact insect. He has shown that haemocyte membranes of Pieris brassicae can be stabilized and the rapid uptake of tyrosine prevented, without interfering with bursicon-enhanced uptake, by addition of 0.1 mM sodium diethyldithiocarbamate t o the incubation medium. He suggested it had an inhibitory effect on tyrosinase released from ruptured haemocytes thus preventing the accumulation of polyphenols which cause membrane deterioration. Inclusion of CaZ+ and Mg2+ in the incubation medium enhances the metabolism of tyrosine about three-fold (Post, 1972). This effect of divalent ions is reminiscent of the permissive effect which CaZ+has on 5-HT and cyclic AMP-stimulated fluid secretion in Calliphora salivary glands (Prince et al., 1972). Injection of cyclic AMP into ligated CaZZiphora (von Knorre et at., 1972) and ligated untanned thoracic segments of Penplaneta (Vandenberg and Mills, 1974) causes darkening of the cuticle. If cyclic AMP is injected together with C-tyrosine there is an eight-fold enhancement in the incorporation of labelled metabolites into cuticle when compared with control insects (Vandenberg and Mills, 1974). Thus cyclic A M P mimics the action of bursicon. Vandenberg and Mills (1974) suggest that the hormone intereacts with an adenyl cyclase in the haemocyte membrane to promote the synthesis of cyclic AMP which in turn increases the permeability of the membrane t o tyrosine. Recently they have confirmed the presence of an adenyl cyclase in the membrane (Vandenberg and Mills, 1975).

5.3.4 Synthesis of dopamine 3-0-sulphate Bursicon, by virtue of its ability to expedite the synthesis of dopamine from tyrosine, may stimulate the synthesis of a number of dopamine derivatives. Newly moulted cockroaches contain dopamine 3-0-sulphate whose concentration decreases sharply as sclerotization proceeds (Bodnaryk and Brunet, 1974). Only the dopamine moiety is incorporated into the new cuticle. It is likely that bursicon will be found to increase the concentration of this metabolite at the time of ecdysis. Its conversion t o N-acetyldopamine-3-0-sulphate has also been demonstrated (Bodnaryk et al., 1974) but it is not known whether it takes place in haemocytes or epidermal cells. The formation of the sulphate derivative is of great importance if its function is t o ensure protection from enzymatic degradation before translocation t o the outer layers of the cuticle. A similar explanation may apply to the finding that 3,4-dihydroxyphenylaceticacid, derived from dopamine, has been found in haemolymph of newly moulted cockroaches where it is present as the 0-glucoside (Koeppe and Mills, 1974). The significance of the diphenolic glucoside is unknown but the fact that glucosylated proteins are transported across membranes may indicate that the glucose acts as an identifying marker.

J. E. STEELE

294

5. 4

CONTROL OF NITROGEN METABOLISM BY THE CORPORA CARDIACA

The mechanism by which CC influence nitrogen metabolism appears to differ from that of the CA. Their removal results in a loss of urates from the fat body of Periplaneta, an effect which can be reversed by the implantation of CC (Bodenstein, 1953). These results have been confirmed in the same species by Bruchhaus (1972) who showed that cardiacectomy of 70-day-old adult females caused a 45 per cent decrease in uric acid stored in the fat body 70 days after the operation. The corresponding figure for males is 35 per cent. Removal of the CA had no effect during this period. In contrast, removal of the medial neurosecretory cells (assumed to be the source of the active factor in the CC) in Locusta reduces total uric acid excreted during the nine days following the operation to 46.4 mg from 86.7 mg in unoperated insects, an effect easily seen on the first day. Concomitant with these changes is an 87 per cent decrease in glutamatepyruvate transaminase (GPT) activity and a 6 3 per cent decrease in glutamate-oxalacetate transaminase (GOT) activity (Mordue and Goldsworthy, 1973). If CC extracts are injected in vivo enzyme activity returns to near normal levels within 3 hours. This effect, where the CA have none, clearly shows that the CC have an entirely different modus operandi on nitrogen metabolism. The question remaining concerns the fate of amino nitrogen when the CC are not present since it cannot be accounted for either as stored or excreted uric acid. 6 Effect of hormones on respiration It is thought that energy metabolism and therefore the rate of respiration is controlled by the level of ADP within the cell. The performance of biological work causes hydrolysis of ATP and a rise in ADP concentration leading to increased oxidative phosphorylation and oxygen consumption. A decrease in the demand for ATP has the opposite effect. The measurement of oxygen consumption following an experimental alteration in hormone titre has been a popular method of studying hormonal effects but is of little value without accompanying data on changes in the rate of chemical reactions or work performed. Only when correlations have been made and analysed can oxygen consumption later be used t o monitor the effect of hormones on specific processes. The data presented in Tables 4-7 showing the effect of CA and CC on oxygen consumption allow the following generalizations. 1. Allatectomy reduces oxygen uptake in whole insects. 2. Implantation of CA, particularly into allatectomized insects, increases oxygen uptake. 3. The fat body is a principal target for CA.

TABLE 4 Effect of corpora allata and juvenile hormone o n respiration in whole insects Species

Leucophaea maderae Adult $’ Adult 9

Operation

Effect o n oxygen consumption

Reference

CA implanted

43%increase 5 days after operation

Sagesser (1960)

CA implanted into allatectomized hosts

66%increase

Luscher and Leuthold (1965)

Carausius morosus Adult Adult

CA implanted into allatectomized hosts AJlatectomy

No effect

Neugebauer (1961)

No effect

Courgeon (1966)

Locusta migratoria

Allatectomy

29% decrease 10 days after operation

Roussel (1963)

Schistocerca gregaria

Allatectomy and cardiacectomy

60%decrease 10 days after operation

Lafon-Cazal and Roussel (1971)

Gryllus domesticus Adult 9 Last instar nymph

Allatectomy

50% decrease 25 days after operation No change

Roussel (1967)

Pywhocoris apterus Adult 9

CA implanted Allatectomy

Roussel (1967) Slama (1964b)

Allatectomy Allatectomy

Decreased. Abolishes cyclical change in respiration coincident with reproductive cycles No effect No effect

S l b a (1 964c) S l h a (1964~)

Pyrrhocoris apterus Last instar nymps

CA implanted

Increase in latter part of instar

Novak and Slima (1962)

Antheraea polyphemus Pupae

Cecropia oil injection

Increase by those pupae which show retention of pupal characters as adults

Steen (1961)

Adult d Adult d

TABLE 4-continued Species

Operation

Effect on oxygen consumption

Reference

~~

Galleria mellonella Last instar larvae

CA implant

Increase

Sehnal and Slima ( 1 9 6 6 )

Leptinotarsa decemlineata

Allatectomy after diapasue broken

20% decrease 35 days after operation

DeWilde and Stegwee (1958)

Tenebrio molitor

Injected with famesyl methyl ether

Depression of 0 2 consumption during pupal development is lessened

Schmialek and Drews (1965)

Calliphora erythrocephala

Allatectomy

24% decrease 7 days after operation 19% increase 2 days after operation Decrease

Thomsen ( 1 9 4 9 )

0

0 0

3 CA implanted

d

Allatectomy

Thomsen ( 1 9 4 9 ) Thomsen ( 1 9 4 9 ) Irn

cn

4rn

r rn

0

0

z

TABLE 5 Effect of corpora allata and juvenile hormone on respiration in intact tissues

-4

a

52 $ I

Species

Leucophaea maderae Adult 9 Adult 0 Adult 0

Nauphoeta cinerea Adult

Pat body incubated with CA

Effect on oxygen consumption 10.8% increase

Reference

-

Luscher and Leuthold (1965)

5

4

D

c*l

Allatectomy Allatectomy

42% decrease in fat body respiration 22% increase in thoracic muscle

CA implanted into decapitated host

78% increase in fat body respiration

Allatectomy

50% decrease in abdominal segments 15 days after operation

Miiller and Engelmann (1968) v)

Samuels (1956) Luscher (1968)

K

9

Pyrrhocoris aptelus Adult

Operation

0

Slima (1965)

-I v)

N

W

03

TABLE 6 Effect of corpora allata and juvenile hormone on mitochondrial and homogenate respiration Species

Operation

Effect on oxygen consumption

Reference

Allatectomy

No effect on fat body mitochondria

CA extract added t o fat body and muscle homogenates

20% increase in muscle; 40% increase in fat body. Substrate: succinate

Allatectomy CA added to fat body and muscle mitochondria Allatectomy

N o effect on mitochondria1 respiration N o effect. Substrate: pyruvate/ malate o r a-glycerophosphate 10-20% increase in fat body and muscle mitochondria. Substrate: succinate

Sckistocerca pegaria

Allatectomy

Results as above

Clarke and Baldwin (1960)

Plodia interpunctella

Mitochondrial respiration in presence of 0.177 mM Cecropia

Inhibition with pyruvate, malate, a-ketoglutarate and glutamate. Stimulation with succinate, aglycerophosphate and ascorbate

Firstenberg and Silhacek (1973)

120% increase

DeWilde and Stegwee (1958)

Blaberus discoidalis Adult Adult 9

Locusta migratoria Adult Adult Adult

JH Leptinotarsa decemlineata Diapausing ?

50 CA per g of whole tissue homogenate

Keeley (1970, 1972), Keeley and Friedman (1969), Keeley and Waddil (1971) Ralph and Matta (1965)

Minks (1967) Minks (1967) Clarke and Baldwin (1960)

rn v)

;rn ;i r rn

I

TABLE 7 Effect of corpora cardiaca on whole body, intact tissue and mitochondria1 respiration Species

Operation

Effect on oxygen consumption

0 10

3 0 Reference

-

Effect of cardiaca extract on muscle homogenate in vitro

10%increase

Ralph and Matta (1965)

Blaberus disco idalis Adult d Adult d

Cardiacectomy and allatectomy

36% decrease in fat body 30 days after operation 50% decrease in fat body mitochondria 38% decrease in succinate-cytochrome C reductase. 3 7% decrease in cytochrome C oxidase activity 31.8% decrease in fat body N o effect

Keeley and Friedman (1967)

Keeley and Waddil (19 7 1) Keeley (1971)

28% increase

Wiens and Gilbert (1967a)

72.2% increase

Luscher and Leuthold (1965)

60% increase

Miiller and Engelmann (1968)

Cardiacectomy and allatectomy

Adult d

Cardiacectomy and allatectomy

Adult d Adult d

Cardiacectomy and allatectomy Effect of cardiaca extract on fat body mitochondria in uitro

Adult ?

Nauphoeta cinerea Adult 9 Locusta mipatoria

?

n

Btaberus cranifer

Leucophaea maderae Adult d Adult ?

z

Effect of cardiaca extract on fat body in uitro Effect of cardiaca extract on fat body in vitro Cardiaca reimplanted into allatectomized and cardiacectomized hosts

Cardiaca implanted into decapitated N o effect hosts Effect of cardiaca extract on Small increase with dipalmitin as flight muscle respiration in uitro substrate-decrease with trehalose

Keeley (1972), Keeley and Friedman (19 69) Keeley (1970, 1971, 1972)

Luscher (1968) Robinson and Goldsworthy (1974)

W

0 0

TABLE 7-continued Species

Operation

Effect on oxygen consumption

Reference

~~

Locusta migratoria

Effect of cardiaca extract on muscle mitochondria in vitro

28% decrease ( n = 1 )

Clarke and Baldwin ( 1 9 6 0 )

S chisto cerca gregaria Adult d?

Cardiacectomy

26.6% decrease in whole body respiration 10 days after operation

Lafon-Cazal and Roussel ( 1 97 1 )

Oncopeltusfascintus

Removal of medial neurosecretory cells

20% reduction in whole body respiration

Conradi-Larsen ( 1 9 7 0 )

Pyrrhocoris apterus

Cardiacectomy and allatectomy

Decrease. Greater than with allatectomy alone

Slima ( 1 9 6 4 b , c)

HORMONAL CONTROL OF METABOLISM IN INSECTS

301

4. The CA have no consistent effect on mitochondrial respiration in vivo or in vitro. 5. CC stimulate respiration in intact fat body. 6. Long-term cardiacectomy depresses the basal level of mitochondrial respiration. There are exceptions to these generalizations, the most notable being the lack of effect by CC on fat body respiration in Nuuphoetu (Luscher, 1968), which may possibly be due to differences in experimental techn:q 1 ue. Respiration during the intermoult period of Rhodnius follows a U-shaped curve (Zwicky and Wigglesworth, 1956), an upward trend not occurring until after the release of MH. Wigglesworth (1957) points out that very shortly after feeding there is an increase in the synthesis of RNA followed almost immediately by an increase in protein and concludes that the respiratory pattern is, in effect, a pattern of protein synthesis. In recent years much additional evidence has been accumulated t o show that MH stimulates the synthesis of RNA and protein. This is considered to be the principal reason for its stimulatory effect on respiration. The role of MH in diapause respiration has already been adequately reviewed (Harvey, 1962) and will not be considered here. The realtionship between JH and protein synthesis, as determined by allatectomy and replacement therapy in larvae and adults, has already been discussed (section 4.1.2). The data show that synthesis of a number of quantitatively important proteins is regulated by JH; moreover, the change in respiration following allatectomy or implantation of CA is in keeping with the highly endergonic nature of protein synthesis. 6.1

ENDOCRINE CONTROL OF RESPIRATION IN ISOLATED TISSUES

Although an association between protein synthesis and oxygen uptake may appear self-evident, modified respiration following a change in hormone titre may be due to other causes. Hormones also stimulate active processes such as gut movement and heartbeat (Brown, 1965), trehalose synthesis in fat body (Steele, 1963), diglyceride release by fat body (Mayer and Candy, 1969) and water reabsorption in the rectum (Wall, 1967). Any change in the rate of these processes will lead to a change in respiration. Unfortunately there are few reports in which the CA have been tested for an effect on respiration in isolated intact fat body. Liischer and Leuthold, (1965) and Wyss-Huber and Luscher (1966) showed that CA caused a 10 per cent rise in respiration but the increase was not statistically significant. In contrast to the CA the CC have a pronoupced activating effect on the respiration of isolated intact fat body which is usually evident after a few minutes (Liischer and Leuthold, 1965; Wiens and Gilbert, 1967).

