Histology and Ultrastructure of Wing

HISTOLOGY AND ULTRASTRUCTURE

OF WING DEVELOPMENT IN

ONCOPELTUS FASCIATUS DALLAS

(HEMIPTERA: LYGAEIDAE)

NIVEDITA MALLELA M. Sc,

A thesis submitted for the degree of Doctor of Philosophy in the University of London

Department of Zoology and Applied Entomology Imperial College of Science and Technology South Kensington LONDON SW 7.

September, 1979. 2

ABSTRACT

The postembryonic development of the fore and hind wings of Oncopeltus fasciatus has been studied by various methods. A morphological account is given of the adult wings, including their venation, basal attachment and axillary sclerites. The development of the tracheal supply to the larval wing pad is described in relation to the venation, with information on tracheal variation and the processes of tracheal retractiōn and degeneration in the adult insect. The bearing of these results on the venational homologies of the Heteroptera is discussed and a system of homologies and terminology is proposed.

Light-microscope histological investigations provide a detailed account of the differentiation of the wing pad at selected intervals throughout the moulting cycle and the inter-moult period. The formation of lacunae is described and it is shown that their development (with which that of the veins is related) precedes the growth of the associated tracheal supply. Paranotal regions of cell proliferation have been discovered in the prothorax and are perhaps serially homologous with the wings, but they regress later in postembryonic life.

Parallel ultrastructural studies were made of the cuticle, epidermis, tracheae, tracheoles and innervation of the developing wing pads and the young adult wings. These supplement and extend the above histological observations and include detailed accounts of changes in cell organelles during apolysis, ecdysis and the intermoult period from the earliest appearance of wing rudiments in the third larval instar. Haemocytes, epidermal glands, macrotrichia and microtrichia are also described. The collapse, retraction and 3

degeneration of the tracheal system, and the degeneration of the wing epidermis after the formation of veins and trabeculae occur in the adult wings within 48 hours of emergence and are described. The work is illustrated with numerous line drawings and electron micrographs. 4

ACKNOWLEDGEMENTS

I am grateful to Professor T. R. E. Southwood. F. R. S. for providing me with a place in the department to undertake this work. I owe a debt of gratitude to my supervisor, Mr IL G. Davies for his invaluable guidance, constructive criticism, constant encouragement and support throughout the course of this study. I am also grateful to him for translating the relevant German literature, and for his care and patience when reading my manuscript. He has proved to be not only a guide but also a great teacher to me. I thank Mr C. L. Meredith, Departmental Superintendent, for providing me with all the necessary technical facilities and for his personal help. I am extremely grateful to all my colleagues, friends and members of the technical staff who provided me with technical and moral support. In particular I thank Dr R. E. Sinden for explaining the techniques of Electron microscopy; Mr N. L. Costa for providing me with facilities in the Life Sciences E.M. Unit; and Miss M. Singh for technical assistance. I also thank Mr I.J. French, Mrs A. Merry, Mr J. Harmer and Mrs J. Yeadon and the other library staff for their co-operation and help. I have also benefited from discussions with Dr T. Srihari and with my husband Dr Rao I am grateful to my husband for constant encouragement and financial support; and to my father and family for their encouragement and helps I am also pleased to thank Mrs A. Meredith for her great care and interest in typing my thesis. I am also grateful to Mr P. Jayasankar and C. K. M. College Committee for their encouragement and sanctioning leave. 5

TABLE OF CONTENTS

Page

ABSTRACT ...... 2 ACKNOWLEDGMENTS ...... 4 CONTENTS ...... 5 GENERAL INTRODUCTION ...... 7

I : MORPHOLOGICAL ASPECTS:

1. The adult wing venation of Oncopeltus fasciatus Dallas ...... 15 2. The basal attachment and articular

S dc rites ...... 23 3. Tracheation of the larval wing pad ...... 29 4. Variation of the cubital trachea , , , , , , 40 5. Tracheal retraction ...... 48

II : HISTOLOGICAL OBSERVATIONS (Light Microscopy): 1. Materials and methods ...... 54 2. Mesothoracic wing development ...... 55 3. Metathoracic wing development ...... 110 4. Discussion ...... 125

III: ULTRASTRUCTURAL OBSERVATIONS: 1. Materials and methods ...... 142 2. Third larval instar wing pads .. , ... 145 3. Fourth larval instar wing pads ...... 163 (a) cuticle ...... 16 3 (b) epidermis 000 000 ...... 179

Page

(d) nerve supply ...... 235 4. Fifth larval instar wing pads ...... 240

(a) cuticle ...... 243

(b) epidermis ...... 248 (c) tracheae and tracheole s ...... 278 (d) nerve supply 292

5. Adult wings ...... 297 (a) Newly emerged adult wings ...... 297

(b) 18-hour adult wings ...... , ...... 321

(d) 48-hour adult wings ...... 353

IV : GENERAL DISCUSSION AND CONCLUSIONS ... 366

V : SUMMARY ...... 387

VI : LIST OF ABBREVIATIONS USED IN ILLUSTRATIONS 390

VII : REFERENCES ...... 394 7 GENERAL INTRODUCTION

The subject of the present study is the postembryonic development of the wings in the Large Milk-Weed Bug, Oncopeltus fasciatus (Dallas). This species is a member of the family Lygaeidae in the series Geocorisae (section Pentatomom'orpha) of the suborder Heteroptera. This, and closely allied families from the Lygaeoidea can be distinguished from the Coreoidea by the somewhat reduced venation of the hemelytral membrane. The species was named Lygaeus fasciatus by Dallas (1852). In 1868 Still transferred it to Oncopeltus, under which it has since stood. It is widely distributed in the Western Hemisphere and five of forty-one known species of Oncopeltus are found in the United States; some occur in South America and some in Australia. Oncopeltus fasciatus is a seed-sucking Heteroptera which feeds on various species of Milk Weed plants (Asclepiadaceae). Like most Heteroptera it passes through five larval instars in its growth to the adult, each instar being easily recognisable through its coloration, wing pad size and other features (Andre, 1934). In the third instar, the mesothoracic wing pads appear externally for the first time as small bag-like structures on the posterolateral angles of the mesothorax. The general biology of Oncopeltus has been reviewed by Andre (1934), and the ease with which it can be reared indoors at room temperature has led to its wide use as an experimental animal (Feir, 1974). An extensive literature deals with the morphology and histology of wing development in insects, but previous studies have mostly dealt with the holometabolous insects and relatively little is known of the simpler and presumably more primitive processes of wing development in hemimetabolous species. The main aims of the present work are, therefore, as follows:- (1) To investigate by simple light-microscope study the venation and tracheation of the wings of Oncopeltus; (2) to re-examine the proposed homologies of the principal veins with the help of developmental data; (3) to compare histological details during the various stages of post- 8 embryonic development with published observations on holometabolous species and the much smaller body of information on hemimetabolous forms; (4) to establish the relationships between the tracheae of the wing pad and the developing venation; (5) to examine the differences between the corium and membrane of hemelytra and to suggest possible causes for the tracheal retraction observed in the membrane; (6) to provide new information on the ultrastructural organisation of the developing wing and the changes it undergoes during post embryonic life; and (7) to consider the findings in the light of other published interpretations of cell and tissue differentiation in the insects. The background of available information on these and allied subjects will be dealt with in detail in the course of the thesis, but it is convenient here to summarise some previous work in the fields most intensively studied by earlier workers. 1. Tracheal homologies: Since 1850 or before, taxonomists have tried to use venational characters in the classification of the Heteropteran. A systematic set of homologies applicable in all orders of insects was first proposed by Comstock & Needham (1898, 1918). This was based on the tracheation, in the belief that the wing tracheae, which precede the veins in ontogeny, also determine their paths. They found that the course of the tracheae in many wings was often more readily interpretable than the subsequent venation and therefore provided evidence of the homologies of the veins. They included a little work on the wing-venation of Pentatomidae, considering them to be a generalised type of Heteropteran wing. They found six principal tracheae, with corresponding veins, in each wing and designated them by the now familiar terms: Costa, Sub- costa, Radius, Media, Cubitus and Anal. Handlirsch (1908) presented evidences for the absence of the costa in many Heteroptera and employed a different terminology. In his figures of fore and hind wings of Lygaeus sp. and in Lygaeus familiaris F. he interpreted the most anterior trachea as the sub- 9 costa (C of Comstock & Needham), followed by R, M, Cu and 2 anal tracheae (lA and 2A) in the fore wings and by R, M, lA and 2A in the hind wings. Metcalf (1913) published an account of the homologies of the veins in certain families of Fulgoroidea and followed the nomenclature of Comstock & Needham. Lameere (1923) discussed the wing venation of Heteroptera, basing his conclusions upon the two allegedly palaeo-hemipterous fossil wings discussed by Handlirsch. Muir (1923) discussed Fulgoroid wing venation and concluded that the costa is not an anterior branch of the sub-costa; that the first branch of radius• is never distinct and free from the remainder; that the vein associated with the claval suture is the second main branch of the cubitus; and that the Y-vein of the clavus is formed by the first and second anal veins. He also noted that in some Fulgoridea a third anal vein is present. In 1926, Hoke published an extensive study of the hind wing venation of Heteroptera. She followed Comstock & Needham's system, naming the first vein as the costa, but this conclusion has not been adopted by most hemipterists, as the absence of costa in Heteroptera had generally been accepted before. Also in 1926 Tanaka, who accepted the view that C is absent, made a comparative study of Heteropteran wing venation and concluded that the general scheme of tracheation is quite similar between the fore and hind wing pads, and also among various different families throughout the order. Tillyard (1926) brought forward additional evidence to support Handlirsch's (1908) interpretation while China & Myers (1929), when reconsidering the classification of the Cimicoid families accepted Tanaka's interpretation of the adult venation though they suspected lA of Tanaka's larval tracheation (Fig. 21-23) should really be Cut. Snodgrass (1935) also considered that the origin and growth of the tracheae of wings are of great importance in the study of wing venation. Fennah (1944) re-examined the morphology of the 10 tegmina and wings of the Fulgoroidea (Homoptera) in the light of palaeontological and morphological evidence and established that the costa of Comstock & Needham is really a branch of Sc, that the claval furrow lies just behind Cu2 and in addition to Cu2 the clavus contains PCU and two anal veins. Slater & Hurlbutt (1957) summarised their veins on the generalised Lygaeid wing and made a comparative study of the metathoracic wing in the family, using a terminology introduced by Leston (1953a), as modified from Tanaka (1926). Davis (1961), re-interpreted the venation and regions of the wings of Hemiptera, basing his views on evidence from tracheation, the relation of veins to the axillary sclerites, and a survey of wing venation in the more primitive Auchenorrhynchan Homoptera. He re-named the first vein in the clavus of Triatoma protracta (Reduviidae) PCU t 1A, continuing it into the membrane as PCU, while in the hind wing PCU and lA are separate veins, sometimes arising from a common stalk. Leston (1962) interpreted the venation of the Lygaeidae and some other Heteroptera, designating the main veins Sc, R, M, Cu and A; he also reported certain tracheal shifts during development, thereby casting some doubt on the rigid use of tracheation in determining venational homologies. The recent work of Kukalova.-Peck (1978) on the origin and evolution of insect wings and their relation to metamorphosis, as documented by the fossil record, has also contributed to studies on the homology of the main veins. She regards the primitive insect wing-venation as consisting of six symmetrical pairs of veins, in each wing (C, Sc, R, M, Cu and A) with the first branch always convex and the second concave. She considered that all are primi- tively composed of two separate branches and tried to harmonise the observations in modern comparative insect morphology and palaeo- entomology. In 1965 Wootton had published a paper on tracheal capture in early Heteroptera, taking Leston's (1962) views into consideration with the help of fossil material, he established a condition 11 within the Heteroptera, where the Cul had two branches, Cil ia and Culb. The vein Cut was thought to have been lost during evolution and the trachea lA crossed the claval furrow and came to lie in the lacuna preceding vein Cuib, whose own trachea was reduced. In 1977, Wootton extended his work on primitive Heteropteran venation when he re-described and re-classified twenty-seven species of Homoptera from the upper Liassic of Mecklenburg and Saxony. He also interpreted the veins of the Lygaeid wing membrane as Sc ? (he was in doubt about this vein), R and M with CuA, forming the fourth and fifth veins. The specialised nature of the wing venation in the living Heteroptera and the reports on tracheal shifts during ontogeny have clearly made it difficult to interpret the Heteropteran venation. The literature relating to the venational homologies of the suborder contain unresolved contradictions and a closer study of the subject seems to be needed. Meanwhile, from a functional viewpoint, Wigglesworth (1954) had demonstrated in Rhodnius prolixus Stal. that local areas of difficient oxygenation in the developing wing result in active migrations and proliferations of nearby tracheoles, the movements of which are sufficient to pull the tracheae out of their normal paths. The experimental work of Smart (1956) on Periplaneta americana (Linn. ) wings has proved similar flexibility in the development of the tracheal system of the wing. Again, Whitten (1962) demonstrated that the wing tracheation may vary not only among individuals of the same species, but even between instars. Most work on wing tracheation in connection with wing venation has paid little attention to the nature and origin of the tracheae. Wootton (1979) says that in some groups like Ephemeroptera (Henke 1953) and Heteroptera (Leston 1962, Wootton 1965), tracheation is misleading and in general it should be treated as no more than a useful aid to vein identification, thus conflicting with the relatively strict adherence to tracheational patterns favoured by Comstock & Needham and by Comstock (1918). 12

2. Histology of the wing: Since the beginning of this century, several papers have been published on the histology of insect wing development, but most of them dealt with Endopterygota. Powell (1904, 1905) made an histological study of the wing development of certain Coleoptera; Marshall (1913) described wing development in Platyphylax designates Walker (Trichoptera); Kōhler (1932) worked on Ephestia kuehniella Zell. , (Lepidoptera); Kuntze (1935) on Philosamia cynthia Drury (Lepidoptera); Behrends (1935), again on Ephestia kuehniella Zell.; Hundertmark (1935) on Tenebrio molitor Linn. (Coleoptera); Waddington (1941) on Drosophila. So far only a smaller body of less detailed work has been carried out on the wing development of exopterygotes; Tower (1903) worked on the first instar larva of

Anasa tristis Degeer (Hemiptera ); Sulc (1911) on Neophilaenus lineatus (Linn.) and Beck (1920) on Blattella germanica (Linn. ). Holdsworth (1940, 1941), made what is the most extensive study on the histology of wing development in Pteronarcys proteus Newport (Plecoptera). Despite the number of works on the histology of wing development, there are still differences of opinion regarding the development of wing lacunae, tracheae and the relations between the two. 3. Ultrastructure: In the past, very few ultrastructural studies have been carried out on the wing tissue and these are mainly on the wing integument. Willis (1966) worked on the micromorphology of the wing of the giant silk moth Hyalophora cecropia (Linn.) but the results are not very impressive, mainly due to poor fixation of the tissue. Detailed observations on the ultrastructure of generalised epidermal cells and of scale-forming and socket-forming cells in the developing wings of Hyalophora cecropia are, however, available from Greenstein (1972a 1972b). Interesting changes were found to take place in the adult wings of Oncopeltus after the final ecdysis, whereby much of the tissue degenerates, leaving only the cuticles. Fristrom (1968) dealt with somewhat similar cellular degeneration 13

in the wing development of the mutant vestegial of Drosophila melanogaster (Meigen). In 1975 Seligman et al., worked on morphogenesis and cell-death in the wing epidermis of Lucilia cuprina (Wied). Full consideration of these and similar processes involve the study of several changes in the wing cuticle and epidermis during the moult and inter moult periods, for which Richards (1951), Neville (1975) and various other modern reviews of insect integument are very useful. Noble-Nesbitt (1963) dealt with the fully formed inter moult and moult cuticle and associated structures of Podura acquatica Linn. (Collembola). Wigglesworth (1933, 1947, 1948, 1970, 1973, 1975, 1977, 1979), gave full details of cuticle, epidermis, basement membrane and haemocytes during various stages of the moulting cycle in Rhodnius prolixus Stā.l (Hemiptera). Locke in a series of papers (1957, 1958, 1961, 1964, 1966, 1969, 1974) on Calpodes ethlius has made many contributions to the study of the cytoplasmic organelles of epidermal cells during the formation of various types of cuticle. Beside these, the works of Filshie & Waterhouse (1961) on the structure and development of a surface pattern on the cuticle of Nezara viridula and of Zacharuk (1972) on Elaterid cuticle, epidermis and fat body, during moulting have proved very useful in relation to this study of Oncopeltus wings. Lawrence (1966b, 1975), Lai-Fook (1970), Nairot & Quennedy (1974) have also dealt with epidermal organelles. The wing tracheae and tracheoles undergo several changes during various stages of wing development in Oncopeltus; but there is no previous ultrastructural investigation of developing wing tracheae. Edwards et al. , . (1958); Edwards (1960); Locke (1966); Miller (1964); Whitten (1969, 1972); Smith (1967) are the main available works on the ultrastructure of insect tracheae and tracheoles. A nerve is present within lacuna of Oncopeltus wing pads and newly emerged adult wings. This is retained in a living condition during adult life inside the dorsal walls of the fully formed veins. 14

A recent paper by Altman et al. (1978) on Locusta migratoria is the only work which deals with the ultrastructure of wing nerves. However, the fine structure of the insect peripheral nerves have been described by Hess (1958), Edwards (1960), Smith & Treherne (1963), Smith (1967). They have shown the relationship between the axons and their sheath is morphologically intermediate between that seen in the myelinated and unmyelinated nerves of vertebrates. This has been confirmed in the present study of those peripheral nerves which enter the developing wing pad. PART I

MORPHOLOGICAL ASPECTS 15

1. The Adult wing venation of Oncopeltus fasciatus (Dallas) The thorax of Oncopeltus bears two pairs of wings; the fore wings are attached to the mesothorax and the hind wings to the metathorax. In Oncopeltus as in other Heteroptera, the two pairs of wings differ in structure. The basal half of the fore wing is coriaceous and tougher, and the distal half is membranous (Fig. 1). This type of wing is usually termed a hemelytron. Whereas the hind wings are entirely membranous (Fig. 3). At rest the wings are folded back flat over the body with the distal portions overlapping and coinciding, usually with the venation remaining distinctly visible. The Fore Wing (Figs. 1 and 2): The fore wing is divided into a proximal corium and a distal membrane. The anal area, which is hardened and separated from the corium by a claval suture, is termed the clavus. The costal margin of the corium has been folded ventrally. Owing to this turning over of the costal margin of the hemelytron, the sub-costal vein in the corium is very indistinct, though the sub- costal trachea is very clear, in the newly emerged imago, and is retained throughout life. The sub-costal vein is present in the membrane only (Fig. • 1). The costal margin forms a vein-like structure, but the costal trachea is absent from the larval wing pad. Just beneath the sub-costa a thick vein is present, in which two closely adjacent trachea run. This vein is therefore apparently formed by the fusion of the radius and media. They become separated towards the middle of the corium and the radius runs straight into the membrane. The medial vein bends away from R + M, runs straight up to the corio-membranic border, then bends costal-wards and runs along the corio-membranic border for a short distance until it approaches R, when it runs straight into the membrane. A cubital vein is present in front of the claval suture. It bends forwards at the corio-membranic border, almost touches the media, then turns into 16

the membrane and runs distally in it. Behind the anal furrow is the first anal vein. It bends costal-wards, and runs along the corio- membranic border, crossing the anal furrow, then bends into the membrane and runs straight distally. In the middle of the membrane Cu and lA are connected by a cross vein, which does not appear to contain a trachea; it lies near the posterior margin of the wing, and ends at the point where it meets vein 1A. (Fig. 2). On approaching the membrane, R, M, Cu and IA, all turn costal-wards; but Sc turns posteriorly and the dividing line between corium and membrane is formed to a greater or lesser extent from their bent parts. In the fore wing of the adult, 2-3 days after its emergence from the fifth-instar larva, the basal half hardens completely, and veins Sc, R, M and. Cu seem to be interrupted near the base of the membrane just after crossing the corio-membranic border; whereas vein lA is interrupted about half way between the border and the cross vein Cu - a (Fig. 1). During my observations I have noticed that the points of interruption correspond to the points where the tracheae have been shifted from their original lacunae after retraction (pp. 48). I assume them to be more weakly sclerotized points in the veins rather than true "dis-connections" as Hoke (1926) calls them. A median furrow is well seen in the basal portion of the fore wing, situated closely behind R + M. The anal furrow is found between Cu and lA (Fig. 1). The costal margin of the corium bears very strong, pointed macrotrichia throughout its length, arranged in several rows. These macrotrichia, with well defined sockets, are abundant on the main veins and also on the membrane of the corium. They are also very prominent at the base and apex of the posterior (anal) border and at the corio-membranic border. A double edge is present on the posterior ventral margin of the clavus, containing hook-like structures which hold the costal margin of the hind wing. 17

Fig. 1. Dorsal view of the right forewing of Oncopeltus fasciatus, showing venation 1A, first anal vein; cl, clavus; cmb, corio-membranal border; cor, corium; cs, claval suture; Cu, cubital; Cu- 1A, cubito-anal crossvein; de, double edge of the wing coupling; M, medial; me,membrane; mac, macrotrichium; R, radial; R + M, radio-medial compound vein; Rs, radial sector; s, suture; Sc, sub-costal; vf, ventral fold.

Fig. 2. Fore wing tracheation of newly emerged adult Oncopeltus (including basal connection of wing trachea. ). 1A, first anal; ZA, second anal; cl, clavus; cmb, corio- membranal border; cor, corium; crg, costo-radial group; cs, claval suture; Cu, cubital; Cuag, cubito-anal group; me,membrane; M, medial; R1, radial one; Rs, radial sector; s, suture; Sc, sub-costal; tbt, transverse basal trachea; tl, tracheole. Rs

FIG.I

FIG.2 2mm. 19

Pesson (1951a) says' that the Heteroptera have a short gutter, edged with a brush of hairs, on the under-side of the clavus which holds the costal margin of the hind wing. The hind wing (Figs. 3 and 4): The hind wings of Oncopeltus are uniformly membranous, and at rest are folded back flat over the body with the distal portions overlapping and coinciding, usually with the venation remaining distinct upon it. The fore wings cover the hind wings completely. The following veins have been observed in the hind wing (Fig. 3): The costa runs along its anterior margin, but contains no trace of trachea in the larval wing pad. The mid costal margin forms a recurved flange, with roughened, tooth-like projections which catch in the turned under hind claval margin of the fore wing when the wings are spread for flight. The sub-costa is united with the costal margin. Behind the sub-costa is the vein R + M; this compound vein is formed by a coalescence of R and M up to about one-third of the distance from the base of the wing. In the newly emerged imago two closely adjacent trachea can be seen very clearly in the same lacuna. At about one- third of the distance from the base of the tip of the wing, they become separated. The vein R takes one or two small curves, then runs straight distally. The medial vein bends towards the posterior side, almost touches the cubital vein, bends anteriorly towards the radial vein and then finally runs distal-wards. In the fully formed hind wings of the 2-3 day old adult, the medial vein has been disconnected near its point of divergence from the compound vein R + M, though it is continuous in the newly emerged adult, as is also the larval trachea. The cubital vein lies behind the medial vein, the base of which has been lost in the fully grown adult. Cu runs straight into the membrane of the wing until it meets the medial vein, then bends slightly towards the posterior side and runs forwards distally. There are two anal veins lying inside 20 the anal fold behind the furrow. The first anal vein has lost its base; and the second anal is a stout vein, bearing large macrotrichia. Two furrows are present behind Cu, separating the anal area from the rest of the wing. The anal area is bilobed (Fig. 3). In the newly moulted adult fore wings there are six principal trachea arising from two tracheal trunks, which are themselves connected by a transverse basal trachea (Fig. 2). The first trachea of the anterior costo-radial trunk is the sub-costa, which enters the base of the wing near the humeral angle and runs behind the costal margin up to the tip of the wing. Behind the sub-costa a common tracheal trunk from the costo-radial trunk runs for a short distance inside the wing, then bifurcates into radial and medial tracheae; on its way to the membrane the radial trachea gives a small anterior branch, at the corio-membrane border (Fig. 2), which runs costal- wards. This small branch appears to correspond to R1 , and the main radial trunk running inside the membrane is, therefore, the radial sector. Tillyard (1926) named the anterior-most second vein of the membrane as Rs without giving reasons. The medial trachea also runs to the tip of the wing. The main costo-radial trunk runs towards the posterior side of the base to form a transverse basal connection; the cubital and two anal tracheae arise posteriorly from the cubito-anal trunk (Fig. 2). The sub-costal trachea of the hind wing is a very short branch (Fig. 4). All the tracheae make a small bend in the middle of hind wings. Bunches of tracheoles are joined to their associated tracheae at definite nodal points, each occupied by a tracheal end cell. The tracheoles arising from the tip of the trachea interdigitate with others along the wing margin, in a manner that recalls an ambient vein similar to that of Auchenorrhyncha of Homoptera. The lacunae around these tracheae become sclerotised within a few hours of emergence to form veins. A similar type of tracheation has been noticed in the wing pads of third, fourth and fifth-instar larval wing pads (Figs. 8, 10, 11, 12 and 13). 21

Fig. 3. Dorsal view of the right hand wing of Oncopeltus fasciatus, showing venation 1A, first anal; ZA, second anal vein; af, anal fold; Cu, cubital; f, furrow; M, medial; R, radial; ur, upturned ridge.

Fig. 4. Hind wing tracheation of newly emerged Oncopeltus fasciatus 1A, 2A, anal tracheae; af, anal furrow; Cu, cubital; crg, costo-radial group; Cuag, cubito-anal group; M, , medial; R, radial; Sc, sub-costal; tbt, transverse basal; tl, tracheoles. u.r.

FIG.3

FIG.4 2mm. 23

2. The Basal attachment and articular sclerites The base of the wing and the thoracic terga are connected by a soft, flexible axillary membrane, in which a group of small sclerites are present, and which permits movement of the wing. (a) Fore wing (Figs. 5 and 6): In the fore wing of Oncopeltus two median plates and four axillary sclerites have been observed (Figs. 5 and 6). The- axillary sclerites articulate with the wing base and with the anterior and posterior notal process of the thoracic terga. The wing also articulates below with the.pleuralwing process, and with an anterior basalar, and a posterior sub-alar sclerite. The direct flight muscles are attached mainly to these articulatory sclerites. Median Plates: In the fore wing of Oncopeltus two large basal plates are associated with the base of the wing and articulate with the axillary sclerites (Fig. 5). The proximal plate is the bigger one. The tip of the proximal median plate is pointed and connects with the fused bases of the radial and medial veins. Its upper anterior margin is attached to the base of the costal margin and humeral plate (Fig. 5). The distal median plate lies behind the proximal plate and joins its proximal portion. All these give the appearance of a compound structure. The humeral plate bears a handle-like structure which articulates with the first axillary. The distal pointed portion of the distal median plate is attached to the vein 1A, and joins a limb of the T-shaped, third axillary sclerite (Figs. 5 and 6). The humeral process connects the anterior base of the wing to the basalare,, which lies just above the pleural wing process. (Fig. 6). First axillary: This lies in the dorsal surface of the membrane (Fig. 5). Its dorsal surface bears a deep groove which receives the proximal border of the second axillary. Its proximal portion articulates with the anterior notal process (Fig. 5). The handle- like process of the humeral plate articulates with it. 24

Second axillary (Figs. 5 and 6): This is a sclerotization in the ventral side of the articulatory membrane which pivots on the pleural wing process. Dorsally its proximal end is joined to the first axillary. A short ventrally projecting posterior process is attached by membrane to the sub-alare. The dorsal border articulates with the arm of the third axillary. Third axillary (Figs. 5 and 6): This is a Y-shaped structure. One arm is connected with the distal tip of the distal median plate, at which point the two sclerites join with the anal vein. A second arm articulates with the second axillary, while a third, transverse, arm is joined to the fourth axillary. Fourth axillary (Fig. 6): The proximal and median ends fuse with the sub-alare. The sclerite lies towards the ventral side. Sub-alare (Figs. 5 and 6): The anterior edge of this sclerite is attached by membrane to the second axillary. The fourth axillary is attached to its median protruberence. The posteriorly prolonged edge is attached to the meso-scutellar process. (b) Hind wing (Fig. 7): The axillary sclerites in the hind wing (Fig. 7), are three in number. In addition a humeral plate is present, which connects the basal anterior region of the hind wing to the thorax. First axillary: This lies between the second axillary and the anterior notal process. Second axillary: This is a sickle-shaped structure. The proximal border is connected to the first axillary. The pleural wing process pivots on the concavity of the slcerite. The distal side is attached by membrane to the sub-alare. Third axillary: This is a V-shaped structure. The anterior arm is connected to the second axillary and the transverse arm to the sub- alare. Sub-alare: This sclerite has three projections. The anterior projection is joined by membrane to the second axillary, a median projection is attached to the third axillary. While the posterior side is attached 25

Fig. 5. Diagram of articulation of the right forewing with the thorax (dorsal side). 1A, anal vein; anp, anterior notal process; axi, ax2, axillary sclerites; axc, axillary cord; axm, axillary membrane; bas, basalar sclerite; Cs, claval suture; Cu, cubital; dmp, distal median plate; hp, humeral plate; pmp, proximal median plate; R + M, compound veinR+M.

Fig. 6. Articulation of the left forewing with thorax (ventral view). 1A, first anal vein; axi, ax2, ax3, ax4, axillary sclerites; axc, axillary cord; axm, axillary membrane; bas, basalar sclerite; Cs, claval suture; Cu, cubital vein; dmp, distal median plate; hp, humeral plate; pmp, ' proximal median plate; pwp, pleural wing process; R + M, compound vein R + M; vf, ventral fold.

FIG.5 2 mm. 27

Fig. 7. A, Articulation of the right hand wing with thorax (dorsal view). B, humeral plate. C, D, F, axillary sclerites (1, 2, 3). E, sub-alar sclerite. 1A, 2A., anal veins; anp, anterior notal process; axl , ax2, ax3, axillary sclerites; axc, axillary cord; axm, axillary membrane; Cu, cubital vein; hp, humeral plate; pnp, posterior notal process; R + M, compound vein R + M; sub, subalar sclerite. B. C -0.

das

E. FIG.7 29

to direct flight muscles. Several campaniform sensilla are present on the basal plates of the fore wings and on the humeral plate of the hind wing. They are relatively large circular structures which are presumably sensitive to distortions of the wing base in particular planes, and thereby involved in the nervous regulation of wing movements during flight. 3. Tracheation of the Larval Wing Pad: I have observed the larval wing-pads in whole mounts at various stages except for the first and second instars where they are not apparent externally. In third-instar larva, the mesothoracic wing pads appear externally and in the fourth and fifth-instar larvae both pairs of wings appear externally. Tracheation is made visible by dissecting fresh larvae in 50% glycerol. Semi-permanent mounts made with glycerine jelly show the detailed structure of the tracheation for several months. Third-instar (Fig. 8G): In the newly moulted third-instar the mesothoracic wing pads appear externally as bag-like structures on the posterolateral angles of the mesothorax, covering a small portion of the metathorax and measuring about 0.4 mm in length. They are delicate, transparent and pale in colour. With a little care whole mounts of isolated wing pads can be made with glycerine jelly to give a clear picture of complete tracheation. These wing pads are provided with four small fine tracheae (with bundles of terminal tracheoles), arising from two tracheal trunks, which are themselves connected by a transverse basal trachea (Fig. 8G). The costo-radial trunk enters the base of the wing pad from its anterior side, and gives rise to three longitudinal tracheae supplying the wing pad: sub-costal, radial and medial. The sub-costa is the anterior-most branch of the costo-radial trunk which enters the wing pad just beneath the humeral angle. The radial and medial tracheae arise as a bifurcation from a common tracheal 30 branch of the costo-radial trunk next to the sub-costal trachea. After giving rise to these three tracheae the costo-radial trunk runs towards the posterior side and joins with the cubito-anal trunk to form a transverse basal connection. After forming the transverse basal connection the cubito-anal trunk runs towards the posterior side of the wing base, giving rise to a small cubital trachea, and two separate bundles of tracheoles. From the tips of the sub-costal, radial, medial and cubital tracheae bundles of tracheoles arise and run throughout the length of the wing pad. Fourth-instar (Figs. 10 and 11): In the newly moulted fourth-instar the mesothoracic and metathoracic wing-pads appear externally. The mesothoracic wing pad covers the metathoracic pad completely. They are transparent and colourless. The mesothoracic wing pads measure approximately 1 mm in length, whereas the metathoracic pair is 0. 5 mm long. In both pairs of wing pads six prominent tracheae are pre sent. In the fore wings of newly moulted fourth.-instar larvae the first five tracheae run through the whole length of the wing pad. The sixth trachea runs parallel to the base of the wing pad; it is weaker and shorter than the others. In the hind wings of the same instar, the sub-costal trachea is a very short and weak branch, but the radial, medial, cubital and first anal run throughout the whole length of wing pad. ZA is again a short branch and runs parallel to the base of the wing pad. The sub-costal trachea, the first branch of the costo-radial trunk, originates near the humeral angle and runs parallel to the costal margin of wing pad. Sometimes instead of arising from the main costo-radial trunk, it originates from the base of the tracheal branch from which the radial and medial tracheae bifurcate (Fig. 11). After emitting the sub-costal trachea, the costo-radial trunk gives off a stout trachea, which enters the wing pad and bifurcates into radial and medial tracheae. The costo-radial trunk 31 then runs posteriorly and joins the cubito-anal trachea to form the transverse basal trachea. The cubito-anal trunk bends a little near the base of the wing pad after joining the transverse basal trachea and then gives off the cubital, first anal and second anal tracheae. All these tracheae, instead of running straight, bend a little in mid course and groups of tracheoles arise from each nodal point of the trachea. The tracheoles from the tips of the tracheae interdigitate with others in front, forming a kind of net-work (Figs. 10 and 11). By this stage of development the pattern of adult tracheation has been established completely. Fifth-instar (Figs. 12 and 13): In the newly moulted fifth-instar the wing pads measure 3 mm in length and are thus twice as big as those found in the fourth-instar. They are triangular, bag-like structures hanging from the notum. There are again six main tracheae arising in two groups from the costo-radial and cubito-anal trunks, which are themselves connected by a transverse basal trachea. Except for their diameter and length, the pattern of tracheation is the same as that of the fourth-instar larva and the newly emerged adult. The sub-costal trachea is the most anterior branch to come off from the main trunk. It enters the humeral angle and runs parallel to the costal margin. The costo-radial trachea gives a second branch, which in turn bifurcates into radial and medial tracheae. The radial trachea then gives off a short anterior branch in mid course; this anterior branch of radial trachea runs towards the sub-costa and apparently corresponds to R1, while the main branch, which continues towards the tip of the wing, is the Radial sector (Fig. 12). The posterior extension of costo-radial trunk joins with the cubito-anal trunk, so that the transverse basal connection is preserved (Figs. 12 and 13). The cubital, first anal and second anal tracheae arise as branches of the cubito-anal trunk (Figs. 12 and 13). Tracheoles arise from each nodal point to supply oxygen to every part of the wing, and the tracheoles from the tips of tracheae interdigitate with others in front. 32

Fig. 8. Schematic diagram to illustrate course of tracheae in developing mesothoracic wings from first to third larval instars. A, First-instar. B, Late Pharate Second-instar. C, Newly moulted second-instar. D, 40 hour second-instar. E, 72 hour-second instar. F, Pharate third-instar. G, Newly moulted third-instar. atr, anterior trachea; c - r, costo- radial; Cu, cubital; Cu- a, cubito-anal; M, medial; ptr, posterior trachea; R, radial; R + M, radio-medial; Sc, sub-costa; tbt, transverse basal; th2, mesothorax; tl, tracheoles; wr, wing rudiment.

a tr, a t r

wr- the

wr ptr 6

c-r., c-r

w r ---- 1c- r — R+M Sc R...... t h 2 M

Cu ~tbt

c u 'a F

FIG.8 G 34

Fig. 9. Schematic diagram to illustrate course of tracheae in developing metathoracic wings from first to fourth larval instars. A, first-instar. B, Late Pharate Second-instar. C, Newly moulted Second-instar. D, Late Pharate third-instar. E, Newly moulted third-instar. F1, F2 , Pharate fourth- instar. G, Newly moulted fourth-instar. 1A, 2A., anal tracheae; atr, anterior trachea; c - r, costo- radial; Cu, cubital; Cu - A, cubito-anal; M, medial; ptr, posterior tracheae; R, radial; R + M, radio-medial; Sc, sub-costal; tbt, transverse basal; th3, metathorax; wr, wing rudiment. a tr atr

th3 wr pt r- A B c -rfi c-r

C-r Sc R M Cu IA 2A cu-a

F, c-r

FIG. 9 36

Fig. 10. Tracheation of right fore wing pad of fourth-instar larva of Oncopeltus (including basal connection) 1A, ZA, anal tracheae; crg, costo-radial; Cu, cubital; Cuag, cubito-anal; M, medial; R, radial; R + M, radio- medial; Sc, sub-costal; tbt, transverse basal.

Fig. 11. Basal connections and tracheae of right hind wing pad of fourth-instar larva. 1A, ZA, anal; crg, costo-radial; Cu, cubital; Cuag, cubito-anal; M, medial; R, radial; R + M, radio-medial; Sc, sub-costal; tbt, transverse basal. FIG.11 38

Fig. 12. Tracheation of fifth-instar fore wing pad (including basal connections). 1A, 2A, anal; crg, costo-radial; Cu, cubital; Cuag, cubito-anal; M, medial; R, radial; R + M, radio-medial; Sc, sub-costal; t, trachea; tl, tracheoles; tbt, transverse basal.

Fig. 13. Tracheation of the hind wing pad of fifth-instar larva of Oncopeltus including basal connections. The sub- costa is a very small trachea. LA, 2A, anal; crg, costo-radial; Cu, cubital; Cuag, cubito-anal; M, medial; R, radial; R + M, radio-medial; Sc, sub-costal; ti, tracheoles; tbt, transverse basal. .wwS *0 40

In all stages the first trachea of the hind wing pad is a very short branch, sometimes difficult to see. Workers like Comstock & Needham (1898) have neglected this trachea in the Hemipteran wing pad they figured. This trachea is the costa of Handlirsch (1908). The position of tracheae in the newly emerged adult and the venational pattern of adult wings are very similar. The costal border of the adult fore wing gives the appearance of a vein, but no trachea occurs in it, either in the larva or in the adult. The sub-costal trachea is present at every stage, but is not accompanied by an adult sub-costal vein in the corium, in the fore wing a vein like structure is present in the membrane; a corresponding trachea is present in the fifth-instar and newly emerged adult, but is lost when tracheal retraction occurs (pp. 48). In the adult a compound vein is formed around the closely adjacent tracheae R and M, some distance from the base. In the adult I have not observed tracheae R and M in the radial and medial veins of the membrane, though they are present in the corium. The cubital and first anal veins are connected by a cross vein in the middle of the fore wing membrane. This cross vein never normally contains a trachea. 4. Variation of cubital trachea I have dissected the wings of 50 mature adults, 30 newly emerged adults and 25 fifth-instar larvae in glycerine; mounted them under glycerine jelly, and observed the trachea and veins. Apart from the instances mentioned below I have not noticed any important variation in tracheation or venation. In four newly emerged adults, however, I have noticed variations in the course of the cubital trachea. My findings are as follows: (a) In one newly moulted adult the cubital trachea does not follow its original lacuna (Fig. 14). Instead of bending at the corio- membranic border, it runs straight and then takes a forward bend proximal to the cross vein Cu - a, subsequently re-entering the original lacuna. This variation was seen in both fore wings. 41

(ii) In another specimen the cubital trachea has shifted from its lacuna as shown (Fig. 15). It enters the cross vein Cu - a, and runs in it for some distance before it bends, and runs parallel to the first anal trachea in the first anal vein. The latter may therefore be said to have captured the cubital trachea. (iii) In the third specimen I have noticed a division of the cubital. trachea. Towards its base the trachea is much thicker than its normal diameter (Figs. 16, 17, 18 and 19); it runs most of its length in the corium, then divides into two branches; both of which run side by side in the same lacuna up to the cross vein Cu - a, after which one runs in its original lacuna, while the other enters first the lacuna of cross vein Cu - a, then that of the first anal vein (Fig. 16). The first anal trachea is shorter and weaker than normal. This variation was noticed in all four wings. (iv) In another specimen I have noticed a similar bifurcation of the cubital trachea, but with a very small posterior branch. In 1962 Le stop drew attention to tracheal capture, where the trachea of vein comes to lie in the lacuna of another, and claimed it to be wide spread in Heteroptera, occuring both as an individual variation and as an established condition. Wootton (1965) has discussed this problematical, cubito-anal area, of the wing of early Hemiptera and tried to show that in ancestral forms like the Actinocytinidae, Cul is supposed to have two branches Cula and Cuib. The trachea of lA, is thought to have crossed the claval furrow and come to lie in the lacuna of Cuib, whose own trachea was reduced. In the evolution of Heteroptera it is suggested that the veins have become more or less fixed, and the trachea have tended to wander to the areas of insufficient oxygen supply. My observations, showing shifting and branching condition of cubital trachea support these theories on tracheal flexibility, and are most consistent with the presence of Wootton's (1965) Cu la and Cu lb in the ancestral forms. Perhaps some individuals in the Heteroptera 42

Fig. 14. Right forewing of Oncopeltus, showing shift of cubital trachea from its lacuna. LA, 2A, anal tracheae; Cu, cubital trachea; M, medial trachea; R 1, radial one trachea; Rs, radial sector trachea; Sc, sub-costal trachea.