302

J. E. STEELE

TABLE 8. Relationship between 0 2 consumption and protein synthesis in fat body of Leucophaea maderue'

Source of fat body

Percentage change in 0 2 uptake

Percentage change in protein synthesis

CA

CC

Brain

CA

CC

Brain

5-Day-old oocyte maturing female

+7

+38

+25

+1

+10

+9

40-Day-old pregnant female

+10

+40

+42

+1

-9

+2

* Data from Liischer et al. (1971). Percentage change in 0 2 consumption and protein synthesis is by comparison with control following 4 h incubation period. An attempt t o correlate protein synthesis .with respiration in the presence of CA, CC and brain has been made by Liischer et al. (1971). The results, summarized in Table 8, indicate that the relationship between protein synthesis and respiration is not a simple one. Protein synthesis is not demonstrably affected by CA during the 5-hour course of the experiment although there is a slight (approximately 10 per cent) increase in oxygen consumption. Brain and CC have essentially similar effects on respiration and protein synthesis, undoubtedly due t o the presence of active factors in the CC originating in the brain. Fat bodies of 5-day-old females containing maturing oocytes show a 20 per cent increase in protein synthesis and a 40 per cent rise in respiration 4 hours after the addition of CC t o the medium. However, fat body taken from 40-day-old pregnant females shows no increase in protein synthesis in response t o the CC but continues t o show the same rise in respiration. Clearly it is dangerous to assume that because a gland has effects on both protein synthesis and respiration the one is dependent on the other. In this instance it appears that the 40-day-old fat body is refractory to that component present in CC responsible for stimulating protein synthesis and the increase in oxygen consumption reflects the activity of another factor in the gland. HGH, whose effect on oxygen uptake has already been mentioned, is probably responsible. Although the release of diglyceride is an active process (Chino and Gilber, 1965a) it would probably not be a factor in determining respiratory rate in these experiments since haemolymph is necessary for release of diglyceride but was not incorporated into the medium. In spite of the fact that CC will induce protein synthesis in Leucophaeu and Schistocerca fat body in uitro (Osborne et al., 1968; Liischer et al., 1971) the

HORMONAL CONTROL OF METABOLISM IN INSECTS

303

findings of Luscher et a[. (1971) make it apparent that changes in respiration due t o a change in protein synthesis are not detectable under in uitro conditions. Since Engelmann (1969) has shown that vitellogenin does not make its appearance in response to JH until about 3 days following treatment it is not surprising that the CA have no effect on respiration which can be attributed to protein synthesis during the relatively short incubation period used by Luscher et al. (1971). The induction period preceding the appearance of vitellogenin and the long-lived mRNA associated with its synthesis (Engelmann, 1971, 1972) probably explains why insects that have been allatectomized or implanted with CA often require a few days or longer to achieve the new respiratory plateau. 6.2

ENDOCRINE CONTROL OF MITOCHONDRIAL RESPIRATION

The reduction in whole body respiration caused by allatectomy naturally suggested that CA may act directly on the mitochondria although there is no conclusive evidence that any of the factors contained in the CA or CC act in this fashion. Clarke and Baldwin (1960) tested CA for an effect on mitochondria prepared from flight muscle and fat body of Locustn and found an 8-20 per cent increase in oxygen consumption. Similarly Ralph and Matta (1965) found that respiration of cockroach muscle and fat body homogenates was increased by 20 per cent and 40 per cent respectively when incubated in the presence of extract prepared from five CA. The respiration of whole body homogenates of Leptinotarsa increased by 120 per cent in the presence of 50 CA (DeWilde and Stegwee, 1958). In each instance the substrate used was succinate. In contrast to these studies Minks (1967) found that CA had no effect on respiration ofLocusta muscle or f a t body mitochondria when a-glycerophosphate or pyruvate-malate was the substrate. Allatectomy some days prior to the removal of the tissues has no effect on endogenous mitochondrial respiration or enzymes and electron acceptors associated with the electron transport system (Minks, 1967). Although respiration is promoted by CA in vitro when succinate is the substrate, a strong inhibition by JH, at least in vitro, can be seen when other substrates such as pyruvate, malate, glutamate and a-ketoglutarate are employed (Firstenberg and Silhacek, 1973). JH appears to have an effect on the electron transport system prior to the entry of electrons at the level of ubiquinone. Aged mitochondria of Plodia lose the ability to oxidize pyruvate-malate but can still oxidize NADH (Firstenberg and Silhacek, 1973). The oxidation of NADH in these mitochondria reflects coupling of electron transport to oxidative phosphorylation as shown by the inhibition of respiration induced by oligomycin (a specific inhibitor of oxidative phosphorylation). Under these conditions JH inhibits respiration as it

304

J. E. STEELE

would in fresh mitochondria with pyruvat,e-malate as the substrate. These results are taken t o indicate that JH inhibits electron transport between NADH and ubiquinone. The results are interesting but do not explain why oxidation of succinate is increased by JH nor do they explain why JH inhibits the oxidation of NADH by mitochondria in vitro but appears to enhance it in vivo, assuming that most substrates are oxidized via NADH in the mitochondria. The effect of allatectomy and CA implantations on respiration manifests itself only after many hours or even days (Table 4). Thus the effect of CA or JH on mitochondrial respiration in vitro must be viewed with some suspicion. The best explanation for the in vitro action of JH seems to be that it alters the mitochondrial membrane such that electron transport between NADH and ubiquinone is disrupted. An increase in permeability towards succinate would explain why it is oxidized at a faster rate in the presence of JH. The studies by Baumann (1968, 1969) showing that JH increases the conductance of the salivary gland cell membrane causing it to become depolarized strongly support the idea of a membrane effect. Additional support is provided by the work of Cohen and Gilbert (1972) showing that JH causes swelling and lesions in the plasma membrane of insect cells growing in culture. Although long-term allatectomy does not induce any change in mitochondrial respiration of Blaberus fat body (Keeley and Friedman, 1969; Keeley, 1970, 1972; Keeley and Waddil, 1971) or Locusta flight muscle (Minks, 1967), long-term cardiacectomy depresses the respiratory activity of Blaberus fat body mitochondria (Keeley and Friedman, 1969). This is thought to result from the decreased activity of succinate-cytochrome C reductase and cytochrome C oxidase (Keeley and Waddil, 1971). In view of this apparent direct action of the CC on the mitochondria it is surprising to find that extracts of these glands have no effect on the respiration of fat body mitochondria of Blaberus when examined in vitro (Keeley, 1971) although Clarke and Baldwin (1960) reported a positive effect on Locusta flight muscle mitochondria in one experiment but none in another. Since long-term cardiacectomy has the result of lessening the amount of succinate-cytochrone C reductase and cytochrome C oxidase in the mitochondria it might be expected that addition of CC t o mitochondria in vitro would oppose this effect. CC extract has no effect on mitochondrial respiration (Keeley, 1971), yet the glands will increase the respiration of intact fat body in vitro by as much as 72 per cent (Luscher and Leuthold, 1965). The explanation for these apparently contradictory results is probably that the hormone or hormones contained in the CC have both long-term and short-term effects. This is indicated by the fact that a reduction in the amount of succinate-cytochrome C reductase and cytochrome C oxidase following cardiacectomy occurs only over a period of

HORMONAL CONTROL OF METABOLISM I N INSECTS

305

weeks. In our studies we have observed that Periplaneta fat body incubated with CC extract has three times as much citrate as control tissues (Coulthart and Steele, unpublished results). Succinate levels have not yet been measured but the metabolic proximity to citrate suggests that it may be affected in a like manner. It is interesting t o speculate that the synthesis of succinate-cytochrome C reductase may be subject to substrate regulation. It might also be expected that the rise in citrate concentration following treatment of fat body with CC extract would explain the short-term effect of that hormone on respiration. Increasing the concentration of NADH or succinate in a tightly coupled system with the high degree of respiratory control found in intact tissue would probably have little effect on mitochondrial respiration unless there was a concomitant change in the concentration of ADP. The most apparent reason for the increase in oxygen consumption by the fat body on treatment with CC extract is because of the hyperglycaemic factor it contains. The synthesis of UDPG required as a glucosyl donor in trehalose synthesis is dependent on a source of ATP as shown in Fig. 6. This at least is one reason for the increase in respiration of fat body when treated with CC extract. The correlation between trehalose synthesis and respiration in Periplaneta fat body in the presence of HGH is highly significant (P < 0.01) (White and Steele, unpublished observations). These results make it clear that the effect of the CC on mitochondrial respiration is an indirect one resulting from an effect by the hormone elsewhere in the cell. There is no convincing evidence that any of the insect hormones have direct effects on the electron transport system. The most likely explanation for the stimulatory effect of hormones on respiration is that it reflects the need for ATP created by the activation of developmental and physiological mechanisms and is not the result of a direct controlling influence on the oxidative phosphorylation machinery.

7 Conclusions There is no evidence to support the notion that any of the known hormones occurring in insects functions as a “metabolic” hormone. The idea that such a hormone exists, and may be one of those already described, has been extant since the metabolic effects of hormones were first described and orginated in the observation that allatectomy decreased the oxygen consumption of whole insects. By analogy with the effect of thyroxine on respiration in vertebrates, the CA were thought to have a similar action. Indeed, one may question the premise on which the idea of a metabolic hormone is based since insects are poikilothermous animals and apparently do not require the type of metabolic regulation provided by

306

J. E. STEELE

thyroxine in homeotherms. The hormones occurring in insects perform a variety of functions including the regulation of protein synthesis and modulation of homeostatic mechanisms such as water and ion transport. It is difficult to believe that any of these hormones is a “metabolic” hormone although an effect on metabolism is inevitable. The principal objection to considering any of the presently known insect hormones as a “metabolic” hormone lies in the fact that their known functions are secondary to the effects on metabolism. Because the supply of energy within the cell is mainly common for all endergonic reactions the hormone could not serve as a regulator of metabolism, whose rate is the net result of all reactions within the cell, and simultaneously as the regulator of a physiological process. It must be obvious that hormones clearly create the demand for energy which is met through feedback or coupling of the mechanism to ATP synthesis. It has long been known that the concentration of ATP in flight muscle remains constant even though the work done by the muscle may change (Sactor and Hurlbut, 1966) suggesting that it is the work done by the muscle that regulates the rate of ATP synthesis. Insects provide excellent model systems for the study of a number of metabolic problems, the importance of which extends far beyond the insects. More specifically, it seems certain that, because synthetic JH is now available, much attention will be directed towards the metabolic aspects of its action, particularly because of its potential use as an insecticide. The interaction of JH with female fat body to initiate the synthesis of vitellogenin in relatively large quantities may well prove a powerful instrument in elucidating the complex mechanisms by which hormones control the synthesis of proteins. The fat body also, by virtue of the readily monitored effect of the hyperglycaemic hormone on trehalose release, may be immensely useful in studying the coupling of energy metabolism to work performed by a physiological mechanism. The most pressing need in the immediate future is to isolate the various hormones and factors which have been shown to be biologically active. In recent years methods for the isolation of peptides and proteins from small amounts of starting material have been devised. Techniques for the structural analysis of these substances are also available. Unless reasonably pure hormones can be obtained for studies at the cellular Ievel it seems certain that our understanding of the mode of action and metabolic effects of these hormones will be greatly hindered. Metabolic studies in which the function of whole glands or crude extracts containing a number of biologically active factors are used are difficult to interpret and can hardly be expected to yield unequivocal results. It is not surprising therefore that, apart from studies with ecdysone and JH, the number of papers reporting effects of hormones on metabolism in insects has dwindled in recent years.

HORMONAL CONTROL OF METABOLISM IN INSECTS

307

Acknowledgements

I am indebted to Drs S. Caveney, W. Chefurka, D. B. McMillan and M. Owen for their critical and constructive comments on the manuscript. Thanks are also due to Mr K. C. Coulthart and Mr W. Peek for their assistance in the preparation of the illustrations and to Dr G. R. Wyatt who kindly provided me with an unpublished manuscript. The work was begun during a sabbatical leave spent in the Department of Zoology, University of Cambridge, and I thank Professor T. Weis-Fogh and Dr J. E. Treherne for their hospitality and facilities provided during that period. Work originating in the author’s laboratory was supported by the National Research Council of Canada.

References Adyodi, K. G. and Nayar, K. K. (1967). Hemolymph proteins and reproduction in Periplaneta americana: The nature of conjugated proteins and the effect of cardiacallatectomy on protein metabolism. Biol. Bull. mar. biol. Lab., Woods Hole, 135,

466-475. Agui, N., Yagi, S. and Fukaya, M. (1969). Effects of ecdysterone on the in vitro development of wing discs of rice stem borer, Chilo suppressaiis. AppL Ent. Zool. 4,

158-159. Applebaum, S. W. and Schlesinger, H. M. (1973). Regulation of locust fat-body phosphorylase. Biochem. J. 135, 37-41. Arking, R. and Shaaya, E. (1969). Effect of ecdysone on protein synthesis in larval fat body of Calliphora. J. Insect Physiol. 15,287-296. Bade, M. L. (1964). Biosynthesis of fatty acids in the roach, Eurycotis floridana. J. Insect Physiol. 10,333-341. Bassi, S. D. and Feir, D. (1971). Effects of juvenile hormone on the rates of protein synthesis in Oncopeltes fasciatus. Insect Biochem. 1,428-432. Baumann, G . (1 968). Zur wirking des juvenilhormons: elektrophysiologische messungen an der zellmembran der speicheldriise von Galleria mellonella. J. Insect Physiol. 4,

1459-1476. Baumann, G . (1969).Juvenile hormone: effect on bimolecular lipid membranes. Nature, Lond. 223, 316-317. Beenakkers, A. M. T. (1965). Transport of fatty acids in Locusta migratoria during sustained flight. J. Insect Physiol. 11, 879-888. Beenakkers, A. M. T. (1969). The influence of corpus allatum and corpus cardiacum on lipid metabolism in Locusta migratoria. Gen. comp. Endocr. 13,492. Beenakkers, A. M. T. (1973). Influence of flight on lipid metabolism in Locusta mk-atoria. Insect Biochem. 3,303-308. Bell, W. J. (1969). Dual role of juvenile hormone in the control of yolk formation in Periplaneta americana. J. Insect Physiol. 15, 1279-1290.

308

J . E. STEELE

Bell, W. J. (1970). Demonstration and characterization of two vitellogenic blood proteins in Periplaneta americana: an immunochemical analysis. J. Insect Physiol. 16, 291-299. Bell, W. J. and Barth, R. E. (1971). Initiation of yolk deposition by juvenile hormone. Nature New Biol. 230, 220-221. Bentz, F. and Girardie, A. (1969). Action de la pars intercerebralis et des corpora allata sur les protiines ovariennes de Locusta mipatoria mz@atorioides (Orthoptera) au cours de la vitellogenese. C. 7. hebd. Stanc. Acad. ScL, Paris Skr. D. 269, 2014-2017. Bentz, F., Cirardie, A. and Cazal, M. (1970). Etude ilectrophoritique des variations de la proteinemie chez Locusta migratoria pendant la maturation sexuelle. J. Insect Physiol. 16, 2257-2270. Bhakthan, N. M. G. and Gilbert, L. I. (1968). Effects of some vertebrate hormones on lipid mobilization in the insect fat body. Gen. comp. Endocr. 11, 186-197. Bhakthan, N. M. G. and Gilbert, L. I. (1971). Effects of epinephrine and lipase on the morphology of insect fat body. Ann. ent. SOC.A m . 64, 68-72. Bodenstein, D. (1953). Studies on the humoral mechanism in growth and metamorphosis of the cockroach, Periplaneta americana. 111. Humoral effects on metabo1ism.J. exp. Zool. 124, 105-115. Bodnaryk, R. P. (1971). Effect of exogenous molting hormone (Ecdysterone) o n 0-alanyl-L-tyrosine metabolism in the larva of the fly Sarcophaga bullata Parker. Gen. comp. Endocr. 16, 363-368. Bodnaryk, R. P. and Brunet, P. C. J. (1974). 3-0-hydrosulphate o-4-hydroxyphenethylamine (Dopamine 3-O-sulphate),a metabolite involved in the sclerotization of insect cuticle. Biochem. J. 138, 463-469. Bodnaryk. R. P. and Levenbook, L. (1969). The role of 0-alanyl-L-Tyrosine (Sarcophagine) in puparium formation in the fleshfly Sarcophaga bullata. Comp. Biochem. Physiol. 30, 909-921. Bodnaryk, R. P., Brunet, P. C. J. and Koeppe, J. K. (1974). On the metabolism of N-acetyldopamine in Periplaneta americana. J. Insect Physiol. 20, 91 1-923. Bounhiol, J. J. (1938). Recherches expirimentales sur le determinisme de la mitamorphose chez les Lipidopttres. Bull. biol. Suppl. 24, 1-199. Bowers, W. S. and Friedman, S. (1963). Mobilization of fat body glycogen by an extract of corpus cardiacum. Nature, Lond. 198, 685. Bowers, W. S . , Thompson, M. J. and Uebel, E. C. (1966). Juvenile and gonadotropic hormone activity of 10, 11-epoxyfarnesenic acid methyl ester. Life Sci. 4, 2323-2331. Brookes, V. J. (1969). The induction of yolk protein synthesis in the fat body of an insect Leucophaea maderae by an analog of the juvenile hormone. Deul Biol. 20, 459-471. Brostrom, C. O., Hunkeler, F. L. and Krebs, E. G. (1971). The regulation of skeletal muscle phosphorylase kinase by CaZ+. J. biol. Chem. 246, 1961-1967. Brown, B. E. (1965). Pharmacologically active constituents of the cockroach corpus cardiacum: Resolution and some characteristics. Gen. comp. Endocr. 5 , 387-401. Bruchhaus, H. (1972). Untersuchung hormonaler Einfliisse auf den Harnsauregehalt im Fettkorper mannlicher und weiblicher Periplaneta americana L. 2001.Jb. (Physiol,) 76, 375-407.