Fig. 15. Right fore wing of Oncopeltus. The cubital trachea has shifted from its lacuna, entered the cross vein Cu - a, and runs parallel to first anal vein. 1A, 2A, anal tracheae; Cu, cubital trachea. 1 1 11 1 1 / 1 1 II , I1 I I I I 11 I I II It rr I 1 / I II 1I II II II II I! 1 1 1 1 II It 1 1 1 11 I1 11 I! 11 I) it 11 it II II Is I I It II 1I I , il r! II II 1 1 ►I r ! 11 II 1 1 I I I/ II I I 1 !r I/ I r / 11 rr 11 // II !, I :-..il II `\ \ -- 1 1 I t:4IIi \// 1I I !I

II \ /, /

11 ~ `1, /// / / I I _ \ \ / / / 11 11 // I , 1 I; // 1 ,1 // / / 11 1 1 ' \\\ I I 1 1 ~ ` 1 ~ II .11 '1 \ Ir II \ I / I r II \ 1t ►I \\ ' 1 I I/ II \\ 1 1 I/ II `\\ ~;,; II

1 44

Fig. 16, 17. Showing bifurcation of fore wing cubital trachea. The posterior branch runs in the lacuna of cross vein Cu- a, and enters the lacuna of the first anal vein; the first anal trachea is reduced. 1A, ZA, anal tracheae; Cu, cubital trachea; M, medial trachea; R1, radial one trachea; Rs, radial sector; Sc, sub-costal trachea.

TE II E 1 1 I/ 1 1 II I/ t l 1, /1 II 1 1 I/ ~1 I fl /r I I II r 1 If I/ 11 j1 ri I II 1 1 1 1 !r 1 1 11 1 l I If I 11 I I 1, 1 1 II 11 I1 11 11 1 I I1 t1 II II I I I II 11 I I 11 I 1 11 11 II 1/ r ► 7 j I I r Ir If / , SI 11 i f f II ~,// r1 11 rr ,■/ I !t e i { s~ I t . /11 •1 II / Ilr t i •' II 1 II I /~ // 1, \ // / 1 1 II t ‘,~ // / / 1 1 ' / / %/ // r \\1 \/ II 1 1 l h \ I/ 11 Ir• \ it 1 I I % `. II I I I/ t+: y r/ t , t !I I I / '',► `0 1 j 1 I I \x\ I II o r 11!//1 11 \ 1 I I 1 1

~I I r I / I Ir

II 1 1 \ r r l II 1 1 Ir / r \ I 1 1 t 1 / 1 1• `I I 1 46

Fig. 18. Right hind wing of Oncopeltus showing bifurcation of Cu trachea 1A, 2A, anal tracheae; Cu, cubital trachea; M, medial trachea; R, radial trachea.

Fig. 19. Showing bifurcation of hind wing cubital trachea. The posterior branch runs in the lacuna of first anal vein; the first anal trachea is reduced. 1A, 2A, anal tracheae; Cu, cubital trachea; M, medial trachea; R, radial trachea. N

I! /1 II // // 48 tend to recapitulate the lost structures or relationships which were once present in their ancestors. 5. Tracheal Retraction In the nymphal wing pads of Oncopeltus, six tracheae are distinct and well developed throughout the third, fourth and fifth-instar larvae (Figs. 80, 10, 11, 12 and 13). All of them run the full length of the wing pad, and the same condition is found in the newly emerged imago, where each trachea is a straight tubular structure enclosed in a vein. After adult emergence, however, a remarkable and interesting change occurs in tracheal distribution. The tracheae start to shift from the tip of specific veins, apparently due to pulling of the tracheae from the base of the wing. This mechanism is suspected because the tracheae attain a wavy course instead of retaining their original straight paths (Fig. 20). The degree of retraction varies from hour to hour as shown in the accompanying figures (Figs. 20, 21, 22, 23 and 24). The whole process takes place over a period of two days, and the tracheae are then retained in an unchanging condition for the rest of the insect's life. The movement of tracheae from their associated veins takes place in the membranous half of the fore wing up to corio-membranal border (Fig. 22). In the hind wing it occurs up to about the middle of the wing (Fig. 24). When they have been displaced from the veins, all the tracheae lie close to each other at a more or less central point, actually between the medial and cubital veins. The tracheae in the corium are very compressed, and in the membranous portion they have all curled up, like bunches of grapes in the middle of the cubitus and media. The same condition has been noticed in the hind wings. Owing to this movement of tracheae the veins in the membranous half of the fore wing have become discontinuous with their corresponding portion in the corium. The causes and significance of tracheal 49

Fig. 20. Fore wing of Oncopeltus one day after emergence, showing tracheal retraction from the membrane. 1A, 2A, anal; Cu, cubital; M, medial; Rs, radial sector; Sc, sub-costal.

Fig. 21. Fore wing of Oncopeltus 1 day after emergence 1A, 2A, anal; Cu, cubital; M, medial; Rs, radial sector; Sc, sub-costa.

Fig. 22. Fore wing of Oncopeltus, 2 days after emergence, showing fully retracted tracheae of membrane. 1A, ZA, anal; Cu, cubital; M, medial; Rs, radial sector; Sc, sub-costa. FI G.2 0

FIG. 21

FIG 22 1mm. 51

Fig. 23. Hind wing of Oncopeltus one day after emergence, showing tracheal retraction from distal half. 1A, 2A, anal; Cu, cubital; M, medial; R, radial; Sc, sub-costal.

Fig. 24. Hind wing of Oncopeltus, 2 days after emergence, showing fully retracted tracheae from distal half. 1A, 2A, anal; Cu, cubital; M, medial; R, radial; Sc, sub-costal.

53 retraction are not clear from observations made under the light microscope. It can hardly be due to the thickening of the corium, since it also occurs in the hind wing. Wigglesworth (1959) experimented on the role of epidermal cells in the migration of tracheoles in Rhodnius prolixus St&.l. and proved that the epidermal cells are responsible for the migration of air filled tracheoles into regions deprived of their normal tracheal supply. The cells give rise to contractile filaments, sometimes 100 urn in length, which become attached to the tracheoles and draw them inwards. In 1962 Leston noticed tracheal shifts in a few Heteropteran hind wings and in the fore wings of a Mirid. He says that the tracheae R, M and Cu have been dragged out of position to supply a white pigment spot on the wing. The above statement is not applicable here. In Oncopeltus all the tracheae from the posterior half of the wings have been dragged out of position. Moreover there is no white pigment spot or other specialised area in the hind wings. Nor there is no tracheal retraction during the fifth-instar similar to the one which Leston reported. PART II

HISTOLOGICAL OBSERVATIONS (Light Microscopy) 54 1. Materials and Methods: The insects were reared in plastic containers, fed with decorticated sunflower seeds and provided with water. The culture was placed in a room where the temperature varied between 24-280c. Under these conditions the fifth larval stage lasts 190-220 hours (8-9 days), the fourth larval stage 120-130 hours, the third instar 100-105 hours and the second instar 96-100 hours. The first-instar usually moults 8-9 days after eclosion, but sometimes earlier than that. Approximate times of apolysis sand ecdysis for each instar are summarised in the table below (hours after the previous ecdysis).

Temp. Larva Apolysis Ecdysis

1st -Instar 5th day 96-100 hrs 8-9th day 190-200 hrs 2nd-Instar 3rd day 70- 75 hrs 5th day 96-100 hrs 24-28°c 3rd-Instar 2nd day 40- 50 hrs 5th day 100-105 hrs 4th -Instar 2nd day 40- 50 hrs . 5-6th day 120-130 hrs 5th -Instar 2-3rd day 50- 60 hrs 8-9th day 190-200 hrs

Instars of various ages were removed from the cultures, and part of the thorax and attached wing rudiments fixed in Bouin's fixative for 18 hours, dehydrated in 70% Alcohol followed by dioxan, embedded in parafin wax, sectioned serially at a thickness of 6 p.m, and stained. Some slides were stained in Mallory's connective tissue stain (Hughesdon's modification), and some in Phosphotungstic Haemotoxylin (Mallory's). Both techniques were found to be good for making out full histological details of the developing Oncopeltus wings at all stages. The staining times in these solutions are subject to variation depending on the age of the insect. In Mallory's Phosphotungstic Haemotoxylin, it is necessary to stain the first, second and third larval stages for 30 minutes, and the fourth and fifth-instar tissues for a period of 40 minutes to get precise staining. In Mallory's connective tissue stain, staining the fourth and fifth larval tissue for 5 minutes, in both solutions gives satisfactory results, (Solution A: 0. 5g. Biebrich Scarlet, 0. lg. orange G and 0. 2ml glacial acetic acid in 55 100m1. distilled water; Solution B: 0. 25g Aniline blue, 0.25m1. concentrated hydrochloric acid, 100m1. distilled water). The tissues of the first three larval instars need only 3 minutes to give good staining. From the stain the sections were taken to absolute alcohol; after a rapid dehydration they were cleared in xylene and mounted in D. P. X. The study of earlier stages is made by reconstructions from the serial sections as described by Holdsworth (1942). 2. Mesothoracic wing development First-Instar: The histological study of the thoracic segments of newly hatched first instars reveals the presence of histologically recognisable wing pads at this stage. The external indication of the wing pads is a minute flange on each side of all three thoracic segments. All these are of the same size, measuring 90-100 ppm in length. Each rudiment appears as a hollow, flattened, out growth of the body wall along the lateral margins of the dorsum following the line where the suture between tergum and pleuron later develops. The rudiments are directly continuous with the tergum. As the wing pad is only a simple outgrowth of the body wall, it consists of an outer cuticle, and an inner layer of epidermal cells bounded by a thin, non-cellular basement membrane. The cuticle covering these wing pads is thin and membranous, not or apparently only weakly sclerotised. As the first stage larva feeds and grows in size the wing rudiments also grow, due to epidermal hyperplasia and the stretching of the cuticle. A few macrotrichia are present on the wing pad, each arising from a cup-like socket; some are long and thin walled, and others short, stumpy and thick walled. The epidermis in the area of the first-instar wing pad is made up of 13-15 cells in transverse section, arranged in a single layer (Fig. 25). The cell boundaries are not very clear between nuclei under the light microscope, so that the tissue appears to be syncytial in structure. The nuclei are ovoidal, measuring 4-5 p.m 56

Fig. 25. T. S. of left me sothoracic wing rudiment of newly moulted first instar (detachment of basement membrane is an artefact). bm, basement membrane; cut, cuticle; ep, epidermis; n, nucleus; t, trachea.

Fig. 26. T.S. of 48-hour first-instar left fore wing pad, showing beginning of cell divisions. bm, basement membrane; cut, cuticle; ep, epidermis;. mit, mitotic division; n, nucleus; oen, oenocyte; t, trachea. (detachment of basement membrane is an artefact). FIG.26 25/um 58 by 5-6 pm across. In my preparations the basement membrane is separate from the wing pad epidermis, but this may be an artefact. The first-instar wing pads are supplied by a single trachea, arising from a large tracheal trunk towards the anterior side of each thoracic segment (Fig. 8A). It enters the base of the wing pad anteriorily and runs posteriorly between the thorax and the base of the wing disk, measures 40-50 pm in length and 1-1.5 pm in diameter. Histological preparations of the first-instar made 2-4 days after eclosion show constant, gradual growth in the size of the wing pad due to the division of cells, and elasticity of the cuticle, but the epidermis maintains its unilaminar arrangement throughout (Fig. 26). Later in the instar, when the cuticle loses its capacity for stretching, the wing pad stops growing in size; but the proliferative divisions of the epidermal cells continue. In the middle of the disk, one or two epidermal cells grow in size and start to divide (Fig. 26). The mitotic figures are very clear because of the size of these cells. The epidermal cells connected with the cuticular hairs also divide mitotically. Approximately 5 days after hatching, apolysis occurs in this region, and the epidermis detaches from the cuticle as an entire sheet (Figs. 27 and 28). A well defined exuvial space is present between cuticle and epidermis. The epidermal cells are naked, without any apparent cuticular covering, but they continue to undergo mitotic division (Fig. 28). In preparations made on the sixth day after hatching the exuvial space contains a foam-like material representing fixed moulting fluid (Fig. 28). The large rounded cells resulting from the division of previously swollen cells are now greater in size than the surrounding cells, and seem to be unicellular glands, which pour out their products and degenerate. Wigglesworth (1933-1934), states that in Rhodnius prolixus the dermal glands are formed anew at each moult from previously undifferentiated epidermal cells, and are 59

Fig. 27. T.S. of a left forewing pad of 120 hour, first-instar showing the onset of apolysis after the cell numbers have increased. bm, basement membrane; cut, cuticle; ep, epidermis; gl, unicellular glands; n, nucleus; oen, oenocyte; t, trachea.

Fig. 28. T. S. of 150 hour first-instar left fore wing pad showing fully formed exuvial space between retracted epidermis and cuticle. The basal cytoplasm of the cells is produced into tall process. bm, basement membrane; cp, cytoplasmic process; cut, cuticle; ep, epidermis; exsp, exuvial space; gl, unicellular glands; icsp, inter cellular space; n, nucleus; oen, oenocyte; t, trachea. FIG. 2 7

FIG. 2i3 25Rm. 61 functional only during moulting. This is confirmed by my observations. The cells of the disk now elongate, and their ends meet those of the opposite surface (Fig. 29). A small intercellular space can be seen towards the anterior region of the wing pad, just in front of the tracheal branch, which enters the base of the wing pad and runs across (Fig. 29). This trachea is probably the future costo-radial trunk. Prior to the first ecdysis, the new cuticle has formed and is thrown into folds (Fig. 30). The epidermal cells become unilaminar in arrangement again, but the fully formed pharate second- instar wing pad is still enclosed in the old cuticle .(Fig. 30). At the time of moulting, the cuticular hairs are strongly cuticularised and amber coloured. Though there is a single trachea throughout the first- instar, in the late pharate second-instar, another branch has been observed to grow out from a large tracheal trunk near the posterior side of the throacic segment (Fig. 8B), and enter the rudiment of the wing from its posterior side; it measures 35-40 dam in length, and 1-1. 5 1Zxn in diameter. The two tracheal branches now lying in the wing pad are therefore growing from opposite directions. The development of the mesothoracic expansion seems to be a little in advance of the prothoracic and metathoracic expansions. The histological study of the prothoracic segment of the newly hatched first-instar of Oncopeltus also reveals the presence of histologically recognisable doro-lateral thoracic expansions, similar to the developing wings of the meso- and metathoracic segments. These rudiments also appear as hollow flattened outgrowths of the body wall, along the lateral margins of the dorsum following the line where the suture between tergum and pleuron later develops. They are also supplied by a very small trachea (Fig. 54). Some carboniferous insects from the Palaeodictyoptera 62

Fig. 29. T. S. of 170 hour first-instar left fore wing pad containing enlarged dermal glands; the cytoplasmic processes of apposed cells are meeting each other. And an intercellular space has formed between them. bm, basement membrane; cp, cytoplasmic process; cut, cuticle; ep, epidermis; exsp, exuvial space; gl, unicellular gland; icsp, inter-cellular space; n, nucleus; t, trachea.

Fig. 30. T. S. of late Pharate second-instar fore wing pad, showing fully formed new cuticle covering the folded epidermis. bm, basement membrane; cp, cytoplasmic process; cutl, old cuticle; cut2, new cuticle; ep, epidermis; exsp, exuvial space; haem, haemocyte; mac, macrotrichium; n, nucleus; t, trachea.

64 and other fossil orders had, in addition to the wings, a pair of small, flat lobes projecting laterally from the prothoracic terga. Similar paranotal structures are reported in Mantids, Lepismatids, and in Hemidercus (Hemiptera) and some Ephemeropteran nymphs. For further discussion see P•14 0 The prothoracic paranotal expansions are supplied with tracheae, which are arranged in a pattern somewhat similar to that found in developing wings (Sūlc, 1927). These facts suggest that at an earlier period, the wings themselves evolved from similar tergal lobes of the mesothorax and metathorax. This paranotal theory, proposed by Crompton (1916) has been supported by Comstock & Needham (1918), Handlirsch (1908) and others. Second-Instar: A batch of newly moulted second-instar larvae were selected from the culture, reared separately, and fixed at intervals of 24 hours, until the last day of the moulting cycle. In the newly moulted second-instar, the wing pads on each thoracic segment are still indicated only as a flange at the junction of pleuron and tergum; but these now measure 120-130 pm length, have, therefore, more than doubled in size. In the fully grown second-instar specimens the wing rudiments look like swollen black areas on the dorso-lateral regions of the thoracic segments. As it is very difficult to separate them from the thorax, the latter was fixed and cut as a whole. On the dorso-lateral regions of the mesothorax the two plates of epidermis constituting the wing pad have become fused internally by this stage, but the prothoracic and metathoracic ones are still separate layers of epidermis, that do not show any advance over the first-instar except for their growth in size and cell number. As the development of the mesothoracic wing pads is more advanced, I have concentrated mainly on describing them. The details which I am presenting here are based largely on the development of the fore-wing and its tracheal supply. Further details of the development of hind wing and prothoracic expansions will be discussed later. 65

In the newly moulted second-instar, the cuticle is very pale, thin and delicate. There has been an increase in the number of macrotrichia, and in addition to them, some dome-shaped cuticular structures can be seen, apparently campaniform sensilla. The .' function of these cuticular structures in larval insects is not under- stood. The epidermis still consists of a single layer of cells (Fig. 31), ostensibly syncytial in nature. The nuclei are large, ovoidal structures, measuring 5-6 pm by 6-7 pm across. Each nucleus occupies part of a tail-like conical process of the cell, that extends to the basement membrane. The basement membranes of apposed layers have come close together, and a middle membrane has formed, but lacunae have not yet developed near the membrane nor are tracheae associated with it. Only a small cavity is present at the base of the wing'pad, just in front of the middle membrane. I assume it to be the beginning of the first formed lacuna (Fig. 31). The two tracheae mentioned above enter from opposite ends of the wing bud and run across the base. The anterior trachea is a large branch, which meausres 60 pm in length and 3-4 pm in diameter, which I identify as the costo-radial trunk. The posterior branch, regarded as the cubito-anal trachea, measures 40 atm in length and 1. 5-2 pm in diameter. They have grown very close together, and are almost in contact, but a transverse basal connection has not yet become established between them. From, the costo-radial trunk, a small branch has been seen growing towards the wing pad for the first time (Fig. 8C). It is only 6-10 atm long at this stage. The wing pads grow in size for a few hours as the cuticle covering them is still soft and flexible. The dividing cells still maintain an unilaminar arrangement for about 20-24 hours after ecdysis, but after that the cuticle becomes thickened (3-4 p.m). The cuticle of the older instar larvae is thicker than that of younger members of the same instar (Klinger, 1936). Towards the dorsal side, the outer half of the procuticle is impregnated with an amber- coloured material, presumably indicating sclerotisation. 66

Fig. 31. T. S. of a newly moulted second instar left fore wing pad, showing thin cuticle, unilaminar epidermis, and a basement membrane between. bm, basement membrane; cut, cuticle; ep, epidermis; mm, middle membrane; n, nucleus; oen, oenocyte; t, trachea.

Fig. 32. T. S. of a 24 hour second-instar left fore wing pad. The cuticle has thickened, the epidermal cells have increased in number. Two small profiles of tracheae are present near the base of the wing pad. bm, basement membrane; cut, cuticle; ep, epidermis; mit, mitotic division; mm, middle membrane; n, nucleus; oen, oenocyte; t, trachea. FIG.31 68

The inner layers are still soft and flexible, and stain blue with Mallory's connective tissue stain. The epidermis has increased in thickness, and the wing pad measures 180 pm in length by this stage. At 40 hours after the first ecdysis the cell numbers have increased due to mitotic division, and the unilaminar arrangement is now lost. The differentiation divisions of cuticular sensilla and swollen unicellular glands can also be seen by this time (Fig. 33). The position of the tracheae is the same as that of the early second- instar, and the newly grown branch of the costo-radial trunk has shown little further extension. At a distance of 18 pm from its origin from the costo-radial trunk, this branch penetrates the wing pad for a further 12-18 pm. It is the first branch to enter the wing pad epithelium, and grows into the space at the base of the wing pad, which is formed earlier than this trachea. The presence of this space in front of the wing pad epithelia has been referred to in the description of the newly moulted second-instar wing above. At 72 hours after ecdysis, proliferative cell divisions can also be seen in the wing pad and tracheal epithelium. The epidermal cells of the wing rudiment are now crowded in huge numbers between cuticle and basement membrane, and resemble a pseudostratified epithelium (Fig. 34). Most of the nuclei occupy part of a tail-like, conical process of the cell that extends to the basement membrane, which is now 0. 5-1 pm thick. Some unicellular gland cells are present near the border between cuticle and epidermis. Within a few hours, the larva enters an apolytic phase. The cuticle slowly becomes detached from the epidermis, which is without a recognisable investement. The exuvial space is filled with foam-like fixed moulting fluid. The unicellular glands seem to have discharged their products, as their number is decreasing. I assume that these unicellular glands pour their secretions, into the exuvial space and degenerate. Small beak-like projections of these gland cells can sometimes be seen towards the exuvial space, 69

Fig. 33. T. S. of 48-hour second-instar right mesothoracic wing pad, showing macrotrichium, and mitotic divisions. bm, basement membrane; cut, cuticle; ep, epidermis; mac, macrotrichium; mit, mitotic division; mm, middle membrane; n, nucleus; t, trachea.

Fig. 34. T.S. of left mesothoracic wing pad from 72 hour second-instar, showing exuvial space between cuticle and epidermis, increased number of cells in epidermis and tracheal epithelia. bm, basement membrane; cp, cytoplasmic process; cutl, old cuticle; cut2, new cuticle; ep, epidermis; exsp, exuvial space; mit, mitotic division; n, nucleus; t, trachea. FIG. 33

I FIG. 34 2514m 71

probably indicating the point at which the secretions are being liberated (Fig. 34). At 90-100 hours after the first ecdysis, the new cuticle of the pharate third-instar is formed. The epidermis by this time has become arranged into a single layer; the nuclei are ovoidal, and each nucleus occupies part of a separate tail-like process directed towards the basement membrane. The basement membranes of the • apposing layers have come together to form a middle membrane. The fully formed pharate third-instar wing pad is now folded inside the old cuticles (Figs. 35 and 36). At 72 hours after ecdysisl the first formed longitudinal trachea, a branch of the costo-radial trunk, has penetrated into the wing pad epithelium to a depth of about 42 p.m. It now bifurcates, with branches 24 iim each in length (Fig. 8E). Just in front of this main branch near the humeral angle of the wing pad, a further fine branch has grown from the costo-radial branch. This is the most anterior tracheal branch of the wing pad and it enters the pad to form the sub-costal trachea. The anterior bifurcation of the next posterior branch (1. e. the first to enter the wing pad) is the radius, and the posterior bifurcation is the media. The costo-radial and cubito-anal trunks have now grown very near to each other (Fig. 8E). Prior to the second ecdysis, that is 96-100 hours after the first ecdysis, the sub-costal, radial and medial tracheae have shown increased growth inside the wing pad. The transverse basal trachea has also been established completely, and a separate cubital trachea has started to grow from the cubito-anal trunk, (Fig. 8F) thus confirming Wiggle sworth's (1954) account of tracheal development in Rhodnius prolixus: "New tracheae and tracheoles arise by the outgrowth of columns of cells from the sides or ends of existing trachea at the time of moulting." The pharate third-instar is now ready to carry out ecdysis. Until this time, growth has also occurred in the prothoracic expansions, which consist of closely connected apposing layers of 72

Fig. 35. T.S. of a 100 hour second-instar mesothoracic wing pad, showing tall basal process of epidermal cells with a trachea entering wing pad for the first time. cp, cytoplasmic process; cut1, old cuticle; cut2, new cuticle; ep, epidermis; exsp, exuvial space; mm, middle membrane; n, nucleus; oen, oenocyte; t, trachea.

Fig. 36. T. S. from base of left mesothoracic wing pad of 96 hour second instar; showing a folded pharate third-instar wing pad inside the thick cuticle of second- instar. cut1, old cuticle; cut2, new cuticle; exsp, exuvial space; mm, middle membrane; n, nucleus; t, trachea. •

* 74 epithelium, with tall, conical cells, bearing long inner tail-like processes. The tracheae have not entered the proliferating area, however, though a single trachea runs in front of its base. The epidermal cells of the two surfaces of the fold are connected by long, tail-like processes (Marshall, 1915; Schūter, 1933). Similar cellular connections are seen in the book-lungs of Arachnids (BOrner, 1904) and in gills of Crustacea (Bernecker, 1909; Leydig, 1878). The growth of the metathoracic expansions is rather slow during the second-instar and the inner surfaces of the two layers of the wing pad have not fused. Third-Instar: In the newly moulted third-instar, the mesothoracic wing pads are pale in colour, delicate and transparent. As the growth of the mesothoracic wing pads has been very rapid in the second-instar, they appear externally in the early third-instar as bag like structures on the posterolateral angles of the mesothorax covering a small portion of the metathorax. With a little care, whole mounts of isolated wing pads can be made with glycerine jelly, and studied to give an accurate picture of tracheal distribution (tracheation), and its changes during the growing third-instar. The mesothoracic wing pad measures about 420 1xm in length. Histological preparations of the wing pads of newly- moulted third-instar larvae show that in all six thoracic expansions, the wing membranes of both surfaces have come to lie very close together, and a fused middle membrane is formed by the apposed basement membranes (Fig. 37). The cuticle covering the wing pad is at first very delicate, soft and flexible, and apparently not hardened. It stains blue with Mallory's connective tissue stain, though an outer layer on the dorsal side does not stain, and is slightly amber-coloured, indicating sclerotisation. The epidermal cells are tall, conical, with oval nuclei, usually towards the base of each cell. The pointed ends of both epidermal surfaces meet at the middle membrane. 75

While most of these pointed ends pass towards the basement membrane, some of the cells do not extend up to the basement membrane. Instead, they are curved to associate with the ends of neighbouring cells, and leave an intercellular space. Some of these intercellular spaces merge to form lacunae which provide cavities for the circulating blood. In the newly moulted third-instar such spaces are present, even extending to the tip of the wing pad (Fig. 37). Five prominent lacunae were found in the middle of wing pad, some time before the appearence of tracheae in them. The tracheae at this stage are still very short, and confined to a small region at the base of the wing pad. Many circular haemocytes, with clear eosinophilac cytoplasm, are present within the lacuna, indicating that some circulation or movement of blood occurs in them. The presence of these empty lacunae shows that, they develop earlier than tracheae, and precede the entrance of tracheae into them (Fig. 37). At this stage the framework of adult tracheation has also been established. There are two tracheal trunks entering the base of the wing pad, from the anterior and posterior sides, and joined by a transverse basal connection (Fig. 8G). The anteriormost branch of the costo-radial trunk is the sub-costa, and it enters the wing pad, just beneath the humeral angle. It is about 120 pm long and bundles of tracheoles arise from it to supply the tip of wing pad. These tracheoles are not visible in some sections, but in others they are very clear. Just behind the sub-costa the posterior branch of the costo-radial trunk enters the wing pad, runs for a distance of 75-80 pm, and then bifurcates. The anterior branch running in the lacuna behind the sub-costa is the radius. While the other branch is the media and enters the third lacuna (Fig. 8G). These two tracheae, radius and media, run inside their lacunae for a distance of 120-130 pm, then give rise to bundles of tracheole s to supply the tip of the wing pad. Behind these branches the costo-radial trunk runs for a short distance and joins with the cubito-anal trunk. The anteriormost branch of the cubito-anal trunk is 6-10 p.m long and 76

Fig. 37. T. S. from a newly moulted third-instar right me sothoracic wing pad, showing lacuna enclosing few tracheoles and another lacuna enclosing a small trachea. Middle membrane is well developed. cp, ctyoplasmic process; cut, cuticle; ep, epidermis; lac, lacuna; mm, middle membrane; n, nucleus; t, trachea; tl, tracheole.

Fig. 38. T.S. of 24 hour third-instar mesothoracic wing pad showing cell divisions and an empty lacuna (detactment of cuticle is an artefact) bm, basement membrane; cp, cytoplasmic process; cutl, cuticle; ep, epidermis; haem, haemocyte; lac, lacuna; mm, middle membrane; n, nucleus; t, trachea.

Fig. 39. T.S. of a 48 hour third-instar, left mesothoracic wing pad showing mitotic cell divisions, lacunae enclosing tracheae and blood cells. bm, basement membrane; cp, cytoplasmic process; cutl, old cuticle; exsp, exuvial space; lac, lacuna; haem, haemocyte; mit, mitotic division; mm, middle membrane; n, nucleus; t, trachea.

78 enters the fourth lacuna of the wing pad as the cubital trachea (Fig. 8G). It gives off bundles of tracheoles, and towards the tip of the cubito-anal trunk, two separate bundles of tracheoles have grown out and entered the remaining lacuna to indicate the future growth of the first and second anal tracheae, thus again confirming Wigglesworth's (1954) observations in Rhodnius prolixus, that the new tracheae arise from a nodal point, where there is an abrupt contraction in size and where a group of tracheoles, the terminal tracheoles of the preceding instar, are given off. The middle membrane is very thin. A few oenocytes with eosinophil cytoplasm have become attached to the main trachea in the vicinity of the wing pad, but are not to be seen entering the wing pad. There is an increase in the number of ma cr otrichia. Approximately 30 hours after the previous ecdysis, the epidermal cells associated with cuticular sensilla, and some swollen epidermal gland cells in the middle of the wing disk, undergo mitotic division, followed by proliferation. Within a few hours, that is 45-50 hours after the second ecdysis, the cuticle has increased in thickness, but not uniformly. It varies from base to the tip. As Richards (1940) points out, "the total thickness of the cuticle shows great variation, not only between different species and groups, but even in different areas on a single specimen." The outer cuticle on the dorsal side is amber coloured, while the inner half and ventral side stain blue with Mallory's connective tissue stain. The cuticle loosens from the epidermis at this stage, but the exuvial space is still free of moulting fluid. By 45-50 hours after the second ecdysis, the epidermis has thickened and, as a result of mitotic division, the number of cells has increased. The epidermis has again lost its unilaminar arrangement, and looks like a pseudostratified epithelium (Fig. 39). The epidermal cells are tall, with oval nuclei, and tail-like processes running to the very thick basement 79 membrane (0. 5-1 pm). By 70 hours after the second ecdysis, moulting fluid appears in the exuvial space, though the new cuticle has not yet become visible under the light microscope. The unicellular glands pour their secretions into the exuvial space through beak-like processes and some of the gland cells are still dividing very actively. The liberation of the secretions of the gland cells and the first appearance of moulting fluid seem to occur at about the same time. The granules in the middle of the exuvial space resemble those in the gland cells, when judged by the simple histological criteria used here. At this time the lacunae are very prominent. The anteriormost four tracheae, regarded here as the sub-costa, radius, media and cubitus have now extended towards the tip of wing pad, only 40-50 pm away from the apex. The fifth trachea runs parallel to the base. Oenocytes with vacuolated cytoplasm have been noticed inside the wing pad, and within the thorax, oenocytes at various stages of development or secretion can be seen. By 72 hours after the second ecdysis the epidermis has expanded to its maximum thickness, as proliferative cell division is still in progress, though it is now about to decline in intensity (Fig. 40). Though they are very much crowded together to a thickness of several layers, each nucleus is associated with a tail like process that runs towards the basement membrane. The dermal glands have decreased in number. A faint line of newly deposited cuticle is visible. The division of the epidermal cells associated with the cuticular sensilla is still in progress; resistant connection of these sensilla with the epidermis extend through the fluid-filled exuvial space. Feuerborn (1927) suggested that these might be nerve processes which maintain the sensory input from the old sensillum when, at the time that moulting fluid is produced, direct cytoplasmic contact between these cuticular structures and the epidermis is relinquished. All five lacunae contain their individual 80

Fig. 40. T. S. of a right mesothoracic wing pad of 72 hour third-instar. An exuvial space containing moulting fluid is present between epidermis and cuticle. Several layers of epidermal cells showing long basal processes; lacuna are prominent and enclose a tracheae. bm, basement membrane; cp, cytoplasmic process; cut, cuticle; ep, epidermis; exsp, exuvial space; gl, unicellular gland; lac, lacuna; mac, macrotrichium; mfl, moulting fluid; mit, mitotic division; n, nucleus; t, trachea.

Fig. 41. T.S. of a fully formed pharate fourth-instar wing pad inside old cuticle. New cuticle has formed; epidermal cells are arranged in a single layer; Lacunae enclosing tracheae. bm, basement membrane; cp, cytoplasmic process; cutl, old cuticle; cut2, new cuticle; ep, epidermis; exsp, exuvial space; lac, lacuna; mm, middle membrane; n, nucleus; t, trachea. ; 4 Iii

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ill1111111111111111/14 0141/ 82 tracheae, which run up to the tip of the wing pad (Fig. 8G). The third-instar undergoes ecdysis 100-105 hours after the previous ecdysis. Prior to the ecdysis, much new cuticle is deposited, and the wing pad has stretched to its full length. The epidermal cells have once again attained a unilaminar arrangement. Each cell has an oval nucleus, accompanied by a tail-like process, which runs towards the basement membrane. Because of the expansion of the wing, the middle membrane becomes attenuated, and is very difficult to observe clearly, except around the lacunae (Fig. 41). The fully developed pharate fourth-instar wing pads are still covered by old cuticle, and because of the constricted space, they are folded into several layers (Fig. 41). During the third instar, an increased number of cell divisions has occurred leading to a greater degree of development in both fore and hind wings. The development of the prothoracic expansions on the other hand is rather slow. In the first and second instars, the epidermal wing disk cells acquire tail-like processes only during the later stages. Whereas in the third-instar the cells are produced into such structures throughout the stage. In the first, second and third instars, oenocytes of several sizes are present in the thorax, attached to a tracheal trunk near the base of the wing pad. In the third instar the oenocytes have for the first time been seen inside the wing pad. Fourth Instar: The meso- and metathoracic wing pads appear externally, with the metothoracic pair nearly covering the metathoracic pair. The mesothoracic wing pads are about 960 1Zm long. Whole mounts of wing pads, prepared by dissecting newly moulted instars under glycerine, and mounts made with glycerine jelly show the detailed structure of wing tracheation (see p. 29). Histological examination of newly moulted fourth-instar wing pads show that the cuticle covering the wing pad is very thin and delicate, and not hardened, but within a short time, the outer 83

Fig. 42. T.S. of a newly emerged fourth-instar mesothoracic wing pad, containing a thin cuticle; epidermis is thin and single layered; well developed lacunae enclosing tracheae and tracheoles. cp, cytoplasmic process; cut, cuticle; ep, epidermis; lac, lacuna; mm, middle membrane; n, nucleus; t, trachea; tl, tracheoles.

Fig. 43. T. S. of 48 hour fourth-instar me sothoracic wing pad, which is greatly expanded. Moulting fluid is present inside the exuvial space; cells are increasing by mitotic divisions. cp, cytoplasmic process; cutl, old cuticle; ep, epidermis; exsp, exuvial space; gl, unicellular gland; haem, haemocyte; lac, lacuna; mac, macrotrichium; mfl, moulting fluid; mm, middle membrane; mit, mitotic division; n, nucleus; t, trachea.

85 half assumes a reddish brown colour in sections, while the non- pigmented inner half stains blue with Mallory's connective tissue stain as usual. The number of cuticular sensilla has increased since the previous instar. The dome-shaped structures are presumably campaniform sensilla, and I assume that the long spine-like structures are mechano-receptors. The short spine-like structures might be termed sensilla chaetica and are • perhaps chemoreceptors. All these innervated cuticular structures comprise four types of cells (Trichogen, Tormogen, Sense cell and a Neurilemma cell). The total number of these cuticular sensilla on the meso-thoracic wing pad of fourth-instar varies from 60 to 80. The epidermis of the wing pad is thin, consisting of a single layer of cells. The cells are conical with a large ovoidal nucleus occupying the tail-like process of the cell running to the basement membrane. The basement membranes of the apposed layers remain close together as a middle membrane, except around the lacunae, where they encircle the tracheae and blood cells (Fig. 42). This basement membrane is very thing. (less than 0. 5 pm). There are five prominent tracheae running throughout the length of the wing pad to about 150 p.m behind the tip. There is also now a sixth lacuna, which runs parallel to the base of the wing pad, and is occupied by a second anal trachea. It is fully grown in the late fourth-instar. All six lacunae correspond in arrangement to the veins of the adult wings, which will be formed later, by differential sclerotisation of the integument adjacent to the lacuna. By this time, therefore, the basic patterns of adult venation and tracheation have been established completely. Groups of tracheoles arise from each nodal point in order to supply air to every part of the wing pad. Approximately 30 hours after the third ecdysis, proliferative cell divisions of the epidermal cells begin again, and some cells in the middle of the epidermal cell layers also divide, to give unicellular gland cells. The division of bristle mother cells is also in progress. Lawrence (1966) says that in Oncopeltus the determination of bristles 86 occurs between the onset of an ecdysis and. proliferative cell divisions of the epidermis. The outer cuticle of the wing pad is strongly sclerotised but thin, whereas the inner side is thick and apparently unsclerotised, since it stains blue with Mallory's stain. The middle membrane is slightly thicker than in the previous instar. The wing pad has now also strated to expand from the apical region like a slowly inflating balloon. At 48-50 hours after the third ecdysis, histological study of the wing pad shows that it has expanded throughout its entire length. The cuticle has now become very thick (5-9 pm), and is already separated from the epidermis, so that apolysis has evidently occurred quite soon after ecdysis (Fig. 43). There are slight traces of moulting fluid in the exuvial space, but the epidermal cells are naked under the light microscope as no visible new cuticle has yet been formed. Active proliferation of epidermal cells leads to an approximate doubling of cell number, and loss of the unilaminar arrangement, so that the wing rudiment looks like a syncytial, stratified epithelium (Fig. 43). The nuclei are larger in. size (measuring 4 by 5 up to 8 by 9 pm across). Each nucleus occupies part of a tail- like conical process of the cell that extends to the basement membrane. The basement membrane is still- relatively thick (0. 5-1 13.m). The unicellular gland cells are still multiplying, even though the early-formed ones are now liberating their products. The cuticular sensilla are associated with resistant connections which run to the epidermis across the exuvial space (Fig. 43). The number of cells per unit area of section varies from the base to the tip of the wing pad. It seems that the cells in the middle region of the wing disk enter mitotic division first, followed by the cells from the sides. There is also division in the tracheal epithelium. At 72 hours after the third ecdysis the exuvial space is filled with a jelly-like moulting fluid containing basophil granules. 87

The unmodified epidermal cells and the gland cells are still dividing, and the distal ends of the gland cells are drawn out into slender neck-like processes, from which the products appear to escape into the exuvial space (Fig. 44), leaving an empty space in the area formerly occupied by the cell. The epidermal cells at this stage are very closely crowded together between the newly forming cuticle and the middle membrane (Fig. 44). Each oval nucleus occurs in the tail-like process. Many of these processes overlap, so that the epithelium has a pseudostratified appearance. The epidermal cells are covered by slight traces of newly deposited cuticle towards the outer side, while towards the inner side they are limited by a thick middle membrane. The sensilla of the old cuticle are still connected with the epidermis, though the divisions of the mother cells leading to the formation of new sensilla has come to an end. Numerous spheroidal blood cells, a trachea and tracheoles are present inside each lacuna. The oenocytes are now very large, with basiphilic, vacuolated cytoplasm and large nuclei and some have now entered the wing-pad. At room temperature the fourth-instar undergoes ecdysis between 120 and 130 hours after the previous moult. A few hours before ecdysis, the newly formed cuticle covering the wing pad stretches to its full length, so allowing the epidermal cells to re-arrange themselves in two layers (Fig. 45). The pharate fifth-instar wing pad has, therefore, now stretched to its full length, and is folded inside the old cuticle, with its cuticle thrown into further little folds, to give the opportunity for further expansion. Each epidermal cell has a single ovoidal nucleus, and is produced into a separate long tail, extending to the basement membrane. The basement membrane has become very thin due to stretching; and for this reason, it is very difficult to see it between the long cellular processes. It can only be seen clearly around the lacunae, which are now larger, containing the tracheae and numerous haemocytes. Towards the base of the wing pad, a 88

Fig. 44. T.S. of a 72 hour, fourth-instar mesothoracic wing pad showing exuvial space, several layers of epidermal cells and dividing unicellular glands. cp, cytoplasmic process; cut, cuticle; ep, epidermis; exsp, exuvial space; gi, unicellular gland; haem, haemocyte; lac, lacuna; mac, macrotrichium; mfl, moulting fluid; mit, mitotic division; mm, middle membrane; n, nucleus; t, trachea.