HORMONAL CONTROL OF METABOLISM IN INSECTS

309

Brunet, P. C. J. (1963). Tytosine metabolism in insects. Ann. N . Y . Acad. Sci. 100,

1020-1034. Brunet, P. C. J. (1965).The metabolism of aromatic compounds. In “Aspects of Insect Biochemistry” (Ed. T. W. Goodwin), pp. 49-77. Biochem. SOC. Symp. No. 25. Academic Press, London and New York. Brunet, P. C. J. and Kent, P. W. (1955).Observations on the mechanism of a tanning reaction in Periplaneta and Blatta. Proc. R o y . SOC. B. 144,259-274. Butenandt, A. and Karlson, P. (1954). Uber die Isolierung eines MetamorphoseHormons der Isekten in Kristallisierter Form. Z. Naturf. 9b,389-391. Butterworth, F. M. and Bodenstein, D. (1969).Adipose tissue of Drosophila melanogaster IV. The effect of the corpus allatum and synthetic juvenile hormone on the tissue of the adult male. Gen. comp. Endocr. 13, 68-74. Candy, D. J. and Kilby, B. A. (1961).The biosynthesis of trehalose in the locust fat body. Biochem. J . 78, 531-536. Cazal, M. (1971). Action des corpora cardiaca s u r la trthalostmie et la glyctmie de Locusta migratoria L. C. r. hebd. Skanc. Acad. Sci., Paris, Sir. D. 272, 2596-2599. Chalaye, D. (1969).La trbhalosemie et son contr6le neuroendocrien chez le Criquet migrateur, Locusta migratoria migratonoides 2. Role de corpora cardiaca et des organes ptrisympathiques. C. 7. hebd. Skanc. Acad. ScL, Paris, S h . D. 268,

31 11-3114. Chino, H. (1957).Carbohydrate metabolism in diapause egg of the silkworm, Bombyx mori I. Diapause and the change of glycogen content. Embryologia, 3, 295-316. Chino, H. (1958). Carbohydrate metabolism in the diapause egg of the silkworm, Bombyx mori. 11. Conversion of glycogen into sorbitol and glycerol during diapause. J. Insect Physiol. 2, 1-12. Chino, H. (1960).Enzymatic pathways in the formation of sorbitol and glycerol in the diapausing egg of the silkworm, Bombyx mori. I. On the polyol dehydrogenases. 1. Insect Physiol. 5 , 1-15. Chino, H. (1961). Enzymatic pathways in the formation of sorbitol and glycerol in the diapausing egg of the silkworm, Bombyx mori. 11. On the phosphatases. 1. Insect Physiol. 6, 231-240. Chino, H. (1963).Respiratory enzyme system of the Bombyx silkworm egg in relation to the mechanism of the formation of sugar alcohols. Archs Biochem. Biophys. 102,

400-415. Chino, H. and Gilbert, L. I. (1965a).Lipid release and transport in insects. Biochim. biophys. Acta, 98,94-110. Chino, H. and Gilbert, L. I. (1965b). Studies on the interconversion of carbohydrate and fatty acid in Hyalophora cecropia. J. Insect physiol. 11, 287-295. Chino, H., Murakami, S. and Harashima, K. (1969).Diglyceride-carrying lipoproteins in insect hemolymph. Isolation purification and properties. Biochim. biophys. Acta,

176, 1-26. Chino, S., Sakurai, S., Ohtaki, T., Ikekawa, N., Miyazaki, H., Ishibashi, M. and Abuki, H. (1974). Biosynthesis of a-ecdysone by prothoracic glands in vitro. Science, 183,

529-530.

Clarke, K. V. and Baldwin, R. W. (1960). The effect of insect hormones and of

310

J. E. STEELE

2:4-dinotrophenol on the mitochondrion, of Locusta migatorM L. J. Insect Physiol. 5, 37-46. Clegg, J. S . and Evans, D. R. (1961). The physiology of blood trehalose and its function during flight in the blowfly. J. exp. Biol. 38, 771-792. Clements, A. N. (1959). Studies on metabolism of locust fat body. J. exp. Biol. 36, 665-675. Cohen, E. and Gilbert, L. I. (1972). Metabolic and hormonal studies on two insect cell lines. J. Insect Physiol. 18, 1061-1076. Coles, G. C. (1965). Studies on the hormonal control of metabolism in Rhodnius prolixus St%l-I.The adult female insect. J. Insect Physiol. 11, 1325-1330. Colhoun, E. H: (1963). Synthesis of 5-hydroxytryptamine in the American cockroach. Experientia, 19, 9-10. Conradi-Larsen, E.-M. (1970). Influence of neurosecretion and nutrition on 0 2 consumption in the bug Oncopeltus fasciatus. J. Insect Physiol. 16,471-482. Cottrell, C. B. (1962a). The imaginal ecdysis of blowflies. The control of cuticular hardening and darkening. J. exp. Biol. 39, 395-411. Cottrell, C. B. (1962b). The imaginal ecdysis of blowflies. Detection of the blood-borne darkening factor and determination of some of its properties. J. exp. Biol. 39, 413-430. Courgeon, A. M. (1966). Etude de l’intensitt respiratoire de Carausius morosus Br., au cour des deux derniers stades larvaires, apres ablation des corporal allata. C.Y. hebd. Shanc. Acad. Sci., Paris, Sir. D. 262, 1272-1275. Dejmal, R. and Brookes, V. J. (1968). Solubility and electrophoretic properties of ovarial protein of the cockroach, Leucophaea maderae. J. Insect Physiol. 14, 3 7 1-381. DeLoof, A. and DeWilde, J. (1970). Hormonal control of synthesis of vitellogenic female protein in the Colorado beetle, Leptinotarsa decemlineata. J. Insect Physiol. 16, 1455-1466. DeWilde, J. and Stegwee, D. (1958). Two major effects of the corpus allatum in the Colorado beetle. Archs. ne‘erl. Zool. 13 (suppl.), 277-289. Doane, W. W. (1961). Developmental physiology of the mutant female sterile (2) adipose of Drosophila rnelanogaster. 111. Corpus allatum complex and ovarian transplantations. J. exp. Zool. 146, 275-298. Doane, W. W. (1973). Role of Hormones in Insect Development. In “Developmental Systems: Insects” (Eds S. J. Counce and C. H. Waddington), vol. 2, pp. 291-497. Academic Press, London and New York. Downer, R. G. H. (1972). Interspecificity of lipid-regulating factors from insect corpus cardiacum. Can. J. 2001.50, 63-65. Downer, R. G. H. and Steele, J. E. (1969). Hormonal control of lipid concentration in fat body and hemolymph of the American cockroach, Periplaneta americana. Proc. ent. SOC.Ontario, 100, 113-116. Downer, R. G. H. and Steele, J. E. (1972). Hormonal stimulation of lipid transport in the American cockroach, Periplaneta americana Gen. comp. Endocr. 19, 259265. Duchiteau-Bosson, C., Jeunieux, C. and Florkin, M. (1962). Contribution i la biochimie du ver A soie-XXV. Variations de la concentration de 151 tyrosine de I’htmolymph au

HORMONAL CONTROL OF METABOLISM I N INSECTS

31 1

cour du developpement de Bombyx mori L. Archs int. Physiol. Biochim. 70, 287-291. Dutrieu, J. and Gourdoux, L. (1967). Le contr6le neuroendocrinien de la trkhaloskmie de Carausius morosus. C. r. hebd. S i h c . Acad. Sci.. Paris, Sir. D. 265, 1067-1070. El-Ibrashy, M. T. and Boctor, I. Z. (1970). Effect of allatectomy upon lipid metabolism of the female moth Spodoptera littoralis. Boisd. 2. vergl. Physiol. 68, 111-116. Emmerich, H. (1970a). Ecdysonbindende Proteinfraktionen in den Speichdriisen von Drosophila hydei. 2. vergl. Physiol. 68, 385402. Emmerich, H. (1970b). Uber die Haemolymphproteine von Pyrrhocoris apterus und uber die Bindung von Ecdyson durch Haemolymphproteine. J. Insect Physiol. 16, 725-747. Emmerich, H., Schmialek, P. and Zahn, A. (1965). Uber die Aktivat einiger Dehydrogenasen und Transaminasen bei Tenebrio rnolitor unter dem Einfluss von Farnesylmethelather. J. Insect Physiol. 11, 1161-1168. Engelmann, F. ( 1 969). Endocrine-stimulated synthesis of sex-specific and non-specific proteins in Leucophaea maderae. Gen. comp. Endocr. 13, 503-504. Engelmann, F. (1970). “The physiology of Insect Reproduction”. Pergamon Press, Oxford. Engelmann, F. (197 1). Juvenile hormone-controlled synthesis of female-specific protein in the cockroach Leucophaea maderae. Archs Biochem. Biophys. 145,439-447. Engelmann, F. (1972).Juvenile hormone-induced RNA and specific protein synthesis in an adult insect. Gen. comp. Endocr. Suppl. 3, 168-173. Engelmann, F. and Penney, D. (1966).Studies on the endocrine control of metabolism in Leucophaea maderae (Blattaria). I. The haemolymph proteins during egg maturation. Gen. comp. Endocr. 7 , 314-325. Firstenberg, D. E. and Silhacek, D. C. (1973).Juvenile hormone regulation of oxidative metabolism in isolated insect mitochondria. Experientia, 29, 1420-1422. Fogal, W. and Fraenkel, G. (1969). The r61e of bursicon in melanization and endocuticle formation in the adult flesh fly Sarcophaga bullata. J. Insect Physiol. 15, 1235-1247. Fraenkel, G . and Hsiao, C. (1962). Hormonal and nervous control of tanning in the fly. Science, N.Y. 138, 27-29. Fraenkel, G. and Hsiao, C. (1963). Tanning in the adult fly: A new function of neurosecretion in the brain. Science, N.Y. 141, 1057-1958. Fraenkel, G. and Hsiao, C. (1965). Bursicon: A hormone which mediates tanning of the cuticle in the adult fly and other insects. J. Insect Physiol. 11, 513-556. Fraenkel, G. and Rudall, K. M. (1947). The structure of insect cuticles. Proc. R . SOC. B 134, 113-143. Fraenkel, G., Hsiao, C. and Seligman, M. (1966). Properties of bursicon: An insect protein hormone that controls cuticular tanning. Science, N. Y. 151, 91-93. Friedman, S. (1967). The control of trehalose synthesis in the blowfly, Phormia regina Meig. J. Insect Physiol. 13, 397-405. Friedman, S. (1968). Trehalose regulation of glucose-6-phosphate hydrolysis in blowfly extracts. Science, N.Y. 159, 110-111. Fuchs, M. S. and Schlaeger, D. A. (1973). The stimulation of dopa decarboxylase activity by ecdysone and its enhancement by cyclic AMP in adult mosquitos. Biochem. biophys. Res. Commun. 54, 784-789.

312

J. E. STEELE

Fukuda, S. (1940). Induction of pupation innsilkwormby transplanting the prothoracic gland. Proc. imp. Acad. Japan, 16,414-416. Fukuda, S. (1941). Role of the prothoracic gland in the differentiation of the imaginal characters in the silkworm pupa. Annotnes Zool. jap. 20, 9-13. Fukuda, S. (1962). Hormonal control of diapause in the silkworm. Gen. comp. Endocr. Suppl. 1,337-340. Gersch, M. (1974). Selektive Freisetzung des hyperglykamischen Faktors aus den Corpora cardiaca von Periplaneta americana in vivo. Experientia, 30, 767. Gersch, M. and Stiirzebecher, J. ( 1 968). Weitere Untersuchungen zur Kennzeichnung des Aktivationshormons der Insektenhautung. J. Insect Physiol. 14, 87-96. Gersch, M. and Stiirzebecher, J. (1970). Experimentelle Stimulierung der zelluliiren Aktivitat der Prothorakaldriisen von Periplaneta americana durch den Aktivationsfaktor. J. Insect Physiol. 16, 1813-1826. Gersch, M., Fischer, F., Unger, H. and Kabitzer, W. (1961). Presence of 5-HT in the nervous system of Periplaneta americana. Z. Naturf. 16b, 351-352. Gilbert, L. I. (1967). Changes in lipid content during the reproductive cycle of Leucophaea maderae and effects of the juvenile hormone on lipid metabolism in vitro. Comp. Biochem. Physiol. 21, 237-257. Gilbert, L. I. (1973). Physiology of growth and development: endocrine aspects. In “The Physiology of Insecta” (Ed. M. Rockstein), 2nd ed., vol. 1 , pp. 249-370. Academic Press, London and New York. Gilbert, L. I. and Chino, H. (1974). Transport of lipids in insects. J. Lipid Res. 15, 439-456. Goldsworthy, G . J. (1969). Hyperglycaemic factors from the corpus cardiacum Qf Locusta migratoria. J. Insect Physiol. 15, 2131-2140. Goldsworthy, G. J. (1970). The action of hyperglycaemic factors from the corpus cardiacum of Locusta migratoria on glycogen phosphorylase. Gen. comp. Endocr. 14, 78-85. Goldsworthy, G. J. (1971). The effects of removal of the cerebral neurosecretory cells on haemolymph and tissue carbohydrate in Locusta migratoria migratorioides R. lk F. J. Endocr. 50, 237-240. Goldsworthy, G. J. and Coupland, A. J. (1974). The influence of the corpora cardiaca and substrate availability on flight speed and wing beat frequency in Locusta. J. cornp. Physiol. 89, 359-368. Goldsworthy, G. J. and Mordue, W. (1974). Neurosecretory hormones in insects. 1. Endocr. 60, 529-558. Goldsworthy, G. J., Mordue, W. and Guthkelch, J. (1972a). Studies on insect adipokinetic hormones. Gen. comp. Endocr. 18, 545-551. Goldsworthy, G. J., Johnson, R. A. and Mordue, W. (1972b). In vivo studies on the release of hormones from the corpora cardiaca of locusts. J. comp. Physiol. 79, 85-96. Goldsworthy, G. J., Coupland, A. J. and Mordue, W. (1973). The effects of corpora cardiaca on tethered flight in the locust. J. comp. Physiol. 82, 339-346. GoltzenC-Bentz, F., Joly, L., BrehClin, M. and Hoffman, J. A. (1972). Influence des corpora allata sur la proteinemie de larves de stade V et d’imago femelles de Locusta

HORMONAL CONTROL OF METABOLISM I N INSECTS

313

migratoria L. (Insecte orthoptire). C. r. hebd. Sianc. Acad. Sci., Paris, S h . D. 274, 1059-1062. Hackman, R. H. (1965). Changes in free amino acids of the blood of blowfly larvae at metamorphosis. Aust. J. biol. Sci. 9 , 400-405. Hart, D. E. and Steele, J. E. (1969). Inhibition of insect nerve cord phosphorylase activity by 5-hydroxytryptamine. Experientia, 25, 243. Hart, D. E. and Steele, J. E. (1973). The glycogenolytic effect of the corpus cardiacum on the cockroach nerve cord. J. Insect Physiol. 19, 927-939. Harvey, W. R. (1962). Metabolic aspects of insect diapause. Ann. Rev. Ent. 7,57-80. Hasegawa, K. (1957). The diapause hormone of the silkworm, Bombyx mori. Nature, Lond. 179, 1300-1301. Hasegawa, K. and Yamashita, 0. (1967). Control of metabolism in the silkworm pupal ovary by the diapause hormone. J. Sericult. Sci. Japan, 36, 297-301. Highnam, K. C., Lusis, 0. and Hill, L. (1963). Factors affecting oocyte resorption in the desert locust Schistocerca gregaria (Forsul). J. Insect Physiol. 9 , 827-837. Hill, L. (1965). The incorporation of C14-glycine into the proteins of the fat body of the desert locust during ovarian development. J. Insect Physiol. 11, 1605-1615. Hill, L. and Izatt, M. E. G. (1974). The relationships between corpora allata and fat body and haemolymph lipids in the adult female desert locust. J. Insect PhysioZ. 2 0 , 2 143-2156. Hill, L., Izatt, M. E. G., Horne, J. A. and Bailey, E. (1972). Factors effecting concentrations of acetoacetate and D-3-hydroxybutyrate in haemolymph and tissues of the adult desert locust. J. Insect Physiol. 18, 1265-1285. Hocks, P. and Wiechert, R. (1966). 20-Hydroxy-Ecdysone, Isoliert aus lnsekten. Tetrahedron Lett. No. 26, 2989-2993. Hoffman, A. G. D. and Downer, R. G. H. (1974). Evolution of I4CO2 from 1 - I C-acetate in American cockroach, Periplaneta americana. Comp. Biochem. Physiol. 48b, 199-204. Horie, Y., Nakasone, S. and Ito, T. (1968). The conversion of 14C-carbohydrates into COz and lipid by the silkworm, Bombyx mori. J. Insect Physiol. 14, 971-981. Huber, R. and Hoppe, W. (1965). Zur Chemie des Ecdysons VII Die Kristall-p. Molekulstrukturanalyse des Insektenverpuppungshormons Ecdyson mit der Automatisierten Faltmolekulmethod. Chem. Ber. 98, 2403-2424. Ichikawa, M. and Ishizaki, H. (1963). Protein nature of the brain harmone of insects. Nature, Lond. 198, 308-309. Ilan, J. and Ilan, J. (1973). Protein synthesis and insect morphogenesis. A . Rev. Ent. 18, 167-182. Ishizaki, H. and Ichikawa, M. (1967). Purification of the brain hormone of the silkworm Bombyx mori. Biol. Bull. mar. biol. Lab., Woods Hole, 133, 355-368. Ito, T. and Horie, Y. (1957). Glycogen in the ligated silkworm pupa (Bombyx mori). Nature, Lond. 179, 1136-1137. Janda, V. and Slima, K. (1965). Uber den Einfluss von Hormonen auf den Glykogen-, Fett- und Stickstoffmetabolismus bei den Imagines von Pyrrhocoris apterus L. (Hemiptera). Zool. Jb. (Physiol.) 71, 345-358. Judy, K. J., Schooley, D. A., Hall, M. S., Bergot, B. J. and Siddall, J. B. (1973).