Fig. 45. A portion of a folded pharate fifth-instar mesothoracic wing pad inside the old cuticles. The new cuticle is fully formed; epidermal cells are arranged in a single layer; basal cytoplasm of cells is produced into long processes. Lacunae are very prominent. bm, basement membrane; cp, cytoplasmic process; cutl, old cuticle; cut2, new cuticle; ep, epidermis; exsp, exuvial space; haem, haemocyte; lac, lacuna; mm, middle membrane; n, nucleus; t, trachea.

90 single lacuna accommodates both radial and medial tracheae, but it bifurcates distally and runs separately around each trachea (Figs. 45 and 10). There is also some limited growth in the prothoracic expansion during the fourthinstar (Fig. 57), but there is no sign of any longitudinal tracheae in them. Only two layers of apposed integument with tall, columnar cells are present. Fifth Instar: In the fifth instar the external wing pads are nearly twice the length of those in the preceding instar, and now cover a portion of abdomen. In the development of these wing pads, the sequence of events is essentially the same as in the fourth instar. Transverse sections of newly moulted fifth-instar larvae show that the cuticle is very thin and delicate. Sclerotisation of the outermost layers of the procuticle is complete, and this layer is almost colourless before staining, but becomes red with a brownish tinge in Mallory's connective tissue stain; as before, the inner layers of the cuticle stain blue. The bristles are strongly cuticularised and amber coloured, and in some preparations the trichogen, tormogen and sense cells are easily seen at the base of these bristles. The campaniform sensilla also include homologues of all these cells. The epidermis of the newly moulted fifth-instar is very thin and made up of a single layer of cells. The cells are conical, with their pointed ends directed towards the basement membrane. The nuclei are ovoidal, arranged basally or centrally or towards the apex (Fig. 46). Both basal membranes of the wing are still close together, forming a middle membrane. The basal membranes are separate only around the lacuna, and are very delicate in the newly formed wing pad. Inside the lacunae, and between the unfused layers of basement membrane, are spherical blood cells with large nuclei and clear eosinophil cytoplasm. There are five longitudinal tracheae running to the tip of wing pad; while the sixth is a short branch, that runs for a little distance, parallel to the base of 91 the wing pad.(Fig. 12). All these tracheae enter the base of the wing pad, in two groups, connected by a transverse basal trachea. Bunches of tracheoles, arising from the tip of the tracheae, come close to the adjacent tracheoles from the trachea lying in front (Fig. 12). All six main tracheae are enclosed in lacunae. Towards their base the radial and medial tracheae share a single lacuna. The presence of a nerve in each lacuna is now very clear; each lies towards the dorsal integument. About 40-50 hours after the fourth ecdysis, the wing pad starts to expand in thickness like an inflating balloon. The thickened apical portion consists of cells with very long processes. The outer layers of the cuticle are amber coloured and the inner layers stain blue. The epidermal cells begin to divide mitotically, and the dividing chromosomes and their re-arrangement are very clear in the enlarged gland moth cells, because of their gigantic size. The resulting daughter-cells are the unicellular glands. It seems that the formation of these gland cells starts towards the base of the wing pad. When the division of epidermal cells is in progress, the cuticle becomes detached from the epidermal layer as a uniform sheet. The 72 hour fifth-instar wing pad shows full expansion throughout its length. It is two to three times thicker than the early wing pad, and by this time the cuticle is completely separated from the epidermal layer. The detached cuticle is very thick, with a black outer procuticle and a blue-stained inner layer. The cuticular sensilla still maintain a resistant connection with the epidermis across the exuvial space, which contains only a little moulting fluid (Fig. 47). When seen in fixed, stained preparations, the moulting fluid is foam-like with a few red-staining particles resembling the chromation elements of the gland cells. The epidermis at this stage is naked, as the new cuticle has not formed to a degree visible with the light microscope. The epidermal cells are dividing and as a 92

Fig. 46. T.S. of a newly moulted fifth-instar mesothoracic wing pad. Sclerotization of outermost layers of procuticle is complete; epidermal cells are thin, conical structures; lacuna encloses trachea, tracheoles and blood cells. cp, cytoplasmic process; cut, cuticle; ep, epidermis; haem, haemocyte; lac, lacuna; mm, middle membrane; n, nucleus; t, trachea; tl, tracheoles.

Fig. 47. T. S. of 72 hour fifth-instar mesothoracic wing pad. It is strongly inflated; with exuvial space present in between epidermis and cuticle; epidermal cells are in several layers. Between ordinary epidermal cells are gland cells and dividing bristle-forming cells. A nerve is now clearly seen inside the lacuna. cp, cytoplasmic process; cutl, old cuticle; ep, epidermis; exsp, exuvial space; gl, unicellular gland; haem, haemocyte; lac, lacuna; mac, macrotrichium; mfl, moulting fluid; mic, mitotic division; mm, middle membrane; n, nucleus; t, trachea.

94 result, the epidermis has lost its single-layered condition, except at the tip where it is still unilaminar. This confirms that cell division begins towards the middle of the wing pad, and proceeds to the sides. The cells, when they lose their unilaminar arrangement, crowd into groups, giving the appearance of a pseudostratified epithelium (Fig. 47). Each nucleus occupies the tail-like connections with the basement membrane. The processes from each group of cells come very close together and overlap, looking like a single, thick process. The basement membrane becomes thicker. The dermal glands are now dividing very actively and some of the early formed ones are fully developed, with slender neck-like ends towards the exuvial space (Fig. 47). As before this neck-like process apparently acts as a duct to release its products. The tracheal epithelium also divides simultaneously with the epidermal cells. The wing pads 96 hours after the fourth ecdysis show progress in digestion of the old endocuticle by the moulting fluid. Slight traces of newly deposited cuticle are now present over the epidermis. In the exuvial space, the fixed moulting fluid occupies most of the space. Cell division has reached its maximum at this time, as has also the number of dermal glands. The division of bristle mother- cells is in progress, but they still maintain their connection with the old bristle. The middle membrane is very thick (approximately 1 pm thick); every epidermal cell sends its own tail-like connection to the middle membrane. Many blood cells, tracheae and tracheoles are present inside the lacunae. At 120 hours after the fourth ecdysis the digestion of the old cuticle by moulting fluid has come to an end, though there is still plenty of moulting fluid within the exuvial space. A Gayer of new cuticle has formed over the epidermis (Fig. 48), and is slightly folded. The formation of new bristles is almost complete. Proliferative division of the epidermal cells has come to an end, and the dermal glands have completely disappeared. 95

Fig. 48. T.S. of 120 hours, fifth-instar mesothoracic wing pad showing exuvial space containing moulting fluid. A thin layer of new cuticle is visible over the epidermis; several layers of epidermal cells are present on either side of basement membrane. cp, cytoplasmic process; cutl , old cuticle; cut2, new cuticle; ep, epidermis; lac, lacuna; mfl, moulting fluid; mm, middle membrane; n, nucelus; nv, nerve, t, trachea; tl, tracheoles.

djt1 mf I cut2

FIG.48 2514m 97

The epidermis appears under the light microscope to be a syncytial epithelium, and contains a large number of ovoidal nuclei, each occupying part of the tail-like conical process of the cell. The tracheae are very prominent, each enclosed in a lacuna. In the pharate adult stage, a well developed nerve is found in each lacuna, but the blood cells seem to be few in number. At this stage some slightly swollen cells bulge inwards and over the adjacent epidermal cells and start to divide. These dividing cells are apparently bristle-forming and socket-forming cells. The histological study of fifth-instar wing pads 8 days after the fourth ecdysis shows the presence of well-formed pharate adult wings inside the old cuticle (Fig. 49). The new cuticle has grown to the full thickness it has on emergence (though it is still heavily folded). The epidermal cells have mostly re-arranged themselves into a single layer, but a few more have still to be arranged. Each ovoidal nucleus occupies one of the very long processes to the basement membrane. As the length of the newly formed wing is greater than the enclosing cuticle, it has been folded into several layers. The fine, non-socketed hairs (microtrichia) have not appeared, but they are formed abundantly in sections of wing pads, after 140 hours from the previous ecdysis (Fig. 50). In preparations made just a few hours before the fifth ecdysis, the fully formed adult wing is present inside the old cuticle, ready to be freed on emergence. The wing has reached its full length; the cuticle is completely covered by microtrichia, and the epidermal cells arranged uniformly into a single layer (Fig. 50). They are conical in shape, and each possesses a single, oval nucleus located in the basal portion. These nuclei are accompanied by a long tail-like process, that runs to the basement membrane. The middle membrane has been attenuated by the stretching of the wing and is again very delicate. It is clearly defined only around the lacuna, but with great care a middle membrane can also be seen 98

Fig. 49. T. S. of 8 day fifth-instar mesothoracic wing pads containing folded pharate adult wings. The cuticle is fully formed, but still very thin. The epidermal cells are arranging themselves into a single layer. cp, cytoplasmic process; cutl, old cuticle; cut2, new cuticle; ep, epidermis; haem, haemocyte; lac, lacuna; mil, moulting fluid; mm, middle membrane; n, nucleus; t, trachea; tl, tracheoles. cp n ep mfl • \ I I 1 I I 1 I 1 , 1

.1 .1

. _

cūt, cūt2 FI G.49 25µm 100

Fig. 50. T.S. of fully formed adult fore wing, while lying folded inside the old cuticle. The cuticle bears microtrichia. Lacuna are very prominent and encloses nerve, blood cells and trachea. cp, cytoplasmic process; cutl, old cuticle; cut2, new cuticle; ep, epidermis; exsp, exuvial space; haem, haemocyte; lac, lacuna; mic, microtrichium; mm, middle membrane; n, nucleus; nv, nerve; t, trachea; tl, tracheoles. haem lac tl~ x / I /

-cp

m ~m

mic CUt2 cut~ exIs P nv

FIG. 50 25j m 102 between the cellular layers. Many spheroidal blood cells are present inside the lacunae. In every instar there is an indication of the prothoracic flange. This shows little growth in size until the fifth larval stage (Figs. 55, 56, 57 and 58). By this time the cell number has increased and the cellular processes of the apposed layers have fused together. I have not seen any trace of a middle membrane, and neither lacunae nor tracheae are present. There is no trace of circulating blood cells in the flange. There is no sign of cell growth and division in the prothoracic flange during the fifth-instar, and later in the period the epidermis degenerates, and little trace of the flange remains. The slight growth of prothoracic expansion in all earlier instars and the subsequent degeneration of the epidermis is presumably connected with the lack of adequate nutrition and oxygen supply. Newly moulted adult (Imago): Both pairs of wings are inflated to their full size by blood pressure, but they are still pale and transparent, since impregnation of the outer layers of the procuticle with sclerotin is incomplete. The integument darkens and hardens within a day or so while additional inner layers of endocuticle are being deposited. A transverse section of a newly moulted adult fore wing shows structural resemblances to the newly moulted fifth-instar wing (Fig. 51). A major difference is that the cuticle of the adult fore wing is covered by serially arranged microtrichia in addition to the bristles and campaniform sensilla. Hairs are present on both sides of the wing, whereas the other cuticular structures are confined to the anterior half of the dorsal membrane. The cuticle is thicker towards the dorsal side. The epidermis consists of tall, conical cells, whose ovoidal nuclei are located in the apical or basal portion of the cells. The basal regions of most cells are produced into long tail-like processes, containing basiphil miof ibrils. Within a few hours after emergence the miofibrils apparently disappear 103

Fig. 51. A portion of a transverse section of newly emerged adult wing corium covered by microtrichia. The dorsal cuticle is thicker than the ventral. The lacuna encloses several blood cells, tracheae and a nerve. bm, basement membrane; cp, cytoplasmic process; cut, cuticle; ep, epidermis; haem, haemocyte; lac, lacuna; mac, macrotrichium; mic, microtrichia; n, nucleus; nv, nerve; t, trachea.

Fig. 52. A portion of a T. S. of newly emerged adult wing membrane. The epidermal cells are very small and show degeneration. The basement membrane is very faint. The cuticle covering the dorsal surface of lacuna is thicker than the rest of the area. bm, basement membrane; cp, cytoplasmic process; cut, cuticle; ep, epidermis; haem, haemocyte; lac, lacuna; mic, microtrichia; mm, middle membrane; n, nucleus; t, trachea.

Fig. 53. Portion of a T.S. from the newly emerged adult hind wing. The cuticle covering dorsal surface of lacuna is very thick. There are no microtrichia. The epidermal cells are very small. bm, basement membrane; cp, cytoplasmic process; cut, cuticle; ep, epidermis; lac, lacuna; n, nucleus; t, trachea.

105 abruptly and the cells retract towards the wing surface with few remaining long processes. The presence of these basal cytoplasmic tails containing basifil miof>ibrils in the wing pads of the newly moulted pupa of Tenebrio molitar Linn. (Coleoptera), and their retraction after 6-8 hours, has been reported by Hundertrnark (1935). The basement membranes of two epithelia come together in certain areas and are fused to form the middle membrane of the wing. The basement membrane is very delicate. There are six prominent lacunae in the wing, each enclosing a central trachea, dorsally placed nerve and circulating blood cells. The haemocytes are spheroidal, with large nuclei and eosinophil cytoplasm. Towards the base of the wing a single lacuna encloses the radial and medial tracheae. In the membrane of the wing, the lateral regions contain thin cuboidal cells and the cuticle lying towards the ventral side of the lacuna is thicker (Fig. 52). Within a day or so the wings have become sclerotised, and the cell layers become very thin, with the cells degenerating irregularly and gaps between them. The two surfaces of the corium of the hemelytra are separated by a blood space running across which are cuticular columns, the trabeculae, arranged in longitudinal rows formed from bundles of cells developed immediately after metamorphosis. These cells push in from the epidermis of the wing and the trabeculae grow from the dorsal integument. Hundertmark (1935), and Reuter (1937) report that trabeculae develop in the mid-pupa, late-pupa or pharate adults of Tenebrio molitar, and Calandra sp. but are seen in adult wings. Transverse sections of the wings obtained between 0 and 30 hours after the final ecdysis still do not show any marked sclerotisation of the integument adjacent to the lacunae (vein-formation). Unfortunately I was unable to section further stages to give a detailed description of vein formation as seen under the light microscope. 106

Fig. 54. T. S. of newly emerged first-instar prothoracic margin. bm, basement membrane; cut, cuticle; ep, epidermis; n, nucleus; t, trachea.

Fig. 55. T. S. of a 72 hour prothoracic expansion. bm, basement membrane; ep, epidermis; haem, haemocyte; mac, macrotrichium; n, nucleus; t, trachea.

Fig. 56. T.S. of a 96 hour second-instar, prothoracic expansion, with epidermis showing long basal processes. bm, basement membrane; cp, cytoplasmic process; cutl, old cuticle; cut2, new cuticle; ep, epidermis; exsp, exuvial space; haem, haemocyte; n, nucleus; t, trachea.

108

Fig. 57. T.S. of a prothoracic expansion of Oncopeltus larva about to moult into fifith-instar. The pharate fifth wing pad is folded inside old cuticle. There are no lacunae or tracheae inside the expansion. bm, basement membrane; cp, cytoplasmic process; cut1, old cuticle; cut2, new cuticle; ep, epidermis; exsp, exuvial space; n, nucleus.

Fig. 58. T. S. of a newly emerged fifth-instar prothoracic expansion. They do not contain lacunae or trachea. bm, basement membrane; cp, cytoplasmic process; cut, cuticle; ep, epidermis; n, nucleus. FIG. 57

FIG. 58 251.1m 110

3. Metathoracic wing development As already described in connection with the mesothoracic wings, the metathorax of newly hatched first-instar larvae bears histologically recognisable wing pads, each indicated externally as a minute flange on the side of the thorax, and measuring 90-100 pm long. The rudiments are directly continuous with the tergum, and formed from a simple integumentary fold. The external changes in the wing rudiment during growth are comparatively slight. There is a gradual increase in the size of the wing in successive larval instars. First-Instar: Histological study of the metathoracic wing rudiments of newly hatched first-instar larvae shows the presence of an outer, delicate, membranous, non-sclerotized cuticle, bearing two or three macrotrichia. The epidermis is made up of a few cells (less than 10 in each section), arranged in a single layer, which appears as a syncytial epithelium under a light microscope. The epidermal cells are bounded internally by a thin, non-cellular basement membrane. Each metathoracic wing disc is supplied by a single trachea, arising from a large tracheal trunk towards its anterior side; this enters the base of the wing disk anteriorly, and runs posteriorly between the thorax and the base of the wing disk. It measures 1 in diameter. The development of the metathoracic wing pads, during the first larval stage is very slow. Three days after eclosion, two or three epidermal cells in the wing disk grow in size and divide mitotically to form unicellular glands. The epidermal cells increase in number by mitotic division, and the instar then enters an apolytic phase, approximately 5 days after hatching. The cuticle detaches from the epidermis and the exuvial space is filled with a foam-like material representing fixed moulting fluid. Prior to the first ecdysis the new cuticle has formed and is thrown into folds. Due to the stretching of the cuticle and re-arrangement of the epidermal cells, the size of the wing rudiments increases. 111

During this period, in addition to the anterior trachea, another branch has been observed to grow out from a large tracheal trunk, near the posterior side of this wing-bearing segment. (Fig. 9B). Second-Instar: The metathoracic wing pads of the newly moulted second-instar are still indicated only as flanges at the junction of pleuron and tergum, but they now measure about 120 pm in length and are more than doubled in area. The two layers of epidermis constituting the wing pad are still separate. The cuticle covering the newly formed second-instar wing pad is soft and flexible; it is 1 p.m thick and stains blue with Mallory's connective tissue stain. A few macrotrichia, each arising from a cup-like socket, are also present on the wing pads. The epidermis still consists of a single layer of cells, apparently syncytial in nature. The nuclei are ovoidal structures measuring 4 by 6 pm, while the basement membrane is 0. 5 pm thick. Oenocytes are present between the separate epithelial layers of the wing pad. The anterior trachea, which I identify as the costo-radial trunk, enters the base of the wing pad anteriorly and runs across the base of the wing pad; it is now 110 pm long and 2.5-3 µm in diameter. The other trachea, which enters the wing rudiment posteriorly is regarded as the cubito-anal trachea; it measures 40 pm in length. These two tracheal trunks now lie within the wing rudiment and therefore supply its tissue from opposite directions (Fig. 9C). The wing pads grow in size a little during the instar as the cuticle covering them is still soft and flexible. By about 20-24 hours after the first ecdysis, the cuticle has become thicker (5-6 pm) and by the time the wing pads enter the apolytic phase (70-75 hours after ecdysis) the cuticle is 9-10 pm thick. The cuticle detaches from the epidermis as an entire sheet. The outer half of this procuticle stains reddish in colour, presumably indicating sclerotisation. The inner layers are still soft and flexible, and stain blue with Mallory's connective tissue stain. The newly apolysed epidermis is naked at first without any apparent cuticular investement. The epidermis has increased in 112

thickness to 12-13 pm due to cell multiplication by mitotic division. The sequence of other events, such as the formation and differentiation of unicellular glands, proliferative divisions of the epidermal cells and tracheal epithelia, apolysis, and ecdysis are essentially the same as in the mesothoracic wing pads, but the growth of the metathoracic wing pads is still slow during the second instar. Prior to the second ecdysis, the two tracheal trunks, mentioned above as entering from opposite sides of the wing pad, have grown very close to each other, and are almost connected. The transverse basal trachea, has in fact been established as a column of cells between them (Fig. 9D). From the costo- radial trunk, a small branch is now noticeable for the first time and grows towards the wing pad; it measures 30 pm in length, and has penetrated into the wing pad epithelium to a depth of about 6 p.m (Fig., 9D). Another trachea has also been noticed to grow from the cubito-anal trunk at about 30-40 pm towards the wing pad and penetrates the wing pad epithelium to a depth of 12 pm. Prior to the second ecdysis, a lacuna has developed as a space distal to the basement membrane. Third Instar: The metathoracic wing pads are still not visible externally. Only histological preparations of the metathoracic segment reveals their presence as dorso-lateral thickenings of the me tatho rax. The epithelia of both surfaces of the rudiment have now become very closely apposed, and a fused middle membrane is formed by the contiguous basement membranes. These small flange- like rudiments now measure 240 pm in length. Their cuticle is very delicate in the newly emerged third-instar metathoracic wing pads (1-2 pm thick). As in the previous instar, the outer half of the procuticle stains reddish in colour, presumably indicating sclerotisation, while the inner side stains blue with Mallory's connective tissue stain. The epidermis still consists of a single layer of cells, apparently syncytial in nature. Each ovoidal nucleus 113

occupies part of a tail-like conical process of the cell that extends towards the basement membrane. The latter is very thin (0. 5 pm). In newly emerged third-instar larvae the transverse basal tracheal connection is fully established between the anterior and posterior trunks (Fig. 9Ē). The first formed longitudinal trachea of the costo-radial trunk has shown further growth (100-120 p.m long), and the branch of the cubito-anal trachea now measures 36-40 p.m in length (Fig. 9E). Both have entered the lacuna of the wing pad, whose formation was described above while dealing with the development of the mesothoracic wing pads. The growth of the metathoracic wing pads is very rapid during the third instar, due to epidermal hyperplasia and stretching of the cuticle. The complete pattern of adult tracheation develops during this stage (Fig. 9F; third-instar, late). A few hours after ecdysis, the cuticle thickens; the unicellular glands appear between the epidermal cells; numerous mitoses appear in the epidermis; the cuticle loosens visibly from the epidermal cells; the new cuticle is secreted (apolysis at 70-75 hours after the previous ecdysis), and cuticular stretching leads to the re-arrangement of epidermal cells as a single layer. During this period the tracheal matrix cells also divide mitotically, and new tracheae arise by the outgrowth of columns of cells from the sides or endings of existing tracheae at the time of moulting. The observations of Wigglesworth (1954) on tracheal development in Rhodnius prolixus have been confirmed by my observations on Oncopeltus fasciatus. In the third instar, during the moulting cycle, the sub- costal trachea has grown from the costo-radial trunk, arising from a nodal point, close to the origin of the previously mentioned first branch of the costo-radial trunk (Fig. 9F1). In a few cases, however, it originates from the base of this first branch, instead of developing from the main branch (Fig. 9F2). It is a very fine trachea, which enters the wing pad closely beneath the humeral 114

angle. The first-formed branch of the costo-radial trunk enters the base of the wing pad and bifurcates. The two branches then enter previously established lacunae in the wing pad. The first branch, which enters the lacuna behind the sub-costa, is regarded as the radial trachea; the posterior bifurcation is the medial trachea. The previously mentioned branch of the cubito-anal trunk runs across the base of the wing pad towards the posterior side; the cubital trachea arises from a nodal point near its origin, and enters the lacuna next to the medial trachea. From the tip of this first-formed branch of the cubito-anal trunk, the first and second anal tracheae grow out and occupy the fifth and sixth lacuna of the wing pad. By 72 hours after the second ecdysis, four prominent lacunae, with tracheae within them, were found in histological preparations of the metathoracic wing pads; the lacunae extend almost up to its tip (Fig. 37). Approximately 90 hours after the second ecdysis three lacunae are present up to the tip of the wing pad, all occupied by bundles of tracheoles and blood cells. The radial, medial and cubital tracheae have been found to occupy them from the base to about 100 im from the tip. The fourth lacuna is occupied by the-first anal trachea, which is shorter than the others. The sub- costal and second anal tracheae are very thin and short branches, occupying the lacuna on each margin of the wing pad. During development, the presence of empty lacunae has been observed (distal to the tracheae), indicating the formation of lacunae, before the tracheae enter the developing wing pad. Fourth Instar: In the newly-moulted fourth instar, the metathoracic wing pads appear externally as bag-like structures, at the posterolateral angles of the metathorax. They are hidden beneath the mesothoracic wing pads, and measure about 540 p.m in length when exposed. Histological preparations of newly moulted fourth-instar, metathoracic wing pads show a very delicate cuticle. A very few macrotrichia are present on it, each arising from a cup-like socket, 115

The epidermal cells are tall, conical with ovoidal nuclei, measuring 4 by 6 to 6 by 7 p.m and they occupy tail-like cell connections with the basement membrane. The middle membrane is very thin (less than 0. 5 }lm). Between the fused layers of integument, four prominent lacunae are present up to the tip of the wing pad (Fig. 59). In addition to these, a short lacuna is present behind the costal margin, and another, sixth lacuna is found towards the posterior margin of the wing pad, running parallel to its base. All these lacunae are occupied by tracheae. By this time the framework of adult tracheation has been established very clearly (Fig. 9G). The two tracheal trunks, entering the base of the wing pad from opposite sides are joined by a transverse basal connection. The anteriormost longitudinal trachea is the sub-costa, a short and delicate branch. The radial and medial trachea are branches of the first formed branch of the anterior tracheal trunk. The branch of the posterior trachea bends a little towards the posterior side of the wing pad; near this bend the cubital trachea is given off. The first and second anal tracheae have grown from the tip of this cubito-anal branch (Fig. 59). Within a few hours of the previous ecdysis, the cuticle has thickened; the unicellular glands appear; active proliferation of epidermal cells leads to an approximate doubling of cell numbers, and the wing pad epithelium loses its unilaminar arrangement. About 40-50 hours after ecdysis the wing pads enter the apolytic phase. While these changes have been taking place, mitoses in the tracheal epithelia lead to the formation of longer tracheae of increased diameter. The newly formed tracheoles are joined to the sides and tips of these tracheae. A few hours before the next (fourth) ecdysis, the newly formed cuticle, covering the wing pads, stretches to its full extent, allowing the epidermal cells to re-arrange themselves into two single layers; the fully grown metathoracic wing pads now stretch to their full length, but remain folded inside the old cuticle (Fig. 45). 116

Fig. 59. T.S. of a newly emerged fourth-instar mesothoracic wing pad. cp, cytoplasmic process; cut, cuticle; ep, epidermis; haem, haemocyte; lac, lacuna; n, nucleus; t, trachea; tl, tracheole. •

Fig. 60. T.S. of a 72 hour fourth-instar metathoracic wing pad. cp, cytoplasmic process; cut, cuticle; ep, epidermis; exsp, exuvial space; gl, unicellular gland; lac, lacuna; mfl, moulting fluid; mm, middle membrane; n, nucleus; t, trachea.

118

Fifth Instar (Fig. 13): In the fifth instar, the metathoracic wing pads, like those of the mesothorax, are nearly twice the length of those in the preceding instar. They cover a portion of the abdomen, and lie between the body, and the mesothoracic wing pads. Histological preparations of newly moulted metathoracic wing pads of fifth-instar larvae, show a delicate cuticle on both main surfaces of the wing pads. Macrotrichia are very few in comparison with the mesothoracic wing pads. The epidermis of the newly moulted fifth instar is very thin and made up of a single layer of conical cells with their pointed ends directed towards the basement membrane. The ovoidal nuclei are arranged basally and the middle membrane is very thin (less than 0. 1 pm thick). The lacuna containing the sub-costal trachea is longer than the trachea, and located underneath the costal margin. The radial, medial and cubital tracheae run through most of the wing pad to about 100 pm from the tip; they measure 2 pm in diameter, and are lodged each inside its own lacuna. The first anal trachea is a little shorter than the above (20 pm), while the second anal trachea, which is still a delicate branch, occupies the lacuna that runs parallel to the posterior margin of the wing pad. From all these tracheae, bundles of tracheoles appear from each nodal point, in order to supply air to every part of the wing pad. During the moulting cycle, the sequence of events is essentially the same as in mesothoracic wing pads, until 100-110 hours after the previous ecdysis. The histology of the metathoracic wing pads of the fifth-instar at about 120 hours after ecdysis reveals the following differences from the fore wings. A considerable number of campaniform sensilla have developed towards the base of the wing pad. The number of macrotrichia is much smaller, and they are confined to a restricted area, adjacent to that occupied by the campaniform sensilla. I have not seen microtrichia at any stage of development in the hind wing pad. Adult hind wings: Transverse sections of newly moulted adult hind 119 wings are structurally similar to those of newly moulted adult fore wing membrane, but differ from it in not possessing microtrichia (Fig. 53). The cuticle is mostly about 1 p.m thick, but it is about 3 p.m, towards the ventral side of the lacuna. The epidermis consists of thin, conical cells, which are already degenerating towards the peripheral parts of the wing. It does not seem to possess basement membrane, except around the lacuna. Within a few hours all the tissue will have degenerated completely. Trabeculae do not develop in the hind wing. •

120

Fig. 61. T.S. of a late pharate adult hind wing showing companiform sensilla. cp, cytoplasmic process; cut, cuticle; ep, epidermis; haem, haemocyte; lac, lacuna; mm, middle membrane; n, nucleus; t, trachea. FIG. 61 2 51.2m.

n c rt

Fig. b2. T.S. of left mesothoracic wing rudiment of newly moulted first instar. bm, basement membrane; cut, cuticle; ep, epidermis. cut

Fig. 63. T.S. of 72-hour fifth-instar left mesothoracic wing pad. cut, cuticle; cp, cytoplasmic process; ep, epidermis; lac, lacuna; t, trachea. Ut

ep • ~~. C p

•

Fig. 64. T. S. of folded pharate adult rnesothoracic wing inside old cuticle. cut, cuticle; cp, cytoplasmic process; ep, epidermis; lac, lacuna; t, trachea. 125

4. Discussion: It is convenient here to summarise some of the general conclusions in relation to comparable work by other authors. (a) Tracheation, Venation and Vein homologies: The above study describes the venation, tracheation and the basal articulations of the adult wings of Oncopeltus. The general scheme of tracheation during the developmental stages and in the newly moulted adult (including the basal connections) has also been established. After making a detailed examination of these structures in comparison with other works dealing with the Heteropteran wing venation, an appropriate system of venational homologies and notation for the wing veins is now proposed. This differs only slightly from some of the previously proposed systems, but considerably from others. The homologies of the wing venation in the Heteroptera is a controversial subject, " and their wing veins have proved hard to interpret, so that accounts in the literature are very conflicting. The homologies of the wing veins in the Heteroptera have been much obscured by the interpretation put upon them by Comstock & Needham (1918), who considered the most anterior trachea of the larval wing pad to be the costa, with the second as the sub-costa, the third radial and fourth medial. The fifth trachea lying posterior to the claval suture, they therefore called Cubital, and considered that in this suborder, alone among insects, Cu lies in the clavus, whereas in all others it lies anteriorly to or in the claval suture. Comstock & Needham also thought that trachea IA of other authors is a branch of Cul . They also overlooked the small anteriormost trachea present in the hind wing pad, and consequently in naming the trachea in the Pentatomid hind wing pad they referred to the first visible anteriormost trachea as the costa, though in fact it represents the radial trachea of the Heteropteran wing. Handlirsch (1908) considered the costa of Comstock & Needham to be the sub-costa, and interpreted the Heteropteran forewing veins accurately as Sc, R, M, Cu, lA and 2A. However, 126 he made a few mistakes in naming the hind wing tracheae of the Lygaeidae, by considering the anteriormost short trachea as a branch of the sub-costa, followed by Sc, R, M, lA and ZA. He also considered that the cubital trachea is not present in this hind wing, and that there is a relatively wide 'cubital space' between the medial trachea and the first anal trachea. Consequently he concluded that the cubital vein must develop without a precending trachea in Lygaeidae, Pentatomidae, Saldidae, and Miridae. A somewhat similar case has been reported by Kohler (1940) inanabberent strain of Lepidoptera Ephestia, where the medial trachea is absent from the early pupa but the adult wing venation is perfectly normal. Wigglesworth (1954) demonstrated experimentally that the wing tracheae are less regular when Rhodnius prolixus is reared under low oxygen concentrations, but the adult venation is quite normal. Though the tracheae are not, therefore, the causal precursors of the adult veins, an adult vein usually contains a tracheae. These abnormalities do occur sometimes in certain individuals depending upon various abnormal environmental factors like oxygen lack and temperature (Henke 1953), but there is no evidence that they occur regularly as a normal condition in many groups of insects. I tend, therefore, to agree with Hoke (1926) in rejecting Handlirsch's proposition concerning the cubital vein when she says that, "if we interpret the trachea in that way, it means the cubital vein in the majority of the Heteropterous group develops without a trachea." There is no need to postulate such an unusual condition. Tillyard (1926) has brought forward evidence in support of Handlirsch (1908) by considering the first vein in the Heteroptera as Sc and the second to fourth as R, M and Cu. He has shown that Ri is absent from a primitive Pentatomid-like type of hemelytron from the upper Triassic Dunstaniidae and subsequently identified the veins from the membrane of Lygaeus singularis as Rs, Ml + 2 M3+ 4, Cu la and Culb . Imms (1925) followed Tillyard in naming the cubital branches Cula , Cuib and Cut. 127

The most detailed description and interpretation of the Heteropteran venation is that of Tanaka (1926) who, in my opinion, correctly figured the veins from the proximal half of the hemelytra. He considered the costa to be absent and also that the so-called first anal vein of Comstock & Needham is in many insects the second branch of the Cubitus , though this had previously been recognised by Tillyard (1919). Tanaka also suggested that the anterior branch of Cui has become obsolete in Heteroptera and the existing Cubital vein is Cu2. But the Cubital branches which he is considering are likely to be Cula and Cuib, because it is already probable that Cu2 is lost in the claval suture. In the same year Hoke (3926) followed Comstock & Needham's terminology and wrongly interpreted the hind wing venation of the Lygaeidae and other Heteroptera as comprising C, Sc, R, M1 + 2, Cu and 1A. According to her, the medial trachea tends to migrate either towards the costo-radial group or towards the cubito-anal group or to remain about the centre of the transverse basal trachea. It is difficult to believe that there are such fundamental differences between the members of the same suborder. The careful dissections of third, fourth, fifth and adult wings, and the histological study of wing tracheal development in the early larval stages of Oncopeltus suggests strongly that the anterior wing tracheae consist of sub-costal, radial and medial trachea, while the posterior trachea gives rise to the cubital and two anals. Both are connected by a transverse basal connection. Slater & Hurlbutt (1957) consider the most generalised Heteropteran hind wing venation is that of the sub-family Lygaeinae and that the group Lygaeini, to which Oncopletus belongs, shows a somewhat specialised condition in that interva.nnals (= ? branches of an anal complex) are absent, though they possess a short, distinct sub-costal vein. Slater & Hurlbutt have taken Leston's (1953) terminology (modified from Tanaka) into consideration and correctly identified the anterior veins of the Lygaeid hind wing as •

128

Sc, R, M and Cu; but they seem to have made an unnecessary compli- cation in referring to the vannal and jugal folds of the hind wing and the veins which they contain as the vannals and jugals. These seem to be simply lA and 2A t as indicated by the basal articulation. Davis (1961) tried to establish the identity of the veins of the fore and hind wings of Heteroptera by a comparative study with those • of Auchenorrhynchan Homoptera. He identified the Heteropteran veins as Sc, R, M, Cu, and PCU+A. His reasons for considering the first vein of the Heteropteran wing as Sc are based on the absence or reduced condition of the costal vein in Auchenorrhyncha and seem quite reasonable. The reason for naming the fifth vein present in the forewing as PCU +A seems to be incorrect, as there is no evidence that it is a compound vein. During the development of Oncopeltus it arises in association with a single lacuna and encloses a single trachea. The sixth trachea of the forewing always lies near the anal margin. Moreover the position these veins occupy in the wing and their relationship to the third axillary sclerite suggests them to be the Anal veins (1A and 2A) in both fore and hind wings. In 1977 Wootton suggested a phylogentic relationship of the Lygaeid wing to that of a Liassic Heteropteran of the family Pachymeridiidae (Pentatomomorpha). He named the veins from the membrane of Lygaeus pandarus as follows: The most anterior vein as Sc ? (he is in doubt); second, R; third, M; and the fourth and fifth veins as branches of Cua (i. e. equivalent perhaps to Cula and Cuib). This notation supports his previous work on tracheal capture. In 1965 he had given evidence for tracheal capture in early Heteroptera. Taking his examples from what appears to be an ancestral form from the family Actinoscytinidae (U. Permian to U. Jurassic), he also figured some hypothetical stages to show how vein Cuib might have captured the trachea lA during evolution. Leston (1962) had previously suggested that tracheal capture was an established fact in the wings of the Heteroptera. The veins, he 129

considered have become more or less fixed, indifferent groups, overlying the lacunae, while the tracheae have tended to wander, filling different lacunae in different groups. Leston reported this kind of evolutionary tracheal captures from the wings of Triatoma infestans (Klug) (Reduviidae), in Acanthosoma haemorrhoidale (L. ) (Pentatomidae) and also, as a developmental process, in Dysdercus superstitiosus (Fabr.) (Pyrrhocoridae). Nevertheless the notation that he proposes for the veins follows that of the included trachea. Among other orders, Holdsworth (1942) appears to have suspected that the radial sector in the forewings of Pteronarcys (Plecoptera) has captured the anterior medial trachea. In the present study, I have noticed some variations in the cubital trachea of a few Oncopeltus wings. In these the cubital trachea appears to have moved out of its lacuna, especially on its approach to the cross vein Cu- a, and tends to ,migrate towards 1A. In two insects the cubital trachea is bifurcate in all four wings, and in such cases the anal trachea is very greatly reduced. In the forewings the posterior branch of the bifurcate Cu enters the cross vein Cu- a and continues in the distal portion of 1A, while in the hind wings the second branch of Cu crosses the furrows present between cubital and anal veins to enter the first anal vein. It is very difficult to visualise possible explanations for these variations. Smart (1958) carried out experiments on Periplaneta americana (Linn.) , which showed that mechanical damage to some areas of the wing pad provoke the development of new tracheal branches; these tend to follow the paths along which the old tracheae passed (i.e. the lacunae) rather than take new paths of their own across the lacunae. In connection with the formation of new trachea, Wigglesworth (1954) has discussed the details of growth and regeneration of the tracheal system in Rhodnius not only in tissues deprived of their original tracheation but also in those subjected to sub-normal oxygen tension. Low oxygen tension resulted in additional tracheation. If some variations in the wing tracheae of 130

Oncopeltus happen to occur through accidental injury to one area (where the branching and shift of trachea has taken place), a similar type of tracheal variation would not take place in all four wings. The branching of the Cubital trachea in all wings, and occupation of the anal vein by a new trachea has occurred in a well organised manner, apparently at normal oxygen tensions. This cubital variation recalls the views of Wootton (1965), who postulates the evolution of the cubital area from a hypotehtical ancestral wing where the cubital has branched into Cu1a and Cuib. It is possible that some individuals may recapitulate the lost characters which were once present in ancestral forms. It is quite clear from previous studies (Tanaka, 1926) that the first branch of Cu (i. e. Cula ) is obsolete in Heteropteran wings and that the second branch, i.e. Cult), is the cubital vein of recent forms. It is quite certain that there is some plasticity in the tracheal system (Smart, 1956; Locke, 1958; Whitten, 1962). The explanation for this must be sought in the development of local areas of higher oxygen requirements (Wigglesworth, 1954). Tracheal captures may well have taken place during the evolution of Heteroptera, and many of the venational homologies are traditionally based on tracheation, with a belief that the position of larval tracheation foreshadows the adult venational pattern. Now, after learning something of the nature and development of the tracheal system in the developing wing, the question is to decide whether to use tracheation as a major criteria in determining venational homologies. In Heteroptera most of the changes (tracheal shifts) have taken place in the membrane (distal half of fore wing) and the venation and tracheation of the proximal half has been left undisturbed. It has been postulated that the terminal branches of wing tracheae are non-homologus, variable structures. However, it is agreed, (Landa, 1948; Henke, 1953; Whitten, 1965) that if no changes occur in the areas to be supplied the tracheae follow a stable, 131

constant developmental pattern. The veins in the adult wings of Oncopeltus are very simple and each contains a tracheae. The tracheation is quite similar in both fore and hind wings and among the wing pads of third, fourth and fifth instars and also among various species of different families throughout the order. In the proximal half of the wing the tracheation is stable enough to provide a guide to the homologies of the respective veins (i. e. there is a concordance between tracheal and lacunar patterns). In order to maintain uniformity, and create less confusion, the veins which form the membrane of the fore wing were named after the trachea which are continuous with those in the corium. The present conclusions on the homologies of Oncopeltus wing venation are, therefore, made with the help of the tracheae and their basal connections, the agreement between tracheal and lacunar patterns, the basal articulation, comparative anatomy and palaeontological evidence (as cited by other authorities). Both pairs of Oncopeltus wings contain six prominent veins here named Sc, R, M, Cu, lA and 2A. The trachea R always gives a small anterior branch near the corio-membranic margin, which is considered as R1 while the branch lying in the membrane is regarded as the radial sector (Rs). The trachea R1 was observed here for the first time; its presence effectively confirms the identification of the radial. Table 2 summarises theories of venational homology in the Heteroptera. I 1

TABLE 2 - THEORIES OF VENATIONAL HOMOLOGY IN THE HETEROPTERON

Comstock lc Slater & Needham Handlirsch Tillyard Tanaka Hoke Hurlbutt Leston Davis Wootton Present Study (1918) (1908) (1925) (1926) (1926) (1957) (1962) (1961) (1977) (Mallela)

Vein F.W. F.W. H.W. F.W. F.W. H.W. H.W. F.W. F.W. F.W. F.W. H.W.

First C C Sc Sc Sc C Sc Sc Sc Sc Sc

M C Sc Rs Sc - Sc ? Sc

Second + Sc+R i R+M Sc+R R+M R+M R+M R+M R+M Third (Compound Vein) Second C Sc R Sc R R Sc R R R R R

M Sc R M1+2 R R R Re Third C Re M It M M R M M M M M

M Re M M3+4 / M M M M

Fourth C M Cu M Cu Cu M1+2 Cu Cu Cu Cu Cu r

M M Cu Cilia Cu Cu Cu Anterior Fifth C Cu IA IA IA IA Cu Vanal 1A PCu+ lA CuA lA IA M Cu IA Cu lb 1A PCu 1 IA 4 J Posterior Sixth C A 2A 2A 2A 2A lA Vane! 2A 2A 2A

M - - - - - 133

(b) Tracheal Shifts and Retraction: Further observations on tracheal shifts and retraction in the distal wing areas are considered here. In the newly emerged adult each vein encloses a straight tubular trachea. On the second day after emergence the wing trachēāe appear rather lā.t and wavy; after 30 hours from emergence they start to move slowly from the tip of their specific veins. And by the third day all the tracheae have been displaced from the veins of the membrane of the forewing and the corresponding distal half of the hind wing. The displaced tracheae and tracheoles coil up and lie between the medial and cubital veins, adjacent to the corio-membranic border. Similar conditions are retained for the rest of the insect's life. Until this study was made, the retraction of trahceae from the distal veins of the wings had not come to the notice of any hemipterists. The absence of the tracheae from the veins of the adult membrane Lygaeid hemelytra had, however, been observed by Tanaka (1926):. and is the reason why he did not name the veins lying in the membrane. A similar distal absence of tracheae was shown by Leston (1962) in the hind wings of Nabis ferus (L.) (Nabidae), and in the forewings of the Mirids Deraeocoris ruber and Lygocoris pabulinus. According to him, in these species the tracheae Sc, R, and M have been dragged out of position to supply a white pigment spot (tracheal capture by an organ), where the tracheae branch and allegedly anastomose. This is not true in Oncopeltus where, though all six tracheae of both pairs of wings have shifted, the hind wings do not possess a pigmented spot nor is there any retraction during the fifth instar to indicate the cause of the changes in the adult wing tracheae. It is very difficult to understand the mechanism and significance of the tracheal retraction seen in Oncopeltus without a much closer study of the subject. It is, however, quite evident from the experiments of Wigglesworth (1954) that in Rhodnius the tracheal system grows during the moulting process by extension of 134

new tracheae and tracheoles from the existing epithelial tubes. Changes in distribution of the tracheae can take place in the absence of any growth since the epidermal cells are responsible for the 'migration' of air filled tracheoles into the regions deprived of a normal tracheal supply. Contractile filaments from the epidermal cells become attached to the tracheoles and draw them back (Wigglesworth, 1959). An ultrastructural study by Wigglesworth (1977) provides further evidence to show how the epidermal cells, deprived of their oxygen supply, give off cytoplasmic processes to attach to the air filled tracheoles in neighbouring areas and draw them into the oxygen-deficient zone. If we assume that the tracheal shifts in the Oncopeltus wings were caused by epidermal cells from a particular area being deprived of oxygen, a further question arises: what might cause a demand for more oxygen? This is not a simple question to answer and it will require further physiological and ultrastructural study to elucidate the cause of tracheal retraction. While studying the circulation in the wings of insects, Clare (1952a) noticed significant movements of the wing tracheae in Euconocephalus extensor, Walk. (Tettigonidae), Megacrania bakeri (Phasmatidae), Tenodera aridifolia, Stoll (Mantidae), and some other Orthopteroids. When the abdomen contracted and forced haemolymph into the anterior wing channels, the large tracheae and tracheal trunks near the base of the wings sometimes collapsed. Tracheae become inflated during abdominal contraction when the thoracic spiracles are closed, but if the spiracles are open the thoracic and wing tracheae are liable to collapse; perhaps depending on the extent of abdominal contractions and the haemolymph pressure in the wing. This tracheal collapse may extend partially into the wings and a "coiling" and resulting "shortening" of the longitudinal tracheae beyond the areas of collapse were also observed. Clare concluded that the tracheae collapsed and retracted during the expansion of some wings in the newly emerging adult and remained in that condition with the aging of the adult. In speaking of types of tracheal tubes, Wigglesworth (1947) 135

says: "In form the tracheae are extremely varied.. Typically they are circular in cross section and prevented from collapsing by their helical folds. Often they are elliptical in cross-section, as in the main tracheae of Dytiscus larvae, or mosquito larvae, and the helical thread then tends to atrophy so that the tubes collapse when the air pressure within them is reduced." Beard (1953) stated that the loss of blood circulation of the wing causes the contained tracheae to collapse and contract. It is also mentioned by Clare (1953) that in some Coleoptera and possibly most of the Heteroptera, there is an incomplete circulation of haemolymph in the wings. Though the principle of inspiration and expiration is the same among various orders of insects, permanent collapse of wing tracheae occurs in only a few orders. It may possibly depend on the structure of the tracheae and the amount of haemolymph present in the particular lacuna surrounding it. (c) Epidermis and Lacunar Differentiation: In the early first instar the epidermis consists of upper and lower layers, corresponding to the dorsal and ventral surfaces of the future wing. The epidermal cells soon increase in size and undergo mitotic division. The divided cells elongate and the inner ends meet those of the opposite surface. During this stage a small intercellular space can be seen towards the anterior region of the wing pad. In the second instar the two layers of epidermis constituting the wing pad have become fused internally and the basement membranes of the apposed layers have come close together and have formed a middle membrane. In the young second instar neither tracheae nor lacunae are to be seen in the wing pad, but a small cavity is present at the base of the wing pad and is assumed to be the beginning of the first-formed lacuna. The epidermal cells undergo mitotic division and increase in number. The divided cells elongate and usually meet the basement membrane. But some of the basal cell-processes, instead of going directly to the basement membrane, merge with the ends of neighbouring cells to leave a 136

space. These small spaces fuse together to form lacunae. The formation of lacunae in the wings of Oncopeltus is quite similar to the same process in Pteronarcys proteus (Holdsworth 1940, 1941). In the first instar a small tracheal branch arises from the anterior side of the wing pad and runs posteriorly between the thorax and the base of the wing pad. In the same way a posterior branch appears in the late pharate second instar. In the second instar, sub-costal, radial and medial tracheae grow from the anterior trachea and enter the previously formed lacunae of the wing pad. The posterior trachea gives a small cubital trachea, and a transverse basal connection is established between the anterior and posterior tracheae. In the third instar there are five prominent lacunae, extending up to the middle of the wing pad and containing blood cells, so indicating the existence of some kind of circulation in the pad. The tracheae at this stage are still very short and confined to a small region at the base of the wing pad. This shows that in Oncopeltus lacunar development precede the entrance of tracheae. The fourth and fifth instar wings contain six lacunae corresponding in arrangement to the main longitudinal veins of the wings. (d) Tracheae and lacuna formation! It is established that the lacunar system corresponds to the venational system. But there are different opinions regarding the developmental relationship of lacuna and trachea. Marshall (1913), studying Platyphylax designatus Walker (Tricheoptera), stated that lacunae develop in the wing pad of the last larval stage and that tracheae do not enter until the wing has everted at pupation. Hundertmark (1935), on the other hand while dealing with the histology of Tenebrio molitor wings, reported that the tracheae grew into the newly everted wing disc and that later lacunae formed about them. Kuntze (1935) in his studies of Philosamia cynthia Drury (Lep.) observed the formation of lacuna earlier than the entrance of tracheae. All the above are 137

Endopterygotes, but Holdsworth (1940, 1941) in his histological sutides of the wing pads of Pteronarcys proteus Newport (Plectoptera) found that the precursors of the veins in the nymphal wing pad are the lacunae, free spaces surrounded by spongy columnar epidermal cells. The tracheae and nerves grow into these channels only after their pattern has been established. The veins are then secreted by epidermal cells through deposition of cuticular material above and below the lacuna at the final adult moult only. This concept of the primary role of blood channels (rather than tracheae) determining the variation was recognised and to some extent supported independently by several authors using the approaches of histology, ontogenetic analysis, comparative morphology and circulatory physiology (Tower, 1903; Holdsworth, 1940; 1941; Smart, 1956; Whitten, 1962; Leston, 1962; Arnold 1964). The same idea is also supported by Palaeontologists (Tillyard, 1926; Martnov, 1925; Carpenter 1971, 1976). Histological study of the thoracic segments of the newly hatched first instar larva of Oncopeltus reveals the presence of minute wing pads in the form of a hollow, flattened outpocketings of the body wall along the lateral margins of the thoracic nota. As described above, lacuna formation in Oncopletus has already occured before they are occupied by tracheae and Holdsworth's conclusions are, therefore, extended to another quite unrelated order of Exopterygotes. Whether the condition found by Hundertmark is a secondary peculiarity of the Coleoptera or whether there is considerable variation in the sequence of lacunar and tracheal development requires further comparative study. (e) Differentiation of cuticle: In the first instar, as seen with the light microscope, the wings are at first covered by a thin transparent cuticle, which stains blue with Mallory's triple stain, except for an outermost thin amber-coloured region. Within a few hours of eclosion, the cuticle between the amber-coloured and inner blue layers stains red. The newly emerged second, third and fourth instars possess an outer 138

amber coloured layer and a thick blue stained inner layer. A few hours after ecdysis the outer half of the cuticle attains a red colour and then slowly turns to black. The newly emerged fifth instar wing pads are already covered by a cuticle with an outer black and an inner blue-staining layer, Applying the nomenclature generally accepted for arthropod cuticles, the outer refractile amber- coloured layer is the epicuticle, the red-staining layer is mesocuticle, the blue layer is endocuticle and the black layer is exocuticle (Schatz, 1952; Lower 1956, 1958). Mesocuticle was originally defined as cuticle which stains red with Mallory's triple stain (Richard, 1951) and is considered by Wigglesworth (1970a) to be untanned exocuticle. All these staining properties show that the cuticle at first covering the first instar larva does not contain tanned exocuticle, which permits further growth of the wing pad before entering the next moulting cycle. The wing pads of the second, third, fourth and fifth instars cannot increase in size without undergoing a moult due to the presence of tanned exocuticle. (f) Apolysis and epidermal hyperplasia: In Oncopeltus the increase in cell numbers and mitotic divisions were followed by the detachment of the cuticles, thus justifying the use here of the term apolysis, introduced by Jenkin & Hinton (1966) to describe the stage in the intermoult/moult sequence when the epidermal cells separate from the old cuticle. Wigglesworth (1973a) has pointed out that apolysis is necessary before the cells can undergo mitosis. Locke (1970), however, found that cell divisions takes place in Calpodes during the period of lamellate cuticle deposition, when the cuticle is firmly attached to the epidermis. Kunkel (1975), too, noted that apolysis in Blatella germanica (L.) occurs after mitoses are no longer seen in the epidermis. Barbier (1971) noted similar instances in the larva and pupa of Galleria. Locke (1979) further confirmed by ultrastructural investigations that epidermal cell division does not require detachment. Thus, although it has been claimed that this is one of 139

the functions of apolysis, the account given for Oncopeltus confirms other evidence that mitosis can precede apolysis. Immediately after apolysis in the wings of Oncopeltus larval instars a foam-like moulting fluid appears between the apical plasma membrane and the old cuticle. Noble-Nesbitt (1963b) also reported that the appearance of a foam-like secretion indicates the onset of the moulting cycle in Podura aquatica (Collembola). (g) Gland cells: Prior to the detachment of the cuticle in each instar of Oncopeltus, some epidermal cells enlarge to form unicellular epidermal glands. They appear to pour their secretions into the exuvial space and then degenerate. Wigglesworth (1933-1934) reported dermal glands in Rhodnius, formed anew at each moult from previously undifferentiated epidermal cells and functional only during the moult. In Oncopeltus no ducts were noted, but sometimes a neck-like process was seen lying towards the exuvial space and apparently acting as an outlet for the secretory products. The liberation of the secretions by these cells and the first appearance of the moulting fluid in the exuvial space seem to occur more or less simultaneously. A few hours after the appearance of the moulting fluid, the number of gland cells decreases. Some glands are even associated with bristle mother cells, but they are not present in the tracheal epithelium. Insect epidermal gland or integ- umental glands are usually each provided with a duct, but there are a few occasions where ductless glands have been reported. Philiptschenko (1907) noted the occurence of unicellular exuvial glands in the epidermis of Collembola, Hoop (1933) in some genera of Diptera, Richards &Korda (1948) in Limnophora. The dermal glands are absentfrom the epidermis of the adult wings of Oncopeltus. (h) Trabecular formation: Regarding the development of trabeculae in the elytra of Tenebrio molitor, and Calandra, Plundertrnark (1935) and Reuter (1937) reported that they develop in the mid pupa, late pupa or 140

pharate adult. In Oncopeltus, however, they arise entirely in the adult wings, presumably because the latter are very much larger than the wing rudiments of the fifth instar larva. (i) Prothoracic paranota: The histological study of the prothorax from the first larval instar reveals the presence of prothoracic expansions. There is a limited growth in these structures till the insect reaches the early fifth larval stage, but later the epidermis degenerates and little trace of a flange remains. During the first instar these rudiments appear as hollow flattened outgrowths of the body wall, comprising cuticle, a few epidermal cells and a basement membrane. In the late second instar the epidermal cells of the two surfaces of the fold are connected by long tail like processes. Such long processes are present during the late pharate stage of every instar and even in the newly moulted fifth instar. But the basement membranes do not develop and lacunae and tracheae never seem to enter these prothoracic expansions. Long tail like processes resembling those of the Oncopeltus prothoracic are seen in the book-lungs of arachnids (Borner 1904) and in gills of Crustacea (Bernecker 1909, Leydig 1878) but it seems likely that these diverse structures have all evolved independently. The tail-like processes may simply be a commonly adopted solution to the causal processes involved in forming a sclerotised bilamellate structure. Para notal structures are reported in adults of some Mantids, Lepismatids, Hemidercus (Hemiptera) and in the nymphs of some Ephemeroptera. All these resemble the prothoracic para notal lobes occuring in some Carboniferous insects from the Palaeodictyoptera and other fossil orders. The tracheation of the prothoracic paranoia of Lepisma saccharina has been shown by Sulc (1927) to resemble that of the meso- and metathoracic paranota, which in turn, have been compared with the tracheation of the mesothoracic wing pad in some Ephemerotera. In other respects, however, the development of the prothoracic paranota has not been studied histologically and the 141 results obtained here for Oncopeltus are the only ones of their kind that are available. PART III

ULTRASTRUCTURAL OBSERVATIONS 142

1. Materials and Methods For the electron microscopic investigations reported here, the material used included the third, fourth and fifth larval instar wing pads. The duration of the various stages has been given earlier while describing the materials and methods used for light microscopy (see p. 54 ). The duration of the larval instars was slightly irregular, owing to relatively small variations in temperature and relative humidity- in the laboratory where the animals were kept. Accurate timing of events within the moulting period was not considered of great importance. Various problems were encountered during the fixation of the material for ultrastructural study. Not only were the wing-pads highly sclerotised, but they had a tendency to float on all the reagents used. It is not always easy to keep the material submerged under the fixatives without exposure of the operator to toxic Os04 or causing undue variation in the temperature of the fixative. Vacuum treat- ment is not suitable for this particular material, as the low pressure above the reagent causes damage to the air-filled tracheae and tracheoles. The following method overcame this difficulty. A plastic disposable hypodermic syringe of 1 ml. capacity had the neddle end closed with dental wax. The plunger was removed and the barrel filled with fixative solution using a pasteur pipette. The material was then introduced, and the syringe barrel placed horizontally on ice. Due to the high interfacial tension, the fixative did not escape and the material remained submerged about half-way along the syringe barrel. After primary fixation, the material can safely be transferred to a narrow specimen tube and will remain submerged if the tube is slightly slanted with the help of a support. The same method can be used up to dehydration. Several fixatives based on Glutaraldehyde and Osmium tetroxide were tried as primary and secondary fixatives, with different combinations of buffers, including Phosphate, Sodium cacodylate, and veronal acetate-. The material seems to require a 143 fixative with rapid powers of penetration. As the wing buds are enclosed in a hard cuticle, the tissues cannot be fixed properly • with a slow-acting reagent. I have obtained satisfactory results by using paraformaldehyde and glutaraldehyde fixative, followed by 1% Osmium tetroxide (Karanovsky, 1965). The Primary fixative is made up with 2 gms Paraformal- dehyde + 25 mgms cac12 heated at 65°c, with 20 mis of distilled water to which 2 drops of N Sodium Hydroxide are added. After cooling to room temperature, 10 mis of 25% Glutaraldehyde are added and the solution made up to 50 mis with 0. 2 M cacodylate buffer at 7.4 PH. Fixation was carried out after storing for an hour at room temperature. This fixative cannot be used when more than 24 hours old. 0.12 M sodium cacodylate buffer is used as a wash buffer. The secondary fixative is made up with equal propor- tions of 5% Osmium tetroxide and 0. 2 M cacodylate buffer. Rapid fixation of tissue was obtained by dissecting off the wing-pads while the whole insect lay under the primary fixative. The material was cut into small pieces by placing it on a sheet of dental wax in a fume cupboard, adding enough fixative to keep the tissue moist, and then cutting the tissue cleanly with a new single edged razor blade. The pieces were then transferred to the syringe containing fixative with the help of a tooth pick. Primary fixation was carried out on ice for 2 hours. In order to remove the aldehydes from the tissue, it was washed twice for half an hour in 0. 12 M cacodylate buffer. From the wash buffer the tissue was transferred to 2. 5% Osmium tetroxide containing 0. 1 M cacodylate buffer and kept on ice for about an hour. It was then rinsed twice for 20 mins in 0. 1 M sodium acetate, and brought to 0. 25 % aqueous Uranyl acetate. After one hour the tissue was taken back to sodium acetate, rinsed, and washed twice, each time for 10 minutes. The tissue may then be dehydrated in graded Ethanol or Acetone. From Sodium acetate it was usually taken to 35% and 50% acetone, for five minutes in each, and gradually changed to 70% 144 Acetone containing 1% uranyl acetate and 1% phosphotungstic acid. It was then left overnight at 4 deg. c after which the tissue can be brought to room temperature. The material from 70% Acetone was transferred to 90% acetone at room temperature for 10 minutes, then dehydrated completely with absolute acetone (3 changes, each for 15 minutes at room temperature). Infiltration with 50% absolute acetone + 50% pure araldite then followed for 6 hours at room temperature on a rotator. Finally the tissue was transferred to pure araldite and left to infiltrate for 24 hours, again on a rotator at room temperature. The tissue was embedded in pure araldite in plastic moulds, which were left in an oven at 60°c for 48 hours to polymerise. The Araldite had been prepared very carefully in the following way, to obtain a suitable hardness'for sectioning strongly sclerotised wings. Araldite mixture - Expoxyresin (CY 212) 50 ml. Hardener (HY 964) - 50 ml. Accelerator (DY 064) 1. 5 ml. Plasticiser (D. B. T.) - 2.5 ml. After the addition of each component the mixture was thoroughly stirred for 5 minutes, and finally for half an hour. This mixture produced very good blocks of moderate hardenss. After polymer- isation the blocks were left at room temperature to complete the curing process. The blocks were sectioned with a cambridge Huxley ultratome using a glass knife after trimming the block face to a 1 mm square pyramid. For light microscopic observation thin sections were cut at i - 1 p.m and stained with 1% toludine blue in 1% borax. Ultrathin sections, of pale gold and silver colour, (90 nrn - 150 nm; 60 um - 90 nm), were obtained by adjusting the ultratome thickness to 0. 075 p.m and speed D. Ultrathin sections floating on 10% acetone were expanded with chloroform and the 145

ribbon was picked up on a copper mesh grid, (G 150, size 3 mm) coated with a formwar film (0. 3 % formwar in chloroform). As the material was stained with uranyl acetate and phosphotungstic acid during dehydration, the ultrathin sections were stained only with lead citrate (Reynolds, 1963), for 7 - 12 minutes, giving a very good contrast, depending upon the age of the tissue. Micrographs were taken at 60 KV, on an AEI EM6B transmission electron microscope. Prints were examined under a low magnification dissection lens, and all measurements made with vernier calipers. 2. Third Larval Instar Wing Pads: In the third instar, the mesothoracic wing pads appear externally for the first time as bag like structures, 0. 5 pm long, on the posterolateral angles of the mesothorax, covering a small portion of the metathorax. Their cuticle is not very strongly sclerotized and their tracheae are very short and confined to a small region at the base of the pad. As the general structure and significant events are essentially the same as in other instars, ultrastructural details of the third instar have been dealt with very briefly and are fully discussed in the fourth instar. Micrographs obtained from 18-hour third-instar wing pads show a cuticle 1. 7 pm thick, of which the outer 0. 8 pm is slightly electron-dense, and the inner 0. 94 pm is electron-lucent. The apical plasma membrane of the epidermis is in close contact with the cuticle and thrown into irregular surface microvilli with dark tips (Fig. 65). The nuclei of the epidermis are irregular in profile, with chromatin masses distributed in the nucleoplasm and arranged peripherally. The cytoplasm contains single free ribosomes, much rough-surfaced endoplasmic reticulum in the form of vesicles and short cisternae, scattered Golgi bodies consisting of dense vesicles, very few mitochondria, scattered microtubules, lysosome-like bodies, multivesicular bodies and electron-dense vesicles (Fig. 65). •

146

Fig. 65. T.S. through 18-hour third-instar wing pad integument. X 20000. cut, cuticle; G, Golgi body; ly, lysosome-like body; m, mitochondria; mt, microtubule; mvb, multivesicular • body; n, nucleus; ver, vesicular rough-surfaced endoplasmic reticulum.

Fig. 66 T.S. of trachea from the base of 18-hour third- instar wing pad. X 17000. bm, basement membrane; cv, coated vesicle; elv, electron- lucent vesicle; ncl, nucleolus; pr, polyribosomes; sd, septate desmosomes; t, trachea. 65

66 148

Fig. 66, from the base of an 18-hour third-instar wing pad, shows a trachea, whose lumen is lined by a dense cuticulin layer, approximately 30 ,nm thick, which is helically folded into prominent taenidia. The cuticulin rests on a less electron-dense cuticle, about 5 nm thick. The taenidial folds are also filled with similar less dense cuticle. Both intra-taenidial and taenidial portions of epicuticle show micropapillate processes, except over the inner surface of the taenidia (Fig. 66). The tracheal epithelium is composed of 2 or 3 epidermal cells in transverse section, arranged in a single layer. Each contains an elongate nucleus with a prominent central nucleous and chromatin masses distributed in the nucleoplasm (Fig. 66).. The intercellular membranes are linked by an apical intermediate junction followed by a continuous zone of septate desmosomes. The cytoplasm contains numerous ribosomes, arranged singly and in clusters, sparse ribosomal endoplasmic reticulum, scattered microtubules, numerous heterogeneous lysosome-like bodies, a very few small scattered Golgi bodies, a few spherical and elongate mitochondria (sometimes aggregated in groups) and electron-lucent vacuoles (Fig. 66 and 67). The tracheal epithelium is bounded by a delicate basement membrane about 50 nm thick. Columns of cells are connected to the trachea (Figs. 67 and 68). The basement membrane lining the trachea is continuous, running over the surface of the columns of cells (Figs. 67 and 68). Wigglesworth (1954) has described, how new tracheae and tracheoles arise by outgrowth of columns of cells from the sides or endings of existing tracheae at the time of moulting. Numerous mitochondria are present where the.columns of cells join the tracheal epithelia. These cells are closely associated with each other by their plasma- membranes, either fused by tight junctions, or separated by a narrow intercellular space, which sometimes encloses "vacuoles" of different sizes (Figs. 67 and 68). Each cell is a columnar structure with an elongate nucleus of irregular profile. The nucleoplasm consists of dense granules; a prominent central •

149

Figs. 67 and 68. T. S. of tracheae and columns of tracheal matrix cells from interior of 18-hour third-instar wing pad. X 26000. bm, basement membrane; t, trachea; tec, tracheal end cell; tep, tracheal epithelia. •

• 151 nucleolus is present, and some chromatin masses are dispersed generally or arranged around the periphery (Figs. 67 and 68). The cytoplasm contains numerous ribisomes, lysosome-like bodies and a few mitochondria; a ribosomal endoplasmic reticulum is rarely found. In 48-hour third-instar wing pads the cuticle has increased in thickness; it now measures 2. 6 11m thick, and still seems to be thickening. The epidermis is in intimate contact with the cuticle and the apical plasma membrane is produced internally into long tubular structures towards the cytoplasmic side (Fig. 69). Some dense vesicles are joined to the bases of these infoldings (tubules). The epidermis is still single layered, but the epidermal cells have increased in size, and the nuclei are rounded up to divide (Figs. 69 and 70). The tall, columnar epidermal cells have conical basal processes (Figs. 70 and 72). Some nuclei which are rounding up possess a prominent nucleolus in the form of thick anastomosing strands (Fig. 69) and some nuclei show large masses of chromatin. The above description shows that in Oncopeltus, mitotic divisions begin before the epidermis has become detached from the cuticle. The cytoplasm contain numerous spiral polyribosomes which are more numerous than in any other instar (Figs. 69 and 70). The cytoplasm also contains circular and elongated profiles of mitochondria, which seem to have increased in number since the early third-instar. Ribosomal endoplasmic reticulum is less abundant than before, and well developed Golgi bodies containing coated vesicles, electron-dense vesicles and electron lucent vacuoles are arranged around the nucleus (Figs. 69 and 70). Similar types of coated vesicles have beenreported by Roth & Porter (1962, 1964) in the mosquito Aedes aegypti, and by Locke (1966, 1969b) in Calpodes (Lep. ). Microtubules, electron- dense vesicles, electron-lucent vacuoles, multivesicular bodies and lysosome-like bodies are also present in the apical and basal cytoplasm (Figs. 69 and 70). Some basal cytoplasm also contains •

152

Fig. 69. of 48-hour third-instar wing pad integument, showing cuticle, apical cytoplasm and nuclei. X 17000. cut, cuticle; dv, dense vesicle; G, Golgi body; ly, lysosome- like body; m, mitochondria; mvb, multivesicular body; n, nucleus; ncl, nucleolus; pi, plasma membrane infolding.

Fig. 70. T. S. of epidermal cells from 48-hour third-instar wing pad showing basal processes. X 13000. mt, microtubules; mvb, multive sicular bo die s; pr, polyribosomes. • 154 beta-glycogen particles. The basal cytoplasm of most of the cells is produced into long processes to meet the basement membrane (Fig. 71). A prominent middle membrane, about 0. 3 p.m thick, is present between the two epidermal surfaces of the third-instar wing pad (Fig. 71). Such a continuous, thick middle membrane is not found in any later instars. The basal cytoplasm of the epidermal cells from the tip of the wing pad is produced into several long, thin proce s se ss which entangle in all directions with similar processes from other cells (Fig. 72) as described by Wigglesworth (1954, 1959, 1977) in Rhodnius prolixus. These processes contain polyribosomes, microtubules, mitochondria. and some ribosomal endoplasmic reticular cisternae. In some places haemocytes are found between the two epidermal layers of the wing pad (Fig. 73). They contain a small nucleus and huge amounts of cytoplasm with spiral polyribosomes, agranular endoplasmic reticulum, elongate mitochodrial profiles, lipid droplets, a very few cisternae of granular endoplasmic reticulum, small Golgi bodies, multivesicular bodies, electron- lucent vacuoles, microtubules and isolation bodies (Fig. 73). Lacunae are very prominent in the third-instar wing pads (Fig. 74), and are lined by a basement membrane 0. 05 FZm thick. Micrographs from the tip of the wing pad show empty lacunae with neither a trachea nor a nerve. Some tracheoles are found close to the basal processes of the ventral epidermis, lying adjacent to the lacuna (Fig. 74). The formation of lacunae, prior to the entrance of trachea has been observed under the light microscope. Marshall (1913), Kuntze (1935), and Holdsworth (1940, 1942) have reported the formation of lacuna before the appearance of trachea in the wing pads. The tracheoles were included within the main body of the tracheoblast or in the prolonged cytoplasmic extensions (Fig. 74). The tracheolar intima is separated from the tracheoblast cytoplasm by a thin limiting membrane (Fig. 74). The tracheolar intima is helically folded into taenidia, and the 155

Fig. 71. T.S. of 48-hour third-instar wing pad showing well defined middle membrane between the two epidermal layers. X 17000. g, glycogen; ler, lamellae of rough- surfaced endoplasmic reticulum; m, mitochondria; mm, middle membrane; n, nucleus.

Fig. 72. 48-hour third-instar wing tip showing cytoplasmic processes from several epidermal cells. X 17000. cp, cytoplasmic process; G, Golgi body. •

•

w.1115,.

11" "cc. 1.2i 72 157 inner walls of the tracheole are smooth, without any micropapilla. The internal diameter of the tracheolar tube is about 0. 7 µm (Fig. 74). The cytoplasmic sheath around the tracheole is provided with more organelles than at any other stage: single, free and clustered ribosomes, small mitochondria, microtubules, a few cisternae of ribosomal endoplasmic reticulum, electron-dense vesicles and electron-lucent vacuoles (Fig. 74). All the tracheoles lie very close to the tracheolated basal cytoplasm of the epidermal cells, their plasma membranes separated by a uniformly narrow intercellular space (Fig. 74). These tracheolated basal regions of the epidermal cells are usually joined to the basement membrane of the lacuna (Fig. 74). Their cytoplasm contains numerous polyribosomes, circular and elongated mitochondrial profiles, well developed Golgi bodies with electron-dense vesicles and electron- lucent vacuoles, a very few cisternae of ribosomal endoplasmic reticulum, some regions filled with alpha-glycogen particles, a few lysosome-like bodies, scattered microtubules, electron-dense vesicles and electron-lucent vacuoles (Fig. 74). Tracheae were examined from the base of the wing pad. The intima of the tracheal tube is now detached from the epidermis. The epidermis contains very large nuclei. There is no evident nucleolus; the chromatin is partly dispersed in the nucleoplasm, but some masses are arranged around the periphery. The intercellular membranes are joined by intermediate junctions and septate desmo some s. The cytoplasm contains numerous spiral polyribosomes, mitochondria, lysosome-like bodies, small scattered Golgi bodies, a few short cisternae of ribisomal endoplasmic reticulum, some alpha-glycogen particles and electron-dense vesicles. Tracheae are bounded externally by a basement membrane and lying adjacent to the tracheal matrix cells there are sometimes large lipid droplets. The epidermis of the third-instar wing pads becomes detached from the cuticle between 50 and 90 hours after the 158

Fig. 73. T.S. of a haemocyte from 48-hour third-instar wing pad. X 17000. elv, electron-lucent vacuole; ep, epidermis; ld, lipid droplet; m, mitochondria; mvb, multivesicular body; n, nucleus.

Fig. 74. T.S. from a part of epidermis, tracheole and empty lacuna from 48-hour third-instar wing pad. X 26000. bm, basement membrane; dv, dense vesicle; er, endoplasmic reticulum; g, glycogen; lac, lacuna; mt, microtubule; pr, polyribosomes; tl, tracheole. 73

lacc

74 160 previous ecdysis. Micrographs obtained from a 96-hour third-instar wing pad show a folded Pharate fourth-instar pad lying inside the old cuticle. A continuous trilaminate cuticle has already formed over the dark tips of the pharate epidermal microvilli (Locke, 1966). The cytoplasm of the epidermis now contains numerous single, free and clustered ribosomes, lysosome-like bodies, well developed Golgi bodies containing dense-vesicles, microtubules, electron- dense vesicles and electron-lucent vacuoles. The exuvial space between the old tracheal intima and its epithelium is well defined and filled with electron-dense, fibrous material (Fig. 75). Similar fibrous material has been described in the newly developing tracheae and tracheoles by Locke (1966, 1969), and homologized by Whitten (1969) with the ecdysial membrane of normal integument. Patches of cuticulin are present over the raised portions of the apical plasma membrane (Fig. 75). In fourth- and fifth-instar wing pads the apical plasma membrane of tracheal matrix cells is thrown into long stacks of microvilli during this period. Locke (1966) found that in the small tracheae and tracheoles the cuticulin arises in small patches directly above the non-villate plasma membrane or sometimes the plasma membrane is slightly raised, and more dense, where the cuticulin is presumed to be about to appear. The cytoplasm contains single, free and clustered ribosomes, short cisternae of ribosomal endoplasmic reticulum, several mitochondria, some mitochondria apparently forming isolation bodies, numerous Golgi bodies containing electron-dense vesicles and electron-lucent vacuoles, a few lysosome-like bodies and electron-dense vesicles (Fig. 75). Micrographs from 120-hour third-instar wing pads also show well-developed pharate fourth-instar wing pads inside the old cuticle. The Pharate fourth-instar wing pad is now lined by a fully- formed cuticle about 0. 55 p.m thick (Fig. 76). The apical plasma membrane is provided with long tubular internal folds. The apical cytoplasm contains numerous large forms of coated vesicles, 161

Fig. 75. T. S. of tracheal epithelia and detached old cuticle (tracheal intima) from 96-hour third-instar wing pad; patches of cuticulin are present over the dense raised portions of the apical plasma membrane. X 17000. cut, cuticle; er, endoplasmic reticulum; G, Golgi body; in, mitochondria; n, nucleus; t, trachea; tl, tracheole.

Fig. 76. T.S. of portion of 120-hour third-instar wing pad showing fully formed new cuticle of pharate fourth-instar. Coated vesicles are present in the apical cytoplasm. X 26000. cv, coated vesicle; mt, microtubule; mvb, multivesicular body. •

•

• • tvl•.! , - 1

.•f4-t • • * •

76 •

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electron-dense vesicles, multivesicular bodies, spiral polyribosomes, circular and elongate mitochondrial profiles, vesicular and short cisternae of ribosomal endoplasmic reticulum, Golgi bodies, microtubules and heterogeneous lysosome-like bodies (Fig. 76). The ecdysial membrane is not found in trachea from 120 hour third-instar wing pads, and, the tracheae are lined by a continuous

• dense cuticular layer, folded into prominent taenidia.. Micropapillae are present on the inner surface of the cuticulin.. The apical plasmamembrane first accompanies the cuticulin as it buckles to form taenidia (Locke 1966). When the taenidial pattern is fully established the plasma membrane retracts towards its original form. The tracheal epithelium contain very large nuclei. The cytoplasm contain ribosomes, short cisternae of ribosomal endoplasmic reticulum, Golgi bodies containing much more numerous dense- vesicles and electron-lucent vacuoles, a few mitochondria of various sizes, microtubules and few lysosome-like bodies. 3. Fourth larval instar wing pads The externally visible wing pads of fourth-instar larvae measure about 1 mm long, and have a fully developed tracheal system. Wing pads from larvae of various ages within the fourth- instar were fixed and ultrathin transverse sections were photographed and examined for various structures during the moult and intermoult stages. (a) Cuticle The newly moulted fourth-instar larval wing pads are covered externally on both sides by a soft, pale, transparent cuticle, varying from 1 to 2. 5 pm in thickness. With Mallory's connective tissue stain, the outer three-fifths of the thickness stains red, and the inner two-fifths appears blue in colour. The micrographs of newly moulted fourth-instar wing pads show a cuticle thickness of 1.1 p.m - 1.2 p.m. The outer third of its thickness is electron dense, and especially under high magnification (Fig. 78), it reveals a closely packed lamellate character; with alternating layers of more •

164

Fig. 77. T.S. of a newly moulted fourth-instar wing pad integument, showing unilaminar arrangement of epidermal cells. X 13000. • cut, cuticle; ic, intercellular junction; ij, intermediate junction; m, mitochondria; mt, microtubule; n, nucleus; ncb, nucleolus; pi, plasma membrane infolding.

Fig. 78. T.S. of cuticle and apical epidermis from a newly moulted fourth-instar wing pad, showing the fusion of dense vesicles with plasma membrane infoldings. X 25000. dv, dense vesicle; G, Golgi body; ly, lysosome-like body; sv, secretory vesicle. •

•

78 166

Fig. 79. T. S. of epidermis from a newly moulted fourth-instar wing pad to show electron-dense and light cells. Lipid droplets are well preserved; fusion between coated vesicles is also shown. X 22000. dep, dense epidermal cell; G, Golgi body; ld, lipid droplet.

Fig. 80. T. S. of a basal process from a newly moulted fourth- instar. X 32000. er, endoplasmic reticulum; m, mitochondria; mt, microtubules. 79

80 168 and less electron-dense material. The remaining inner two- thirds of the cuticle is mainly electron-lucent with widely spaced darker laminae. The outermost layer that is clearly visible in transverse sections of fully formed intermoult cuticle is cuticulin (Locke 1958, 1960, 1961; cf. Wigglesworth, 1933). It is an uniformly electron- dense layer, about 34 nm thick. The inner epicuticle with close lamellations is 0.3 pm thick (Fig. 77 and 78). It is laminate when first formed and homogeneous later, as also reported in Elateridae (Coleoptera) by Zacharuk (1972). The laminae are parallel to the surface of the cuticle, alternating with less dense interlaminar regions. Within the epicuticle lies an electron-lucent lamellate procuticle, consisting of alternating thin electron-dense laminae and thicker homogeneous electron-lucent zones. Only four electron-dense laminae have been secreted in newly moulted fourth-instar larvae. The procuticle is then about 0. 7 - 0. 8 pm thick. Successive laminae are separated by intervening less dense zones which are 0. 17 p.m - 0. 18 p.m thick. The innermost layer of cuticle lying close to the epidermis is slightly more electron dense, about 0. 14 p.m thick, and probably represents the sub-cuticle, reported in different orders (Schmidt 1956; Locke, 1961; Taylor & Richards, 1965; Rinterknecht & Levi, 1966; Delachambre, 1967; Filshie (1970b). The micrograph measurements, showing the thickness of epicuticle and pro-cuticle disagree with those recorded in the light-microscopical observations. The explanation may possibly be the same as, Zacharuk, (1972) put forward in his observations on Elaterid cuticle: "The first few sub-epicuticular lamillae, appear to be transitional in nature between the dense layer of epicuticle and fibrillar lamillae of procuticle. This is believed to be a region of transformation, deposited at the time when epidermal cells are changing from epicuticular to procuticular secretion and is perhaps sclerotized at a later stage. This transitional zone between epicuticle and procuticle is as thick as the •

169

Fig. 81. T.S. of dorsal integument from 24-hour fourth- instar wing pad. Perinuclear Golgi bodies, long tubules of rough- surfaced endoplasmic reticulum and numerous alpha-glycogen deposits in the basal cytoplasm are shown. X 17000. cut, cuticle; G, Golgi body; g, glycogen; ler, lamellae of rough-surfaced endoplasmic reticulum; m, mitochondria; n, nucleus.