314

J. E. STEELE

Chemical structure and absolute configuration of a juvenile hormone from grasshopper corpora allata in Vit70. Life Sci. 13, 1511-1516. Jungreis, A. M. and Wyatt, G. R. (1972). Sugar release and penetration into insect fat body: relations to regulation of haemolymph trehalose in developing stages of Hyalophora cecropia. Biol. Bull. mar. biol. Lab., Woods Hole, 143, 367-391. Kageyama, T. and Ohnishi, E. (1971). Carbohydrate metabolism in the eggs of the silkworm, Bombyx mori. I. Absence of phosphofructokinase in the diapausing egg. Devl. Growth Different. 13, 97-106. Kageyama, T. and Ohrishi, E. (1973). Carbohydrate metabolism in the eggs of the silkworm Bombyx mori. 11. Anaerobiosis and polyol formation. Devl. Growth Different. 15, 47-55. Karlson, P. (1960). Zum Tyrosinstoffwechsel der Insekten. 111. h e r den Einbau von Tyrosin-Umwandlungsprodukten in das Puppentonnchen der Schmeissfliege Calliphora erythrocephala. Hoppe-Segler’s 2. physiol. Chem. 318, 194-200. Karlson, P. (1974). Mode of action of ecdysones. In “Invertebrate Endocrinology and Hormone Heterophylly” (Ed. W. J. Burdette), pp. 43-54. Springer-Verlag, New York, Heidelberg and Berlin. Karlson, P. and Herrlich, P. (1965). Zum Tyrosinstoffwechsel der Heusschrecke Schistocerca gregaria Forsk. J. Insect Physiol. 11, 79-89. Karlson, P. and Schweiger, A. (1961). Zum Tyrosinstoffwechsel der Insekten, IV. Das Phenoloxydase-System von Calliphora und seome Beeinflussung durch das Hormon Ecdyson. Hoppe-Segler’s Z. Phys. Chem. 323, 199-210. Karlson, P. and Sekeris, C. E. (1962a). Zum Tyrosinstoffwechsels der Insekten. IX. Kontrolle des Tyrosinstoffwechsels durch Ecdyson. Biochim. biophys. Acta, 63, 489-495. Karlson, P. and Sekeris, C. E. (1962b). N-Acetyl-dopamine as sclerotizing agent of the insect cuticle. Nature, Lond. 195, 183-184. Karlson, P., Hoffmeister, H., Hummel, H., Hocks, P. and Spitteller, C. (1965). Zur Chemie des Ecdysons. VI. Reaktionen des Ecdysonmolekiils. Chem. Ber. 98, 2 394-2402. Keeley, L. L. (1970). Insect fat body mitochondria: endocrine and age effects on respiratory and electron transport activities. Life Sci. 9 , 1003-1001. Keeley, L. L. (197 1). Endocrine effects on the biochemical properties of fat body mitochondria from the cockroach, Blabems discoidalis. /. Insect PhysioL 17, 1501-15 15. Keeley, L. L. (1972). Biogenesis of mitochondria: neuroendocnne effects on the development of respiratory functions in fat body mitochondria of the cockroach, Blaberus discoidalis. Archs Biochem. Biophys. 153, 8-15. Keeley, L. L. and Friedman, S. (1967). Corpus cardiacum as a metabolic regulator in Blaberus discoidalis Serrille (Blattidae). I. Long-term effects of cardiatectomy on whole body and tissue respiration and on trophic metabolism. Gen. comp. Endocr. 8 , 129-134. Keeley, L. L. and Friedman, S. (1969). Effects of long-term cardiatectomy-allatectomy on mitochondria1 respiration in the cockroach, Blaberus discoidalis. J. Insect Physiol. 15, 509-518. Keeley, L. L. and Waddil, V. H. (1971). Insect hormones: evidence for a neuroendocrine factor affecting respiratory metabolism. Life Sci. 10, 737-745.

HORMONAL CONTROL OF METABOLISM IN INSECTS

31 5

King, D. S. (1972). Metabolism of a-ecdysone and possible precursors by insects in vivo and in vitro. Gen. comp. Endocr. Suppl., 3, 221-227. King, D. S. and Marks, E. P. (1974). The secretion and metabolism of a-ecdysone by cockroach (Leucophaea maderae) tissues in vitro. Life Sci. 15, 147-154. King, D. S. and Siddall, J. B. (1969). Conversion of a-ecdysone to Pecdysone by crustaceans and insects. Nature, Lond. 221, 955-956. King, D. S., BoUenbacher, W. E., Borst, D. W., Vedeckis, W. V., O’Connor, J. D., Ittycheriah, P. I. and Gilbert, L. I. (1974). The secretion of a-ecdysone by the prothoracic glands of Manduca sexta an vitro. Proc. natn. Acad. S c i U.S.A. 7 1 , 793-796. Kirimura, J., Saito, M. and Kobayashi, M. (1962). Steroid hormone in an insect Bombyx mori. Nature, Lond. 195, 729-730. Kobayashi, M. (1967). An action of insect hormones on carbohydrate metabolism in insects. J. Sericult. Sci. 36, 302-308. Kobayashi, M. and Kimura, S. (1967). Action of ecdysone on the conversion of 14C-glucose in dauer pupa of the silkworm, Bombyx mori. J. Insect Physiol. 13,

545-552. Kobayashi, M., Kimura, S. and Yamazaki, M. (1967). Action of insect hormones on the fate of ‘‘C-glucose in the diapausing, brainless pupa of Samea Cynthia pryeri (Lepidoptera: Saturniidae). Appl. Entomol. 2001. 2, 79-84. Koeppe, J. K. and Mills, R. R. (1974). Possible involvement of 3,4-dihydroxyphenyl-acetic acid as a sclerotization agent in the American cockroach. J. Insect Physiol. 20, 1603-1609. Kopek, S. (1922). Studies on the necessity of the brain for the inception of insect metamorphosis. Biol. Bull. mar. biol. Lab., Woods Hole, 42, 322-342. Kroeger, H. (1963). Chemical nature of the system controlling gene activities in insect cells. Nature, Lond. 200, 1234-1235. Lafon-Cazal, M. and Roussel, J.-P. (197 1). Modifications metaboliques aprhs ablation simultanee des corpora allata et des corpora cardiaca chez Schistocerca gregaria et Locusta migratoria. C. r. hebd. Skanc. Acad. Sci, Paris, Sir. D. 273, 26132615. Laufer, H. (1960). Blood proteins in insect development. Ann. N.Y. Acad. Sci 89, 490-515. Lea, A. 0. and Van Handel, E. (1970). Suppression of glycogen synthesis in the mosquito by a hormone from the medial neurosecretory cells. J. Insect Physiol. 16, 3 10-321. Levenbook, L. and Dinamarca, M. L. (1966). Free amino acids and related compqunds during metamorphosis of the blowfly Phormia regina. J. Insect Physiol. 12, 1342-1362. Lezzi, M. and Gilbert, L. I. (1970). Differential effects of & and Na+ on specific bands of isolated polytene chromosomes of Chironornus tentans. J. Cell Sci. 6 , 615-628. L’Helias, C. (1953). Role des corpora allata dans le metabolisme des flucides, de l’azote et des lipides chez le phasme Dixippus morosus. C. r. hebd. Skanc. Acad. Sci., Paris, S k . D. 236, 2164-2166. Liu, T. P. (1973). The effect of allatectomy on the blood trehalose in the female housefly, Musca domestica L. Comp. Biochem. Physiol. 46a, 109-113.

316

J. E. STEELE

Liu, T. P. (1974).The effect of allatectomy on glycogen metabolism in the fat body of the female housefly, Musca domestica L. Comp. Biochem. Physiol. 47b, 79-86. Locke, M., Condoulis, W. V. and Hurshman, L. F. (1965). Moult and intermoult activities'in the epidermal cells of an insect. Science, 149,437438. Louloudes, S. J., Kaplanis, J. N., Robbins, W. E. and Monroe, R. E. (1961).Lipogenesis from C"-acetate by the American cockrdach. Ann. ent. SOC.A m . 54,99-103. Lunan, K. D. and Mitchell, H. K. (1969).The metabolism of tyrosinc 0-phosphate in Drosophila. Archs Biochem. Biophys. 132,450-456. Liischer, M. (1968). Hormonal control of respiration and protein synthesis in the fat body of the cockroach Nauphoeta cinerea during oocyte growth. J. Insect Physiol.

14,499-511. Liischer, M. and Leuthold, R. (1965). h e r die hormonale Beeinflussung des respiratorischen Stoffwechsels bei der Schabe Leucophaea maderae (F.) Revue suisse Zool.

72,618-623. Liischer, M., Moesch. K., Scheurer and Wyss-Huber, M. (1971).Hormonal control of protein synthesis in the fat body of Leucophaea maderae. Endocr. Exper. 5, 69-72. Maddrell, S. H. P. and Treherne, J. E. (1967).The ultrastructure of the perineurium in two insect species (Carausius morosus and Periplaneta americana). J. Cell Sci. 2 ,

119-128. Mansingh, A. (1972). Effects of farnesyl methyl ether o n carbohydrate and lipid metabolism on the tent caterpillar, Malacosoma pluviale. J. Insect Physiol. 18,

2251-2263. Mayer, R. J. and Candy, D. J. (1967). Changes in haemolymph lipoproteins during locust flight. Nature, Lond. 215,987. Menon, M. (1965).Endocrine influences on yolk deposition in insects. J. Anim. Morph. PhysioL 12, 76-80. Meyer, A. S., Schneiderman, H. A., Hanzmann, E. and KO, J. H. (1968).The two juvenile hormones from the Cecropia silkmoth. Proc. natn. Acad. Sci. U.S.A. 60, 853-8 60. Mills, R. R. and Nielsen, D. J. (1967).Hormonal control of tanning in the American cockroach-V. Some properties of the purified hormone. J. Insect Physiol. 13,

273-280. Mills, R. R. and Whitehead, D. L. (1970).Hormonal control of tanning in the American cockroach: changes in blood cell permeability. J. Insect Physiol. 16, 331-340. Mills, R. R., Greenslade, F. C. and Couch, E. F. (1966).Studies on vitellogenesis in the American cockroach. J. Insect Physiol. 12, 767-779. Mills, R. R., Lake, C. R. and Alworth, W. L. (1967).Biosynthesis of N-acetyldopamine by the American cockroach. J. Insect Physiol. 13, 1539-1546. Minks, A. K. (1967). Biochemical aspects of juvenile hormone action in the adult Locusta migratoria. Archs ne'erl. Zool. 17, 175-258. Mitchell, H. K. and Lunan, K. D. (1964). Tyrosine-0-phosphate in Drosophila. Archs Biochem. Biophys. 106,219-222. Mordue, W . and Goldsworthy, G. J. (1969). The physiological effects of corpus cardiacum extracts in locusts. Gen. comp. Endocr. 12, 360-369. Mordue, W. and Goldsworthy, G. J. (1973). Transaminase levels and uric acid production in adult insects. Insect Biochem. 3, 419-427.

HORMONAL CONTROL OF METABOLISM I N INSECTS

317

Morohoshi, S. and Fugo, H. (1971):The control of growth and development in Bombyx mori. X. Effect of the suboesophageal ganglion hormone on the incorporation of 4C-glucose into the lipids of the fat body, haemolymph and ovary. Proc. imp. Acad. Japan, 47,416-421. Morohoshi, S . and Fugo, H. (1972). The control of growth and development in Bombyx mori. XI. Effect of the corpus allatum, suboesophageal ganglion, brain and ecdysterone on the incorporation of l 4 C-U-glucose into pupal fat body and ovary lipids. Proc. imp. Acad. Japan, 48, 127-132. Miiller, H. P. and Engelmann, F. (1968). Studies on the endocrine control of metabolism in Leucophaea maderae (Blattaria). 11. Effect of the corpora cardiaca on fat body respiration. Gen. comp. Endocr. 11, 43-50. Miiller, P. J., Masner, P. and Trautmann, K. H. (1974). The isolation and identification of juvenile hormone from cockroach corpora allata in vitro. Life Sci. 15, 915-921. Murphy, T. A. and Wyatt, G. R. (1965). The enzymes of glycogen and trehalose synthesis in silkmoth fat body. J. bid. Chem. 240, 1500-1508. Natalizi, G. M. and Frontali, N. (1969). Purification of insect hyperglycaemic and heart accelerating hormones. J. Insect Physiol. 12, 1279-1287. Nathanson, J. A. and Greengard, P. (1973). Octopamine-sensitive adenylate cyclase: evidence for a biological role of octapamine in nervous tissue. Science, N.Y. 180, 308-3 10. Neufeld, G. J., Thomson, J. A. and Horn, D. S. H. (1968). Short-term effects of crustecdysone (20-hydroxyecdysone) o n protein and RNA synthesis in third instar larvae of Calliphora. J. Insect Physiol. 14, 789-804. Neugebauer, W. (1961). Wirkung der Extirpation und Transplantation der Corpora allata auf den Sauerstoffverbrauch, die Eibildung und den Fettkorper von Carausius (Dixippus) morosus. Arch EntwMech. Org. 153,314-352. Nohel, P. and Slama, K. (1972). Effect of a juvenile hormone analogue on glutamatepyruvate transaminase activity in the bug Pyrrhocoris apterus. Insect Biochem. 2, 58-66. Normann, T. C. and Duve, H. (1969). Experimentally induced release of a neurohormone influencing hemolymph trehalose level in Calliphora erythrocephala (Diptera). Cen. comp. Endocr. 12,449-459. Novdk, V. J. A. and Slima, K. (1962). The influence of juvenile hormone on the oxygen consumption of the last larval instar of Pyrrhocoris apterus L. J. Insect Physiol. 8, 145-153. Oberlander, H., Berry, S. J., Krishnakumaran, A. and Schneiderman, H. A. (1965). RNA and DNA synthesis during activation and secretion of the prothoracic glands of Saturniid moths. J. exp. 2001.159, 15-32. Odhiambo, T. R. (1966). The metabolic effects of the corpus allatum hormone in the male desert locust. I. Lipid metabolism. J . exp. Biol. 45,45-50. Orr, C. W. M. (1964). The influence of nutritional and hormonal factors on the chemistry of the fat body, blood and ovaries of the blowfly, Phormia regina Meig. 1. Insect Physiol. 10, 103-119. Osborne, D. J., Carlisle, D. B. and Ellis, P. E. (1968). Protein synthesis in the fat body of the female desert locust, Schistocerca gregaria Forsk., in relation to maturation. Cen. comp. Endocr. 11, 347-354.

318

J. E. STEELE

Pan, M. L. and Wyatt, G. R. (1971). Juvenile hormone induces vitellogenin synthesis in the Monarch butterfly. Science, N.Y. 174, 503-505. Patterson, D. S. P. (1957). Qualitative and quantitative changes observed in the free a-amino nitrogen fraction of Tenebrio molitor pupal tissues during metamorphosis. Biochem. J. 65, 729-735. Peled, Y. and Tietz, A. (1973). Fat transport in the locust, Locusta migratoria: the role of protein synthesis. Biochim. biophys. Acta, 296,499-509. Pfeiffer, I. W. (1945). Effect of the corpora allata on the metabolism of adult female grasshoppers. J. exp. 2001. 99, 183-233. Post, L. C. (1972). Bursicon: its effect on tyrosine permeation into insect haemocytes. Biochim. 6iophys. Acta, 290,424-428. Post, L. C. and DeJong, B. J, (1973). Bursicon and the metabolism of tyrosine in the moulting cycle of Pieris larvae. J. Insect Physiol. 19, 1541-1546. Pratt, G. E. and Davey, K. G. (1972). The corpus allatum and oogenesis in Rhodnius prolixus (Stal.). I. The effects of allatectomy. J. exp. BioL 56, 202-214. Pratt, G. E. and Tobe, S. S. (1974). Juvenile hormones radiobiosynthesised by corpora allata of adult female locusts in uitro. Life Sci. 14, 575-586. Price, G. M. (1968). Factors affecting protein synthesis by the fat body of the larva of the blowfly Calliphora erythrocephola. Biochem. J. 108, 19-20P. Price, G. M. (1973). Protein and nucleic acid metabolism in insect fat body. Biol. Rev. 48, 333-375. Price, W. T., Berridge, M. J. and Rassmussen, H. (1972). The role of calcium and cyclic AMP in the secretory response of the blowfly salivary gland to 5-hydroxytryptamine. Proc. natn. Acad. Sci. U.S.A. 69, 553-557. Pryor, M. G. M. (1940). On the hardening of the cuticle of insects. Proc. R . SOC.B . 128, 393-407. Rall, T. W. and Sutherland, E. W. (1958). Formation of a cyclic adenine ribonucleotide by tissue particles. J. 6iol. Chem. 232, 1065-1076. Ralph, C. L. and Matta, R. J. (1965). Evidence for hormonal effects on metabolism of cockroaches from studies of tissue homogenates. J. Insect Physiol. 11, 983-991. Ralph, C. L. and McCarthy, R. (1964). Effects of brain and corpus cardiacum extracts on haemolymph trehalose of the cockroach, Periplaneta americana. Nature, Lond. 203, 1195-1196. Robbins, W . E., Kaplanis, J. N., Louloudes, S. J. and Monroe, R. E. (1960). Utilization of l-CI4-acetate in lipid synthesis by adult houseflies. Ann. ent. SOC. A m . 53, 128-129. Robertson, H. A. (1970). Effects of some amines on insect nerve cord phosphorylase activity. M.Sc. Thesis, University of Western Ontario. Robertson, H. A. (1975). Studies on the distribution and biosynthesis of octopamine in the insect-nervous system. Proc. int. SOC.Neurochem. V Int. Mtg., Barcelona, p. 62. Robertson, H. A. and Steele, J. E. (1972). Activation of insect nerve cord phosphorylase by octopamine and adenosine 3',5'-monophosphate. J. Neurochem. 19, 1603-1606. Robertson, H. A. and Steele, J. E. (1973a). Octopamine in the insect central nervous system: distribution, biosynthesis and possible physiological role. J. Physiok 237, 34-35P.

HORMONAL CONTROL OF METABOLISM IN INSECTS

319

Robertson, H. A. and Steele, J: E. (1973b).Effect of monophenolic amines on glycogen metabolism in the nerve cord of the American cockroach, Periplaneta americana. Insect Biochem. 3, 53-59. Robinson, N. L. and Goldsworthy, G. J. (1974). The effects of locust adipokinetic hormone on flight muscle metabolism in vivo and in vitro. J. comp. Physiol. 89,369-377. Roller, H., Dahm, K. H., Sweely, C. C. and Trost, B. M. (1967).The structure of the juvenile hormone. Angew. Chem. (Int. Ed.) 6,179-180. Roussel, J:P. (1963). Consommation d’oxygtne apres ablation des corpora allata chez des femelles adultes de Locusts migatoria L. J. Insect Physiol. 9, 721-725. Roussel, J.-P. (1967). Fonctions des corpora allata et contrBl de la pigmentation chez Gryllus bimaculatus De Geer. J. Insect Physiol. 13, 113-130. Sacktor, B. and Hurlbut, E. C. (1966).Regulation of metabolism in working muscle in vivo. XI. Concentrations of adenine nucleotides, arginine phosphate, and inorganic phosphate in insect flight muscle during flight. J. biol. Chem. 241, 632-634. Sagesser, H. (1960).Uber die Wirkung der Corpora allata auf den Sauerstoffverbrauch bei der Schabe Leucophaea maderae (F.). J. Insect Physiol. 5, 264-285. Sahota, T. S. and Mansingh, A. (1970).Cellular response t o ecdysone: RNA and protein synthesis in larval tissues of oak silkworm Antheraea pernyi. J. Insect Physiol. 16,

1649-1654. Samuels, A. (1956).The effect of sex and allatectomy on the oxygen consumption of the thoracic musculature of the insect, Leucophaea maderae. Biol. Bull. mar. biol. Lab., Woods Hole, 110,179-183. Scheurer, R. (1969). Endocrine control of protein synthesis during oocyte maturation in the cockroach Leucophaea maderae. J. Insect Physiol. 15, 1411-1419. Schlaeger, D. A. and Fuchs, M. S. (1974).Dopa decarboxylase activity in Aedes aempti: A preadult profile and its subsequent correlation with ovarian development. Devl Biol. 38, 209-219. Schlaeger, D. A., Fuchs, M. S. and Kang, S.-H. (1974).Ecdysone-mediated stimulation of dopa decarboxylase activity and its relationship to ovarian development in Aedes aegypti. J. Cell Biol. 61,454-465. Schmialek, P. and Drews, G. (1965).Wirkung von Farnesylmethylather auf die Atmung von Tenebrio molitor puppen. Z. Naturf. 20b, 214-215. Schooley, D. A., Judy, K. J., Bergot, B. J., Hall, M. S. and Sidddl, J. B. (1973). Biosynthesis of the juvenile hormones of Manduca sexta: labelling pattern from rnevalonate propionate and acetate. Proc. natn. Acad. Sci. U.S.A. 70, 2921-2925. Sehnal, F. and Slirna, K. (1966).The effect of corpus allatum hormone on respiratory metabolism during larval development and metamorphosis of Galleria mellonella. J. Insect Physiol. 12,1333-1342. Sekeris, C. E. (1964). Sclerotization in the blowfly imago. Science, N.Y. 144,419-420. Sekeris, C. E. (1974). Molecular action of insect hormones. In “Invertebrate Endocrinology and Hormonal Heterophylly” (Ed. w. J. Burdette), pp. 55-78.SpnngerVerlag, New York, Heidelberg and Berlin. Sekeris, C. E. and Karlson, P. (1966). Biosynthesis of catecholamines in insects. Pharmacol. Rev. 18, 89-94. Seligman, M., Friedman, S. and Fraenkel, G. (1969). Bursicon mediation of tyrosine

320

J. E. STEELE

hydroxylation during tanning of the adult cuticle of the fly Sarcophaga bullata. J. Insect Physiol. 15, 553-562. Shaaya, E. and Bodenstein, D. (1969). The function of the accessory sex glands in Periphneta americana (L.). 11. The role of the juvenile hormone in the synthesis of protein and protocatechuic acid ghcoside. J. exp. Zool. 170,281-292. Shaaya, E. and Sekeris, C. E. (1970).The formation of protocatechuic acid-4-0,P-glucoside in Periphneta americana and the possible role of the juvenile hormone. J. Insect Physiol. 16, 323-930. Shigematsu, H. (1960). Protein metabolism in the fat body of the silkworm, Bombyx mori L. Bull. Seric. Ewp. Sta. Japan, 16, 141-170. Siakotos, A. N. (1960).The conjugated plasma proteins of the American cockroach. I. The normal state. J. gen. Physiol. 43, 999-1013. Slima, K. (1964a). Hormonal control of haemolymph protein concentration in the adults of Pyrrhocoris apterus L. (Hemiptera).J. Insect Physiol. 10,773-782. Slima, K. (1964b). Hormonal control of respiratory metabolism during growth, reproduction and diapause in female adults of Pyrrhocoris apterus. J. Insect Physiol. 10,283-304. Slima, K. (1964~).Hormonal control of respiratory metabolism during growth, reproduction and diapause in male adults of Pyrrhocoris apterus L. (Hemiptera). Biol. Bull. mar. biol. Lab., Woods Hole, 127,499-510. Slima, K. (1965). Effect of hormones on the respiration of body fragments of adult Pyrrhocoris apterus L. (Hemiptera). Nature, Lond. 205,416-417. Sridhara, S. and Bhat, J. V. (1964).Incorporation of [ l-'4C]-acetate into the lipids o f the silkworm, Bombyx mori L. Biochem. J. 91,120-123. Steele, J. E. (1961).Occurrence of a hyperglycaemic factor in the corpus cardiacum of an insect. Nature, Lond. 192, 680-681. Steele, J. E. (1963).The site of action of insect hyperflycaemic hormone. Gen. comp. Endocr. 3,46-52. Steele, J. E. (1964). The activation of phosphorylase in an insect by adenosine 3',5'-phosphate and other agents. A m . 2001. 4,328. Steele, J. E. (1969).A relationship between ionic environment and hormonal activation of phosphorylase in an insect. Comp. Biochem. Physiol. 29, 755-763. Steen, J. B. (1961). The effect of juvenile hormone on the respiratory metabolism of silkworm pupae, as recorded with a new semi-micro device. Acta physiol. scand. 51,

275-282. Stephen, W. F. and Gilbert, L. I. (1970).Alterations in fatty acid composition during the metamorphosis of Hyalophora cecropia: correlations with juvenile hormone titre. J. Insect Physfol. 16, 851-864. Stevenson, E. and Wyatt, G. R. (1964).Glycogen phosphorylase and its activation in silkmoth fat body. Archs Biochem. Biophys. 108,420-429. Strong, R. E. (1963). Studies on lipids in some homopterous insects. Hilgardia, 34,

43-61. Strong, L. (1968a). The effect of enforced locomotor activity on lipid content of allatectomized males of Locusta migratoria migmtorioides. J. exp. Biol. 48, 625-634. Strong, L. (1968b). Locomotor activity, sexual behaviour, and corpus allatum hormone in males of Locusta. J. Insect Physiol. 14,1685-1691.

HORMONAL CONTROL OF METABOLISM IN INSECTS

321

Takahashi, S. Y. (1972). Accumulation of 4-0-P-D-glucoside of protocatechuic acid in the left colleterial gland of the cockroach, Periplaneta americana. 11. Hormonal regulation. Devl. Growth Different. 14,25-35. Thomas, K. K. (1972). Studies on the synthesis of lipoproteins during larval-pupal development of Hyalorphora cecropia. Insect Biochem. 2 , 107-118. Thomas, K. K. and Gilbert, L. I. (1968). Isolation and characterization of the haemolymph proteins of the American silkmoth, Hyalophora cecropia. Archs Biochem. Biophys. 127, 512-521. Thomas, K. K. and Gilbert, L. I. (1969).The hemolymph lipoproteins of the silkmoth Hyalophora gloven’: studies on lipid composition, origin and function. Physiol. Chem. Physics, 1, 293-311. Thomas, K. K. and Nation, J. L. (1966a).Control of a sex-limited haemolymph protein by corpora allata during ovarian development in Periplaneta americana. Biol. Bull. mar. biol. Lab., Woods Hole, 130, 254-264. Thomas, K. K. and Nation, J. L. (1966b). RNA, protein and uric acid content of body tissues of Periplaneta americana (L) as influenced by corpora allata during ovarian development. Biol. Bull. mar. biol. Lab., Woods Hole, 130,442-449. Thomsen, E. (1949). Influence of the corpus allatum on the oxygen consumption of adult Calliphora erythrocephala Meig. J. exp. Biol. 26, 137-149. Thomsen, E. (1952). Functional significance of the neurosecretory brain cells and the corpus cardiacum in the female blowfly, Calliphora erythrocephala Meig. J. exp. Biol. 29,137-172. Tietz, A. (1961). Fat synthesis in cell-free preparations of the locust fat body. J. Lipid lies. 2 , 182-187. Trautmann, K. H., Schuler, A., Suchy, M. and Wipf, H.-K. (1974).Eine Methode zur qualitativen und quantitativen Bestimmung von drei Juvenilhormonen von Insekten. Nachweis von 10,ll-Epoxy-3,7,11-trimethyl-2-trans-6-trans-dodecardiensauremethylester in Melolontha melolontha. 2. NaturJ 29C, 161-168. Vandenberg, R. D. and Mills, R. R. (1974). Hormonal control of tanning by the American cockroach: cyclic AMP as a probable intermediate. I. Insect Physiol. 20,

623-627. Vandenberg, R. D. and Mills, R. R. (1975). Adenyl cyclase in the haemocytes of the American cockroach. J. Insect Physiol. 21, 221-229. Van Handel, E. and Lea, A. 0. (1965).Medial neurosecretory cells as regulators of glycogen and triglyceride synthesis. Science, N. Y . 149, 298-300. Van Handel, E. and Lea, A. 0. (1970).Control of glycogen and fat metabolism in the mosquito. Gen. comp. Endocr. 14,381-384. Van Handel, E. and Lum, P. T. M. (1961).Sex as regulator of triglyceride metabolism in the mosquito. Science, N.Y. 134, 1979-1980. Vejbjerg, K. and Normann, T. C. (1974).Secretion of hyperglycaemic hormone from the corpus cardiacum of flying blowflies, Calliphora erythrocephala. J. Insect Physiol. 20, 1189-1192. Vogt, M. (1949). Fettkorper und Oenocyten der Drosophila nach Exstirpation der adulten Ringdriise. 2. Zellforsch. mikrosk. Anat. 34, 160-164. Von Knorve, D.,Gersch, M. and Kusch, T. (1972). Zur Frage der Beeinflussung des “tanning”-Phanomens durch zyklisches 3’,5’-AMP. Zool. Jb. (Physiol.) 76, 434-440.

322

J. E. STEELE

Vroman, H. E., Kaplanis, J. N. and Robbins, W.,E. (1965). Effect of allatectomy on lipid biosynthesis and turnover in the female American cockroach, Periplaneta americana (L). J. Insect Physiol. 11, 897-904. Walker, P. R. and Bailey, E. (1971a). Effect of allatectomy on fat body lipid metabolism of the male desert locust during adult development. J. Insect Physiol. 17, 81 3-821. Walker, P. R. and Bailey, E. (1971b). Effect of allatectomy on the growth of the male desert locust during adult development. J. Insect Physiol. 17, 1125-1137. Walker, P. R. and Bailey, E. ( 1 9 7 1 ~ ) .Effect of allatectomy on fat body lipogenic enzymes of the male desert locust during adult development. J. Insect Physiol. 17, 1359-1 369. Wall, B. J. (1967). Evidence for anti-diuretic control of rectal water absorption in the cockroach, Periplaneta americana J. Insect Physiol. 13, 565-578. Wang, S.-Y. and Dixon, S. E. (1960). Studies on the transaminase activity of muscle tissue from allatectomized roaches, Periplaneta americana. Can. J . Zool. 38, 275283. Weis-Fogh, T. (1952). Fat combustion and metabolic rate of flying locusts (Schistocerca gregaria Forskal). Phil. Trans. R . SOC.Ser. B. 237, 1-36. Wellington, W. G. and Maelzer, D. A. (1967). Effects of farnesyl methyl ether on the reproduction of the western tent caterpillar, Malacosoma pluviale: some physiological, ecological and practical implications. Can. Ent. 99, 249-263. Welsh, J. R. and Moorhead, M. (1960). The quantitative distribution of 5-HT in the invertebrates, especially in their nervous system. 1.neurochem. 6 , 146-169. Whitehead, D. L. (1969). New evidence for the control mechanism of sclerotization in insects. Nature, Lond. 224, 721-723. Whitehead, D. L. (1970). L-dopa decarboxylase in the haemocytes of diptera. FEBS Lett. 7, 263-266. Whitehead, D. L., Brunet, P. C. and Kent, P. W. (1960). Specificity in vitro of a phenoloxidase system from Periplaneta americana. Nature, Lond. 185, 610. Wiens, A. W. and Gilbert, L. I. (1965). Regulation of cockroach fat body metabolism by the corpus cardiacum in vitro. Science, N.Y. 150, 614-616. Wiens, A. W. and Gilbert, L. I. (1967a). Regulation of carbohydrate mobilization and utilization in Leucophaea maderaw. J. Insect Physiol. 13, 779-794. Wiens, A. W. and Gilbert, L. I. (1967b). The phosphorylase system of the silkmoth Hyalophora cecropia. Comp. Biochem. Physiol. 21, 145-160. Wigglesworth, V. B. (1936). The function of the corpus allatum in the growth and reproduction of Rhodnius prolixus (Hemiptera). Q. Jl mictosc. Sci. 79, 91-121. Wigglesworth, V. B. (1940). The determination of characters at metamorphosis in Rhodnius prolixus (Hemiptera).J. exp. Biol. 17, 201-222. Wigglesworth, V. B. (1952). Hormone balance and the control of metamorphosis in Rhodnius prolixus (Hemiptera).J. exp. Biol. 29, 620-631. Wigglesworth, V. B. (1957). The action of growth hormones in insects. Symp. SOC.exp. Biol. 11, 204-227. Wigglesworth, V. B. (1960). The nutrition of the central nervous system in the cockroach Periplaneta americana L. The role of perineurium and glial cells in the mobilization of reserves. J. exp. Biol. 37, 500-512. Williams, C. M. (1952). Physiology of insect diapause. IV. The brain and prothoracic