Fig. 82. 24-hour fourth-instar wing pad epidermis showing several thin cytoplasmic filaments and glycogen deposits in the basal cytoplasm. X 15000. cp, cytoplasmic process; icsp, intercellular space. Z8

18 171 dense layer of epicuticle and because `these two layers were not differentiated by the stains used in light-microscopic preparations, the transitional zone could easily be interpreted as part of dense epicuticle layer; perhaps it should be regarded as an inner epicuticle. On the ultrastructural level it appears to be permeated during the sclerotization process after ecdysis by a homogeneous material similar to that of dense layer of epicuticle. " The plasma membrane of the epidermal cells is in intimate contact with the cuticle, and appears folded locally into coarse tubules, with adjacent vesicle-like structures (Figs. 77, and 78). These are interpreted as profiles of secretory material derived from the Golgi bodies and in the process of transfer to the apex of the cell, fusing with the plasma membrane, and thus contributing to the cuticular material (Fig. 78). The micrographs taken from an intermoult fourth-instar larva, 24-hours after ecdysis, show a cuticle, 1. 3 pm thick, from the ventral integument, and a cuticle 2. 5 pm thick from the Dorsal surface (Fig. 81). The thickness of the cuticle varies from one area to another, but the dorsal side of the wing-pad is always thicker than the ventral side. There is a clear demarcation between the outer and inner epicuticles. The dense outer epicuticle is about 40 nm thick. The dorsal cuticle of the wing has an inner epicuticle, 0. 93 pm thick, and a procuticle with increased lamellations measuring 1.45 pm thick. The ventral integument has an inner epicuticle of 0. 17 pm thick, and the procuticle is 1 pm thick. The sub-cuticle is not very prominent. The infoldings of the adjacent epidermal plasma membrane are somewhat less numerous. By 48 hours after the previous ecdysis, some areas of the fourth-instar wing pad have begun to enter the apolytic phase, though this is not complete everywhere until about 100 hours. The apical plasma membrane is slightly loosened from the cuticle and produces prominent slender microvilli (Figs. 83 and 84). Their tips appear more electron-dense due to the concentration there of •

172

Fig. 83. T. S. of a 48-hour fourth-instar wing pad integument, showing detachment of epidermis. X 13000. elv, electron-lucent vacuole; G, Golgi body; ly, lysosome- • like body; pr, poly ribosomes.

Fig. 84. A micrograph from section of a 48-hour fourth-instar wing pad integument, showing dense tipped microvilli of apical plasma membrane and swollen vesicles of rough-surfaced endoplasmic reticulum. X 26000. cut, cuticle; er, endoplasmic reticulum; my, microvillus; mvb, multivesicular body. r

84 174 ribosomes. The areas from which I have taken micrographs do not show an exuvial space. At this time the cuticle has attained a thickness of 2. 7 - 3 p.m, the electron-dense epicuticle being 0. 82 pm thick, and the endocuticle, which is heavily lamellated, with approximately 10 laminae, 2. 28 pm thick. In the micrographs taken 2 days after apolysis has begun, that is when the fourth-instar larva is 96-hours old, the epidermis has completely withdrawn from the cuticle, and an exuvial space has developed between it and the old cuticle. The exuvial space is filled with a foam like moulting fluid, and vesicles of different shapes and sizes (Fig. 85). Some of these vesicles are enriched with ribosomes (Fig. 85). The vesicles are formed as out-pushings of the plasma membrane, which are then pinched off, leaving the vesicles bounded by membrane and free from the cell surface (Fig. 85) (cf. Noble-Nesbitt, 1963a). Several clusters of electron- dense granules, giving a foam like appearance under the light microscope, are present near the cuticle, and seem to be digesting it (Fig. 85). In some sections the apical plasma membranes of the epidermal cells were produced into finger-like microvilli as long as 0. 75 pm, projecting into the exuvial space. Some of the epidermal cells have started to secrete cuticular-like domes on the dark tips of the microvilli (Fig. 86). Further details of the exuvial space and moulting fluid of the wing pads, are not available for later stages, as the pharate fifth-instar wing pads, separate readily from the old cuticle of 120-hour fourth-instar larvae during fixation. The micrographs of the wing pads of this pharate instar, show further advances in the secretion of cuticulin over the tips of uniformly spaced microvilli to form a continuous layer, though a few gaps still remain to be filled (Fig. 87). A micrograph from another specimen of the same age shows regularly spaced microvilli with dark tips, but the cuticulin has not been secreted over them. From sections of 140-hour fourth-instar larvae, the pharate fifth-instar wing pads show a new cuticle about 0. 7 pm •

175

Fig. 85. 96-hour fourth-instar wing pad integument, showing well developed exuvial space, is filled with moulting fluid. • X 17000. dsv, electron-dense secretory vesicle; exsp, exuvial space; G, Golgi body; mfl, moulting fluid.

Fig. 86. 96-hour fourth-instar wing pad: apical epidermis, with patches of cuticulin over dense areas of microvilli. X 26000. cut, cuticle; m, mitochondria; pm, plasma membrane. a

86 •

177

Fig. 87. 120-hour fourth-instar wing pad; apical portion of epidermis with cuticulin over dense tips of microvilli. Large vacuoles are present between plasma membranes. • X 26000. cut, cuticle; my, microvilli; v, vacuole.

Fig. 88. T.S. of portion of pharate fifth-instar wing pad integument, a few hours before ecdysis. X 35000. G, Golgi body; m, mitochondria; rer, rough-surfaced endoplasmic reticulum. 87

88 •

179

thick, over the epidermal layer (Fig. 88). The apical cytoplasm close to the cuticle contains numerous free ribosomes and small vesicles resembling those associated with the Golgi bodies. The plasma membrane, which is in close contact with the cuticle, exhibits small infoldings into the apical cytoplasm; some times 2 small infoldings will meet to isolate an apical portion of • cytoplasm, that may contribute to the cuticle (Fig. 88). Micrographs of the fourth-instar larval wing pad, just before ecdysis show cuticles from the dorsal integument, and from the folded and unfolded ventral integuments, 1. 9 pm, 0. 67 pm and 0. 25 Fm thick respectively (Fig. 108). (b) Epidermis The newly moulted fourth-instar larval wing pad consists of a single layer of epidermal cells from the dorsal and pleural sides of the thorax, surrounding a system of haemolymph- filled lacunae. These epidermal cells are tall columnar structures, with broad apical an&conical basal ends (Figs. 77 and 80). These details were the same in both light- and electron-microscopical observations. Typical cells measure about 7. 6 jm long and 5. 3 dam broad (Fig. 77). Each epidermal cell possesses a very large nucleus, with a characteristic shape. Some nuclei are elongate structures with a hemispherical basal end, while others are lobed towards the apical side (Fig. 77). The nucleus is situated basally in some cells, and in others, it lies near the apical surface. A prominent nucleolus is present, and is sharply demarcated from the surrounding nuceloplasm (Fig. 77). Condensed chromatin is distributed around the periphery in small clumps separated by short intervals (Figs. 77 and 78) and further small clumps of chromatin are widely scattered throughout the nucleoplasm. The nucleus is enclosed by an envelope consisting of two clearly visible membranes separated from each other by a space 22-25 nm wide. Each membrane measures approximately 7. 5 urn across (Figs. 78 and 79). These 180 perinuclear cisternae are perforated by pores of about 45 nm diameter (Figs. 78 and 79). The outermost membrane of the nuclear envelope is studded externally with ribosomes. The plasma membrane bounding the apical side of the cell is in intimate contact with the cuticle. In newly moulted, fourth-instar wing pads (Figs. 78 and 79), it has folded towards the cytoplasm to form inwardly directed tubules with associated vesicles,. The plasmamembrane of the dark (relatively electron-dense) cell shown in Fig. 79, possesses more of these structures than does that of an adjacent light cell. The electron opacity of the cytoplasm varies from cell to cell depending partly, if not entirely, upon the density of the ribosomes. Vesicles similar to those adjoing the plasma membrane are abundant in and near the Golgi bodies and are scattered in the apical cytoplasm (Figs. 78 and 79). Some apparently coalesce into larger vesicles in the apical cytoplasm and towards the base of the microvilli (Figs. 78 and 79). Their contents seem to be released into the tubular infoldings to add further cuticular substance (Lock, 1969b). The cytoplasm contains many single free ribosomes, and spiral polyribosomes. Rough-surfaced endoplasmic reticulum is organised into several vesicles, some of which join together to form a few small lamellae. Mitochondria with circular profiles are more numerous in the apical cytoplasm, though a few elongate ones are found among them. The mitochondria are of the usual cristate type. Small, scattered Golgi bodies with many electron dense vesicles and fewer electron-lucent vacuoles occur in the apical cytoplasm (Figs. 78 and 79). In the apical cytoplasm several large membrane-bounded vesicles, about 0. 25 p.m in diameter, and resembling lysosomes are present. In addition to the above organelles there may occur several scattered bundles of microtubules (Figs. 77, 78 and 80); scattered glycogen particles in the form of alpha-particles or rosettes; and a few multivesicular bodies containing variable numbers of microvesicles embedded in a low-density fibrous matrix. Some areas with lipid droplets (0. 12 - 1. 6 pm in diameter) were also seen in the apical cytoplasm (Fig. 79). 181

The cell boundaries are very clear between the apical two-thirds of the cells. The membranes of the adjacent cells are parallel and separated for much of their length by an intercellular space 16 - 17 nm wide (Fig. 79). A faintly visible intermediate junction (Macula adhaerens) lies just below the apical boundary. Proximal to this the cell boundaries show simply apposed plasma membranes and occasional zones with septate desmosomes (Palade, 1963, 1965; Locke, 1969; Greenstein, 1972a). In some cells the basal cytoplasm is produced into a small process containing ribosomes and vesicular rough endoplasmic reticulum; other cells are each produced into a long process, supported by longitudinally directed microtubule bundles (Fig. 80). Between the microtubles several polyribisome s, and a few short lamellae of rough endoplasmic reticulum are aggregated. The basement membrane has been observed, only around the lacunae of the wing-pads. The basal surfaces of the epidermal cells of the remaining area do not seem to be bounded by any specialised structure. They remain free inside the wing-pad (Figs. 77 and 80), until the haemocytes secrete the basal lamina (Wigglesworth, 1956; 1973). Between the conical basal processes of the cells of the intermoult wing pads there is present a tremendous volume of intercellular space, as shown in both my light-microscopical and ultrastructural observations (p. 84). In 24-hour old fourth-instar larvae the pre-apolysis wing pads show a single thin layer of epidermal cells in each integument, which appear more columnar than they were a few hours previously (Figs. 81 and 82)..The plasma membrane lying close to the cuticle still possesses inwardly directed folds but they have decreased in number. The nucleus lies in the centre of the cell, which is an elongate structure with an irregular outline. There is no evident nucleolus; the chromatin is condensed into clumps in the nuceloplasm and adjacent to the nuclear envelope (Figs. 81 and 82). The cytoplasm possesses abundant, single free ribosomes a

182

and polyribosomes, organised into spirals. The granular endoplasmic reticulum is in the form of small vesicles and short lamellae. A few stacks of elongate ribosomal endoplasmic reticulum are present, lying towards the sides of the cell (Fig. 81). The Golgi bodies are now arranged around the nucleus, and are larger, being formed of abundant dense secretory vesicles and electron-transparent vacuoles. The vesicles are more electron-dense than at an earlier stage (Fig. 81). Some of the vesicles enclosing dense secretions are visible in Fig. 81. Mitochondria with circular and elongate profiles (up to 2.4 p.m long) were noticed in the cells. The basal cytoplasm of many cells contains electron-lucent areas filled with glycogen alpha- particles. Each alpha-particle or rosette consists of a mass of closely packed beta-particles, measuring 60 nm - 65 nm in diameter. Each glycogen containing area contains about a hundred such alpha particles in any one micrograph. The apical cytoplasm also contains bundles of microtubules, electron-lucent vacuoles, heterogeneous lysosome-like bodies, and large electron-dense granules of various sizes. (0. 18 p.m - 0. 38 Tim in diameter); see Fig. 81. The plasma membranes of adjoining cells are very prominent. The intermediate junction lying below the apical border is very clearly visible as a button-like structure consisting of two dense plaques on the apposed cell surfaces; proximal to this the cell junctions show simply apposed plasma membranes and occasional zones with septate desmo- somen as described above, though here the apposed plasma membranes are wider apart than before, leaving a larger intercellular space. This appears to be associated with the onset of cell division in the developing wing pad. The basal cytoplasm of the cell has now altered its form. Some of the cells are produced into delicate processes, containing one or two mitochondria, and a few short lamellae of rough surfaced endoplasmic reticulum, other cells have only a short conical basal ending, that contains a large zone of glycogen granules and ribosomes (Fig. 81). Still further cells possess a stout basal process filled with microtubules and endoplasmic •

183

reticulum. Cytoplasmic processes from the lateral regions of the cells are now formed and entangle in all directions with similar processes from other cells, so that the entire intercellular space thus attains the condition of a spongy network (Wiggle sworth 1977, 1953; and see Fig. 82). The intercellular space contains some huge, highly vacuolate structures enclosed by a fibrillar

• membrane (Figs. 81 and 82). Some of these are empty, but others are filled with ribosome-like bodies (Fig. 81). They may represent degenerate or degenerating haemocytes or coagulated extracellular exudate. The basal portions of the epithelial cell processes rest on a basement membrane, made up of poorly defined filamentous material. Towards the epidermal side, parallel to the basement membrane, small membrane bound vacuoles containing coated vesicles and polyribosomes are present. They are probably of the same nature as the above mentioned vacuolar structures. In 48-hour old fourth-instar larva, (early phase of apolysis), the epidermis has thickened to a greater extent and the wing-pad does not contain large intercellular spaces between the basal areas of the epidermal cells (Fig. 93). In places the epidermis seems to have a double layer of cells, though elsewhere it is still a single layer. The electron microscopic evidence of the course of the plasma membranes shows that the epidermis is still essentially composed of a single layer of cells, but their nuclei are arranged at two more or less distinct levels (Fig. 83). The epidermal cells exhibit a variety of shapes. The different shapes, sizes and increased number of epidermal cells in the 48-hour fourth-instar wing pads appear to reflect the increased mitotic division occuring before the onset of apolysis (i. e. about 30 hours after ecdysis). At this time the plasma membrane of the epidermal cells is still in intimate contact with the base of the old cuticle. Electron micrographs show, however, that it is thrown into a

184

numerous tubular infoldings, with electron dense material near their sites of invagination (Figs. 84 and 91). These dense areas are very close to, but distinct from, the lower-most lamella of the cuticle. The manner of infolding of the plasma membrane is the reverse of that found in the formation of micro-villi in moulting Lepidoptera (Willis, 1966; Greenstein, 1972a). In Fig. 83, the plasma membrane adjoining the cuticle forms patches of long microvillate folds, and a has started to withdraw from the old cuticle. The nuclei of the epidermal cells are also variously shaped, mainly with lobed or smooth profiles. Most of the columnar (conical) cells possess an elongate nucleus whose long axis is perpendicular to the cuticular surface, and which is centrally disposed within the cell (Fig. 93). Some micrographs (Figs. 89 and 91), show intermediate stages of nuclear transformation from elongate-oval to more spherical shapes. They contain condensed masses of chromatin (Fig. 89). The nucleus is oblong with a lobed apical side; it possesses two prominent nucleoli and uniformly distributed chromatin masses. An epidermal cell adjacent to the nerve, shown in Fig. 90, has a nucleus with six lobes, and contains a prominent nucleolus. The variously shaped nuclei with regular or irregular profiles and the presence or absence of nucleoli perhaps indicates different stages of synthelic activity in the cells, specially in relation to nucleic acid metabolism (see discussion, p. 370). The cytoplasmic content varies from one cell to other, even in adjacent cells. A micrograph (Fig. 83) from epidermis in the process of detachment, shows a cell with cytoplasm containing abundant polyribosomes, a few small tubular cisternae of granular endoplasmic reticulum, a few scattered mitochondria with circular and oval profiles, numerous lysosome-like bodies and a few electron-lucent vacuoles. During apolysis there is an increase in the number of Golgi bodies. Each of these contains several moderately dense vesicles and some vesicles of smooth surfaced endoplasmic reticulum and encloses very dense secretions located 185

Fig. 89. T.S. of wing pad epidermal cells from 48-hour fourth- instar wing pad, showing apically lobed oblong. nucleus and bundles of microtubules. X 13000. • G, Golgi body; g, glycogen; ler, lamellae of ribosomal endoplasmic reticulum; m, mitochondria; mt, microtubules; n, nucleus.

Fig. 90. Epidermal cell from a 48-hour fourth-instar wing pad, showing lobed nucleus, with a nerve lying towards one side. X 17000. bm, basement membrane; lac, lacuna.

• • 187 between the nucleus and the cuticle. While the epidermis is becoming detached from the cuticle there is a small increase in the extent of the lateral plasma-membranes. Some columnar cells (Fig. 84) show an apical cytoplasm with abundant mitochondria, numerous polyribosomes, small tubules of lamellate rough-surfaced endoplasmic reticulum and a very few small Golgi bodies. Stacks of cisternae of ribosome-covered endoplasmic reticulum occur in the basal cytoplasm, and sometimes also around the nucleus (Fig. 93). Between these cells the plasma membrane has not yet increased in complexity as mentioned above. Lysosome-like bodies are very few. The cell shown in Fig. 84, has an oval nucleus and contains apical cytoplasm with abundant vesicles of granular endoplasmic reticulum with a diameter of 0. 06 to 0. 3 pm; polyribosomes and mitochondria with circular profiles are also present, together with a few lysosome- like bodies and electron-dense bodies. In the basal cytoplasm polyribosomes are present and rough-surfaced endoplasmic reticulum in the form of vesicles and small lamellae with distended ends; mitochondria with circular profiles can also be seen, and numerous well developed Golgi bodies are present in the basal and apical cytoplasm. Some form flat sacculi lying parallel to each other and are associated with a few vesicles of agranular endoplasmic reticulum, others are provided with dense vesicles. Vacuoles with dense secretions can also be seen in Figs. 90 and 92. All cell boundaries are very clear, with an apically situated adhesion zone followed by simply opposed plasma membranes connected by electron-opaque septa bridging the intercellular space. Some of the epidermal cells contain a very stout conical basal ending (Fig. 91). These are irregular in arrangement, and emit several cytoplasmic processes from all sides. Such processes entangle in all directions with similar processes from other cells (Figs. 91 and 93), and even with the tracheal epithelia. The basal cytoplasm of such cells contains polyribosomes, free ribosomes, numerous mitochondria and granular endoplasmic reticulum in the •

188

Fig. 91. Epidermal cell from 48-hour fourth-instar wing pad. Basal portion of the cell is produced into a stout process with several thin processes given off from it. X 13000. S cp, cytoplasmic process; dsv, electron dense secretory vesicle; g, glycogen; ler, lamellae of rough-surfaced endoplasmic reticulum; m, mitochondria; n, nucleus.

Fig. 92. Basal process of an epidermal cell from 48-hour fourth-instar wing pad; rosettes of glycogen granules occupy a major portion. X 22000. G, Golgi body; ly, lysosome-like body; n, nucleus. •

92 190 form of vesicles and short lamellae (Fig. 91); some of these lamillae are swollen. Dense bodies and lysosome-like bodies are present in the basal and lateral cytoplasms. The cytoplasm of the cell processes contains several polyribosomes and a few small lamellae of rough-surfaced endoplasmic reticulum. The apical region of the cell contains polyribosomes, a few mitochondria and several tubules of rough-surfaced endoplasmic reticulum. Wigglesworth (1977) says that in epidermal cells deprived of oxygen, the cytoplasmic contents tend to be concentrated in the basal region and extend into the conical process which grow out to form a strand running in the direction of air-filled trachea. My observations on the cells producing processes during the moulting period of the wing may suggest a response by dividing cells to a possible oxygen deficiency. Some of the cells possess bundles of microtubules along their lateral surface regions (Fig. 89). Glycogen is present in the basal part of some cells, in the form of rosettes (Figs. 89 and 92). Empty profiles of extracellular membranes, noticed in the previous stage (p. 169 ), are still present in the intercellular space. They are never found free inside the wing bud but are always entangled with some part of the cell or its processes. The basal lamina is well developed, and measures 0. 1 )im thick adjacent to the lacuna (Fig. 90). In the 72 hours old fourth-instar larva, i.e., on the second day after apolysis has started, the epidermal cells of the wing pad are still undergoing division. The apical plasma membrane is now strongly microvillate, and most of the epidermis has become detached from the cuticle. The newly divided cells are much smaller than before and contain nuclei with irregular profiles (Fig. 95). The longitudinal axis of the nucleus is parallel, not perpendicular, to the apical and basal ends of the cell (Fig. 94). It contains a large nucleolus and peripherally distributed chromatin. The cytoplasm possesses small stacks of granular endoplasmic reticulum; profiles of mitochondria and numerous clusters of •

191

Fig. 93. Several cytoplasmic processes containing long tubules of rough-surfaced endoplasmic reticulum, shown in a micrograph taken from 48-hour wing epidermis. X 13000. cp, cytoplasmic process; er, endoplasmic reticulum.

Fig. 94. Nucleus and stout basal process of an epidermal cell of a 72-hour fourth-instar wing pad. X 17000. G, Golgi body; ler, lamellae of rough-surfaced endoplasmic reticulum; n, nucleus; ncl, nucleolus. a

94 0 •

193

ribosomes. The Golgi bodies are small and less numerous than at the previous stage. They contain sacculi of smooth-surfaced endoplasmic reticulum and moderately electron-dense vesicles. Similar vesicles are also found independently in the apical cytoplasm. Membrane-bounded lysosome-like bodies, dense bodies of various sizes, and vacuoles are present in the apical cytoplasm. In some dividing cells, the chromatin is in the form of anastamosing strands (Fig. 95). The perinuclear cytoplasm is very sparse; it includes single free ribosomes, vesicles and small lamillae of rough-surfaced endoplasmic reticulum, swollen locally, and a very few mitochondria (Fig. 95). The nuclear envelope appears to be reconstituted by the coalescence of vesicular elements of endoplasmic reticulum. Near the apical surface of these cells autophagic vesicles and small, dense secretory vesicles are also present. The ultrastructural findings suggest a certain amount of cytoplasmic degeneration during cell division, with accompanying reorganisation. The basal lamina has completely disappeared except from some areas of the lacunae (Fig. 96). Extracellular membranes are present in the lacunae (Fig. 96). In 96-hour old fourth-instar larvae the wing bud epithelium reaches its maximum number of cells, arranged in several layers (Fig. 97). Each cell is now similar in size, and most of its volume is occupied by a large, almost oblong nucleus, with its longitudinal axis perpendicular to the apical and basal axis of the cell (Figs. 97 and 98). The epidermis and cuticle are completely separated from each other. An exuvial space has developed between the plasma-membrane of the epidermis and the old cuticle. The exuvial or extracellular space contain huge masses of foam-like material resembling aggregations of fine granules (Fig. 85). The exuvial space also contains several vesicles of different sizes, filled with ribosome like contents and surrounded by smooth membranes. At first I supposed these to be •

194

Fig. 95. T.S. of integument from a 72-hour fourth-instar wing pad. X 13000. • av, autophagic vesicle; er, endoplasmic reticulum; dsv, electron-dense secretory vesicle; ij, intermediate junction; ly, lysosome-like body; m, mitochondria; n, nucleus.

Fig. 96. 72-hour fourth-instar wing pad, showing lacuna, basement membrane, extracellular exudate and some portion of epidermis. X 13000. bm, basement membrane; lac, lacuna. 4,7* , • • - ligiciv A44 • • !

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Fig. 97. Post-apolytic epidermis from a 96-hour, fourth- instar wing pad. X 20000. exsp, exuvial space; G, Goi i body; ib, isolation body; m, mitochondria; mvb, multivesicular body; pm, plasma membrane; sd, septate desmosome. •

197

Fig. 98. T.S. of epidermis from a 96-hour fourth-instar wing pad; a dark and light cell are present. X 17000. • dep, electron-dense epidermal cell; lc, light cell.

Fig. 99. T. S. of a 96-hour fourth-instar wing pad. Apical cytoplasm containing numerous lysosome-like bodies, mitochondria and abundant plasma membranes. X 17000. cut, cuticle; er, endoplasmic reticulum; ly, lysosome-like body; my, microvillus; mvb, multi vesicular body; pm, plasmamembrane; r, ribosomes; sv, secretory vesicle. •

•

99 199 the tips of microvilli projecting irregularly into the exuvial space and cut in profile when tissue was sectioned. It now appears from some electron-micrographs (Figs. 85 and 97), however, that vesicles are formed by out-pushings of the apical plasma membrane, which are then pinched off, leaving vesicles bounded by smooth. membranes, free from the cell (cf. Noble-Nesbitt, 1963). Ultimately, these vesicles liberate their contents to form the foam-like granular secretion of the exuvial space. The plasma membrane between the epidermal cells and exuvial space shows variations; probably representing stages which may pass one into another. The apical plasma membrane, adjacent to the cuticle is less folded (Fig. 97). In some cells, the apical plasma membrane has folded into microvilli. The micrograph shows further advances in the formation of cuticulin over the tips of microvilli. All these ultrastructural observations amplify my light microscopical findings, that the cell division,separation of cuticle from epidermis and secretion of new cuticle occur gradually in the wing pads, proceeding slowly from their base to the tip. There is rapid growth among the intercellular plasma membranes of the apical cytoplasm (Figs 97 and 99). This involves the apparent extension of some of the intercellular membranes, which give the impression of invading the neighbouring cytoplasm and dividing the apical region of the cells into "compartrnents" (Figs 97 and 99). Considered in three dimensions, however, it seems that this process is really due to the extension and folding of cell processes so that the "compartments" really represent profiles of interdigitating cell extensions. Such areas are very frequent in these early pharate larval wing pads (Fig. 97). The apical cytoplasm contains a few small lamellae of rough-surfaced endoplasmic reticulum, many single free ribosomes and a few polyribosomes (Fig. 85). In the apical cytoplasm well developed Golgi bodies are frequent, comprising long stacks of smooth- surfaced endoplasmic reticular lamellae, 200

electron- lucent vacuoles of about 0. 07 to 0. 4 p.m in diameter and electron-dense vesicles (Fig. 85). The electron-dense vesicles and vacuoles are also found free in the apical cytoplasm. In addition to the above organelles, mitochondria with circular profiles are present in lesser numbers. The cells are separated by adjacent plasma-membranes; specialised cell junctions are present only in the apical portion of the cell. In the apical cytoplasm of some cells vesicular rough-surfaced endoplasmic reticulum is more abundant than the lamellate form (Fig. 97). There is a division and remarkable increase in number of mitochondria (Figs. 97 and 99). Isolation bodies and multivesicular bodies are rarely present. The lysosome-like bodies are not found -in the cytoplasm of every cell (Figs. 97 and 102); when they are present (Fig. 99) they may occur in large numbers. Some epidermal cells of the wing bud at this stage possess very few organelles (Fig. 98). The generally electron-lucent appearance of the cell is due to the limited number of ribosomes; very few single ribosomes, 4 or 5 mitochondria per section, small amounts of rough-surfaced endoplasmic reticulum, and a small, basally located Golgi body with dense vesicles, and a few vacuoles of agranular reticulum are present. The basal portion of most cells is produced into a long process (Figs. 100 and 101). Where the processes lie close to each other, they are separated by normal plasma membranes (Fig. 101). The basal tips of the cell also maintain such contacts with the processes from the opposite integument (Fig. 101). The processes were seen under the light microscope as a thick bundle of cytoplasmic processes separated from another group of cytoplasmic tails by wide intercellular spaces. Each cytoplasmic tail or elongated basal process has a very well developed Golgi complex, enclosing several electron-dense secretory vesicles of about 0.05 p.m diameter and large vacuoles of smooth-surfaced endoplasmic reticulum. They are enriched with ribosomes, which may lie singly or in clusters. Mitochondria of different sizes, but very few in number, are also present with a few or several tubules of Fig,. 100. Basal cytoplasmic processes from 96-hour fourth- instar wing pad, showing large electron lucent vacuoles and glycogen deposits. X 26000. er, endoplasmic reticulum; g, glycogen; m, mitochondria; v, vacuole. •

Fig. 101. Several basal cytoplasmic processes from 96-hour, fourth-instar wing pad epidermal cells. They lie in contact to the adjacent and apposed processes by their plasmamembrane. X 28000. cp, cytoplasmic process; er, endoplasmic reticulum; G, Golgi body; g, glycogen; icsp, intercellular space; m, mitochindria; v, vacuole. •

203

ribosomal endoplasmic reticulum. Scattered microtubules and large vacuoles are very common. The basal areas of some cells enclose very large areas with alpha and beta-glycogen particles. A few isolation bodies are also present, in the basal portions of some cells. Between the normal epidermal cells of the wing pad lie several large cells of uncertain nature which have been seen under the electron and light microscope. With Mallory's triple stain, the central lumen stains red, suggesting it contains a secretory product, but I could not find any duct. Under the electron microscope,. the central lumen is surrounded by long finger like microvilli (Figs. 102 and 103). Towards the basal side the cytoplasm is traversed by apposed plasma membranes, indicating that a form of cellular invagination has taken place. The cell junctions are simple during the early stages, but later the space between the membranes is compartmented by septate desmosomes. The cytoplasm of the cell contains mitochondria, ribosomes, vesicles of rough surfaced endoplasmic reticulum and scattered microtubules. A few microbodies and isolation bodies are also found in the cytoplasm. A bundle of fibre-like structures, encircled by dense material, and several small fibrous structures are present within the lumen. These cells are probably same type of unicellular exuvial glands as mentioned by Wigglesworth (1933, 1947, 1948b), Way (1950) and Wolfe (1954a, b). Further details of these apparently glandular cells will be discussed later. Between normal epidermal cells some large cells with a small cytoplasmic volume and large nucleus containing several chromatin masses are present. The exuvial space adjacent to such cells has a few electron-dense droplet-like bodies. These are perhaps due to a certain amount of cell death, with disintegration of nuclei and the formation of "chromatin droplets" (Wigglesworth 1942; Lawrence 1966). In 120 hour old fourth- instar larvae, the late pharate fifth-instar larval wing pads usually separate from the old cuticle during fixation, which make it •

204

Fig. 102. T. S. of a unicellular gland from 96-hour fourth-instar wing pad. The lumen is surrounded by microvilli. X 12000.

er, endoplasmic reticulum; ib, isolation body; ij, intermediate • junction; 1, lumen; m, mitochondria; mt, microtubules; my, microvillus.

Fig. 103. Unicellular gland from 96-hour fourth-instar wing pad. X 12000. 1, lumen; my, microvillus; sd, septate de smosome s.

• •

•

206

impossible to give any further details of the exuvial space and its contents. Each epidermal layer of the wing pad possesses enormous numbers of cells arranged in several layers, and resembling a stratified epithelium, the cells of which are smaller and less columnar than at previous stages of development. The cell surface is thrown into folds and some parts of this expanding wing have begun to form transverse folds (Fig. 106). The cells compressed in • these folds have flat apical surfaces while cells in unfolded portions of the wing are lobed or rounded (Fig. 105). The interior of each epidermal cell is mostly occupied by a large, oblong nucleus; only a small volume is left for the cytoplasm (Fig. 105). The apical plasma membrane is folded into closely packed villi (Fig. 106). Fig. 87 shows the newly secreted cuticulin, 0. 6 p.m thick, over these structures. In the apical cytoplasm there is a prodigious increase in the extent of the plasma membrane (Fig. 104). The cytoplasm thus gives the appearance of being divided into several "compartments" as discussed above (Fig. 106). This extensive development of plasma membranes and construction of cytoplasmic "chambers" represents a major form of histological reorganisation and reorientation of the cells during the folding and expansion of the wing buds of the pharate larva. Fig. 87 shows the enclosure of extracellular space inside the folded edges of the apical cytoplasm (Fig. 104). The apical cytoplasmic cell processes forming the "chambers" are bounded by intercellular plasma membranes and contain the normal organelles to be expected; there are several mitochondria with circular and elongate profiles, some dividing mitochondria, polyribosomes and single ribosomes, electron-transparent vacuoles, moderately dense vesicles, multivesicular bodies and scattered microtubules. A few small Golgi bodies, associated with small electron-dense contents and vesicles of smooth- surfaced endoplasmic reticulum are found in the apical cytoplasm. A few short swollen lamellae of rough-surfaced endoplasmic reticulum are also sometimes •

Fig. 104. T. S. of epidermis from 120-hour fourth-instar• wing pad. X 17000. elv, electron-lucent vacuole; dv, electron- dense vesicle; exsp, exuvial space; G. Golgi body; ic, intercelluar junction; ij, intermediate junction; m, mitochondria; mt, microtubules; my, microvillus; mvb, multivesicular body.

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Fig. 105. T.S. of epidermis from 120-hour fourth-instar wing pad from unfolded? area; it is in several layers. X 10000. m, mitochondria; n, nucleus; pin, plasma membrane. Fig. 106. T.S. of epidermis from 120-hour fourth-instar wing pad, showing first division of bristle mother cell and a unicellular gland. X 10000. dgc, degenerating cell; gl, gland cell; mc, bristle mother cell. •

210

present (Fig. 105). At times electron-dense bodies of various sizes are found, but no signs of lysosomes. The apical cytoplasm of each normal epidermal cell, basal to the "compartmental" region, possesses almost the same organelles, except for the multivesicular bodies. The basal area of the cell is limited, and it is not usually produced into long processes. The plasma membrane of the apical regions contains intermediate junctions and septate desmosomes r (Fig. 104). The more basal regions of each cell are separated by simple, apposed plasmamembranes (Fig. 105). Some specialised cells (exuvial glands, setal cells), are still undergoing division at this time. A few isolation bodies are recognised in the wing bud epidermis (Fig. 106). They contain invaginated plasma membranes, mitochondria, ribosomes and a few vacuoles. In the wing bud epidermis dividing bristle cells are found among the ordinary epidermal cells (Fig. 106). Towards the apical border of the wing pad epidermis, a large epidermal cell (measuring 12.5 Tim by 5. 1 1m in Fig. 106) contains a large nucleus of approximately triangular outline, and encloses a cell and a unicellular gland cell. Structurally this unicellular gland resembles the one which I have mentioned before. In 140-hour fourth-instar larvae immediately before ecdysis, i. e. late pharate fifth-instar larvae, the wing-pads become separated from the old cuticle during dissection or fixation. The integument of these wing buds is very highly folded within the old cuticle. The plasma membrane of the epidermis lies immediately beneath the new cuticle and is in close contact with it (Fig. 88). It is no longer microvillate, but has a slightly lobed profile. Towards the cytoplasmic side it produces infoldings, which will meet to isolated apical portions of the cytoplasm and so release their contents into the newly secreting lamella of the cuticle. Most of the epidermal cells are organised into a single layer. Each epidermal cell contains a single large oblong nucleus (Fig. 108), the surface of which is sometimes quite irregular. Each nucleus contains a prominent nucleolus or uniformly distributed chromatin (Fig. 108). •

211

Fig. 107. Section of basal processes of epidermal cells from 140-hour fourth-instar wing pad; numerous transversely cut profiles of microtubules are present. X 5000. • cp, cytoplasmic process; ic, intercellular junction; ,mt, microtubule.

Fig. 108. T. S. of an integument from pharate fifth-instar wing pad, immediately before moult. X 13000. cut, cuticle; er, endoplasmic reticulum; G, Golgi body; g, glycogen; ly, lysosome-like body. 0

• • •

213

The apical cytoplasm (Fig. 88) contains numerous polyribosomes, small stacks of granular endoplasmic reticulum, several mitochondria with circular profiles and a few elongate forms. Numerous well-developed Golgi bodies with electron-dense and electron-lucent vesicles are also found. Similar vesicles are present free in the apical cytoplasm. Scattered microtubules were • found everywhere, and bundles of microtubules are also present in the lateral and basal portions of the cell (Figs. 88 and 107). They are straight and variable in length (Fig. 109), with an external diameter of 25 nm. At the magnification employed each tubule has a simple thick electron dense wall and a centre of low density, giving it the usual 'hollow' appearence (Fig. 107). The basal' cytoplasm of each cell is produced into a conical process (Fig. 107). These are in contact with similar processes from neighbouring cells, the adjacent plasma membranes being separated by small intercellular spaces (Fig. 107). The processes contain polyribosomes and longitudinally directed bundles of microtubules. In some cells the processes contain only ribosomes (Fig. 107). Micrographs of the wing buds before ecdysis show an electron-dense appearance due to the closely packed free ribosomes (Fig. 108). These dense pre-ecdysial epidermal cells contain oblong nuclei with a narrow or lobed end towards the cuticle and a well developed nucleolus or condensed chromatin distributed around the periphery and the centre (Fig. 108). The nuclei lie close to the apex, while the basal regions of most of the cells are each produced into a long process (Figs. 109, and 110). The apical cytoplasm contains a few stacks of granular endoplasmic reticular cisternae and a few mitochondria. The cytoplasm also contains several electron-transparent vacuoles filled with glycogen particles. Such glycogen filled areas are abundant in micrographs taken before ecdysis (Fig. 108). Several Golgi bodies consisting of electron-dense and transparent vesicles are also present (Fig. 108). Similar electron-dense vesicles are also found free in,the basal and apical cytoplasm. In the apical 214

Fig. 109. Long stout basal processes of epidermal cells from late pharate fifth-instar wing pad, supported by bundles of microtubules. X 8000. cp, cytoplasmic process; er, endoplasmic reticulum; mt, microtubules.

Fig. 110. Stout basal processes of epidermal cells from late pharate fifth-instar wing pad. X 17000. dsv, electron-dense secretory vesicle; G, Golgi body; m, mitchondria; nit, microtubules; v, vacuole. 109

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cytoplasm there are a few electron-dense bodies encircled by membranes, lysosomes and a few areas containing ribosomes and encircled by mitochondria (Fig. 108). Because of the folding of the wing bud epithelium, the intercellular boundaries are not very clear, though some plasma membranes could be identified (Fig. 108). The basal regions of most cells are produced into long, a stout processes; those from opposite sides remaining free in the middle of the wing bud, and apparently supported by longitudinally directed microtubules (Fig. 109). Some of the processes contain a • very well developed Golgi apparatus with several electron-dense vesicles of 0. 15 pm diameter and a few vacuoles of smooth surfaced endoplasmic reticulum (Fig. 110). The electron-dense secretary vesicles are abundant in the basal cytoplasm (Fig. 110). In addition to the microtubules the basal processes of the cells contain numerous free ribosomes, a few tubules of ribosomal endoplasmic reticulum and a few mitochondria (Figs. 109, 110). Some processes near the trachea contain glycogen-filled areas towards their tips. (c) Trachea and Tracheoles In the newly moulted fourth-instar larva there are five prominent tracheae running throughout the whole length of the fore wing pad, each lying inside a haemolymph filled lacuna; which is encircled by basement membranes of the integumentary layers. Ultrastructurally the wing pad treachea is almost circular in cross- section (Fig. 111) in which may be seen the tracheal intima, tracheal epithelium and basement membrane. The lining of the tracheal tube consists of an electron-dense epicuticle, 40 nm thick, which is arranged helically, and strengthened by taenidial folds. On its inner side the epicuticle bears small tubercles (micropapillae), except over the inner face of the taenidia (Figs. 111 and 112). In transverse section the trachea is ringed by two epithelial cells, arranged in a single layer (Fig. 111). The

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Fig. 111. T.S. of trachea from newly moulted fourth-instar wing pad. X 17000. • e. cut, epicuticle; mvb, multivesicular body; p, micropapillae; t, trachea; tep, tracheal epithelia; tn, taenidia.