HORMONAL CONTROL OF METABOLISM IN INSECTS

323

glands as an endocrine system in the Cecropia silkworm. Biol. Bull. mar. biol. Lab., Woods Hole, 97, 111-114. Williams, C. M. (1956). The juvenile hormone of insects. Nature, Lond. 178, 212-213. Williams, C. M. (1967). The present status of the brain hormone. In “Insect Physiology” (Eds J. W. L. Beament and J. E. Treherne), pp. 133-139. Oliver and Boyd, Edinburgh. Willis, J. H. and Brunet, P. C. J. (1966). The hormonal control of colleterial gland secretion. J. exp. Biol. 44, 363-378. Wright, J. E. and Rushing, D. D. (1973). Glycogen in pupal and adult stable flies as affected by a juvenile hormone analogue. Ann. ent. SOC. A m . 66,274-276. Wright, J. E., Crookshank, H. R. and Rushing, D. D. (1973). Glycogen phosphorylase activity in pharate adults of the stable fly and the effects of a juvenile hormone analogue. J . Insect Physiol. 19, 1575-1578. Wyatt, G. R. (1972). Insect hormones. In “Biochemical Actions of Hormones” (Ed. G. Litwack), vol. 2, pp. 385-490. Academic Press, London and New York. Wyatt, G. R. and Kalf, G. F. (1957). The chemistry of insect haemolymph. 11. Trehalose and other carbohydrates. J. gen. physiol. 40, 833-847. Wyatt, G. R. and Meyer, W. L. (1959). The chemistry of insect hemolymph. 111. Glycerol. 1.gen. physiol. 42, 1005-1011. Wyss-Huber, M. and Liischer, M. (1966). h e r die hormonale Beeinflussbarkeit der Proteinsynthese in vitro im Fettkorper von Leucophaea maderae (Insecta). Revue suisse Zool. 73, 517-521. Yamashita, 0. and Hasegawa, K. (1964). Studies on the mode of action of the diapause hormone in the silkworm, Bombyx mori L. IV. Effect of diapause hormone on the glycogen content in ovaries and the blood sugar level of silkworm pupae. J . Sericult. Sci. 33, 407-416. Yamashita, 0. and Hasegawa, K. (1966). The response of the pupal ovaries of the silkworm t o the diapause hormone with special reference to their physiological age. J. Insect Physiol. 12, 325-330. Yamashita, 0. and Hasegawa, K. (1967). The effect of the diapause hormone on the trehalase activity in pupal ovaries of the silkworm (A preliminary note). Proc. imp. Acad. Tokyo, 43, 547-551. Yamashita, 0. and Hasegawa, K. (1970). Oocyte age sensitive to the diapause hormone from the standpoint of glycogen synthesis in the silkworm, Bombyx mori. J. Insect Physiol. 16, 2377-2383. Yamashita, O., Hasegawa, K. and Seki, M. (1972). Effect of the diapause hormone on trehalse activity in pupal ovaries of the silkworm, Bombyx mori L. Gen. comp. Endocr. 18, 515-523. Yamazaki, M. and Kobayashi, M. (1969). Purification of the proteinic brain hormone of the silkworm, Bombyx mori. J. Insect Physiol. 15, 1981-1990. Zebe, E. and McShan, W. H. (1959). Incorporation of I4C-acetate into long chain fatty acids by the fat body of Prodenia eridania. Biochim. biophys. Acta, 31, 51 3-5 18. Ziegler, R. and Wyatt, G. R. (1975). Cold activated phosphorylase and glycerol production in diapausing silkmoth pupae. Nature, Lond. 254, 622-623. Zwicky, K. and Wigglesworth, V. B. (1956). rhe course of oxygen consumption during the moulting cycle of Rhodnius prolixus. Proc. R. ent. SOC. L o n d A31, 153-160.

This Page Intentionally Left Blank

Subject Index A

Accessory glands, and JH, 243 Acheta domestica egg size, 133 embryogenesis, 135, 176, 205 longitudinal body pattern, 136155 neurosecretory cells, brain, 88 protocerebral, 77 Acrididae coloration, 102 neurosecretory cells, 75, 84, 87 Acronycta, protocerebral neurosecretory cells, 8 1 Activation centres, embryonic pattern specification, 155-160, 206-207 Adelphocoris, protocerebral neurosecretory cells, 79 Adipokinetic hormone, 246-247, 283-286 Aedes, neurosecretory cells brain, 9 3 protocerebral, 76, 81 stomatogastric ganglia, 72 Aedes aegypti, medial NSC hormone, 268 Aedes solicitans, medial NSC hormone, 268 Aedes taeniorhynchus glycogen metabolism and JH, 250 lipid metabolism and JH, 27 1 medial NSC hormone, 268 325

Aeschna, protocerebral neurosecretory cells, 77 Aeschna cyanea, embryogenesis, 155 Agrion, protocerebral neurosecretory cells, 77 Amino acids incorporation in egg, 224 metabolism, endocrine control, 286-294 bursicon, 291-293 juvenile hormone, 288-291 moulting hormone, 287-288 nitrogen metabolism and CC, 294 synthesis of lipid from, 279 Anacridium, protocerebral neurosecretory cells, 78 Anatomy, neurosecretory system, 65-99 see neurosecretory system Andrena, protocerebral neurosecretory cells, 82 Animals other than insects Amphibia, embryogenesis, 126, 208,215 Annelida, nervous organization, 64 Annelida, polychaete, cerebral NSCs, 86 Aplysia, NSC coupling, 106 Arthropoda nervous organization, 64 ecdysone extraction, 17-62

326

Animals other than insects-cont. Ascaris lumbricoides, ecdysone concentration, 23 Balanus balanoides, ecdysone, 22, 25,44 Callinectes sapidus, ecdysone concentration, 23 Cherox destructor, for ecdysone bioassay, 34 chick wing, embryogenesis, 201 coelenterates, nervous organization, 64 Crangon vulgaris, ecdysone concentration, 22 crayfish, ecdysone extraction, 24 Crustacea ecdysone, 20, 22-23 for ecdysone bioassay, 34 neurosecretory cells, 99 X organ, 103 echinoderms, nervous organization, 64 Haemonchus contortus, ecdysone concentration, 23 Homarus americanus, ecdysone concentration, 23 Jasus lalandei, ecdysone, 23, 25, 27,34 leech, neurosecretory cells, 100 mammals, phosphorylase activation, 262, 263 Mollusca ecdysone, 20, 23 nervous organization, 64 neurosecretory cells, 99, 100 Mytilus edulis, ecdysone concentration, 23 Nematoda ecdysones, 20, 23 nervous organization, 64 Plasmodium, cleavage, 131 termite, embryogenesis, 136

SUBJECT INDEX

vertebrates adipokinetic hormones, 285 chromaffin cells, 73 glucagon, 260 nervous organization, 64 neurophysins, 100 neurosecretory cells, 99, 100, 103 thyroxine and respiration, 305 Anisolabis, neurosecretory cells protocerebral, 76, 79 volume, 105 Anoplura, protocerebral neurosecretory cells, 79 Ant embryogenesis, 187 NSCs during life history, 98 Anterior region involvement, embryogenesis, 1 72-184 Antheraea, neurosecretory cells, 81,96 An theraea perny i brain hormone, 245 ecdysone concentration, 21 Antheraea polyphemus choriogenesis, proteins, 10 effect of CA on respiration, 295 vitellogenin synthesis and JH, 278 Aphidae, neurosecretory cells, 7 3, 84,92 Aphis, neurosecretory cells, 80, 103 A p is differentiation centre, 131 embryogenesis, 133, 134, 1 76, 185-187, 203 neurosecretory cells during life history, 97 protocerebral, 82 volume, 105 Atrachya menetriesi, 133, 160, 182184,201,206,221

SUBJECT INDEX

327

Aulacophora, protocerebral neurosecretory cells, 83

B Bacillus, protocerebral neurosecretory cells, 78 Bee, honey, neurosecretory cells, 86 Beetle brain neurosecretory cells, 93 embryogenesis, body pattern cytological aspects, 20, 22, 23 longitudinal body pattern, 155, 160, 172-184, 190, 203 transverse body pattern, 209, 210,211,215 Beetle, Colorado, mitotic waves, 222 Belostoma, neurosecretory cells protocerebral, 80 volume, 105 Belostomatidae, protocerebral neurosecretory cells, 80 Bioassay, for ecdysones, 33-35, 54 Biogenic amines, 247 Blaberus cranifer effect of CA on respiration, 299 Blaberus discoidalis effect of CA on respiration, 298, 299, 304 hyperglycaemic hormone, 260 Blaps, neurosecretory cells anatomy, 109 brain, 90 during life history, 97 protocerebral, 83 total, 93 Blastoderm cell boundaries, 2 2 3 mitotic waves, 221-222 pattern specification, Diptera, 198 Blood, volume, 105

Blowfly cuticle tanning, 246, 291 longitudinal body pattern specification, 194, 198 mitotic waves, 222 tyrosine metabolism, moulting hormone, 287 Body pattern, specification in embryogenesis, 125-238, see Embryogenesis Bombus, flight motor neurones, 104 Bombyx mori body pattern specification, 21 1, 217-218 ecdysones concentration, 2 1 extraction, 18 GLC of, 48 large-scale extraction, 20 mass fragmentography of, 49 hormones brain, 245 diapause, 245, 254,255, 258 juvenile, 243,273,278 moulting, 248 labial glands, cellular metamorphosis, 2-4 neurosecretory cells brain, 89, 90, 94 during life history, 96 protocerebral, 8 1 total, 92 volume, 105 Brachycera, protocerebral neurosecretory cells, 82 Brain hormone, 244-245 Brain neurosecretory cells, 71-72, 87-94 Bruchidius obtectus, body pattern specification egg size, 133

328

SUBJECT INDEX

Bruchidius obtectus-cont. longitudinal, 173-184, 187, 189, 190,203 transverse, 215 Bursicon, 102, 103, 146, 291-294 C

Caliseta, NSCs during life history, 97 Calliphora ery thro cephala body pattern specification, 133, 196-197, 205 ecdysones concentration, 21 use for bioassay, 20, 34, 35 flight motor neurons, 104 hormones bursicon, 293 CA and respiration, 296 5-HT, 270 hyperglycaemic hormone, 260, 267 juvenile hormone, 250, 252, 271 medial neurosecretory cell hormone, 268 moulting hormone, 287 neurosecretory cells, 82, 93 Calliphora stygia, ecdysones, 21, 25, 35 Calliphora vicina, ecdysones, 21, 27 Calliphorid flies, optic lobe NSCs, 71 Callosobruchus maculatus, body pattern specification, 180-181, 209,211 Caliseta, NSCs during life history, 9 7 Calopteryx, protocerebral neurosecretory cells, 77 Calpodes, endocuticle deposition, 242

Camponotus body pattern specification, 187 neurosecretory cells, 82,97 Carausius morosus hormones CA and respiration, 295 hyperglycaemic response, 260 juvenile hormone, 25 0, 27 1 longitudinal body pattern specification, 160 neurosecretory cells protocerebral, 7 8 total, 91 Carbohydrate conversion to lipid, 278-279 metabolism, endocrine control, 247-270 diapause hormone, 254-259 5-hydroxytryptamine, 27 0 hyperglycaemic hormone, 259268 juvenile hormone, 249-254 medial neurosecretory cell hormone, 268-269 moulting hormone, 247-249 octopamine, 269-270 Cecropia moth juvenile hormone, 243 mid-gut, differentiation, 4 Cell polymorphism, sequential, 1-15, see Polymorphism Cellular aspects, embryonic pattern specification, 220-226 Chaetodacus, protocerebral neurosecretory cells, 82 Chiro suppressalis use in ecdysone bioassay, 34, 35 induction of apolysis, 242 Chironomid midges, embryonic pattern specification, 208,224 225

SUBJECT INDEX

Chironomus, chromosome puffing, 244 Chironomus dorsnlis, embryonic pattern specification, 189-192 Chironomus thummi, embryonic pattern specification, 189 Choriogenesis, follicular epithelial cells, 10-11 Chromatography, ecdysones gas-liquid, 38-49, 54, 55 electrophore, 44-45 preparation for ECD, 45-48 trimethyl silyl ethers, 40-43 trimethyl silyl heptafluoroborates, 43-44 high-pressureliquid, 50-53,54,55 liquid-liquid partition, 52-53 liquid-solid absorption, 53 reversed-phase, 5 1-52 thin-layer, 27, 29-33,54 Chromatophore, neurosecretory control of, 76 Chromosome puffing and ecdysone, 20 initiation in culture, 12 Na* and K*, 244 Chrysomelid beetles, neurosecretory cells, 73, 86 Chrysomeloidea, protocerebral neurosecretory cells, 83, 85 Clitumnus, neurosecretory cells, 7 8, 91 Clonal analysis, embryonic pattern specification, 219-220 Cockroach hormones adipokinetic hormone, 285, 286 bursicon, 246, 291-292 haemolymph lipids, 246 hyperglycaemic hormone, 282 juvenile hormone, 288-289

329

octopamine, 247 tanning, 288-289,291-292 neurosecretory cells anatomy, 109 brain, 94 extraganglionic, 74 during life history, 94, 98 optic lobe, 71 protocerebral, 76 Cocoon spinning, hormonal control, 111 Cocoonase organdes, sequential polymorphism, 5-9 Coleoptera egg size, 133 neurosecretory cells, 86, 87 brain, 87, 89 during life history, 9 7 protocerebral, 82, 85 total, 93 uniqueness of secretion, 104 Colleterial glands, glucoside synthesis, 288-289 Coloration, temporal factors, 102 Corpus cardiacum neurosecretory cells, 72, 90-93 nitrogen metabolism, 294 Cossus, protocerebral neurosecretory cells, 81 Cricket embryonic pattern specification differentiation of nuclei, 222 egg, RNA synthesis, 224 longitudinal pattern, 136-155, 199,202 mutants, 218 transverse pattern, 209 neurosecretory cells, 74, 7 6 Cricket, camel, embryonic pattern specification, 161, 2 11 Culex pipiens, embryonic pattern specification, 191

330

SUBJECT INDEX

Culicidae embryonic pattern specification, 191 extra ganglionic NSCs, 75 Culiseta, neurosecretory cells brain, 89 during life history, 99 protocerebral, 81 Cuticle deposition, endocrine control, 242 tanning, endocrine control, 246, 28 6-288 bursicon, 29 1-293 Cyclic AMP and hyperglycaemic hormone, 2 64 and moulting hormone, 288 and octopamine, 269-270 Cyclorrhapha, protocerebral neurosecretory cells, 8 2 D Dacus tryoni, embryonic pattern specification, 196-197, 205 Damsel fly, embryonic pattern specification, 155-160 Danaus, JH and protein synthesis, 275 Dasychiru, protocerebral neurosecretory cells, 81 Dendrolimas, neurosecretory cells, 81,96 Dermaptera, protocerebral neurosecretory cells, 79 Derrnestes, protocerebral neurosecretory cells, 83 Dermestes frischii, embryonic pattern specification, 174-175, 183, 206,207,209, 221, 223 Deutocerebrum, neurosecretory cells, 7 1

Diapause hormone, 245-246 in carbohydrate metabolism, 254259 in lipid metabolism, 281-282 Dicranura, protocerebral neurosecretory cells, 81 Dictyoptera, neurosecretory cells brain, 88 during life history, 95 protocerebral, 78 total, 91 Differentiation centre, embryonic pattern specification, 155-160, 205, 212-216 Diglyceride haemolymph, 246 release, endocrine control, 301 Diptera cyclorrhaphous, cuticle tanning, 287 embryonic pattern specification egg size, 133 higher, 128, 192-200 lower, 189 moulting hormone assay, 33-35 neurosecretory cells brain, 89, 93 extraganglionic, 7 3 during life history, 96 protocerebral, 81, 85, 86, 87 total, 93 uniqueness of secretion, 101, 103 Diuretic hormone, 103 DNA synthesis, silk gland metamorphosis, 4 Dociostuurus maroccanus, ecdysone concentration, 22 Dopamine, site of synthesis, 292 Dopamine-3-O-sulphate, synthesis of,293