Fig. 112. T.S. of a portion of trachea and epidermis from newly moulted fourth-instar wing pad. X 22000. bm, basement membrane; ep, epidermis; er, endoplasmic reticulum; G, Golgi body; icsp, intercellular space; m, mitochondria; mt, microtubules. ZII 219 epithelial cells are squamous structures with a large elongated nucleus occupying most of the area between the cuticular ring and basement membrane (Fig. 112). The chromatin masses are distributed in the centre and periphery of the nucleus. Cell boundaries are very clear, and are linked by intermediate junctions (Zonula adherens), which lie below the apical boundary, and cover an area 200 nm long (Fig. 111). The lateral membranes are linked to this by a series of septate desmosomes, followed by a zone of loosely apposed plasma membranes. The tracheal epithelium also contains dark and light cells, depending on the density of the ribosomes (Figs. 111 and 113). The cytoplasm contains small mitochondria with circular or oval profiles, many single free ribosomes and a few spiral polyribosomes, scattered rough surfaced endoplasmic reticulum in the form of vesicles and short cisternae. Golgi bodies are well developed and are present towards the sides of the cells with many electron-dense vesicles and sacculi of agranular- endoplasmic reticulum (Figs. 111, 112 and 113). Electron dense vesicles similar to the components of Golgi bodies are found free in the cytoplasm. Electron-lucent vacuoles and microtubules are present in the tracheal epithelium (Fig. 112). The trachea is surrounded by a delicate basement membrane, 50 nm thick. The tracheal epithelium lies very close to the epidermal cells or nerves, separated by an intercellular space, sometimes with a gap of 20 nm; in such areas the basement membranes have disappeared (Fig. 112). Micrographs from 24 hour old fourth-instar wing pads show a tracheal tube, lined by an epicuticle 40 nm thick (Fig. 114). The epicuticle is uniformly folded into taenidia. Taenidia are filled with less electron-dense material. Between the epicuticle of the trachea and the apical plasma membrane of the tracheal epithelial cells a continuous ring of moderately dense material, 70 rim thick, is present, probably representing the pro-cuticle. (According to the observations of Locke, 1964, and Smith, 1968, there is no procuticle layer between taenidia and epithelium. •

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Fig. 113. Section from a portion of trachea and adjacent tracheoles from 24-hour fourth-instar wing pad. X 22000. t, trachea; tl, tracheole. •

Fig. 114. Section of a trachea, tracheal end cell, tracheoles and lacuna from a 24-hour fourth-instar wing pad. X 12000. bm, basement membrane; e. cut, epicuticle; p, micropapillae; procut, procuticle; tec, tracheal end cell; tn, taenidia. •

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Richards (1951); Miller (1962); Wigglesworth (1965); and Whitten (1969) showed a solid layer of procuticle). The epithelium contains numerous polyribosomes, circular and elongate mitochondrial profiles, and a little rough-surfaced endoplasmic reticulum in the form of vesicles and small, elongate cisternae. There is an increase in size and number of Golgi bodies (Fig. 114), which are now arranged around the nucleus. Numerous microtubules were found, running parallel with the cuticular tube. Membrane- bounded electron-dense lysosome-like bodies are also present (Fig. 114). The basement membrane forming the outer most coat of trachea is 60 nm thick. In 48-hour old fourth instar larval wing pads, tracheae were found at various stages of pre- and post apolysis. As in the wing bud integument, so also in the tracheal epithelium, events like cell division and separation of cuticle from epidermis take place successively from one end of the trachea to the other, rather than simultaneously at all points. One micrograph from a pre apolytic trachea shows a fully formed procuticle, 0. 5 p.m thick with an epicuticle 40 nm thick. The diameter of the trachea is 4. 4 p.m, and the tracheal tube is enclosed by three squamous epithelial cells, with a large nucleus occupying most of the cell (Fig. 116). There is no evident nucleolus, the chromatin is dispersed in small masses in the nucleoplasm. The cells emit several cytoplasmic processes which meet processes of the epidermal cells (Fig. 116). The cytoplasm of the tracheal epithelia contains cisternae of ribosomal endoplasmic reticulum in the form of small lamellae and vesicles, polyribosomes, mitochondria, a few scattered Golgi bodies, microtubules, and dense lysosome-like bodies (Fig. 116). Tracheoles with a smooth intima are enclosed inside the tracheal end cells (Fig. 116). The cytoplasm of the tracheal end cell is separated from the tracheal matrix cell by intercellular membranes. Another micrograph from the same material shows •

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Fig. 115. L. S. of tracheoles and cytoplasmic extension of tracheal end cell from 24-hour fourth-instar wing pad. X 17000. t, trachea; t1, tracheole; to c, tracheal end cell. •

Fig. 116. T.S. of a trachea and adjacent epidermis from 48-hour fourth-instar wing pad. Cytoplasmic processes are given off by both epidermis and tracheal epithelia. X 8000. cp, cytoplasmic process; ep, epidermis; g, glycogen; pm, plasma membrane.

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a tracheal intima detached from the tracheal matrix cells after apolysis; a well developed exuvial space is present with small vesicles and moulting fluid (Fig. 117). The tracheal matrix cells of different electron density and size contain abundant polyribosomes, a few cisternae of rough-surfaced endoplasmic reticulum, numerous circular profiles of mitochondria, a few scattered Golgi bodies, electron-dense vesicles and microtubules; very dense lysosome- like bodies are also present. Some electron transparent areas with rosettes of glycogen deposits are visible. A post apolytic condition of the trachea, with some degenerating tracheal matrix cells, is seen during this stage. Presumably the capacity of the tracheal matrix cells for division, regeneration and morphogentic moulding will help to increase the diameter of the trachea in every instar. Wigglesworth (1954) says that, when the fever of growth and decay is over, the remaining cells and nuclei assume the orderly arrangement needed to refine the form of the next instar. The cytoplasm of the remaining epithelial cells contain polyribosomes, very little rough-surfaced endoplasmic reticulum, a few elongate forms of mitochondria, small Golgi bodies, electron- vacuoles, and electron dense vesicles. In 74-hour fourth-instar wing pads the procuticle of the tracheal tube has completely dissolved and the exuvial space is filled with moulting fluid, giving the appearance of a continuous membrane between the old cuticle and tracheal epithelia (Fig. 118). The cytoplasm of the tracheal matrix cells contain single free ribosomes, spiral polyribosomes, a few small cisternae and vesicles of rough-surfaced endoplasmic reticulum, spherical mitochondria, coated vesicles, electron-lucent vacuoles, microtubules and a few lysosome-like bodies (Fig. 119). There is an increase in the number of Golgi bodies, which consist of electron-dense vesicles and electron-lucent vacuoles. A micrograph showing a trachea in close association with a nerve is included in Fig. 119. Both lie within a lacuna. The basement •

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Fig. 117. T.S. of a post apolytic trachea from 48-hour fourth- instar wing pad. X 13000. dv, dense vesicle; exsp, exuvial space; G, Golgi body; g, glycogen; ij, intermediate junction; m, mitochondria; mfl, moulting fluid; mt, microtubules; t, trachea.

Fig. 118. 72-hour fourth-instar wing pad, with a nerve and trachea. X 24000. a, axon; bm, basement membrane; gls, glial sheath; ma, mesaxon; nv, nerve; ti, tracheal intima. •

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membrane of the trachea is in contact with the basement membrane enclosing the nerve. Fig. 119 shows the fused basement membranes of trachea and nerve. Micrographs of tracheae from the 96-hour old fourth-instar wing-pads show a very wide exuvial space filled with an opaque material. Profiles of tracheoles are also present inside the same exuvial space (Fig. 120). The diameter of the trachea has now increased and the tracheal matrix is formed of several epithelial cells. Each tracheal matrix cell contains a large nucleus, with the nuclear membrane irregularly folded; one or two nucleoli and condensed chromatin is present in the nucleus (Fig. 120). Inter- cellular membranes are provided with an apical zonula adherens type of intermediate junction; septate desmosomes, tight junctions and simple apposed plasma membranes are more clearly visible than before (Fig. 120). The apical plasma membrane of the tracheal epithelium is slightly raised into electron-dense areas, which indicate the beginning of new cuticle secretion (Fig. 120). In the cytoplasm very well developed Golgi bodies are situated around the nuclei, composed of electron-dense vesicles, vesicles of agranular endoplasmic reticulum and some dense secretions (Fig. 120). Many circular and elongate mitochondria, elongated and vesicular forms of rough-surfaced endoplasmic reticulum, polyribosomes, single free ribosomes, microtubules, electron- dense vesicles and coated vesicles are also present in the cytoplasm (Fig. 120). It contains many electron-lucent vacuoles. A "compartmental" appearance of the apical cytoplasm is also seen, due to the enlargement of lateral intercellular plasma membranes, as described for the wing bud integument, and shown in Fig. 120. A tracheoblast cell with a very large nucleus and several profiles of tracheoles within the cytoplasm, is shown in some micrographs. Lysosome-like bodies, which are very numerous in the previous stage, are absent from 96-hour fourth-instar tracheal sections. Late pharate fifth-instar tracheal micrographs show a fully •

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Fig. 119. 72-hour fourth-instar wing pad, lacuna enclosing a trachea and a nerve. X 17000.

a, axon; bm, basement membrane; . glc, glial cell; • ma, mesaon; mfl, moulting fluid; nv, nerve; p, micropapillae; t, trachea; tl, tracheole.

Fig. 120 T.S. of trachea from 96-hour fourth-instar wing pad. X 17000. cv, coated vesicle; er, endoplasmic reticulum; ij, intercellular junction; mt, microtubules; my, microvillus; sd, septate desmosomes; ti, tracheal intima. •

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formed new tracheal tube (Fig. 121). The epicuticle lining the tracheal tube is 40 nm thick and it is folded into regularly spaced taenidia. The inner side of the epicuticle is already micro-papillate except on the taenidia. Electron opaque material representing procuticle is present between the tracheal intima and the epidermal microvilli, but there is no such material between the taenidia and epidermis (Fig. 121). In Fig. 122, secretions from the microvilli entering the taenidial fold were noticed. Another advanced stage of solid taenidia filled with less dense material is shown in some micrographs. At this stage the apical plasma-membrane of the wing pad tracheal epithelia is microvillate, with long narrow stacks enclosing large vacuoles between them (Fig. 122),;(cf. Locke, 1966). The epithelia contain cisternae of rough-surfaced endoplasmic reticulum, a few mitochondria, single free ribosomes, several well developed Golgi bodies with electron-dense secretions, and free electron dense vesicles. The basement membrane lining the outer surface of the trachea is very delicate (Fig. 121). A tracheoblast cell enclosing several profiles of tracheoles and a large lobed nucleus is shown in some micrographs, taken from a late Pharate fifth-instar wing pad. The basement membrane of the tracheal epithelia is continuous with the tracheal end cell. The inner lining of the tracheole is formed of a dense, smooth epicuticle, 20 nm thick (Fig. 121); (cuticulin of Locke, 1957, 1960, 1961, 1964); which is helically folded. The taenidia of the tracheolar tubes are smaller in breadth and regularly arranged. At this stage another layer of less electron-opaque cuticle lies between the dense inner lining of the tracheole and the surrounding cytoplasm. The cytoplasm of the tracheal end cell contains electron-lucent vacuoles, single free ribosomes, elongate and spherical forms of mitochondria, a little rough-surfaced endoplasmic reticulum, Golgi bodies, electron-dense vesicles, multivesicular bodies and microtubules (Fig. 121). Fig. 121. shows •

Fig. 121. Portion of late pharate fifth-instar wing pad; trachea, tracheal end cell and tracheoles. X 15000. elv, electron-lucent vacuole; G, Golgi body; my, microvillus; pm, plasmamembrane; t, trachea; tl, tracheole; tn, taeridiurn. •

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Fig. 122. Pharate fifth-instar wing pad, trachea and tracheole, to show their connection. X 16000. my, microvillus; t, trachea; tl, tracheole; connection between trachea and tracheole was shown by an arrow.

Fig. 123. Micrograph from newly moulted fourth-instar wing pad showing a portion of epidermis; nerve and lacuna. X 25000. a, axon; bm, basement membrane; gl, glial cell; lac, lacuna; n, nucleus; mesaxon invagination was shown by an arrow. 411

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plasma membranes enveloping the tracheoles. The outer and inner plasma membranes connect by folds, rather in the way that a nonmyelinated axon is suspended within a schwann cell. Edwards et al. (1958) called this plasma membrane a "mestracheon". Further developmentsof the tracheolar intima is shown in some other micrographs, in which the tracheole is lined by a single layer of more dense epicuticle. Tracheoles are surrounded by a considerable amount of tracheoblast cytoplasm and the intima is separated from it by a thin layer of plasma membrane; several tracheoles lying in their own cytoplasmic sheaths drawn out from the tracheoblast and a nucleus with granular, compact material is also shown in some micrographs. The cytoplasmic sheath of the tracheole is very thin. The connection between trachea and tracheole is shown in Fig. 122. The epicuticular lining of the trachea is continuous with that of the tracheole, but the tracheal lining has a rough appearance due to the presence of micropapillae, whereas the inner surface of the tracheole is much more heavily folded into regularly spaced taenidia. The cytoplasm of the tracheal matrix cells is separated from the cytoplasm of the tracheoblast by plasma membranes with junctions of the septate desmosome type. The apical plasma membrane of tracheole does not possess long stacks of microvilli (Fig. 122). (d) Nerve Supply In fourth-instar larval wing pads of Oncopeltus fasciatus, the five longitudinally directed lacunae, each contain a fine nerve in addition to the trachea. I have not paid much notice to the nerves while dealing with the histology of wing development, as they are so delicate as to provide little information. With Mallory's connective tissue stain the nervous tissue stains a violet colour while accompanying nuclei, larger in diameter than the axons, stain red. The presence of nerves in the lacunae of the newly emerged fourth-instar larva has been confirmed in •

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electron-microscope sections. Under the electron microscope, the nerves of the newly emerged fourth-instar wing pads resemble any other multiaxonal peripheral nerve branches of insects (Figs. 123, 124). A bundle of approximately 15 axons, circular in profile, with a diameter less than 0. 5 pm lies inside a glial sheath. From the surface of the glial cell membrane, prominent mesaxonal invaginations arise; at the points of invagination the plasma membranes are connected by septate desmosomes (Fig. 124). These mesaxons make several loose turns around the axons, but the membranes are not fused, and there is no myelin deposition in them. While folding around the axons, the mesaxons follow an irregular and branching course, and the folds are always separated by cytoplasm of the glial cells (Figs. 123 and 124). This condition, which is intermediate between myelinated and unmyelinated fibres is termed tunicated. The glial cells contain a very large nucleus towards one side of the axon bundle (Fig. 123). The cytoplasm of the glial cell contains polyribosomes, mitochondria, a little rough- surfaced endoplasmic reticulum in the form of vesicles and short cisternae, dense secretory vesicles and microtubules lying parallel with the axon (Figs. 123 and 124). The axoplasm contains small mitochondria, neurotubules and vesicles of agranular-endoplasmic reticulum (Fig. 124). At this stage the basement membrane is not yet developed around the nerve (Fig. 124). In Fig. 123, the basement membrane of the lacuna lines one surface of the nerve as it lies in contact with the integument. The nerve fibre lies in close contact with the epithelia, separated only by a narrow intercellular space. Fig. 90, from 48-hour fourth-instar wing pads provide an opportunity to examine the innervation of basal portions of the wing pad epithelium. In some places the epithelial cells are innervated by a delicate nerve fibre consisting of one or a few axons (Fig. 90); in others there may be a multiaxonal nerve fibre. •

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Fig. 124. T.S. of a nerve from a newly moulted fourth-instar wing pad. X 25000. Invaginations of mesaxon are connected by septate s desmosomes denoted by arrow. a, axon; bm, basement membrane; lac, lacuna; ma, mesaxon; nv, nerve.

Fig. 125. Section of a nerve from a 72-hour fourth-instar wing pad; basement membranes of nerve and epidermis are fused together in places. X 13000. ep, epdiermis; G, Golgi body; nv, nerve. t7 Z I

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The axon or axons lie inside the glial cytoplasm. There is a marked increase in the glia during the fourth-instar. Some axons are surrounded individually by glial cytoplasm and some lie in a common glial envelope, while a few separate axons are wrapped only in a single layer of glial cytoplasm (Fig. 90). The axoplasm contains mitochondria and abundant neurotubules. The glial cytoplasm contain numerous polyribosomes, spherical and dividing forms of mitochondria and microtubules (Fig. 90). Figs. 118, 119 and 125 show well developed nerve fibres lying inside the lacuna of the 72- hour, fourth-instar wing pads. A nerve fibre lying between the basement membranes of the lacuna and trachea is shown in Fig. 118. It contains a well developed glial cell and a group of a few axons wrapped in glial cytoplasm with several lamellae. In Fig. 119, only a few axons, each with an individual glial envelope, are encircled from one side by the large nucleus of a glial cell. In Fig 125, a nerve fibre is seen lying within the lacuna, contains several bundles or individual axons, separated by individual glial sheaths and two small nuclei placed towards the sides of the glial cell. The glial cytoplasm of 72-hour, fourth-instar winga pad nerves contains an increased number of circular, elongated and dividing profiles of mitochondria (Fig. 118); individual ribosomes, a few short cisternae of rough-surfaced endoplasmic reticulum and microtubules are also present (Figs. 118 and 125), as well as a few small Golgi bodies (Fig. 125). The axoplasm contains neurotubules and sparsely distributed mitochondria. During this period the nerve fibres are encircled by a well developed basement membrane, approximately 95 mu thick; which lies very close to the trachea and basement membrane encircling the lacuna. In some places the basement membranes of nerve and trachea are confluent (Figs. 118 and 119). There is an increase in the number and size of axons in the nerve fibre in micrographs taken from the wing pad of a 96-hour old fourth-instar larva. There is also a marked increase in the size of the glial cells. A large nerve fibre now contains a bundle of •

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approximately 80 axons of different sizes, all less than 1 j.m in diameter each and all sharing a single glial sheath. Processes of the glial cell, covered by their plasma membranes, twist and turn around the axon bundle, separated by glial cytoplasm. The glial cytoplasm contains numerous microtubules, polyribosomes, individual ribosomes and mitochondria. Rough-surfaced endoplasmic reticulum, in the form of vesicles and short and long cisternae, • is also present. Though not abundant, there is more glial cytoplasm than was present at the previous stage. The axoplasm of small axons also contains a few mitochondria and vesicles of agranular endoplasmic reticulum. In a micrograph taken from a later stage wing pad (120-hour fourth-instar), the nerve fibre resembles in every respect those of the previous stage, but the glial sheath encloses several electron-lucent vacuoles. The nerve fibre from late pharate fifth-instar wing-pads contains large axons, with a diameter of more than 0. 5 pm. They lie inside the glial cytoplasm; some are ensheathed singly, some in groups of two or three, by a common glial envelope (Fig. 126). The glial cytoplasm contains single free ribosomes, mitochondria, neurotubles and Golgi bodies. During this period cisternae of granular endoplasmic reticulum are more abundant than in the previous stages. A few cisternae of agranular reticulum are also present. The axoplasm contains mitochondria, agranular reticular cisternae and neurotubules (Fig. 126). The nerve fibre is closely associated with the basal processes of the epithelial cells, separated by intercellular space without any basement membranes between them, but the neurones from which these axons arise have not been identified. Towards its other side the nerve is encircled by a basement-membrane continuous with that lining the lacuna. 4. Fifth Larval Instar Wing Pads: In the fifth-instar larva of Oncopeltus fasciatus, the externally visible wing-pads are twice as long as those found in •

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Fig. 126. Pharate fifth-instar wing pad immediately before moult, showing several basal processes of epidermal cells and nerve. X 17000. • a, axon; ep, epidermis; er, endoplasmic reticulum; m, mitochondria; mt, microtubules; nr, neurotubules; v, vacuole.

Fig. 127. T. S. of newly moulted fifth-instar wing pad, dorsal integument. X 12000. bm, basement membrane; G, Golgi body; lac, lacuna; n, nucleus; pm, plasma membrane; t, trachea; tl, trache ole . 126

127 243 the fourth-instar. There are five longitudinal tracheae running to the tip of the wing-pad, and a sixth branch runs parallel to the base of the wing-pad. For ultrastructural investigations, wing-pads of various ages within the fifth-instar were dissected under fixative and sliced into pieces about 1 mm long, as the intact pads of fifth-instar larvae are 3 mm. long and therefore too large to allow penetration of fixatives. Material was carefully oriented in the embedding medium to obtain ultrathin transverse sections. (a) Cuticle: The cuticle enclosing the newly moulted fifth-instar larval wing-pads is thicker than in the newly moulted fourth-instar. Under the light microscope the cuticle shows an approximate thickness of 3 - 4 a.m. With Mallory's connective tissue stain its outer half stains red, with a slight brownish tinge, and the inner half takes on a blue colour. Ultramicrographs taken from newly moulted fifth-instar wing-pads, show a cuticle thickness of 1.6 - 2. 8 p.m from the dorsal integuments (Fig. 127), and 0. 7 - 1. 4 am from various regions of the ventral integuments (Fig. 128). The outermost zone of the dorsal integument is composedaf electron-dense, homogeneous cuticle measuring 0. 25 pm thick. Under higher magnification (30, 000) an outer, uniform dense cuticular layer, 34 tun thick could be resolved (cf. Locke 1958, 1960, 1961). The ventral integument is covered only by a cuticulin layer (Fig. 128); the inner epicuticle seems to be absent. Internal to the epicuticle of the dorsal integument lies a lamellate procuticle, composed of more than 10 lamellae, with alternating more or less electron-dense zones (Fig. 127). It is similar in general appearance and fine structure to the lamellate "night-zone", cuticle of locust integument (Neville, 1965a, 1965b). At this stage the procuticle, from the dorsal integument is 2.55 dam thick and 0. 77 µm from the ventral cuticle (Figs. 127 and 128). Measurements of epicuticle and •

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Fig. 128. T. S. of newly moulted fifth-instar wing pad, ventral integument. The basal portions of the epidermal cells are in contact with a tracheal end cell, which encloses tracheoles. X 15000. cut, cuticle; G, Golgi body; haem, haemocyte; mt, micro- • tubules; n, nucleus; pm, plasma membrane; tec, tracheal end cell; tl, tracheole.

Fig. 129. Newly moulted fifth-instar wing pad, transverse section showing the basal processes of epidermal cells, profiles of tracheoles and processes of haemocytes. X 8000. ep, epidermis; haem, haemocyte; tl, tracheole. •

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and procuticle in ultra micrographs taken from the various areas of the pads show a uniform disagreement with the measurements recorded in the light microscopical observations. Histological preparations always show a thicker epicuticle than the electron- dense epicuticle obtained in the electron micrographs. Similar results were obtained while studying the newly emerged fourth-instar wing-pad cuticles of Oncopeltus fasciatus; they are discussed on • p. 367 , and have also been noted in Elaterid cuticle by Zacharuk (1972). The plasma membranes of the epidermal cells are in close contact with the cuticle; they are folded towards the cytoplasm to form inwardly directed tubules with associated vesicles. These are somewhat less numerous than in the late pharate fifth-instar and in the newly emerged fourth-instar wing-pads. In 24-hour, fifth-instar larvae the cuticle is strongly sclerotized and 3 pm thick. It consists of an outer thick electron- dense epicuticle 0. 75 pm thick and an inner thick lamellate procuticle 3 pm thick. The epicuticle still contain more than 10 laminae. The apical plasma membrane lying adjacent to the cuticle is similar to that of the previous stage. In the 48-hour old pre apolysis fifth-instar wing-pad, the cuticle is fully sclerotized, and the lamellae of the procuticle have almost vanished (Fig. 137). The apical epidermal plasma membrane is raised into irregular superficial microvilli; their tips appear dense due to concentration there of ribosomes. In some areas the cuticle seems to be retracting from the epidermis, but there is no visible exuvial space in between them, such as would show clear detachment (Fig. 137). The next stage was obtained from 76-hour, apolytic wing- pads of fifth-instar larva as is represented in Fig. 139, where different stages of cuticular detachment can be seen. In Fig. 139, the exuvial space has formed through retraction of the epidermal cells from the fifth-instar cuticle. The moulting fluid in the

• exuvial space appears as small electron-lucent vesicles with 247 diameters varying from 0. 2 1m to 0. 5 p.m, surrounded by smooth membranes covered by ribosome-like particles and numerous dense vesicles (Fig. 139). The exuvial space also contains polyribosomes. Electron lucent vesicles covered by ribosomes and resembling those in the ecdysial space, are also present within those epidermal cells which are just detaching from the cuticle. The moulting fluid was interpreted by Noble-Nesbitt (1963a) as originating in the form of vesicles formed by eva.ginations of the plasma. membrane, which are then constricted basally and cut off, leaving the vesicles bounded by membrane and free from the cell surface (Fig. 139). My findings confirm this interpretation. The micrographs from 120-hour, fifth-instar larval wing- pads show an exuvial space with vesicles of different sizes containing ribosomes, and dense granules of various sizes (Fig. 140; Noble-Nesbitt, 1963b). Cells similar to those noted in the ecdysial space of moulting Collembola (Barra, 1969) and in Elateridae (Zacharuk, 1972) occur in the ecdysial space of 120-hour fifth-instar wing-pads of Oncopeltus fasciatus (Fig. 140). Barra interpreted them as ecdysial haemocytes, but see my discussion p. 375. Some areas of the wing pad integument still have un- detached cuticle at this stage. Where cuticular detachment has occurred, the apical plasma membrane lying close to the moulting fluid is produced into microvilli with dense tips (Fig. 140), but there is no evidence of new cuticular secretion over these tips. The mid-pharate adult wings are very highly folded inside the old cuticle, as in the 150-hour old fifth-instar wing-pads (Fig. 143). At this stage, the folded pharate adult Swings are covered externally by a continuous, dense cuticulin layer 34 nm thick, formed in the way mentioned by Locke (1958, 1960, 1961, 1966). At some places the cuticular investment is accompanied by cytoplasm which has folded into externally projecting, spine-like microtrichia (Fig. 147). While cuticulin secretion is taking place, the dark tips of the microvilli lie very close to the cuticulin; when it is fully formed the microvilli slowly move away from it. 248

Fig. 144 shows a gap of 25 nm between the cuticulin and the dark tips of the microvilli. In another micrograph (Fig. 145), the apical plasma membrane has folded into small tubules, towards the cytoplasmic side. Micrographs obtained from the late pharate adult wings, just before ecdysis, show fully formed adult wings covered by a thick cuticle, varying in thickness from 0. 7 - 2. 2 Fm; these usually lie folded inside the old cuticle, but separate readily from it during fixation. Micrographs taken from the base of the wings show that development is more advanced than at the apical region of the wing. The plasma membrane adjacent to the cuticle contains long stacks of microvilli with dark tips as shown in Fig. 148, taken from the base of the wings. The plasma membrane from the non-microvillate areas produces infoldings towards the cytoplasmic side; such infoldings meet to isolate apical portions of the cytoplasm and so release their contents into the newly secreting lamella of the cuticle (Fig. 148). (b) Epidermis: In the newly emerged fifth-instar wing pads the integument consists of a thin layer of epidermis, formed of a single layer of columnar cells, each with a broad apex and conical basal portion (Fig. 127). The apical side of the cell is bounded .by cuticle, but the basal processes usually lie free inside the haemocoele, though sometimes the tips of the basal processes from the apposed integumentary layers lie close to each other, and are separated only by plasma membranes (Fig. 130). These plasma membranes are thicker than the normal ones. Ultrastructurally most of the area in the middle of the wing-pad integuments does not show a basement membrane, though one is present mainly around the lacuna (Fig. 127). The long filamentous processes of the haemocytes and the bent portions of the basal processes of the epidermal cells lie together in a line and appear like a middle membrane under the light microscope (Fig. 129). The plasma membrane lying close to the cuticle produces 249

small tubular infoldings into the apical cytoplasm; these tubules isolate apical portions of cytoplasm that may contribute to the cuticle (Figs. 127, and 128). The apical cytoplasm contains free single ribosomes, and a very little rough-surfaced endoplasmic reticulum in the form of vesicles and short cisternae; some of these rough vesicles are joined together to form small lamellae (Fig. 128). The apical cytoplasm also contains spherical and ovoidal mitochondria; Golgi bodies with electron-dense vesicles and agranular endoplasmic reticulum arranged around the nucleus (Fig. 127); abundant microtubules (Fig. 128); some areas containing rosettes of glycogen particles; a very few membrane-bounded, lysosome-like bodies; electron-lucent vacuoles, multivesicular bodies and electron-dense vesicles (Figs. 127 and 128). The lateral portions of adjacent cells are associated by plasma membranes separated by an intercellular space. The intercellular membranes are linked by an apical, zonula adherens type of intermediate junction, running as a ring around them, and followed by septate desmosomes and simple, apposed plasma membranes (Fig. 127). Each epidermal cell contains an ovoidal nucleus lying basally in some cells, and near the apex of others. In some cells the nucleus is lobed towards the cuticular side of the cell (Fig. 128). A prominent central nucleolus is present in many nuclei at this stage. The nucleoli resemble thick anastomosing strands (Fig. 127); condensed chromatin is either distributed around the periphery in small clumps or widely scattered throughout the nucleoplasm (Figs. 127 and 128). The basal portion of some cells forms a small conical process, or a long thing filament, or at times it is a very broad, stout process (Fig. 127), but these various profiles probably indicate only the plane of sectioning; The basal cytoplasm contains single free ribosomes, rough surfaced endoplasmic reticulum in the form of short cisternae and vesicles, a few mitochondria, electron-lucent vacuoles, some beta-glycogen containing areas, and abundant microtubules (Fig. 130). The basal portions of many cells obtain oxygen directly through tracheoles (Figs. 127 and 128): The •

250

Fig. 130. Portion of a T. S. of a newly moulted fifth-instar wing pad showing desmosomal attachment between basal processes

of apposed cells. X 26000. • cp, cytoplasmic process; d, desmosome.

Fig. 131. T. S. of 24-hour fifth-instar wing pad epidermis showing plasma membrane infoldings and apical cytoplasmic organelles. X 30000. cut, cuticle; cv, coated vesicle; elv, electron-lucent vacuole; G, Golgi body; pi, plasma membrane infolding; pm, plasma membrane. S

130

I 31 252

plasma membrane of the tracheal end cell bounds the cytoplasm accompanying individual tracheoles and lies adjacent to the basal plasma membrane of the epidermal cell, separated only by a small intercellular space (Fig. 128). Haemocytes also lie very close to the basal surfaces of the epidermal cells (Fig. 128), whose basal processes usually lie free inside the wing pads. Sometimes the basal processes from apposed integuments are associated by the adjacent plasma membranes of their tips, which are separated by an intercellular space (Fig. 130). The basal processes of some cells bend near their middle and lie parallel to the basement membrane. A wide extracellular space is present, between the basal portions of the cells. This extracellular space contains haemocytes and membrane-bounded vesicles of different sizes with diameters up to 2. 25 pm. The membrane-bounded vesicles are filled with ribosomes and smaller vesicles (Fig. 127). In the 24-hour fifth-instar larva, the epidermis consists of a single layer of narrow, columnar cells. The nuclei usually occupy the central portion of the cells and are irregular in outline, with a large nucleolus and chromatin distributed inside the nucleoplasm. Fig. 131 shows an epidermal cell with a basal nucleus and apical cytoplasm containing numerous microtubules arranged in bundles. The apical plasma membrane is folded internally to form tubules and associated vesicles (Fig. 131). The apical cytoplasm contains individual ribosomes and spirally arranged polyribosomes. There is a marked increase in the content of granular endoplasmic reticulum, compared to the previous stage. It is mainly in the form of small cisternae, but a few vesicles are still present (Fig. 131). The cytoplasm also contains mitochondria with circular and elongate profiles, the latter measuring up to 1. 13 pm in length. Well developed Golgi bodies are present around the nucleus and consist of electron- dense vesicles and agranular reticulum in the form of electron- lucent vesicles or flattened tubules (Fig. 131). The apical cytoplasm also contains scattered microtubules, electron-dense •

253

vesicles, coated vesicles, electron-lucent vacuoles and, very rarely, lysosome-like bodies. The apposed lateral cell membranes are linked by intermediate junctions, septate desmosomes and in some places the two membranes may be quite separate or are partly fused. Some flat, conical epidermal cells give rise to a fine basal cytoplasmic filament. In some cells, the basal cytoplasm forms a • stout process containing bundles of microtubules, a few elongate mitochondria, and single free ribosomes (Fig. 132). These processes lie free inside the wing-pad. Fig. 133, shows a stout basal process, joined by plasma membranes to a similar process from the apposed integument. These plasma membranes are relatively thick and in some places they are fused. The cytoplasmic processes contain microtubules, mitochondria, individual ribosomes, cisternae of rough-surfaced endoplasmic reticulum, electron-dense vesicles and electron-lucent vacuoles (Fig. 133). Well developed Golgi bodies are sometimes present inside the basal cytoplasm. Fig. 134 shows the basal cytoplasm of some cells, which give out several cytoplasmic processes from all sides. These entangle in all directions with similar processes from other cells, occupying most of the extracellular space of the wing-pad, and imparting a spongy appearance to it. The basal and apical cytoplasm of these cells consists mainly of rough- surfaced endoplasmic reticular cisternae in the form of elongated tubules, and spiral polyribosomes. The extracellular space of the wing-pads contains membrane-bounded vesicles measuring up to 5 rm in diameter (Fig. 134). They are filled with single free ribosomes, spiral polyribosomes (Fig. 134), rough-surfaced endoplasmic reticulum in the form of vesicles and short tubules, Golgi bodies, electron- lucent vacuoles and electron-dense vesicles. In some the central area contain very little cytoplasm (Fig. 134). These problematical structures are discussed on p. 377. During this stage the lacunae are lined by basement membranes, measuring up to 0. 14 p.m thick. Fig. 136 shows the haemocytes which lie on the surface of a basement membrane and •

254

Fig. 132. T.S. of basal cytoplasmic processes from 24-hour fifth-instar wing pad, showing microtubules and a long

mitochondrion. X 45000. • m, mitochondria; mt, microtubules; pr, polyribosomes.

Fig. 133. Portion of basal cytoplasmic process from a 24-hour fifth-instar wing pad. The basal tips of two epidermal cells from apposed integuments are in contact by desmosomal attachments. X 26000. cp, cytoplasmic process; d, desmosome. 1 3 2

1 3 3 •

256

Fig. 134. T.S. of 24-hour fifth-instar wing pad, showing epidermal cells and their basal processes. X 12000. G, Golgi body; m, mitochondria; n, nucleus; ncl, nuceolus.

Fig. 135. T. S. of 24-hour fifth-instar wing pad lacuna, showing the basement membrane and a mass of membrane - bounded extracellular exudate. X 15000. bm, basement membrane; er, endoplasmic reticulum; r, ribosomes. •

134

135 •

258

Fig. 136. 24-hour fifth-instar wing pad, showing basement membrane lining thelacuna and an adjacent haemocyte. X 12000. bm, basement membrane; haem, haemocyte; lac, lacuna.

Fig. 137. T. S. of cuticle and a portion of apical cytoplasm of epidermis from 48-hour fifth-instar wing pad, showing apical plasma membrane in contact with cuticle is showing microvilli (with dark tips). X 15000. my, microvillus. 136

137 s

260

seem to be discharging their inclusions, which are merging into the surface of the membrane as described by Wigglesworth (1972). Apart from the regions near the lacuna, the wing pad does not seem to possess any middle membrane. By 48 hours after the previous ecdysis, most of the epidermal cells of the fifth-instar wing pads have increased in • depth and have become more columnar than before. The nuclei are elongated perpendicular to the cuticular surface, and are more centrally disposed in the cell. The nuclear membranes are irregular in outline; some nuclei possess a prominent nucleolus and some contain large dispersed chromatin masses, with no signs of a nucleolus. Some cells are now rounding up to divide. In the majority of cells the apical plasma membrane is in close contact with the cuticle, and is thrown into microvilli, with electron dense tips (Fig. 137). There is still no evidence of a clear exuvial space. The apical cytoplasm of the epidermal cells from the pre- apolysis areas of the wing bud contain groups of single ribosomes, spirally arranged polyribosomes, rough-surfaced endoplasmic reticulum in the form of vesicles and short cisternae with swollen areas, ovoidal and elongated mitochondria and microtubules; well developed Golgi bodies associated with electron-dense vesicles and vesicles enclosing dense secretions around the nucleus are also found. The apical cytoplasm also contains scattered dense vesicles, other membrane-bounded vesicles with dense secretions similar to the contents of the Golgi bodies, and lysosome-like bodies. There is an increase in the extent of the lateral plasma membranes, as mentioned in the fourth-instar. Some of the micrographs from this 48-hour fifth-instar wing-pads show nuclear structure during the onset of moulting. During the growth period the columnar epithelial cells contain elongated nuclei, which are irregular in outline, and will round up and divide. While doing so, the nucleoli disappear, and the chromatin is more uniformly distributed in the nucleoplasm. • r

261

During this period, the apical cytoplasm of the cells contain single and polyribosomes, rough-surfaced endoplasmic reticulum, with swollen areas or fully swollen cisternae, Golgi bodies containing dense secretions, spherical mitochondria, electron-lucent vacuoles, electron-dense vesicles, and a few lysosome-like bodies. The apical cytoplasm of the cell, at the onset of apolysis • shows an enormous number of swollen vesicles of rough-surfaced endoplasmic reticulum and a few cisternae. The extra cellular space between the basal areas of the cells is now very reduced. The plasma membrane covering the tip of the basal area of the cell lies close to the same structure in the integument of the opposite side of the wing pad (Fig. 138). There seems very rarely to be small patches of basement membrane between them. More often these plasma membranes are separated by a narrow intercellular space (Fig. 138). The basal cytoplasm contains single ribosomes (either free or in groups), short cisternae of rough- surfaced endoplasmic reticulum, spherical mitochondria, numerous well developed Golgi bodies containing electron-dense vesicles and electron-lucent vesicles with dense secretions; microtubules, large and small forms of electron lucent vacuoles, and a few lysosome-like bodies are also present (Fig. 138). The basement membrane is present only around lacunae and is up to 0. 6 pm thick. In histological preparations of 72-hour, fifth-instar wing- pads, during the first and second day of apolysis, the epidermis in some places apparently has a stratified appearance, arranged in several layers, though elsewhere, it is still a single layer. The electron microscopic evidence of the course of plasma membranes shows, however, that the epidermis is still essentially a single layer of cells, though their nuclei are arranged at several distinct levels. The epidermis consists of tall columnar cells with an elongate, lobed or ovoid nucleus. While the cells are actively dividing, some are simultaneously degenerating. Active mitotic division and cell degeneration during apolysis was noticed •

262

Fig. 138. T. S. of epidermal layers from 48-hour fifth-instar wing pad. The basal processes contain numerous Golgi bodies, some elongated, branched profiles of mitochondria and microtubules. X 15000. elv, electron-lucent vacuole; G, Golgi complex; m, mitochondria; mt, microtubules.

Fig. 139. T. S. of 72-hour fifth-instar wing pad integument showing detachment of epidermis; exuvial space filled with ribosomes and ribosome coated vesicles. X 12000. cut, cuticle; ep, epithelium; exsp, exuvial space; n, nucleus. s

138

139 •

264

by Wigglesworth (1954). Before apolysis the apical plasma membrane lying close to the cuticle is folded to form long stacks of microvilli with their dark tips towards the cuticle. The apical and lateral cytoplasm contains single free ribosomes, cisternae (mainly short with a few elongate profiles) of rough- surfaced endoplasmic reticulum and a few vesicles; but in general, there is not much rough surfaced endoplasmic reticulum. Spherical and elongate mitochondria, and Golgi bodies seem to be less numerous than at the previous stage. Electron-dense vesicles, electron-lucent vacuoles, autophagic vesicles and microtubules are also present in the apical and lateral cytoplasm. Lysosome-like bodies are not common in 72-hour, fifth-instar wing-pads, whereas they were very abundant in 72-hour fourth-instars. The nuclei contains a prominent nucelolus, resembling thick anastomosing strands, with diffuse chromatin, also present in the nucleoplasm. In the dividing cells, the nuclear envelope appears to be reconstituted by the coalescence of vesicular elements of endoplasmic reticulum. The daughter cells are at first much smaller than their parents. The basal cytoplasm of the cells contains single free ribosomes, a few spirally arranged polyribosomes, spherical and elongate mitochondria, and cisternae of rough-surfaced endoplasmic reticulum; they usually also contain one or two well-developed Golgi bodies with more electron-dense vesicles and electron-lucent vesicles, containing dense secretions; microtubules, electron-dense vesicles and electron lucent vacuoles can also be seen.' The basal ends of apposed integumentary cells lie close together, with their thickened plasma membranes separated by an intercellular space. During this stage, small patches of basement membrane are visible between the apposed epidermal cells and a thick layer of basement membrane forms a lining to the lacuna. The extra cellular spaces of the wing pad integument contain membrane-bounded vesicles of different sizes, filled with ribosomes, a few cisternae of rough-surfaced endoplasmic reticulum, electron-dense vesicles and electron lucent vacuoles. These vesicles have a diameter of up to 3. 7 pm. The 265

cytoplasm of some retracting epidermal cells with a round nucleus, contains large vesicles coated by ribosomes. Such structures are found free in the fully formed exuvial space (Fig. 139). By 120 hours, the fifth-instar larval wing-pad epidermis is almost completely detached from the old cuticle, except for a few areas. The epidermal cells are arranged in several layers; they are smaller in size and less columnar than before. Most of the cell is occupied by an oblong nucleus. Among the basal processes of the wing-pad epidermal cells, only a little extracellular space is now left because of the increase in cell number and their re-arrangement (Fig. 140). There are no traces of basement membranes between the apposed cell layers, except around the lacunae. The apical plasma membrane of some cells is folded into long stacks of microvilli with dark tips. Small vacuoles appear in the intercellular boundary, some distance below the intermediate junction, in the way Wigglesworth (1975) described, arising between the two lateral plasma membranes. The apical cytoplasm usually contains groups of single ribosomes, spiral polyribosomes, numerous spherical and elongate mitochondria, microtubules, electron-lucent vacuoles, electron-dense vesicles, cisternae of ribosomal endoplasmic reticulum in the form of moderately long tubules, multivesicular bodies, some electron-lucent areas containing alpha-glycogen particles, in close association with Golgi bodies. The latter consist of electron-dense vesicles, large electron-lucent vacuoles and longer stacks of flattened sacculi. Fig. 140 shows a large basal area, which contains clusters of single ribosomes, a few spiral polyribosomes, oval and elongate forms of mitochondria, microtubules, microbodies and smaller electron dense vesicles, large electron-lucent vacuoles and a very little granular endoplasmic reticulum. Among the ordinary epidermal cells, there occur large flask shaped cells with a small cytoplasmic volume and large •

266

Fig. 140. T. S. of 120-hour fifth-instar wing, showing cuticle, epidermis, basement membrane, tracheoles and exuvial space containing isolated cells and dense secretions. X 5000. • bm, basement membrane; cut, cuticle; ep, epidermis; exsp, exuvial space; tl, tracheoles.

Fig. 141 T.S. of 120-hour fifth-instar wing pad epidermis, showing degeneration of an epidermal cell. X 8000. dgc, degenerating cell; ep, epidermis; n, nucleus. •

•

140

141 268 nucleus containing several chromatin masses. The cytoplasm of these cells contains single ribosomes arranged in groups, very little rough-surfaced endoplasmic reticulum, microtubules, scattered mitochondria, perhaps one or two Golgi bodies, electron-dense vesicles and multivesicular bodies. Similar cells are present in Fig. 141, but the nuclear envelope around the chromatin seems there to have disappeared. These are perhaps degenerating cells, in which the formation of "chromatin droplets" is followed by disintegration of the nuclei in the way observed by Wigglesworth (1942) and Lawrence (1966). Fig. 142 shows a newly differentiating bristle, containing a shaft-forming trichogen cell, and a socket-forming tormogen cell. The apical trichogen cell is smaller than the tormogen cell, and contains an ovoidal nucleus, with large masses of centrally and peripherally arranged chromatin; the cytoplasmic volume is very small, but it contains polyribosomes and a few mitochondria. The tormogen cell, embraces the basal and lateral regions of the trichogen cell, from which it is separated by intercellular membranes. Most of the tormogen cell is occupied by a large, U-shaped nucleus containing masses of chromatin, organised in the centre and periphery. The cytoplasm is very limited, but contains polyribosomes, microtubules and mitochondria (Fig. 141). The basal surfaces of these cells are associated with axons, but the neurones from which these originate could not be located. On the seventh day after ecdysis, the pharate adult wings are fully formed and folded within the old cuticle (Figs. 143 and 144). A cuticulin layer about 34 nm thick lines the external surface of the wings (Fig. 143). The epidermal cells are mostly arranged in a single layer. Fig. 144 shows the three-layered nature of the cuticulin; the outer two layers are electron-dense, and the middle one is least dense (Locke, 1966). The apical microvilli have now withdrawn from the cuticulin by a gap of 25 nm. Micrographs taken from the base of the wings show the microvillate condition of the apical plasma membranes (Figs. 144 and 147), 269

Fig. 142. T.S. of trichogen and tormogen cells from 120-hour fifth-instar wing pad. X 13000. a, axon; ton, tormogen nucleus; trn, trichogen nucleus. •

Fig. 143. Low power micrograph of pharate adult wing, showing their heavily folded nature while lying inside old cuticles. X 5000. cut, cuticle; ep, epidermis; m, mitochondria; n, nucleus.

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and some other photographs obtained from the middle and apical portions of the wings show that the apical plasma membrane, adjacent to the cuticle, is folded towards the cytoplasm to form inwardly directed tubules with associated vesicles (Fig. 145). Locke (1969) says that the apical plasma membranes undergo a transition at the onset of protein epicuticular secretion. The cells are now less columnar than before and usually contain an oblong nucleus with a prominent nucleolus and dispersed chromatin in the nucleoplasm (Figs. 144 and 147). The longitudinal axis of the nucleus may lie parallel or perpendicular to the apical and basal axis of the cell, depending on the area of the fold it occupies; it usually lies near the base of the cell. The apical cytoplasm contains single free ribosomes, polyribosomes, rough- surfaced endoplasmic reticulum in the form of swollen vesicles and short cisternae, spherical and elongate mitochondria, Golgi bodies lying between the cuticle and nucleus and associated with flattened cisternae of agranular reticulum, electron-dense vesicles and coated vesicles (Figs. 145 and 147). The apical cytoplasm also contains large and small forms of electron-lucent vacuoles, electron-dense vesicles, microbodies, many coated vesicles, autophagic bodies and microtubules (Figs. 144 and 145). Fig. 144 shows a folded apical area of the cell containing very large electron-lucent vacuoles. The basal regions of the cells are not produced into long stout processes, supported by microtubules, as in the 140-hour fourth- instar wing pad epidermal cells. Some of the epidermal cells give off cytoplasmic processes from their basal surfaces. These contain ribosomes, mitochondria, a few short cisternae or vesicles of rough-surfaced endoplasmic reticulum (some of them swollen), microtubules and dense vesicles (Fig. 146). These processes invade the adjacent inter-cellular space, and become entangled in all directions with similar processes from other cells (Wigglesworth, 1953, 1959, 1977) (Fig. 146). At some places, where two apposed cytoplasmic strands come in contact Fig. 144. T. S. of folded pharate adult wing. X 20000. cut, cuticle; dsv, electron dense secretory vesicle; elv, electron-lucent vacuole; G, Golgi body; m, mitochondria; my, microvillus; pm, plasma membrane; rer, ribosomal endoplasmic reticulum. •

273

Fig. 145. T.S. of pharate adult wing integument showing a continuous layer of outer epicuticle, plasma membrane infoldings, electron-dense vesicles, coated vesicles, polymorphic mitochondria and distended rough-surfaced endoplasmic reticulum. X 20000. cut, cuticle; cv, coated vesicle; dv, dense vesicle; G, Golgi body; ij, intermediate junction; in, mitochondria; pi, plasma membrane infolding; ver, vesicular endoplasmic reticulum.

Fig. 146. T. S. of pharate adult wing integument, with lacuna and basement membrane. The epidermis has given off several thin filamentous cytoplasmic strands. X 13000. bm, basement membrane; cp, cytoplasmic process; ep, epidermis; lac, lacuna; n, nucleus. 145 I

■■

i • 146 iii •

•

Fig. 147. T. S. of pharate adult wing integument to show microtrichia. X 20000.

cv, coated vesicle; G, Golgi body; nn , mitochondria; mic, n:icrotrichium; my, microvilli; n, nucleus. 276

with each other, the plasma membranes become thicker than before and the intercellular space contains electron-dense desmosome- like material. Wigglesworth (1977) reports that desmosomes appear where cytoplasmic strands come in contact with one another and sometimes where they come in contact with the basement membrane. The latter, however, is present only around the lacunae, where it is about 0. 05 im thick. The fully formed, highly folded late pharate adult wings are still lying inside the old cuticle in 10-day fifth-instar larvae (i. e. late pharate adults). The new cuticle covering the wings is now fully formed, and consists of epicuticle and procuticle. The microtrichia are simple cuticular folds, resembling in some ways the taenidia of the trachea in structure and formation. In the earlier stages the microtrichia develop as simple extensions of the cuticulin, accompanied by cytoplasm; the cytoplasm slowly withdraws, leaving an empty cavity, which is later filled by lamellate cuticle to give a solid spine-like microtrichia (Fig. 148). The plasma membrane lying close to the cuticle is microvillate, with dark tips (Fig. 148). In some micrographs the apical plasma membrane is raised into small dense areas. The cytoplasm is now more electron-dense, and consists of single free ribosomes, a few spiral ribosomes, rough-surfaced endoplasmic reticulum in the form of small lamellae and vesicles, scattered mitochondria (mainly spherical with a few elongate forms), a few Golgi bodies containing mainly electron-dense vesicles, microtubules, electron-dense vesicles, multivesicular bodies, microbodies, electron-lucent vacuoles, large areas filled with rosettes of glycogen particles and some degenerating membranes (Fig. 148). The basal portions of the cells are produced into long stout processes, containing bundles of microtubules, a few short cisternae and vesicles of rough-surfaced endoplasmic reticulum, ribosomes, Golgi bodies with dense and electron-lucent vesicles, mitochondria, and electron-dense vesicles (Fig. 149). The • •

Fig. 148. T.S. of late pharate adult wings, immediately prior to ecdysis; showing fully developed epicuticle and procuticles. The secretory vesicles are now less abundant, and some electron- lucent areas are filled with glycogen deposits. X 15000. cut, cuticle; elv, electron-lucent vacuole; G, Golgi body; ib, isolation body; ler, lamellae of ribosomal endoplasmic reticulum; my, microvilli; n, nucleus.

• 278

processes from the apposed integuments remain free in the haemocoele, apparently supported by longitudinally directed microtubules (Fig. 149). The tips of processes from the apposed integuments sometimes lie close together, with their plasma- membranes separated by a narrow intercellular space (Fig. 149). In some areas the cells are degenerating, with disintegration of the nucleus. The lacunae and tracheae are encircled by thin basement • membranes 0. 04 p.m thick. (c) Tracheae and Tracheoles The newly emerged fifth-instar wing-pads possess six well developed tracheae. Though the pattern is similar to the preceding instar, there has been a great increase in the length and diameter of the tracheae. Each trachea consists of a cuticular intima comprising two layers. The cuticulin forming the inner lining of the tubes. is 30 nm thick and is helically folded into taenidial thickenings. The tracheal tube is not smooth, but raised into small tubercles and micropapillae. A continuous procuticle about 150 nrn thick is present between the cuticulin and epithelial ring and is less electron- dense than the cuticulin. According to Locke (1964) in Calpodes ethlius (Lepidoptera), there is no procuticle layer between taenidia and epithelia, but see the discussion on p. 379. The tracheal epithelia contain single free ribosomes, rough-surfaced endoplasmic reticulum in the form of moderately long cisternae, a few short cisternae and vesicles, spherical and elongate mitochondria, Golgi bodies containing a few electron-dense vesicles and vesicles of agranular endoplasmic reticulum, microtubules, microbodies, autophagic vesicles and electron-lucent vacuoles. The cells are bounded by intercellular membranes with an apical adhesion zone followed by a zone with septate desmosomes and simple, apposed plasma-membranes. The tracheoles are surrounded by considerable tracheoblast cytoplasm, but separated from it by a thin limiting plasma membrane. The outer and inner plasma membranes of the tracheal matrix cells connect by folds in much the s

279

Fig. 149. T.S. of basal cytoplasmic processes of late pharate adult wings. X 26000. cp, cytoplasmic process; G, Golgi body; icsp, intercellular • space; m, mitochondria; mt, microtubules.

Fig. 150. T.S. of central portion of 24-hour fifth-instar wing pad, showing trachea and tracheoles. The tracheal epithelium is dividing automatically. X 8000. n, nucleus; t, trachea; tep, tracheal epithelia; tl, tracheole.

•

281

same way that a nonmyelinated axon is suspended within a schwann cell. For this reason, Edwards et al. (1958) called the connecting plasma membrane a "mestracheon". Some tracheae lie close to the dorsal integument (Fig. 127), without an intervening basement membrane between the tracheal matrix cells and the epidermis. The basement membrane lining the lacuna, however, is closely apposed to the remaining portion of the tracheal matrix cells and is 0. 05 pm thick. One micrograph shows a portion of trachea with a closely apposed nerve; the basement membranes of the nerve and trachea are continuous here and separate the united nerve and tracheal matrix cell from the adjacent lacuna. The relations between lacuna, epidermis and nerve are discussed below (p. 381). The lining of the tracheoles consists of a single cuticulin layer which is helically folded into taenidia. The walls are smooth and do not bear micropapillae. The tracheoles obeserved were included within the main body of a tracheoblast (Fig. 127) or its prolonged cytoplasmic extension. The tracheoles, accompanied by tracheoblast extensions, lie close to the epidermal cells (Figs. 127, 128 and 129), separated by plasma membranes. In 24-hour fifth-instar wing-pads, the tracheal epithelia have detached from the cuticle (Fig. 151). The exuvial space contains small vesicles and a little fibrous material. The tracheal tube is lined by a cuticulin layer. There are no signs of taenidia or endocuticle; presumably the moulting fluid has already digested the endocuticle. The tracheal epithelia in,Fig. 150, perhaps shows a mitotic division of the cells and simultaneous cell destruction. Wigglesworth (1954a) says that the phenomena of growth and decay are responsible for cellular reorganisation and increase in diameter of the tracheal tube. The dividing nucleus in Fig. 150 is very prominent, with two nucleoli, one at each side of a narrow constriction in the middle; the nucleus occupies most of the area in the cell profile. The cytoplasm contains single free ribosomes and a few polyribosomes. The rough-surfaced endoplasmic •

282

reticulum is much more developed in length and abundant, though only a few more vesicular forms are still present; spherical and elongate mitochondria, small scattered Golgi bodies, coated vesicles, electron-dense vesicles, some electron-lucent areas with alpha- glycogen particles, and also microtubules are present (Fig. 150). A tracheal end cell is shown, with a large number of tracheoles lying in the cytoplasmic sheath drawn out from the tracheoblast into long processes lying parallel to the basement membrane around a lacuna. Fig. 151 shows tracheole formation with invasion by the tracheoles into the intercellular space between tracheal matrix cell and the main body of an adjacent tracheoblast. This gives rise to the "vacuolated" appearance discussed by Wigglesworth (1973). Some "vacuoles"(apparently portions of the intercellular space) seem to' be bounded by these plasma membranes, perhaps adding their secretions to the cuticulin of newly forming tracheoles. The mechanism of tracheole formation in insects is, however, obscure and is discussed below. In 48-hour fifth-instar wing pads, the exuvial space of the trachea is occupied by an ecdysial membrane (cf. Passaneau & Williams, 1953; Locke, 1952, 1958, 1964, 1966; Whitten, 1969). The cytoplasm of the tracheal epithelia contain single free ribosomes, numerous polyribosomes in the form of spirals, very few cisternae of rough- surfaced endoplasmic reticulum, numerous mitochondria, a few Golgi bodies with electron-dense vesicles, microbodies, and microtubules. Trachea is surrounded by a portion of tracheoblast cytoplasm, separated by intercellular plasma membranes. The nucleus of the tracheoblast is more compact than those of the tracheal epithelium. Inside the main body of the tracheoblast, several profiles of tracheoles are present. The cytoplasm contains polyribosomes, a few mitochondria, electron-dense vesicles and microtubules. The trachea is encircled by a basement membrane 90 nm thick. In 72-hour fifth-instar wing-pads the tracheal epithelium

•

283

has not yet become organised into a cylindrical tube after undergoing cell division and decay. The exuvial space, containing an ecdysial membrane, is wider than before (Fig. 152). The tracheal epithelium contains single free ribosomes, and spiral polyribosomes, but there has been an increase in the amount of rough-surfaced endoplasmic reticulum (which is in the form of vesicles, short cisternae and long

• tubular cisternae). Well developed Golgi bodies are more numerous than before and consist of electron-dense vesicles, flat tubular cisternae and vesicles of agranular reticulum (Fig. 152). The cytoplasm also contains microtubules, mitochondria, coated vesicles, electron-dense vesicles and multivesicular bodies (Fig. 152). A tracheal end cell enclosing several tracheoles with mestracheon connections is shown in some micrographs. The basement membrane lining the trachea is now 70 nm thick. In micrographs of the 120-hour fifth-instar wing pads, the tracheal epithelial cells have arranged themselves around the exuvial space as a continuous cylinder consisting of a single layer of 2- 3 cells in each cross-section. The trachea is also surrounded by a considerable portion of tracheoblast cytoplasm delimited by a plasma membrane. The exuvial space of the trachea contains an ecdysial membrane and some electron-dense droplets (Fig. 153). The apical plasma membranes of the tracheal epithelia is produced into some raised portions and microvilli with dark tips (Fig. 153), where the adult cuticulin is presumably about to appear (Locke, 1964). The nuclei are elongate, with their longitudinal axis parallel to the apico-basal axis of the cell (Fig. 153); there are no evident nucleoli, but the chromatin is uniformly distributed in the nucleoplasm. The cytoplasm contains single ribosomes, either free or in clusters, spiral polyribosomes, mitochondria, a few cisternae of rough-surfaced endoplasmic reticulum, well developed Golgi bodies (mainly composed of dense vesicles associated with electron-lucent vacuoles), scattered microtubules, some electron- lucent areas with alpha-glycogen particles, and electron-dense vesicles (Fig. 153). The lateral plasma membranes are linked by •

284

Fig. 151. T.S. of a portion of trachea, with newly developing tracheoles,, some old tracheoles and lacuna. X 12000. bm, basement membrane; G, Golgi body; lac, lacuna; • p, plasmamembrane; t, trachea; tl, tracheole.

Fig. 152. T.S. of a trachea and profiles of tracheoles from 72-hour fifth-instar wing pad, showing exuvial space with some damaged portions of ecdysial membrane. X 15000. exsp, exuvial space; m, mitochondria; mt, microtubule. •

151

1 52 • •

153. T.S. of a portion of trachea from 120-hour fifth- instar, showing exuvial space, tracheal epithelia, tracheal end cell and newly forming tracheole. X 25000. exsp, exuvial space; G, Golgi body; mt, microtubules; nt, neurotubules; pm, plasma membrane; t, trachea. •

287

an apical intermediate junction, followed by septate desmosomes for a little distance and then continue as simple, apposed membranes until they join the basal membranes. The tracheae are lined by a basement membrane 70 nm thick. Tracheoblasts were seen in the interestitial space, closely accompanying the epidermal cells supplied by the tracheoles. Tracheoles are present within the main body of the tracheoblast and its sheath-like cytoplasmic extensions • (Figs. 140 and 154). The cuticular lining of the tracheoles is a single electron-dense membrane; wider than the normal intima and presumably representing both intima and plasma membrane. Newly forming tracheoles associated with invaginated plasma membranes, in the form of "vacuolated" intercellular space is again shown in Figs. 153 and 154. This area is closely associated with a Golgi apparatus containing a large number of electron-lucent vacuoles (Fig. 153). The formation of the tracheolar lumen has never been satisfactorily described. Locke (1966) says, "it could be that in some tracheoles the lumen arises by the fusion of vacuoles from the Golgi appartus, or it may be formed entirely by growth of plasma membrane facing the tracheolar lumen." In the micrographs of 160-hour fifth-instar wing pads the exuvial space of the trachea still contains an ecdysial membrane (Fig. 155). In some micrographs (Fig. 155), profiles of tracheoles are also present within the exuvial space. The apical plasma membrane is uniformly raised into small folds with dark tips which are withdrawn from the ecdysial membrane; traces of dense patches of cuticulin occur over the raised areas (Fig. 155). The general surface of the wing pad is now covered by a continuous layer of cuticulin. The intercellular boundaries are visible at the apex of the cells; a conspicuous adhesion zone lies just below the apical boundary, followed by septate desmosomes. There does not seem to be the usual run of simple apposed plasma membranes, most of the adjacent cell boundaries being linked up with septate de smosome s. The cytoplasm of the tracheal epithelia contains •

Fig. 15:. T.S. of tracheal end cell containing several profiles of tracheoles. Micrograph taken from 120-hour fifth-instar wing pad. X 30000. ler, lamellae of rough-surfaced endoplasmic reticulum; n, nucleus; sv, secretory vesicle; tec, tracheal end cell; tl, tracheole•; v, vesicle. 289

single free ribosomes and ribosomes in clusters, a few scattered mitochondria, rough-surfaced endoplasmic reticulum in the form of swollen vesicles and short cisternae, microtubules, multivesicular bodies, electron-dense vesicles, coated vesicles, microbodies and Golgi bodies with electron dense vesicles (Fig. 155). The mitochondria show some peculiar forms during this stage, as I have already mentioned above when dealing with the late pharate fifth- instar (p. 216 ). They show a thin filamentous construction in the middle and gradually bend around the adjacent cytoplasm and encircle it completely in transverse section (Fig. 155). The cytoplasm also contains electron-opaque areas. Fig. 155 shows a trachea, the formation of new tracheoles, a tracheal end cell, and an old tracheal intima. The formation of the tracheolar lumen is quite clear in this picture; the inter-cellular membranes have separated with the formation of a large channel between them. The plasma membrane towards the lumen side are then raised into a small dense area in the same way as the plasma membrane of the tracheal epithelia, during the secretion of the cuticulin. Over these dense areas a thin film of new cuticulin patches become visible. During this period the trachea is not lined by basement membrane. A tracheoblast with several tracheoles lying close to the basement membrane of a lacuna is shown in some micrographs. Micrographs of tracheae from fifth-instar wing pads, prior to ecdysis show the fully formed tracheal tube of the adult enclosing the old cuticle and undigested parts of the ecdysial membrane (Fig. 156). The epicuticle lines the tracheal tube with its characteristic helical taenidia. The epicuticle is raised 'into small tubercles except over the inner face of the taenidia. A relatively dense cuticular-like material, similar to that present within the taenidial fold, occurs between the tracheal lining and epithelium, but it is not present below each taenidial fold during this period; within a short time, when taenidia are completely filled by a dense material, the trachea contain a layer of endocuticle Fig. 155. T.S. of trachea and tracheal end cells from a pharate adult wing, showing the secretion of cuticulin over the raised dark areas of apical plasma membrane, also formation of new tracheole. X 13000. cut, cuticle; t, trachea; tl, tracheole. 1 1 •.

Fig. 156. T.S. of fully formed trachea from pharate adult wings and old tracheal intima. X 20000. bm, basement membrane; my, microvilli; n, nucelus; p, micropapillae; pm, plasma membrane; ti, tracheal intima; tn, taenidia. 292

(Fig. 156). Edwards et al. (1958) reported that, "external to the limiting membrane occurs a second component of low density equal to procuticle. " The apical plasma membrane is produced into long stacks of microvilli (Fig. 156). The tube is enclosed in transverse section by two or three epithelial cells; they are very elongate structures with long, irregular nuclei. The nucleolus is a very compact structure with further peripherally arranged masses of chromatin. The membrane covering the cuticular intima is confluent with the plasma membrane covering the outer surface of the tracheal cell via a folded ''mestrctcheon" (Fig. 156). The cytoplasm is very dense and contains clusters of single ribosomes, individual ribosomes, small profiles of mitochondria, scattered rough-surfaced endoplasmic reticulum in the form of short cisternae, Golgi bodies with electron-dense vesicles and electron- lucent vacuoles, microtubules, electron-opaque vesicles and electron-lucent vacuoles (Fig. 156). A tracheal end cell containing one or more tracheoles occupies a considerable portion of a tracheal epithelial ring. A basement membrane 30 nm thick now encircles the external surface of the tracheal epithelium and is continuous with the basement membrane lining the lacuna. A small portion of the trachea in close contact with the epidermis is separated from it by an intercellular space (Fig. 156). (d) Nerve Supply In the fifth-instar wing pad there are six prominent lacunae running throughout its length, each accompanied by a nerve and a trachea. Though the pattern is similar to that seen in the fourth-instar, there is a great increase in the diameter of the nerves. The sub-costal nerve lies inside the lacuna and is surrounded by a basement membrane (Fig. 157). The radial, medial, cubital and anal nerves lie inside the extra cellular space of the dorsal integument, closely applied to the basal portions of the epidermal cells towards one side; the ventral side is bounded by the basement membrane surrounding the lacuna (Fig. 158). The a sub-costal nerve, on the other hand is completely encircled by a •

293

Fig. 157. T.S. of a nerve from a 24-hour fifth-instar wing pad lacuna. X 30000. a, axon; av, autophagic vesicle; bm, basement membrane; • dv, dense vesicle; m, mitochondria; ma, mesaxon; nt, neurotubules; rer, ribisomal endoplasmic reticulum.

Fig. 158. Section of a nerve from 24-hour fifth-instar wing pad. X 15000. G, Golgi body. bm

r 157

e •

295

basement membrane which is separate from the basement membrane encircling the lacuna. A micrograph from the newly emerged fifth-instar wing pad shows the sub costal nerve to consist of several axons ensheathed by neuroglial cells, and surrounded externally by a basement membrane 150 nm thick. The mesaxon coils around the axon are separated by glial cytoplasm which contains ribosomes, a few i cisternae of rough-surfaced endoplasmic reticulum, numerous microtubules, spherical and elongate mitochondria and a well developed Golgi body containing electron-dense vesicles and agranular reticulum. The axoplasm contain neurotubules, small vesicles and mitochondria. The nerve and trachea lie in the centre of the lacuna. The basement membrane of the nerve is fused with that of the trachea where they are in contact. The axon associated with the trachea runs longitudinally in a groove in the tracheal matrix cell. This is lined by plasma membrane, the nerve and trachea being enclosed by a common basement membrane. A micrograph. from 24-hour fifth-instar wing pad shows the sub-costal and radial nerves and their arrangement in the lacuna. (Fig. 157). The peripheral nerve entering the sub-costal lacuna lies in the centre of the lacuna, encircled by its own basement membrane 2 dun thick. This multiaxonal nerve contains 14 large axon profiles lying inside the glial cytoplasm; some ensheathed singly and some axons in a group (Fig. 157). Edwards (1960) says an axon must be over 1 pm in diameter to qualify for its own glial sheath and larger axons have more elaborate glial sheaths. Smith & Treherne (1963) say that minute fibres are aggregated in bundles sharing common glial cells. The mesaxons are prominent, making two or three turns about the axons, but these membranes never become myelinated; the coils are separated by the cytoplasm of the glial cell. The glial cytoplasm contains microtubules, single free ribosomes, spiral polyribosomes, a few cisternae of ribosomal endoplasmic reticulum, moderately dense vesicles, and electron-lucent vacuoles (Fig. 157). Some mito- •

296

chondria are very large, some are elongate structures with a thin filament-like construction in the middle. These bend around the cytoplasm and eventually form what appear to be closed autophagic bodies (Fig. 157). The axoplasm contains a large number of neurotubules, but few vesicles and mitochondria. The radial nerve fibre of a 24-hour fifth-instar wing pad lies between the basement • membrane of the lacuna and the dorsal epidermis (Fig. 158) and contains several axons aggregated in three or four bundles. Each bundle is encircled by glial cytoplasm. Glial nucelus is elongate with dispersed chromatin. The extracellular space between the nerve and epidermis contains some electron-dense and electron- lucent vacuoles. In Fig. 158 a small dendritic process is present between the nerve and basement membrane of the lacuna. The radial nerve is not enclosed in a basement membrane. In 48-hour fifth-instar wing pads, the glial cell enveloping the nerve possesses a very long nucleus (approximately 6 1im long), which contain chromatin masses. The glial cytoplasm contains microtubules, spiral polyribosomes, mitochondria, large vesicles of rough-surfaced endoplasmic reticulum, and electron-dense vesicles. The axoplasm contain neurotubules, large vesicles and more mitochondria. In the 72-hour fifth-instar wing pad some nerves contain a glia showing the small recently divided cells in T. S. , with a small irregular nuclei containing masses of chromatin. Panov (1962) has shown that the glial cells divide in cyclic fashion in relation to the moult. An electron micrograph obtained from the pharate adult wing base prior to ecdysis shows a nerve containing several hundred axons, arranged in bundles separated by glial cytoplasm. The glia between them is densely packed with a mass of membranes with a few large areas of cytoplasm containing much electron-dense material and electron-lucent vacuoles. The nerve fibre is in close contact with treachea and epidermis. f

297

5. Adult Wings After ecdysis into the adult stage the wings are expanded and

flattened by increased blood pressure due to contraction of skeletal. muscles. The newly emerged adult Oncopeltus wings are 3- 4 times longer than the fifth-instar wing pads, and are pale, transparent structures containing six longitudinally directed lacunae. Each lacuna encloses a trachea, nerve and circulating blood cells. The a development of adult veins has not been completed in the pharate adult and newly emerged adult wings. The deposition of cuticle around the lacuna to form adult veins takes place a few hours after emergence. The ultrastructure of the mesothoracic wing pads was studied in this investigation. The membranous portion of the fore wings and the hind wings appear to undergo similar changes. The hemelytra of Oncopeltus show a special ultrastructural interest associated with the development of trabeculae, the corio-membranic border, the veins and the peculiar process of tracheal retraction in the membrane (p. 48 ). I have noted conditions in the corium, corio- membranic region and membrane of adult wings when newly emerged and at 18, 32 and 48 hours after ecdysis. (a) Newly emerged adult wings; membrane: Micrographs of a transverse section of membrane show a cuticle 0. 75 pm thick dorsally and 0. 35 p.m ventrally. The cuticle of the newly emerged wing is less electron dense (except for the outer epicuticle) than at later stages. The histological and ultrastructural appearance of the epidermal cells at adult emergence resembles that of newly moulted fifth-instar wing pads. The epidermis consists of a thin layer of flattened, more or less conical cells, which are broader than long and interdigitate laterally (Figs. 159, 161 and 162). The basal surfaces of the cells are free inside the wing, supported by bundles of microtubules lying in the long axis of the cells (cf. the microfibrils of Hundertmark, 1935; and the tonofibrils of Holdsworth, 1942). a

298

Fig. 159. T. S. of newly moulted adult wing showing dorsal integument from the tip of membrane. X 12000.

cut, cuticle; elv, electron-lucent vacuoles; G, Golgi •. body; m, mitochondria; mt, microtubules; mv, microvilli; n, nucleus.

Fig. 160. T.S. of newly moulted adult wing, dorsal integument from tip of membrane to show coated vesicles and multivesicular body in the epidermis; haemocyte with long processes is present in centre of the wing membranes. X 17000. cv, coated vesicle; er, endoplasmic reticulum; haem, haemocyte; mvb, multivesicular body. . ,, ••: •

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Adjacent to the lacunae, basal processes of the apposed cell layers come into contact with each other by their plasma membranes and are usually joined through a desmosomal attachment; Wigglesworth, 1977. There does not appear to be any continuous basement membrane over the irregular inner surfaces of the cells, except where their processes surround the lacunae (Fig. 162). The lacunae in the tip of the wing do not contain trachea, but they enclose one or two tracheoles and a small nerve fibre (Fig. 162). The apical surfaces of adjacent cells are in contact with each other by their plasma membranes and are joined by apical intermediate junctions, followed by a zone of septate de smosomes. The areas occupied by nuclei and basal processes of cells are separated by large extracellular spaces. The epidermal cells enclose small elongate nuclei which are irregular in outline, containing a prominent nucleolus and a dense mass of chromatin. The nuclei of the epidermal cells often lie within cytoplasmic processes (Fig. 162). A micrograph of the dorsal integument from the extreme tip of the membrane shows an apical plasma membrane closely applied to the cuticle by its microvillate folds (Fig. 159). The remainder of the dorsal integument and all the ventral integument has apical plasma membranes in close contact with the cuticle, but with inwardly directed tubular infoldings associated with adjacent vesicles (Figs. 161 and 162). The apical cytoplasm contains single free ribosomes, polyribosomes, vesicular and short cisternae of rough-surfaced endoplasmic reticulum, circular and oval mitochondrial profiles aggregated in groups, well developed Golgi bodies (consisting of electron-dense vesicles and electron-lucent vacuoles), coated vesicles, microtubules, electron- dense vesicles, isolation bodies, a few lysosome-like bodies and small and large electron-lucent areas (Figs. 159, 160, 161 and 162). The basal cytoplasm is occupied by bundles of microtubules, a well developed Golgi body, a few mitochondria, ribosomes, granular endoplasmic reticulum and usually large electron-lucent • vacuoles (Figs. 159 and 162). The latter recall a report by •

301

Fig. 161. T. S. of newly moulted adult wing, ventral integument from tip of membrane. X 17000.

cut, cuticle; G, Golgi body; mt, microtubules; n, nucleus; I pi, plasma membrane infolding.

Fig. 162. T. S. of epidermis, basement membrane, nerve and lacuna from newly moulted adult wing (dorsal side). X 25000. bm, basement membrane; ep, epidermis; G, Golgi body; mt, microtubules; nv, nerve.

303

Seligman & Filshie (1975) that the first ultrastructural indication of epidermal degeneration in Lucilia cuprina is the appearance of large vacuoles and extracellular channels penetrating the cells. Golgi bodies containing electron-dense secretions and electron-dense vesicles in the basal cytoplasm, where the basal cell processes from the apposed epidermal layers are joined by desmosomal attachments are among other features of the epidermis. A fine nerve, lying towards the dorsal side of the lacuna, innervates the tip of the wing; one or two axons lying in the centre of the nerve are encircled by 4 or 5 layers of mesaxon (Fig. 162). The glial cytoplasm separating the mesaxon folds contains numerous microtubules and a few ribosomes. The glial cell lying around the axons and mesaxons contains a very well developed Golgi body composed of numerous electron-dense vesicles, inner saccules, electron- lucent vacuoles, numerous mitochondria, single free ribosomes, polyribosomes and very few short cisternae of granular endoplasmic reticulum. The nerve is interposed between the basal surface of the epidermal cell and a basement membrane 50 nm thick encircling the lacuna. Micrographs from the wing membrane 100 p.m basal to the tip show lacunae enclosing several tracheoles lying inside a cytoplasmic sheath and covered by a plasma membrane (Fig. 164). This arrangement of tracheoles gives a bunch-like appearance under the light microscope. The cytoplasmic sheath contain very little cytoplasm, but includes a few polyribosomes, a few short cisternae of rough- surfaced endoplasmic reticulum, a well developed Golgi body consisting of electron-dense vesicles, isolation bodies, smooth membrane-bounded vesicles, a very few mitochondria, and scattered microtubules. The inner diameter of the tracheolar lumen is 0. 5 pm. Fig. 164 shows the plasma membrane connection between two tracheoles. In these newly formed tracheoles the intima and encircling plasma membranes are very clear, and the latter are joined by intercellular membranes •

304

(Fig. 164), indicating the development of tracheoles within the plasma membranes. In the wing of a newly emerged adult, six longitudinal tracheae, each enclosed in a lacuna, are very clearly seen by light microscopy. They end about 150 pm basal to the tip of the membrane, giving off bunches of tracheoles to the tip itself. In the membrane, the ventral surface of the trachea lies close to the • epidermis, separated by a narrow intercellular space, bounded by their plasma membranes; the lateral and dorsal surfaces of tracheal profile are bounded by the same basement membrane as lines the lacuna. The lumen of the tracheal tube is lined by a cuticulin layer 40 nm thick, followed by less dense cuticle 80 nm thick. Micro- papillae are present on the inner surface of the cuticulin (Fig. 163). The cytoplasm of the tracheal epithelia contains single free ribosomes, spiral polyribosomes, numerous circular and oval mitochondrial profiles, microtubules, electron-dnese vesicles, multivesicular bodies, and short cisternae and long tubular cisternae of rough-surfaced endoplasmic reticulum; Golgi bodies consisting of electron-lucent vacuoles filled with dense secretions are present near the sides of the nuclei (Fig. 163). Profiles of tracheoles lying inside the mestracheon folds are present within the tracheal epithelia. The lateral surfaces of the epithelial cells are bounded by apical intermediate junctions followed by septate desmosomes and simple apposed plasma membranes. Newly emerged adult wings; corio-mernbranic border: Micrographs from the dorsal integument show cuticle 1. 2 am thick, while on the ventral integument it is 0. 8 lam thick. Microtrichia are present on both sides (Fig. 165). The epidermal cells are usually tall and columnar, with large, irregularly lobed nuclei. A prominent central nucleolus is present (Fig. 166), and chromatin masses are arranged around the periphery. The basal cytoplasmic processes from opposite sides of the wing remain free in the middle of the wing, and are again apprently supported by longitudinally 305

Fig. 163. T. S. of trachea and epidermis from portion of newly moulted adult wing membrane, showing their relationship. X 26000. A ep, epidermis; icsp, intercellular space; m, mitochondria; mt, microtubules; t, trachea; tep, tracheal epithelia.

Fig. 164. Group of tracheoles from membrane of newly moulted adult wing; a plasma membrane connection occurs between two tracheoles. X 25000. G, Golgi body; pm, plasma membrane; tl, tracheole. 163

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Fig. 165. T.S. of newly moulted adult wing integument (ventral), adjacent to corio-membranic border. X 17000.

cut, cuticle; dv, dense vesicle; elv, electron-lucent vacuole; • G, Golgi complex; m, mitochondria; mic, inicrotrichiutu; mt, microtubule; pi, plasma membrane inf olding.

Fig. 166. Micrograph of a portion of epidermis and trachea from newly moulted adult wing, adjacent to corio- membranic border. X 26000. ep, epidermis; p, micropapillae; t, trachea; ti, tracheal intima. •

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directed microtubules. These processes from apposed cells sometimes come into contact by a desmosomal attachment (Fig. 167). A delicate and discontinuous (or fenestrated) middle membrane, at least partly secreted from thin processes of the haemocytes, is present between the two epidermal layers. The basement membrane lining the lacunae is slightly thicker than these

• fragments of middle membrane. During this period, haemocytes are found in the middle of the wing membranes and inside the lacunae. The apical plasma membrane of the epidermal cell, which is in close contact with the cuticle, is produced internally into long tubules associated with adjacent vesicles and sometimes in direct contact with the Golgi bodies (Fig. 166). The apical cytoplasm contains numerous large, well developed Golgi bodies associated with more numerous vesicles filled with dense contents. Single free ribosomes, polyribosomes, numerous circular and elongate mitochondrial profiles, vesicular and short cisternae of rough-surfaced endoplasmic reticulum, microtubules, electron- dense vesicles, lysosome-like bodies and electron-lucent vacuoles of different sizes are also present (Figs. 165 and 166). A considerable portion of the basal cytoplasmic process is occupied by bundles of microtubules and a few cytoplasmic organelles may also be found there (Figs. 165 and 167). The basal portions of some cells are produced into long thin processes, running towards the lacuna (Figs. 168 and 169). The basal cytoplasm contains single free ribosomes, microtubules, mitochondria, rough- surfaced endoplasmic reticulum (very sparse), regions filled with beta-glycogen, electron-lucent vacuoles and lysosome-like bodies (Figs. 168 and 169). The basal processes entangle in all directions with those from other cells (Fig . 168; cf. Wigglesworth 1954, 1959, 1977), or are attached to the basement membrane. The basement membrane of lacuna is well developed, 0. 2 pm thick. The tracheae from the corio-membranic border of the wing of newly emerged adults are oval in cross-section, and 310

Fig. 167. Basal processes of apposed epidermal cells, joined by desmosome connections. X 26000. cp, cytoplasmic process; d, desmosomes; mt, microtubules. •

Fig. 168. Micrograph showing thin filament-like cytoplasmic processes, basement membrane, lacuna and haemocyte from a newly emerged adult wing corio-membranic area. X 8000. bm, basement membrane; cp, cytoplasmic process; g, glycogen; haem, haemocyte. •

•

167

• 168 •

312

Fig. 169. T. S. of a portion of newly moulted adult wing, corio- membranic border, showing a thin process of epidermis, basement membrane and. lacuna. X 17000. a bm, basement membrane; cp, cytoplasmic process; G, Golgi body; lac, lacuna; m, mitochondria; mt, microtubules.

Fig. 170. T. S. of a portion of trachea from newly moulted adult wing, corio-membranic border. X 26000. ep, epidermis; t, trachea; ti, tracheal intima; tl, tracheole; tn, taenidia. •

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Fig. 171. Portion of a trachea from newly moulted adult wing, corio-membranic border; to show swollen ribosomal endoplasmic reticulum. X 26000. • bm, basement membrane; edv, electron-dense vesicle; p, micropapillae; rer, ribosomal endoplasmic reticulum; t, trachea; tn, taenidia.

Fig. 172. T.S. of integument from corium of newly moulted adult wing. X 17500. elv, electron-lucent vacuole; G, Golgi body; mt, microtubule. 4J

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316

closely applied to the ventral integument by their fused basal membranes. Desmosome attachments sometimes occur between their plasma membranes. The tracheal tube is lined by a dense cuticulin layer bearing superficial micropapillae except over the inner face of the taenidia. The micropapillae in this area seem to be more numerous than over the trachea of the membrane (Fig. 170). The taenidial

w folds of the tracheae seem to be helical (Fig. 171), and their dense epicuticle is followed by a less dense cuticle similar to the taenidial material, and here measuring 150 nm thick (Fig. 171). In transverse section, each trachea is enclosed by three or four squamous epithelial cells, arranged in a single layer and containing a nucleus of irregular outline. There is no evident nucleolus, but chromatin is dispersed in the nucleoplasm (Fig. 170). The cytoplasm contains single free ribosomes, vesicles and short cisternae of rough- surfaced endoplasmic reticulum. The numerous mitochondria are concentrated towards the ventral side of the tracheal epithelium, where it is closely applied to the lower epidermis. Very small Golgi bodies are present around the nuclei and consist of a few dense vesicles (Fig. 170). Microtubules running parallel with the long axis of tracheal cell, electron-dense vesicles, and electron- lucent vacuoles are also present in the cytoplasm (Figs. 170 and 171). The tracheoles enclosed within their tracheoblast membranes are enveloped inside tracheal matrix cells (Fig. 171). The outer surface of the tracheal epithelia is covered by a thin basement membrane (Fig. 172). Newly emerged adult wing; corium: The cuticle from the dorsal integument is 1.4 p.m thick, and from the ventral integument 0. 9 p.m (Fig. 172). Microtrichia about 1 p.m long and 0. 4 p.m in diameter are present on both surfaces of the wing. The epidermis consists of tall columnar cells with large nuclei, found at various levels between the apical and basal ends of the cell. The latter is variously shaped and elongate; lobed and spheroidal forms occur (Figs. 172 and 173). Only a few nuclei appear to contain small nucleoli. Most of them show chromatin a

317

Fig. 173. T.S. of epidermis and adjacent nerve of corium, from a newly moulted adult wing. X 17000. bm, basement membrane; cv, coated vesicle; G, Golgi body; m, mitochondria; mt, microtubule; n , nucleus; nv, nerve.