331

SUBJECT INDEX

Dragonfly embryonic pattern specification, longitudinal, 155-160, 202, 210 protocerebral neurosecretory cells, 85 Drepanosiphum, neurosecretory cells, 80, 92 Drosophila melanogaster ecdysone determination gas-liquid chromatography, 46 radioimmunoassay, 38 reversedphase chromatography, 52 embryonic pattern specification clonal analysis, 2 19 double abdomens, 224-225 egg size, 133 longitudinal pattern, 192-4, 197, 198-200 mutants, 217,222 nuclear transplantation, 222 hormones JH and glycogen metabolism, 25 0 JH and glycogen synthesis, 253 JH and lipid metabolism, 27 1 MH and tyrosine metabolism, 288 neurosecretory cells brain, 93 during life history, 96 protocerebral, 82, 85 volume, 105 Dye injection, NSC anatomy, 106109 Dysdercus, protocerebral neurosecretory cells, 79 E Ecdyonurus, protocerebral neurosecretory cells, 7 7

Ecdysone description, 241-243 JH, 102 sequential cell polymorphism, 3, 6 Ecdysones, extraction and determination, 17-62 analytical methods, 53-56 large-scale extraction, 20-29 concentration, 25-26 initial extraction, 24-25 isolation, 26-29 microdetermination, 29-53 bioassay, 33-35 gas-liquidchromatography, 3849 high-pressure liquid, 50-53 mass fragmentography, 49-50 optical spectroscopy, 35-36 radioimmunoassay, 36-38 thin-layer chromatography, 2933 Egg size, 133 Electron capture detector, for GLC of ecdysones, 40, 45-48, 54-55 Electron transport system, effect of hormones, 305 Embioptera, protocerebral neurosecretory cells, 77 Embryogenesis, body pattern specification, 125-238 cytological and molecular, 220226 blastodermal cell boundaries, 223 differentiation of nuclei, 222 mitotic waves, blastoderm, 221-222 molecular, 223-226 oocyte, 220-221 early development, modes, 133136

332

Em bry ogenesis- cont. elements and origin, 128-133 genetic studies, 216-220 clonal analysis, 219-220 mapping of foci, 2 19 mutants, 216-219 longitudinal body pattern, 136208 activation and differentiation centres, 155-160 anterior region involvement, 172-184 beetle eggs, 172-184 cricket eggs, 136-155 dragonfly eggs, 155-160 generalizations, 20 0-2 08 gradient concept, 163-172 growing blastema, 160-163 higher Diptera, 192-200 Hymenoptera and Lepidoptera, 184-189 leaf hopper, 163-172 lower Diptera, 189-192 mirroring heads and abdomens, 189-192 potency regions, 184-189 short germ-type, 160-163 transverse bilateral pattern, 208216 blastoderm, 208-212 differences from longitudinal, 212 differentiation centre, 212-216 Endocrine control of metabolism, 239-323, see Hormones Enzymes, effect of JH on, 279 Ephemera, protocerebral neurosecretory cells, 77 Ep hemeroptera, pro tocerebral neurosecretory cells, 77 Ephestza, neurosecretory cells, 8 1, 92,93

SUBJECT INDEX

Ephestia kuehniella, embryonic pattern specification, 188 Epidermis, neurosecretory innervation, 74 Epinephrine, adipokinetic effect of, 285-286 Eudia, protocerebral neurosecretory cells, 81 Eumenes, protocerebral neurosecretory cells, 82 Euroleon, protocerebral neurosecretory cells, 80 Euycnema, neurosecretory cells protocerebral, 78 volume, 106 Euscelis plebejus, embryonic pattern specification activation centre, 207 egg size, 133 gap phenomenon, 176, 190 longitudinal pattern, 163-172, 187,202,203,205 Extraction of ecdysones, 17-62, see Ecdy sones Extraganglionic neurosecretory cells, 73-75 F Farnesyl methyl ether, lipogenic effect, 278 Fat body and hyperglycaemic hormone, 260-261 lipid synthesis and JH, 271-273 protein synthesis and JH, 273276 Flame ionization detector, for GLC of ecdysones, 40 Flight and adipokinetic hormone, 283286

SUBJECT INDEX

333

and hyperglycaemic , hormone, 283 motor neurons, number, 104 Fluorescence spectroscopy, of ecdysones, 5 4 Foci, developmental, mapping of, 219 Follicular epithelial cells, sequential polymorphism, 9-11 Forficula, protocerebral neurosecretory cells, 79 Formica, neurosecretory cells during life history, 97 protocerebral, 82 Formicid queens, neurosecretory cells, 86

G Galea, silkmoth, sequential polymorphism, 5-9 Galeruca, protocerebral neurosecretory cells, 83 Galleria, neurosecretory cells brain, 8 9 , 9 4 during life history, 95 protocerebral, 81, 85 total, 92 Galleria mellonella, effect of CA on respiration, 296 Gap phenomenon, embryogenesis, 171, 175-176 Genetic studies, embryonic pattern specification, 216-220 Glossina, neuro secretory cells protocerebral, 82 volume, 105 Glucose, haemolymph, and hyperglycaemic hormone, 265 Glucoside synthesis, role of JH, 288-29 1

Glycogen effect of diapause hormone, 254255 formation of trehalose from, 260 in nerve cord, and octopamine, 269 synthesis, and JH, 249-253 synthesis, post-hormonal, 265266 Glycogenolysis,and hyperglycaemic hormone, 266 Gonadotropic hormone, lampyrid, 103 Gradient concept, embryonic pattern specification, 163-172 Grasshopper, neurosecretory cells ocellar nerve, 7 1 protocerebral, 76 Grylloblattodea, neurosecretory cells brain, 88 protocerebral, 76, 7 7 , 8 6 Gryllotalpa, protocerebral neurosecretory cells, 7 7 Gryllus, ocellar nerve neurosecretory cells, 71 Gryllus domesticus, effect of CA on respiration, 295 Gut movement, endocrine control, 301 neurosecretory innervation, 74 H Haemocytes, effect of bursicon on, 292-293 Heart endocrine control, 30 1 neurosecretory innervation, 74 Hemiptera embryonic pattern specification, 129

334

Hemiptera-cont. neurosecretory cells brain, 88, 9 3 extraganglionic, 74 median, 84 protocerebral, 79, 86 total, 92 uniqueness of secretion, 101 lierse, neurosecretory cells during life history, 99 protocerebral, 8 1 Homoptera egg size, 133 neurosecretory cells brain, 9 3 protocerebral, 80, 84, 8 6 total, 92 uniqueness of secretion, 101, 103 Hormonal control of metabolism, 239-323 amino acid metabolism, 286-294 bursicon, 291-293 juvenile hormone, 288-291 moulting hormone, 287-288 nitrogen metabolism, and CC, 294 carbohydrate metabolism, 247270 diapause hormone, 254-259 5-hydroxytryptamine, 270 hyperglycaemic hormone, 259268 juvenile hormone, 249-254 medial neurosecretory cell hdrmone, 268-269 moulting hormone, 247-249 octopamine, 269-270 hormones, 241-247 adipokinetic hormone, 246 biogenic amines, 247 brain hormone, 244-245 bursicon, 246

SUBJECT INDEX

diapause hormone, 245-246 hyperglycaemic hormone, 246 juvenile hormone, 243-244 moulting hormone, 241-243 lipid metabolism, 270-286 adipokinetic hormone, 283-286 diapause hormone, 2 81-282 hyperglycaemic hormone, 282283 juvenile hormone, 271-281 respiration, 294-305 isolated tissues, 301-303 mitochondria, 303-305 saturniid labial gland, metamorphosis, 3 uniqueness of secretion, 102-106 Housefly, JH and trehalose level, 253 Hyalophora cecropia hormones adipokinetic, 285-28 6 hyperglycaemic, 263 juvenile, 272, 273, 278 moulting, 249 phosphorylase activity, 258 neurosecretory cells anatomy, 109 protocerebral, 8 1 Hydrous, protocerebral neurosecretory cells, 8 3 5-Hydroxytryptamine, and carbohydrate metabolism, 247, 270 Hymenoptera embryonic pattern specification, 133, 184-187 neurosecretory cells brain, 9 4 during life history, 9 7 protocerebral, 82, 85, 86, 87 uniqueness of secretion, 104 Hyperu, protocerebral neurosecretory cells, 8 3 Hyperglycaemic hormone, 103, 246

SUBJECT INDEX

335

in carbohydrate metabolism, 259- Katydid, protocerebral neuro268 secretory cells, 76 in lipid metabolism, 282-283 Hypoglycaemic factor, 103 L Hypolipaemic factor, 246-247 Labial gland, cellular metamorphosis saturniid, 2-4 I sphingid, 4-5 Labidula, protocerebral neuroIchneumonid wasp, embryonic secretory cells, 79 pattern specification, 187 Lampyrids,gonadotrophic hormone, Imaginal discs, embryonic pattern 76,103 specification, 198-200 Lampyris, neurosecretory cells Iphita, neurosecretory cells protocerebral, 8 3 protocerebral, 79 staining for, 66 volume, 105 Leaf hopper, longitudinal pattern Ischnura elegans, embryonic pattern specification, 147, 155, 163specification, 157 172, 187, 190, 203, 205, 207, Isoperla, protocerebral neurosecre211,220 tory cells, 78 Isoptera, protocerebral neurosecre- Lepidoptera effect of farnesyl methyl ether, tory cells, 77 278 embryohic pattern specification, 188-189, 211 J neurosecretory cells Juvenile hormone brain, 89,94 in amino acid metabolism, 288diversity, 86, 87 29 1 during life history, 95 in carbohydrate metabolism, 249protocerebral, 8 1, 85 254 total, 92 in lipid metabolism, 271-281 uniqueness of secretion, 101 and nucleic acid and protein synvolume, 105 thesis, 240-241 Leptinotarsa, neurosecretory cells and respiration, 294-305 brain, 7 1, 89 and sequential cellpolymorphism, protocerebral, 83 3, 6 volume, 105 and uniqueness of secretion, 102 Leptinotarsa decemlineata embryonic pattern specification differentiation centre, 2 12-216 K egg size, 133 Kqilotermes fL’avicollis, embryonic longitudinal pattern, 177, 178, pattern specification, 160, 222, 179, 180, 182, 205, 206, 224 209.210

336

Leptinotarsa decemlineata-cont. nuclei, 222 hormones CA and respiration, 296, 298, 303 juvenile hormone, 274, 275 Leptothorax, neurosecretory cells, 97 Leucophaea, neurosecretory cell anatomy, 108 Leucophaea maderae ecdysone determination, 38, 52 hormones adipokinetic, 2 85-28 6 CA and respiration, 295, 297, 299 hyperglycaemic, 261, 262, 264 juvenile, 251, 273, 274, 275 respiration and protein synthesis, 302 Lipid metabolism, endocrine control, 270-286 adipokinetic hormone, 283-286 diapause hormone, 2 8 1-282 hyperglycaemic hormone, 28228 3 juvenile hormone, 271-281 Locust, hormones adipokinetic, and flight, 283-286 bursicon, 29 1 haemolymph diglyceride, 246 hyperglycaemic, 282-28 3 juvenile, 290 Locusta, neurosecretory cells anatomy, 108, 109 during life history, 9 4 protocerebral, 7 7 Locusta mipatoria, hormones CA and respiration, 295 CC and nitrogen metabolism, 294 hyperglycaemic, 260, 263, 265, 266,267

SUBJECT INDEX

juvenile and lipid metabolism, 271 and phosphorylase activity, 254 and protein synthesis, 25 1, 271,274,275 transaminase activity, 29 1 medial neurosecretory cell, 268, 269 Locusta migratoria migratorioides, embryonic pattern aberration, 218 Lucilia, brain neurosecretory cells, 89,93 Luminescent organ, neurosecretory innervation, 74 Lygeidae, protocerebral neurosecretory cells, 79 L ymantria brain hormone, 244 neurosecretory cells, 81, 96

M Malacosoma, protocerebral neurosecretory cells, 81 Malacosoma pluviale, effect of farnesyl methyl ether, 278 Mallophaga, protocerebral neurosecretory cells, 79 Malpighian tubules, differentiation, 4 Manduca, neurosecretory cells dye injection, 108 during life history, 96, 98 total, 92 Manduca sexta cellular metamorphosis, 4-5 ecdysone determination, 21, 25, 38 juvenile hormone, 244

337

SUBJECT INDEX

Mass fragmentography of ecdysones, 49-50, 54 Mating behaviour, hormonal control of, 11 Mayfly, protocerebral neurosecretory cells, 85 Mealworm, tyrosine metabolism, 287 Mecoptera, protocerebral neurosecretory cells, 80 Medial neurosecretory cell hormone, 268-269 Melanoplus, neurosecretory cells, 78,105 Melanoplus differentialis, lipid metabolism, 2 7 l Melasoma populi, embryonic pattern specification, 180 Melolontha, juvenile hormone, 244 Metabolism, hormonal control of, 239-323, see Hormones Metamorphosis, cellular, labial gland saturniid, 2-4 sphingid, 4-5 Microdetermination of ecdysones, 29-53 Mid-gut, differentiation, 4 Mimas, neurosecretory cells, 93 Miridae, protocerebral neurosecretory cells, 79 Mirroring heads and abdomens, 189192 Mitochondria embryonic pattern specification, 226 endocrine control of respiration, 303-305 Mitotic waves, blastoderm formation, 221-222 Mosaic development, embryogenesis, 205

Mosquito embryonic pattern specification, 218 moulting hormones, 288 Moth embryonic pattern specification, 21 1 neurosecretory cells, 7.4,93, 110 Moulting hormone in amino acid metabolism, 287288 in carbohydrate metabolism, 247249 effect on respiration, 301 Musca domestica amino acid incorporation, egg, 224 for ecdysone bioassay, 34, 35 glycogen metabolism, 250 Muscle, skeletal, neurosecretory innervation, 74 Mutants, embryogenesis of, 216219 Myzus, neurosecretory ceIls, 73, 92

N

Nauphoeta cinerea effect of CA on respiration, 297, 299,301 JH and protein synthesis, 25 1 Nebria, neurosecretory cells, 82,105 Necrobia rufipes, embryonic pattern specification, 176, 181, 184, 2 02 Nematocera, protocerebral neurosecretory cells, 81 Neomyrma, neurosecretory cells during life history, 97 Neoptera, protocerebral neurosecretory cells, 80

338

SUBJECT INDEX

Nepidae, protocerebral neurosecretory cells, 80 Nerve cord, glycogen metabolism hyperglycaemic hormone, 266 octophamine, 269 Neurosecretory system, unique identifiable neuron concept, 63-123 anatomy, 65-99 distribution, 7 1-75 diversity, 75-99 morphological studies, 70-71 recognition, 65-67 “specific” staining techniques, 67-70 identifiable neuron concept, 99106 constancy, uniqueness and reduplication, 100-106 neurosecretory cells and other neurons, 99-100 techniques, 106-111 cobalt staining, silver precipitation, 107-109 filling with dye, 106-107 intracellular current injection, 110-111 Neuroptera, optic lobe neurosecretory cells, 7 1 Nazuru, protocerebral neurosecretory cells, 79 Nitrogen metabolism, control by CC, 294 Nuclei, functional differentiation of, 22 2 Nucleic acid synthesis, role of hormones, 240-241, 243 0

Ocellar nerve, neurosecretory cells, 71

Octopamine, in carbohydrate metabolism, 247, 269-270 Odonata chromatophore control, 76 egg size, 133 protocerebral neurosecretory cells, 77 Oecanthus pellucens, egg size, 133 Oligoneoptera, neurosecretory cells brain, 71, 80 protocerebral, 80, 82, 84-85, 86 uniqueness of secretion, €04 Oncop el tus fascia t us effect of CA on respiration, 300 embryonic pattern specification, 220 juvenile hormone and protein synthesis, 252 neurosecretory cells brain, 87, 88 protocerebral, 79 stomatogastric ganglia, 72 volume, 105 Oocyte, cytoarchitecture, 220-22 1 Ootheca, tanning, endocrine control, 288-291 Optic lobe neurosecretory cells, 7 1 Orgya, protocerebral neurosecretory cells, 81 Ort hoptera embryonic pattern specification, 133,209,211 neurosecretory cells brain, 71, 88, 94 constancy, 100 coupling, 11 extraganglionic, 74 during life history, 95 protocerebral, 76, 77, 85, 86, 87 staining, 70 total, 91

SUBJECT INDEX

339

uniqueness of secretion, 103 VNC, type A, 75 volume, 105 Ostrinia, neurosecretory cells, 9 3 Ovary, control by JH, 243, 281 Oviposition, hormonal control, 111 P Palaeoptera, protocerebral neurosecretory cells, 76, 77, 86 Panorpa, protocerebral neurosecretory cells, 80 Paraneoptera, protocerebral neurosecretory cells, 79, 84, 86 Pentatomidae, , protocerebral neurosecretory cells, 79 Periplaneta americana hormones adipokinetic, 247 brain, 245 bursicon, 292,293 CC and nitrogen metabolism, 294 CC and respiration 5-HT, 247,270 hypergly caemic, 2 46, 2 5 9-26 1, 264,265,267,282 hypolipaemic response, 286 JH and lipid metabolism 277 JH, naturally occurring, 244 JH and protein synthesis, 251, 274,275 JH, transaminase activity, 29029 1 JH and uric acid production, 290 octopamine, 269 neurosecretory cells brain, 71, 88 during life history, 95 protocerebral, 78

total, 91 volume, 105 Phalaera, protocerebral neurosecretory cells, 8 1 Phasmida chromatophore control, 76 neurosecretory cells brain, 87, 94 extraganglionic, 7 3 protocerebral, 78,84,85, 86 total, 91 Phklosarnia, neurosecretory cells, 81, 96 Philosamia Cynthia, JH and protein synthesis, 2 7 3 Phormia regina, hormones hyperglycaemic, 260, 261, 264265 JH and glycogen metabolism, 250 JH and protein synthesis, 274 moulting hormone, 248 Phosphofructokinase, and diapause hormone, 256-259 Phosphorylase activity hyperglycaemic hormone, 262264 juvenile hormone, 253-254 octopamine, 269 Physical properties, ecdysones, 2729 Pieris, protocerebral neurosecretory cells, 8 1 Pieris brassicae bursicon, 293 moulting hormone, 287 Pimpla turionellae, embryonic pattern specification, 187 Platyenemis pennipes, embryonic pattern specification egg size, 133 longitudinal pattern, 155-160, 161,183, 202 nuclei, 222

340

Plecoptera, protocerebral neurosecretory cells, 78 Polymorphism, sequential, 1-15 saturniid labial gland, 2-4 silkmoth follicular epithelial cells, 9-11 silkmoth galea, cocoonase organules, 5-9 sphingid labial gland, 4-5 Polyneoptera, protocerebral neurosecretory cells, 76, 77, 84, 86 Polyol formation, diapause, 256259 Plodia interpunctella, CA and respiration, 298, 303 Potency regions, embryogenesis 184-189, 206 Proteins in choriogenesis, 10-11 synthesis and JH, 251-253 and respiration, 303 role of hormones, 240-241, 243 Prothoracicotropic hormone, 7 5, 103 Prothoracic gland, neurosecretory innervation, 74 Protocerebral neurosecretory cells, 71, 76-87 Pro tophormia, longitudinal body pattern specification, 176, 195, 197 Psocoptera, protocerebral neurosecretory cells, 79 Pyrrhocoridae, protocerebral neurosecretory cells, 79 Pywhocoris apterus, hormones effect of CA on respiration, 295, 297, 300 juvenile hormone and glycogen metabolism, 250

SUBJECT INDEX

and lipid synthesis, 2 79 and protein synthesis, 274 transaminase activity, 29 1

Q Quinones, metabolism cuticle, 286-288 ootheca, 288-291 role of bursicon, 291-293

R Radioimmunoassay, ecdysones, 3638, 54, 55 Ranatra, neurosecretory cells, 80, 105 Reduviidae, neurosecretory cells, 79, 93 Respiration, endocrine control, 294305 isolated tissues, 301-303 mitochondria, 303-305 Retrocerebral complex, neurosecretory cells, 72 Rhodnius prolixus hormones brain, 244 juvenile CA, 243 lipid synthesis, 279 protein synthesis, 274 uric acid production, 290 moulting, 18, 301 neurosecretory cells brain, 88 extraganglionic, 75 protocerebral, 79, 84 total, 92 volume, 105

SUBJECT INDEX

341

Ribosomes, and embryonic pattern specification, 226 RNA synthesis, cricket egg, 224 mRNA, choriogenesis, 11 S Salivary gland, neurosecretory innervation, 74 Samia Cynthia, hyperglycaemic hormone, 263 Samia Cynthia pryeri, carbohydrate metabolism, 249 Sarcop haga hormones bursicon, 246, 292 moulting, 288 tyrosine metabolism, 288, 29 2 neurosecretory cells during life history, 9 7 ocellar nerve, 7 1 protocerebral, 82 total, 9 3 Sarcophaga peregrina, for ecdysone bioassay, 34 Saturniid moth labial gland, cellular metamorphosis, 2-4 neurosecretory cells optic lobe, 71 stomatogastric ganglia, 72 volume, 105 Scu tellera, protocerebral neurosecretory cells, 79 Sc his tocerca gregaria ecdysone determination concentration, 22 for bioassay, 34, 35 gas-liquid chromatography, 4548 initial extraction, 25, 26

embryonic pattern specification, 160, 162-163, 201, 206 hormones adipokinetic, and flight, 284 CA and respiration, 295, 298, 300 CC and protein synthesis, 302 hyperglycaemic, 265, 267 JH and glycolytic enzymes, 279 JH and lipid metabolism, 271272,277 JH, naturally occurring, 244 JH and protein synthesis, 252, 274 octopamine, 247 neurosecretory cells blood volume, 105 brain, 8 8 , 9 0 dye injection, 107-109 during life hisory, 94, 95 flight motor neurons, 104 protocerebral, 78 total, 91 volume, 105, 106 Schizodactylus, neurosecretory cells, 77, 88 Sequential cell polymorphism, 1-15, see Polymorphism Short germ-type pattern, embryogenesis, 160-163 Silk gland, cellular metamorphosis, 2-4 Silkmoth, sequential polymorphism cocoonase organules, 5-9 follicular epithelial cells, 9-1 1 Silkworm, hormones diapause, and lipid metabolism, 28 1 moulting, and tyrosine metabolism, 287 Silylation, ecdysones, 40-48

342

Siphonaptera, protocerebral neurosecretory cells, 80 Sitophilus, protocerebral neurosecretory cells, 8 3 Skeletal muscle, neurosecretory innervation, 74 Smittia, embryonic pattern specification double abdomen, 189-190, 221, 225-226 egg size, 133 gap phenomenon”, 176 Spectroscopy, optical, ecdysones, 35-36 Sphingid moth, neurosecretory cells, 72 Sphinx, neurosecretory cells, 81, 99 Sphodoptera, lipid metabolism and JH, 271 Spilosoma, protocerebral neurosecretory cells, 81 Staining, “specific”, for neurosecretory cells, 66, 67-70 Stilbocoris, neurosecretory cells protocerebral, 79 total, 92 volume, 105 Stick insects, embryogenesis, 136 Stomatogastric ganglia, neurosecretory cells, 72 S t o m o x y s calcitrans, phosphorylase activity, 253 Strepsiptera, protocerebral neurosecretory cells, 82 Suboesophageal ganglion, neurosecretory cells, 72-73 Sympetrum, protocerebral neurosecretory cells, 77 Synagris, protocerebral neurosecretory cells, 82 (6

SUBJECT INDEX

T Tabanus, protocerebral neurosecretory cells, 82 Tachycines asynamonus, embryonic pattern specification, activation centre, 201 longitudinal pattern, 161-162, 205,206 transverse pattern, 209, 21 1 type of development, 134 Tenebrio molitor embryonic pattern specification longitudinal pattern, 174, 182, 202 type of development, 129,134 hormones adipokinetic, 284 bursicon, 291 CA and respiration, 296 JH, transaminase activity, 29 1 Tettigia, protocerebral neurosecretory cells, 80 Thysanura, neurosecretory cells, 79, 86 Tineola biseliella, embryonic pattern specification, 188-189, 2 1 1 Tipula, protocerebral neurosecretory cells, 82 Transaminase activity, effect of JH, 290-291 Transverse bilateral pattern, embryogenesis, 208-216 Trehalose metabolism, endocrine control, 301 diapause hormone, 254, 255256 hyperglycaemic hormone, 246, 259-260, 264 juvenile hormone, 253 moulting hormone, 248, 249 Trichoptera, protocerebral neurosecretory cells, 80

SUBJECT INDEX

343

Trimethylsilyl ethers, ecdysones, 40-

43 Trimethylsilyl heptafluoroborates, ecdysones, 43-45 Tritocerebrum, neurosecretory cells, 72 Tyrosine metabolism endocrine control, 286-288 bursicon, 291-293

Vespa, embryonic pattern specification, 185 Vitellogenesis, silkmoth, 9-10 Vitellogenin synthesis, endocrine control farnesyl methyl ether, 278 glucoside synthesis, 290 JH, 243,275,276 Volume, neurosecretory cells, 105-

106 U Ultraviolet detector, ecdysone chromatography, 50-51, 56 Unique identifiable neuron concept, 63-123, see Neurosecretory system. Uric acid, regulation by JH, 290

W Water reabsorption, endocrine control, 301 Wax secretion, endocrine control,

242 Wyeomyiu smithii, embryonic pattern specification, 191, 218

V

Vunessu, protocerebral neurosecretory cells, 81 VNC ganglia, neurosecretory cells, 72-73, 87-94

Z Zonaptera, protocerebral neurosecretory cells, 7 7 Zymogen organule formation, 6-9

This Page Intentionally Left Blank

Cumulative List of Authors Numbers in bold face indicate the volume number of the series

Aidley, D. J., 4, 1 Andersen, Sven Olav, 2, 1 Ashini, E., 6, 1 Ashburner, Michael, 7, 1 Baccetti, Baccio, 9, 315 Barton Browne, L., 11, 1 Beament, J. W. L., 2, 67 Berridge, Michael J., 9, 1 Boistel, J., 5 , 1 Brady, John, 10, 1 Bridges, R. G., 9, 5 1 Burkhardt, Dietrich, 2, 13 1 Bursell, E., 4, 33 Burtt, E. T., 3, 1 Carlson, A. D., 6, 5 1 Catton, W. T., 3, 1 Chen, P. S., 3 , 5 3 Colhoun, E. H., 1, 1 Cottrell, C. B., 2, 175 Crossley, A. Clive, 11, 1 1 7 Dadd, R. H., 1 , 4 7 Dagan, D., 8 , 9 6 Davey, K. G., 2 , 2 1 9 Edwards, John S., 6 , 9 7 Eisenstein, E. M., 9, 111 Fraser Rowell, C. H., 8, 146 Gilbert, Lawrence I., 4, 69 Goodman, Lesley, 7, 97 Harmsen, Rudolf, 6, 139 Harvey, W. R., 3, 133 Haskell, J. A., 3, 133 Hinton, H. E., 5 , 65 Hoyle, Graham, 7, 349

Fotis c., 12, 1 Kilby, B. A., 1, 111 Lawrence, Peter A., 7, 197 Lees, A. D., 3, 207 Linzen, Bernt, 10, 117 Maddrell, S. H. P., 8 , 200 Michelsen, Axel, 10, 247 Miles, P. W., 9, 183 Miller, P.'L., 3, 279 Morgan, E. David, 12, 1 7 Narahashi, Toshio, 1, 175; 8, 1 Neville, A. C., 4, 213 Nocke, Harold, 10,247 Parnas, I., 8, 96 Pichon, Y., 9, 257 Poole, Colin F., 12, 1 7 Prince, William T., 9, 1 Pringle, J. W. S., 5 , 163 Riddiford, Lynn M., 10, 297 Rowell, Hugh Fraser, 12, 63 Rudall, K. M., 1 , 257 Sacktor, Bertram, 7, 268 Sander, Klaus, 12, 125 Shaw, J., 1, 315 Smith, D. S., 1, 401 Steele, J. E., 12, 239 Stobbart, R. H., 1 , 3 1 5 Telfer, William H., 11, 223 Thomson, John A., 1 1 , 3 2 1 Treherne, J. E., 1, 401; 9, 257 Truman, James, W., 1 0 , 2 9 7 Usherwood, P. N. R., 6, 205 Waldbauer, G. P., 5 , 229 KdfdtOS,

345

346

Weis-Fogh, Torkel, 2, 1 Wigglesworth, V. B., 2, 247 Wilson, Donald M., 5, 289

CUMULATIVE LIST OF AUTHORS

Wyatt, G . R., 4, 287 Ziegler, Irmgard, 6,139

Cumulative List of Chapter Titles Numbers in bold face indicate the volume number of the series

Active Transport and Passive Movement of Water in Insects, 2, 67 Amino Acid and Protein Metabolism in Insect Development, 3, 5 3 Biochemistry of Sugars and Polysaccharides in Insects, 4, 287 Biochemistry of the Insect Fat Body, 1 , 1 1 1 Biology of Pteridines in Insects, 6, 139 Biophysical Aspects of Sound Communication in Insects, 10, 247 Cells of the Insect Neurosecretory System: Constancy, Variability, and the Concept of the Unique Identifiable Neuron, 12, 63 Cellular Mechanisms Underlying Behaviour-Neuroethology, 7, 349 Chitin Orientation in Cuticle and its Control, 4, 213 Chitin/Protein Complexes of Insect Cuticles, 1, 257 Choline Metabolism in Insects, 9, 51 Colour Discrimination in Insects, 2, 131 Comparative Physiology of the Flight Motor, 5, 163 Consumption and Utilization of Food by Insects, 5, 229 Control of Polymorphism in Aphids, 3, 207 Control of Visceral Muscles in Insects, 2, 219 Cytophysiology of Insect Blood, 11, 117 Development and Physiology of Oocyte-Nurse Cell Syncytium, 11, 223 Effects of Insecticides in Excitable Tissues, 8, 1 Electrochemistry of Insect Muscle, 6, 205 Excitation of Insect Skeletal Muscles, 4, 1 Extraction and Determination of Ecdysones in Arthropods, 12, 17 Excretion of Nitrogen in Insects, 4, 33 Feeding behaviour and Nutrition in Grasshoppers and Locusts, 1, 47 Frost Resistance in Insects, 6, 1 Function and Structure of Polytene Chromosomes During Insect Development, 7, 1 Functional Aspects of the Organization of the Insect Nervous System, 1, 40 1 Functional Organizations of Giant Axons in the Central Nervous Systems of Insects: New Aspects, 8 , 9 6 Hormonal Control of Metabolism in Insects, 12,239 347

348

CUMULATIVE LIST OF CHAPTER TITLES

Hormonal Mechanisms Underlying Insect Behaviour, 10, 29 7 Hormonal Regulation of Growth and Reproduction in Insects, 2, 247 Image Formation and Sensory Transmission in the Compound Eye, 3, 1 Insect Blood-Brain Barrier, 9, 257 Insect Ecdysis with Particular Emphasis on Cuticular Hardening and Darkening, 2, 175 Insect Sperm Cells, 9, 315 Learning and Memory in Isolated Insect Ganglia, 9, 11 1 Lipid Metabolism and Function in Insects, 4, 69 Major Patterns of Gene Activity During Development in Holometabolous Insects, 11, 321 Mechanisms of Insect Excretory Systems, 8, 200 Metabolic Control Mechanisms in Insects, 3, 133 Nervous Control of Insect Flight and Related Behaviour, 5, 289 Neural Control of Firefly Luminescence, 6, 51 Osmotic and Ionic Regulation in Insects, 1, 315 Physiology of Insect Circadian Rhythms, 10, 1 Physiological Significance of Acetylcholine in Insects and Observations upon other Pharmacologically Active Substances, 1, 1 Polarity and Patterns in the Postembryonic Development of Insects, 7, 197 Postembryonic Development and Regeneration of the Insect Nervous System, 6, 97 Properties of Insect Axons, 1, 175 Regulation of Breathing in Insects, 3, 279 Regulation of Intermediary Metabolism, with Special Reference to the Control Mechanisms in Insect Muscle, 7, 268 Regulatory Mechanisms in Insect Feeding, 11, 1 Resilin. A Rubberlike Protein in Arthropod Cuticle, 2, 1 Role of Cyclic AMP and Calcium in Hormone Action, 9, 1 Saliva of Hemiptera, 9, 183 Sequential Cell Polymorphism: A fundamental Concept in Developmental Biology, 12, 1 Specification of the Basic Body Pattern in Insect Embryogenesis, 12, 125 Spiracular Gills, 5, 65 Structure and Function of the Insect Dorsal Ocellus, 7 , 9 7 Synaptic Transmission and Related Phenomena in Insects, 5, 1 Tryptophan + Ommochrome Pathway in Insects, 10, 117 Variable Coloration of the Acridoid Grasshoppers, 8, 146