Fig. 174. T. S. of a portion of a trachea and epidermis, from corium of newly moulted adult wing; tracheal epithelia and epidermal cells have given off cytoplasmic processes which are in contact with each other. X 35000. cp, cytoplasmic process; d, desmosome; ep, epidermis; t, trachea. •

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319

dispersed in the nucleoplasm and peripherally distributed (Figs. 172 and 173). The basal cytoplasm of the cells is produced into long processes, which meet the similar process from the apposed integument and are separated from them by a narrow intercellular space (Fig. 172). Desmosomal attachments are sometimes found. The extracellular space is less abundant in the corium than in the membranous portion of the wing. There is no continuous basement membrane between integuments, except around the 1acuna.e. Bent portions of the thin basal processes of the epidermal cells, desmosomal attachments and a discontinuous or fenestrated basement membrane secreted by haemocyte processes are responsible for the ostensibly continuous middle membrane seen by light microscopy. The apical plasma membrane of the epidermis is folded internally into numerous long tubular structures associated with dense vesicles (Figs. 172 and 173). The cytoplasm is strongly electron-dense and contains single free ribosomes, spiral polyribosomes, rough- surfaced endoplasmic reticulum (in the form of short cisternae and vesicles), circular and elongate mitochondrial profiles and a few scattered Golgi bodies associated with dense vesicles; numerous electron-dense vesicles, lysosome-like bodies and electron-lucent vacuoles are also present (Figs. 172 and 173). Numerous scattered microtubules and bundles of microtubules running from the cuticle, to the basal end of the epidermal cell are shown in Fig. 172. This also illustrates two basal cytoplasmic processes from apposed integuments, containing microtubules and joined by a thick desmosomal attachment. On its approach to a trachea, the basal processes of the epidermal cells give off several processes to meet the tracheal epithelium. The cytoplasm of these basal processes contains a few individual ribosomes, short cisternae of ribosomal endoplasmic reticulum, mitochondria, electron-dense vesicles and microtubules running parallel to the long axis of the cell (Fig. 174). Some stouter basal processes also contain elongate cisternae of rough-surfaced endoplasmic reticulum, very large Golgi bodies with more 320 numerous vesicles and a few saccules, isolation bodies and multivesicular bodies. The basement membrane lining the lacuna are up to 0. 25 p.m thick. A transverse section from the base of the wing shows cuticular deposits around the ventral surface of the lacuna. Tracheae from the corium are also oval in cross section, with an electron-dense cuticulin layer lining the lumen and folded helically into taenidia. The luminal surface of the dense cuticulin bears numerous micropapillae except over the inner surface of the taenidia (Fig. 174). Dense cuticulin is followed by a less dense cuticle up to 150 nm thick (Fig. 174). The tracheal intima is encircled by a thin layer of epidermis consisting in T. S. of 3 or 4 epidermal cells. The apical half of their plasma membranes are linked by septate desmosomes, followed by a zone containing only simple apposed membranes (Fig. 174); the latter are separated locaclly (Fig. 174). The trachea occupies the ventral portion of the lacuna and is encircled by a thin basement membrane. Towards its ventral side the tracheal epithelium produces several cytoplasmic processes (Fig. 174). Where this occurs, the basement membrane of the trachea is interrupted and the cytoplasmic processes of the trachea comes in contact with the basal processes of the epidermal cells and their plasma membranes are then joined by de smosomal attachments (Fig. 174). The cytoplasmic processes of a trachea contain ribosomes, short cisternae of endoplasmic reticulum and microtubules (Fig. 174). The tracheal epithelium contains individual ribosomes, a few spiral polyribisomes, as well as vesicular and short cisternae of ribosomal endoplasmic reticulum, most of which are dilated (Fig. 174). A few mitochondria, several microtubules, a few Golgi bodies (consisting of dense vesicles), multivesicular bodies, electron-dense vesicles and electron-lucent vacuoles are also present in the tracheal epithelia (Fig. 174). The cytoplasm is generally very electron-dense. During this period mitochondria are more numerous in the tracheal epithelium from the membrane of the wing than in the corium. a

321

(b) 18-hour adult wing; membrane: The cuticle of the wing has increased in thickness during the 18-hours since ecdysis; it is now from 1. 0 to 1. 3 p.m thick in different areas of the dorsal integument (Figs. 175 and 176), and 0. 5 - 0. 6 p.m thick in the ventral integument. These measurements show that cuticular thickening is greater to the dorsal than to the ventral sides of the wing and that more thickening occurs near the base of the membrane than at its tip. The melanic cuticle on the dorsal surface is now very electron-dense, and is clearly visible as outer dense cuticulin, followed by less dense inner epicuticle, very dense exocuticle and a less dense mesocuticle. The exocuticle occupies three-quarters of its thickness (Figs. 175 and 176). The cuticle on the ventral surface is invested by an electron dense epicuticle followed by less dense procuticle. Long pointed microtrichia are present on both surfaces. The apical plasma membrane of the ventral integument is slightly folded, but does not seem to . have developed any microvilli or tubular infoldings. The apical plasma membrane of the dorsal integument from the base of the wing membrane is thrown into irregular surface microvilli (Fig. 176), and the rest of the membrane possesses tubular infoldings (Fig. 175). The epidermis has increased in thickness due to an increase in the size of the cells which are now more columnar than before (Fig. 176). The nuclei are elongate, lying perpendicular to the cuticular surface, and are centrally disposed in the cell. The nuclear walls are usually lobed towards the sides of the cell, or sometimes towards the apical and basal surfaces (Fig. 177). A nucleolus and chromatin dispersed in the nucleoplasm is shown in Figs. 175 and 176. The basal cytoplasm of most of the cells is produced into long, stout processes, supported by microtubules. These lie free inside the wing and sometimes the processes bend towards the sides. Adjacent to the lacuna the processes from the apposed integuments come close together, separated by a narrow intercellular space, except where they are occasionally attached by desenosomes (Fig. 177). There is no evident basement membrane •

322

Fig. 175. T.S. of dorsal integument from 18-hour adult wing membrane, showing autophagic vesicles. X 17000. av, autophagic vesicles; cut, cuticle; G, Golgi body; • ler, lamellae of ribosomal endoplasmic reticulum; mt, microtubule.

Fig. 176. T. S. of a 18-hour adult wing, dorsal integument from membrane. X 8000. cv, coated vesicle; my, microvilli; mvb, multivesicular body. 175

176 •

324

Fig. 177. T.S. of epidermis from 18-hour adult wing membrane showing desmosome connection between apposed cells. X 13000. • cp, cytoplasmic process; d, desmosome; ecsp, extracellular space; G, Golgi body; icsp, intercellular space.

Fig. 178. T. S. of trachea from 18-hour adult wing membrane. X. 17000. bm, basement membrane; ler, lamellae of ribosomal endoplasmic reticulum; m, mitochondria; mt, microtubule; mvb, multivesicular body; t, trachea; tl, tracheole. 00 r-- I-0 v■■

• • ct- a

326

in the middle of the wing, except for a delicate one around the lacunae. The epidermal intercellular membranes are linked by an intermediate junction (Zonula adherens), very near the apical border; this is followed by a zone of septate desmosomes. The basal third of the cell boundary consists of simple, apposed plasma membranes. The epidermal cytoplasm contain a few individual ribosomes, clusters of polyribosomes and rough-surfaced endoplasmic reticulum in the form i of elongated, short cisternae and a few vesicles with some swollen areas (Fig. 175). The rough-surfaced endoplasmic reticulum is more abundant than before. Well developed Golgi bodies are more numerous than previously and contain more numerous dense vesicles and a few saccules; they are present around the nucleus and sometimes more in the basal cytoplasm (Fig. 175). Circular and elongate mitochondrial profiles are scattered in the basal and apical cytoplasm, instead of accumulating in the apical cytoplasm as they did previously (Fig. 175). The cytoplasm also contains microtubules, multivesicular bodies, isolation bodies, cytolysosomes, electron-dense vesicles (large and small) and electron-lucent vacuoles (Figs. 175 and 176). Transverse sections of 18-hour adult wings show collapsed tracheae (Fig. 178), possibly due to changes in blood pressure in the wing after ecdysis, as also reported by Clare (1952a) in Euconocephalus extensor. Miller (1964) points out that tubes with an oval cross-section collapse readily under pressure. The cytoplasm of the tracheal epithelium contains numerous microtubules, a few single free ribosomes, spiral polyribosomes, circular and oval mitochondrial profiles (sometimes swollen), very few ribosomal endoplasmic reticular cisternae, small Golgi bodies containing dense vesicles and electron-lucent vacuoles, multivesicular bodies, agranular reticulum and electron-dense vesicles (Fig. 178). Profiles of tracheoles enclosed within tracheoblast cytoplasm and surrounded by mestracheon folds are enclosed locally in the tracheal epithelium (Fig. 178). The cytoplasm of the tracheoblast cell, surrounding the tracheole contains microtubules, ribosomes, dense

• 327 vesicles, multivesicular bodies, a very few cisternae of endoplasmic reticulum, both granular and agranular (Fig. 178). Fig. 178 also shows the connection between outer and inner plasma membranes of the trachea. These plasma membrane folds are seen diviating from each other when they approach tracheoles and are continuous over long distances (Fig. 178). Up to this stage, the trachea has been encircled by a delicate basement membrane (Fig. 178). 18-hour adult wing; corio-membranic border: The micrographs were taken from the posterior part of corium, near the corio-membranic border. By this stage the cuticle on the dorsal surface has thickened to a maximum of 2. 8 pm and on the ventral integument to about 1.2 1.m. There is a great increase in cell size. The basal extremities of the epithelial cells from both dorsal and ventral integument are produced into complicated cytoplasmic processes which extend into the interior of the wing pad and join by desmosomal attachments (Fig. 179). Because of these structures the interior compartment of the wing pad gives the impression, in transverse section, of being separated into small chambers (Fig. 179). The spaces between the joined processes are filled with membrane-bounded vesicles, isolation bodies, vesicular endoplasmic reticulum coated with ribosomes, clusters of ribosomes, dense granules, degenerating nuclei and haemocytes, (Fig. 179). In the area where the two surfaces are joined by their processes, a cuticular thickening from the dorsal integument grows towards the ventral integument. This marks the beginning of trabecular development. Micrographs from the same area also show some fully developed cuticular columns.. When the trabeculae reach the ventral side of the wing, the epithelium from which it has developed covers the surface of the trabeculae. Hundertmark (1935), and Reuter (1937) say that the trabeculae develop in the mid pupa or pharate adult of Tenebrio and Sitophilus, but in Oncopeltus the development of trabeculae clearly takes place in the adult wing after the final ecdysis. •

328

Fig. 179. T. S. of 18-hour adult wing, corio-membranic border. X. 8000. cut, cuticle; cp, cytoplasmic process; g, glycogen; icsp, intercellular space; ler, lamellae of ribosomal endoplasmic reticulum.

Fig. 180 T. S. of portion of cuticle, epidermis and nerve from 18-hour adult, corio-membranic border. X 17000. ib, isolation body; m, mitochondria; nit, microtubule; mvb, multivesicular body; nv, nerve; sd, septate de smo some . •

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330

The apical plasma membranes of the epidermal cells lying between two lacunae are produced into small apical microvilli, containing dense tips, whereas the apical plasma membrane of the epidermis lining the lacunae shows only internal tubular infoldings towards the cytoplasm (Figs. 181, 182 and 183). Cell boundaries opposite lacunae are clearer than at earlier stages and also clearer than those from the membrane of the same wing. The apical third of the intercellular membrane is linked by intermediate junctions, and a zone of septate desrvosomes; the remaining two thirds is separated by intercellular space between the apposed plasma membranes (Fig. 180). The cytoplasm contains abundant ribosomal endoplasmic reticulum, mainly in the form of long cisternae, though short cisternae and vesicular forms are still present. Some of the rough-surfaced endoplasmic reticulum is dilated; spiral polyribosomes are present, together with numerous mitochondria with circular, oval or elongate profiles. Some branched forms occur, and some others isolating cytoplasm are shown in the Fig. 180. The cytoplasm also contains areas filled with alpha-glycogen particles, small Golgi bodies containing electron-dense vesicles and inner and outer saccules, isolation bodies, lysosome-like bodies, autophagic vesicles, electron-lucent vacuoles and microtubules (Figs. 179 and 180). The epidermis surrounding the lacuna consists of squamous or columnar cells (Figs. 181 and 182), with elongate nuclei, lying parallel or perpendicular to the cuticular surface (Figs. 181 and 182). Some nuclei possess an electron-dense nucleolus; in others chromatin is dispersed in the nucleoplasm. The cytoplasm contains polyribosomes, numerous mitochondria, rough-surfaced endoplasmic reticulum with swollen areas, numerous Golgi bodies containing saccules and more dense vesicles, lipid droplets, multivesicular bodies, cytolysosomes, electron-lucent vacuoles, microtubules, glycogen filled areas (rosettes), lysosome- like bodies and electron-dense vesicles (Figs. 181, 182 and 183). A basement membrane is still present around the lacuna. f

331

Fig. 181. T.S. of epidermis lying adjacent to lacuna from 18-hour adult wing, corio-membranic border. X 17000. bm, basement-membrane; cut, cuticle; G, Golgi body; g, glycogen; lac, lacuna; ler, lamellae of endoplasmic reticulum; m, mitochondria.

Fig. 182. T.S. of epidermis towards one side of lacuna, from 18-hour adult wing, corio-membranic border. X 26000. elv, electron-lucent vacuole; G, Golgi body; g, glycogen; m, mitochondria; n, nucleus. 181

182 •

333

Micrographs of the trachea from the corium adjacent to the corio-membranic border shows degeneration of the tracheal epithelium (Fig. 184). The degenerating epidermis undergoes shrinkage and condensation, and its apical plasma membrane has withdrawn from the tracheal intima (Fig. 184). Phagocytosis of the degenerating cell occurs (Fig. 184). The greater part of the cytoplasm from the degenerating cell has disappeared; what is left • contains ribosomes, dense vesicles, a very few mitochondria and large myelin bodies. The remaining portion of the tracheal epithelium is also becoming detached from the tracheal intima. The nuclei are very small, there is no nucleolus, the chromatin is dispersed in the nucleoplasm, and some has accumulated around the periphery. The cytoplasm contains polyribosomes, mitochondria, a few cisternae of ribosomal endoplasmic reticulum, very small Golgi bodies containing a few dense vesicles, microtubules, electron-dense vesicles and multivesicular bodies (Fig. 185). A thin basement membrane is present around the trachea. Micrographs from the corium adjacent to corio- membranic border show a lacuna enclosing a trachea and a large and small nerve (Fig. 185). The large nerve contains more than 100 axons, arranged into several bundles, nearly all lying within one mass of glial cytoplasm. Some large axons however are surrounded by their own glial cytoplasm. The mesaxons are very prominent, and make two or three loose turns around the axon bundles, which are separated by glial cytoplasm; here and there the apposed membranes of the mesaxon are joined by septate desmosomes. The nucleus of the glial cell does not possess nucleoli, but large masses of chromatin are accumulated in its centre and periphery. The glial cytoplasm lying within the axon bundles contains very numerous microtubules running parallel to the long axis of the nerve. Outside the axon bundle the glial cytoplasm contains fewer microtubules, numerous spiral polyribosomes, mitochondria, a few short cisternae of ribosomal

• endoplasmic reticulum, a small Golgi body containing dense •

334

Fig. 183. T.S. of cuticle, epidermis and lacuna from 18-hour adult wing, corio-membranic border, to show lipid droplets. X 8000. M lac, lacuna; ld, lipid droplet; m, mitochondria.

Fig. 184. T.S. of 18-hour adult wing, lacuna from corio- membranic border showing collapsed, degenerating trachea, nerve and haemocytes. X 8000. bm, basement membrane; G, Golgi body; haem, haemocyte; n, nucleus; nv, nerve; t, trachea. 183

184 •

336

Fig. 185. Portion of trachea and nerves from lacuna of 18-hour adult wing, corio-membranic border. X 13000. a, axon; bm, basement membrane; ep, epidermis; • icsp, intercellular space; mt, microtubule; mvb, multivesicular body; nv, nerve; t, trachea.

Fig. 186. Corium of 18-hour wing, showing the beginning of trabecula formation. X 26000. cut, cuticle; ep, epidermis; trab, trabeculae. •

•

185

186 •

338

vesicles and electron-lucent vacuoles. The axoplasm contains mitochondria, vesicles and neurotubules. The nerve is encircled by a basement membrane of 0. 2 Tim thick. The nerve and trachea lie very close together, but are separated by their basement membranes (Fig. 185). The nerve is also closely associated with the basal surface of the epidermis, being separated from it by a narrow intercellular space (Fig. 185). s 18-hour adult wing; base of corium: In this region, the secretion of cuticular columns has almost come to an end. Several micrographs show the beginnings of trabecular growth, from the dorsal integument (Figs. 186 and 187). In Fig. 186 a solid finger-like ingrowth from the dorsal lamellate endocuticle is seen growing towards the inner surface of the wing. Fig. 187 shows further additions of cuticle by the trabecular epidermis which grows until it reaches the ventral integument. Here the epidermal layers. become attenuated so that the trabecular and ventral cuticles meet, though without fusing. The two surfaces of the corium are separated by a blood space, across which run the longitudinal rows of trabeculae. A certain amount of cuticle has been deposited in the corium, and the greater part of the cytoplasmic substance of the epidermal cells has disappeared; what remains is more electron- dense than before. The epidermal cells lining the ventral surface of the lacunae are now secreting the cuticle, which forms the ventral wall of the tubular vein. During this period there is still some intercellular space at the base of the epidermal cells. In this cavity, extra cuticle is deposited to form part of a vein. While depositing the cuticle, some epidermal cells degenerate, lose their cell- junctions and detach from the cuticle. The outer nuclear membranes and its ribosomes are lost, the cytoplasm breaks up into isloation fragments and many of the organelles become unrecognisable. Some areas of the epidermis still contain a few organelles such as mitochondria, a few ribosomes, ribosomal endoplasmic reticulum in the form of short cisternae and swollen vesicles, Golgi bodies, isolation 339

Fig. 187. T. S. of corium from 18-hour adult wing, showing fully formed trabecula. X 8000. cut, cuticle; ep, epidermis; trab, trabecula. •

Fig. 188. Micrograph of corium of 18-hour adult wing showing degeneration of trachea and epidermis. X 26000. av, autophagic vacuole; G, Golgi body; m, mitochondria; mt, microtubule; mvb, multivesicular body; n, nucleus; rer, ribosomal endoplasmic reticulum; t, trachea. 187

188 •

341

bodies, multivesicular bodies and autophagic vesicles (Fig. 188). The epithelium of the collapsed trachea is also undergoing degeneration in the same way as mentioned above (Fig. 188). (c) 32-hour adult wing; membrane: Micrographs from the posterior half and the sides of the membrane show no traces of epidermis or trachea, but contain only separated cuticular layers. Within a few hours these will become • closely pressed together. Snodgrass (1935) and others have pointed out that when wing development is complete the epidermis has largely disappeared, the mature wing is an almost entirely cuticular structure. Bland & Nutting (1969) showed that this generalisation can be applied to the Holometabola. Tracheae and adjacent epidermis are seen in micrographs of the central region of the membrane, though even in this area most of the epidermis has become detached from the epidermis (Figs. 188, 190 and 191). A micrograph of a partly detached epidermal cell shows an elongate nucleus with its long axis perpendicular to the cuticle. There is no nucleolus, the chromatin is condensed into small masses, arranged around the periphery and in the centre (Fig. 190). The cytoplasm contains single free ribosomes; rough-surfaced endoplasmic reticulum is in the form of short cisternae and vesicles (some of which are dilated). Circular and very elongate mitochondrial profiles and Golgi bodies containing electron-dense vesicles and electron-lucent vacuoles are present around the nucleus. The cytoplasm also contains microtubules, large electron-dense vesicles, multivesicular bodies, membrane bounded vesicles and isolation bodies (Fig. 190). The detached epidermis is degenerating, and shows a nucleus with the outer nuclear membrane detaching, and the cytoplasm broken up into small fragments. This degenerating cytoplasm contains a few polyribosomes, numerous swollen vesicles of rough- surfaced endoplasmic reticulum, swollen mitochondria, a few Golgi bodies containing dense vesicles, vesicles bounded by smooth membranes, electron-dense vesicles, multivesicular bodies,

•

342

Fig. 189. T. S. of 32-hour adult wing membrane showing detached epidermis and trachea. X 13000.

bm, basement membrane; ep, epidermis; lac, lacuna; • m, mitochondria; n, nucleus; t, trachea; tl, tracheole.

Fig. 190. Detaching epidermis from 32-hour adult wing membrane. X 26000. G, Golgi body; ib, isolation body; m, mitochondria; mt, microtubule; mvb, multivesicular body; n, nucleus; re r, ribosomal endoplasmic reticulum. •

189

190 •

344 isloation bodies and autophagic vesicles (Fig. 192). Some organelles are unrecognisable. The plasma membranes rupture, and cell contents are liberated. Histolysis seem to take place by autophagy and is followed or accompanied by phagocytosis. The lacunae at this stage are still lined by a basement membrane up to 0.12 1m thick, with huge masses of similar material • attached to it in places. A nerve is seen in Fig. 191 lying close to the dorsal epidermis, but separated by intercellular space. The dorsal epidermis is already detached from the cuticle, but still contains cytoplasmic organelles. Many well-developed Golgi bodies containing more numerous dense vesicles are present in the cytoplasm; it also contains a few ribosomes, rough-surfaced endoplasmic reticulum in the form of vesicles and short cisternae, mitochondria, microtubule s, multive sicular bodies, isloation bodies, and autophagic vesicles (Fig. 191). The nerve visible in this section is somewhat larger and contains 10- 15 axon profiles. The mesaxons are very prominent and make four or five loose turns around the axon bundle, separated by glial cytoplasm. The axon bundle itself consists of a few large and a few small axons enclosed in glial cytoplasm. The latter contains numerous microtubules, a few ribosomes and electron-lucent vacuoles. The glial cytoplasm lying between the mesaxon folds, and also that outside them, contains microtubules, single free ribosomes, mitochondria and a very little ribosomal endoplasmic reticulum. A small Golgi body containing dense vesicles and electron-lucent vacuoles is also illustrated. The axoplasm contains neurotubules and dense vesicles. The nerves lie between the dorsal epidermis and the basement membrane of the lacuna (Fig. 191). There are no signs of nerve degeneration in the membrane portion of the wing. The trachea lying in close association with the degenerating epidermal cytoplasm is separated by a narrow intercellular space. The tracheal epithelium is still apparently alive, showing no signs of degeneration (Fig. 189). The intercellular membranes of the tracheal epithelia are linked by intermediate junctions and septate •

345

Fig. 191 T.S. of detaching cuticle, with epidermis, nerve, basement membrane and lacuna from 32-hour adult wing membrane. X 25000. • bm, basement membrane; cut, cuticle; er, endoplasmic reticulum; G, Golgi body; m, mitochondria; mt, microtubule; nv, nerve.

Fig. 192. Trachea and degenerating epidermis from membrane of a 32-hour adult wing. X 26000. ep, epidermis; met, mestracheon; t, trachea. •

1 I I. , I

-- 4

347 desmosomes; its cytoplasm contains ribosomes, mitochondria, dense vesicles, multivesicular bodies, microtubules, very few rough- surfaced endoplasmic reticular cisternae, and electron-lucent vacuoles (Fig. 189). Fig. 192 shows profiles of tracheoles surrounded by the tracheoblast sheath and lying inside the mestracheon folds of the tracheal epithelia. The tracheoblast cytoplasm contains microtubules, small vesicles surrounded by smooth membranes and some multivesicular bodies. The outer surface of the tracheal epithelium is surrounded by a basement membrane, 70 nm thick (Fig. 192). 32-hour adult wing; corio-membrane border: Some trabeculae are still growing in the corium, at the edge of the corio-membrane border (Fig. 193). The photograph shows columns of cells joining the upper and lower integuments of the wing, prior to the formation of trabeculae (Fig. 193). The cells contain very large, elongate and lobed nuclei. A nucleolus is either present or divided into chromatin masses. The apical cytoplasm of the dorsal epidermis is produced into microvilli, responsible for secreting the cuticular pillar from the dorsal side. The cytoplasm is filled with long tubular cisternae of rough-surfaced endoplasmic reticulum; some larger forms are even folded. Very well developed Golgi bodies, containing electron-lucent vesicles, dense secretions and electron-dense vesicles, lie close to the rough-surfaced endoplasmic reticulum (Fig. 193); the ends of some rough-surfaced endoplasmic reticular cisternae are swollen into vesicles (Fig. 193). The cytoplasm also contains polyribosomes, scattered mitochondria, regions filled with alpha-glycogen, lysosome-like bodies, isolation bodies and electron-lucent vacuoles (Fig. 193). At an advanced stage of trabecular secretion the cytoplasm shows swollen vesicles of rough-surfaced endoplasmic reticulum, and degenerating nuclei. Transverse sections taken from the inter lacunar spaces show two layers of integument, internally lined by basement membranes separated by extracellular space. The apical plasma membrane is •

348

Fig. 193. T.S. of epidermis from corium of 32-hour adult wing, showing developing trabeculae. X 13000. cut, cuticle; G, Golgi body; g, glycogen; m, mitochondria; n, nucleus; trab, trabecula.

Fig. 194. T.S. of cuticle, epidermis, basement membrane, lacuna and portion of trachea from 32-hour adult wing (posterior side of corium). X 17000. bm, basement membrane; ep, epidermis; lac, lacuna. t, trachea. 193

194 •

350

produced into microvillate folds. Some epidermal cells have withdrawn from the cuticle, and completely degenerated, others contain a condensed nucleus, losing its outer membrane, and the intercellular membranes are disappearing. The cytoplasm of such cells contains ribosomes, vesicles surrounded by ribosomes, lysosome-like bodies, but other organelles are becoming unrecognisable. The cytoplasm of some cells contains polyribosomes, vesicular and tubular cisternae • of rough-endoplasmic reticulum, profiles of swollen mitochondria, Golgi bodies containing more numerous electron-dense vesicles than electron-lucent vacuoles, dense bodies, lysosome-like bodies and multivesicular bodies. It seems that most of the epidermis adjacent to the corio-membranic border is degenerating. The lacunae are lined by a basement membrane 0. 3 Am thick. The epidermis adjacent to the trachea still appears healthy. The apical plasma membranes of these cells lie close to the cuticle by their microvillate folds (Fig. 194). The epidermal nuclei are elongate and the cytoplasm possesses spiral polyribosomes, oval and circular mitochondrial profiles, a few short cisternae of ribosomal endoplasmic . reticulum, scattered Golgi bodies consisting of a few dense vesicles and electron-lucent vacuoles, large areas filled with alpha-glycogen particles, electron-dense vesicles, electron-lucent vacuoles and lysosome-like bodies (Fig. 194). Parts of the tracheal epithelium from the corio-membranic border of 32-hour wings have been completely lost (Fig. 195), but other parts are in contact with the tracheal intima and still apparently alive, though both epithelia and its nuclei have under- gone shrinkage (Fig. 194). The cytoplasm contains single free ribosomes, spiral polyribosomes, a few cisternae of rough-surfaced endoplasmic reticulum, a reasonable complement of mitochondria, microtubules, a few small Golgi bodies (containing dense vesicles and sometimes associated with electron-lucent vacuoles), multivesicular bodies and electron-dense vesicles. The tracheae are encircled by a basement membrane. In some areas the tracheae are in close contact with the ventral integument, separated by only a w

351

Fig. 195. Degenerating trachea from 32-hour adult corio-membranic border. X 26000. ep, epidermis; t, trachea; ti, tracheal intima. •

Fig. 196. T.S. of epidermis, nerve and portion of lacuna from 32-hour adult wing (corio-membranic border). X 17000. a, axon; bin, basement membrane; ep, epidermis; G, Golgi body; g, glycogen; lac, lacuna; mt, microtubule; nt, neurotubule. t

195

196 •

353

narrow intercellular space and in some cases a basement membrane lies between them (Fig. 194). Fig. 196, shows a nerve in a lacuna near the corio-membranic border; it contains several groups of axon bundles, each separated by glial cytoplasm. Prominent mesaxons make loose turns around the axon bundles. The glial cytoplasm lying among the axons contains more numerous microtubules while the remaining external glial cytoplasm contains a few microtubules, polyribosomes, more numerous mitochondria, a few cisternae of ribosomal endoplasmic reticulum, and a few Golgi bodies containing electron-dense vesicles and electron-lucent vacuoles. The axoplasm contain more numerous microtubules and mitochondria than in the previous stage and a few small vesicles. One surface of the nerve is in contact with the epidermis and the other side is lined by a basement membrane which borders the lacuna. Here and there small vesicles are present outside the nerve (Fig. 196). The dorsal integument, in contact with the nerve, appears to be alive and contains several organelles, apparently in the process of synthatic activity (Fig. 196). (d) 48-hour adult wing; corium: Micrographs obtained from the middle parts of the corium of 48-hour wings show fully developed and developing veins. By now, larger portions of cytoplasm and the associated organelles from epidermal cells lying around the lacuna and from detached tracheal epithelial cells have been replaced by intracellular cuticular deposits, but some of the cells still possess a nucleus containing condensed chromatin masses. The intracellular cuticular deposits appear to differ ultrastructurally from normal cuticle; they are unlaminated, granular in texture, and rather less electron-dense. The remains of the cytoplasm is occupied by a few organelles such as single free ribosomes, polyribosomes, mitochondria, microtubules, isolation bodies, multivesicular bodies, electron-dense vesicles and electron-lucent vacuoles (Fig. 197). Rough-surfaced endoplasmic reticulum mostly takes the form of larger vesicles, e

354

Fig. 197. T.S. of cuticle and epidermis from corium of 48-hour adult wing, dorsal side of lacuna. X 13000. bm, basement membrane; cut, cuticle; er, endoplasmic • reticulum; elv, electron-lucent vacuole; G, Golgi body; m, mitochondria; n, nucleus.

Fig. 198. T. S. of corium of a 48-hour adult wing corium, showing fully formed vein. X 8000. cum, cuticular material; nv, nerve; t, trachea; V, vein. •

•

197

-4

.111.04Aralla. 198 •

356

but some cisternae have swollen areas, and some is isolating in the cytoplasm (Fig. 197). Numerous Golgi bodies containing electron- dense vesicles and electron-lucent vacuoles are present in the basal cytoplasm (Fig. 197). Micrographs from the same area even show fully developed veins (Fig. 198). The formation of veins takes place through the deposition of cuticle around the lacuna. Fig. 198 shows thicker cuticle deposition on the ventral surface, and only a • weak deposit on the dorsal side. Most of the cytoplasm of the cells lying around the lacuna has disappeared and even what remains is becoming unrecognisable. Inside the vein is the intima of a collapsed tracheal tube. The detached epithelium of the tracheal tube is also sclerotized and is firmly attached to the dorsal surface of the vein (Figs. 198 and 199). The vein also encloses a nerve in its dorsal wall. The latter is still apparently alive, and axons from it extend to the tracheal matrix layer and dorsal integument (Fig. 198). A few axon profiles are present in the nerve, lying inside glial cytoplasm. Mesaxons are very prominent, separated by glial cytoplasm; they make three or four loose turns around the axon profiles. The glial cytoplasm contains polyribosomes, vesicular rough-surfaced endoplasmic reticulum, mitochondria, microtubules and electron-dense vesicles. The glial cells in the branches of the nerve contain single free ribosomes and mitochondria. The axoplasm contain neurotubules and vesicles (Fig. 199). The formation of veins occurs out of cuticle adjacent to blood-filled lacunae, without depending directly on tracheae. The epidermal cells lying in the inter-vein areas of the wing still contain cytoplasmic organelles though cuticle deposition is taking place. The extracellular space lying between upper and lower epidermal layers is also filled with cuticle. The epidermal cells contain nuclei which are losing their ribosomes- coated outer membrane (Fig. 197). The cytoplasm contains polyribosomes, very few cisternae and vesicles of rough-surfaced •

357

Fig. 199. T. S. of corium of 48-hour adult wing, showing dorsal side of vein. X 16000.

a, axon; m, mitochondria; ma, mesaxon; mt, microtubule; • nv, nerve; r, ribosomes; t, trachea; tep, tracheal epithelium; V, vein; v, vacuole.

Fig. 200. T.S. of portion of corium of a 48-hour adult wing, showing the impregnation of cuticular material in the inter vein areas of epidermis. X 17000. cp, cytoplasmic process; cut, cuticle; cum, cuticular material; dv, dense vesicle; ib, isolation body; mt, microtubule; r, ribosomes. •

199 •

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endoplasmic reticulum, mitochondria, Golgi bodeis contain more numerous dense vesicles, microtubules, electron-dense vesicles, electron-lucent vacuoles, lysosome-like bodies, isolation bodies and autophagic vesicles (Fig. 200). 48-hour adult wing; membrane: By now degeneration of most of the epidermal cells in the membrane of the wing is complete. The few remaining cells, accompanied by tracheae, have moved up to the border with the corium, and remain between the medial and cubital veins until further degeneration of cells has taken place (Fig. 201). Light microscopy shows the existance of five bunches of coiled trachea in this region. Leston (1962) reported tracheal retraction in some heteroptera and gave some possible reasons for this. It is very difficult to give an exact interpretation of the structures found in this area as the coiled trachea and tracheoles and accompanying epidermal cells lie one above the other and make it difficult to obtain satisfactory sections. Fig. 202 shows a bunch of degenerating cells lying loosely between its companions. The degenerating cell first shrinks and condenses and the cell junctions disappear. The cells then undergo further condensation and lie loosely enclosed in plasma membranes until they are eventually phagocytosed, apparently by neighbouring epidermal cells - a most unusual situation. Even after being engulfed by another cell, the degenerating cell retains its plasma membranes (Fig. 203). Goldsmith (1966) says the death of the cell is accomplished economically with the digestion and reutilisation of its old components. At an advanced stage the degenerating cell appears as an intracellular inclusion of the phagocytotic epidermal cell, bounded by a single membrane and containing organelles in various stages of disintegration (Figs. 202 and 203). Sometimes only the possession of a nucleus in the form of a homogenous mass identifies it as the remains of a cell. Similar results were reported by Fristrom (1968) in Drosophila melanogaster. Two or three degenerating cells are sometimes included inside the cytoplasm of a single phagocytotic cell. •

Fig. 201. Micrograph from 48-hour adult wing membrane, showing retracted tracheae and degenerating epidermis. X 5000. cut, cuticle; ep, epidermis; t, trachea; tl, tracheole. •

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Eventually the plasma membrane of the degenerating cell breaks down and its organelles begin to cytolyse. The cytoplasm of epidermal cells also contains isolation bodies and autophagic vesicles (Fig. 202). Almost every epidermal cell from this area contains one or two dead cell inclusions; only a few cells do not seem to have been involved in phagocytotic attack. Such cells contain a very elongated nucleus; the nuclear membrane is lobed towards the sides and the nucleus possesses large masses of chromatin in the centre and periphery. The cytoplasm contains numerous polyribosomes, a few mitochondria, a few short cisternae of ribosomal endoplasmic reticulum, microtubules, small Golgi bodies containing electron-dense vesicles, free dense vesicles, electron-lucent vacuoles and autophagic vesicles. The cytoplasm of phagocytic cells contains single free ribosomes, polyribosomes, numerous circular and elongated mitochondrial profiles, very few cisternae of ribosomal endoplasmic reticulum and vesicles. Golgi bodies containing more numerous dense vesicles and saccules, scattered microtubules, isolation bodies, autophagic vesicles, dense vesicles and electron-lucent vacuoles are also present (Fig. 203). Histolysis of epidermal cells in the membrane occurs both by autophagy and phagocytotic attack by other epidermal cells, which themselves later degenerate so that the entire membrane epidermis eventually disappears. Surrounded by dead, dying and livivng epidermal cells, the coiled masses of trachea and tracheoles adjacent to the corium are still apparently alive (Fig. 204). The degeneration of trachea in this region takes place as the last major event in wing development. At this stage the cytoplasm of the tracheal epithelium contains a few spiral polyribosomes, cisternae of ribosomal endoplasmic reticulum, mitochondria, Golgi bodies with more electron-dense vesicles and a few electron-lucent vacuoles as well as microtubules (Fig. 201). In some areas, electron-dense vesicles, electron-lucent vacuoles, multivesicular bodies and autophagic vesicles are also present. •

•

Fig. 202. A group of degenerating cells from membrane of 48-hour adult wing. X 17000. av, autophagic vacuole; dgc, degenerating cell; ib, isolation body; mvb, multivesicular body; n, nucleus. •

Fig. 203. 48 hour adult wing membrane, showing degeneration of epidermal cells by autolysis and phagocytotic attack by neighbour cells. A dead cell is enclosed inside the cytoplasm of another cell. X 17000. av, autophagic vacuole; dgc, degenerating cell; edv, electron-dense vesicle; elv, electron-lucent vacuole; G, Golgi body. •

Fig. 204. Micrograph showing retracted and degenerating trachea and tracheoles from membrane of a 48-hour adult wing. X 8000. t, trachea; ti, tracheal intima; tl, tracheole; n, nucleus. •

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Fig. 204 shows profiles of tracheoles which have been cut longitudinally and are apparently those which appear under the light microscope like bunches of grapes. The cytoplasmic sheath of the tracheoblast covering the tracheole is in a normal living condition; its cytoplasm contains polyribosomes, microtubules, Golgi bodies, dense vesicles, multivesicular bodies and autophagic vesicles. • Sections taken from a similar area of an older wing (probably 6 or 7 days old) do not show any tracheae or tracheoles. The light microscopical appearance of trachea might possibly be due to persistent coiled portions of the tracheal intima after degeneration of the tracheal epithelium. The major changes observed in the adult wings between emergence and 48 hours later may be summarised as follows: 1. The tracheae of the wings collapse somewhat between 2 18 hours after emergence. 2. While the epidermal cells of the corium are engaged in secreting cuticular columns, and in vein formation and cuticular deposition, the tracheal epithelia degenerates before any other wing tissue as noticed, for example, in 18 hour old wing sections. 3. Trabeculae formation, deposition of cuticle around the lacuna and in the epidermis of interlacunar spaces all take place in the adult wing between 0 and 48 hours after emergence, starting from the base and proceeding to the apex of corium. 4. Degeneration of the epidermis of the membrane occurs by autophagy and phagocytosis between 0 and 48 hours, starting with the tip of the membrane and proceeding to the border with the corium. During this period the living tracheae of the membrane move up to the border with the corium, accompanied by the retracted epidermis. After histolysis of all epidermal cells in the membrane is complete, the tracheal epithelium degenerates, and the intima of the tracheal tube remains coiled, adjacent to corio-membranic border for the rest of the insect's life. PART IV

GENERAL DISCUSSIONS AND CONCLUSIONS • •

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The present investigations deal with the fine structure of Oncopeltus wing pads during the third, fourth and fifth instar larvae and in the adult wings. The subject is complicated for various reasons. The various areas of the wing do not all develop at the same rate (Kohler, 1932); in some cells the mitotic divisions occur very early (Bowers & Williams, 1964). Observations on scale-forming cells, socket-forming cells, gland cells and degenerating cells could not always be made satisfactorily at the earlier stages by electron microscopy, so that some reported changes in the ultrastructure may be associated with cell division and differentiation rather than with the biochemical or metabolic maturation that is also in progress (Willis, 1966). For convenience it is proposed to summarise and discuss some aspects of the work under sixteen main headings. (a) Cuticle: Histologically epicuticle, exocuticle and endocuticle are well distinguished by their structure and staining properties. The wing pads are covered by a uniformly dense outermost layer of epicuticle which is homologus with the trilaminate cuticulin layer of Locke (1966); this is not comparable to the cuticulin of W iggle sworth (1933, 1947), who proposed the term to denote the main lipoprotein substance of the epicuticle. During post-apolytic stages the outer epicuticle, i. e. cuticulin of Locke, appears initially as discontinuous trilaminate 'plaques' occupying small areas over dense patches of microvilli projections of the apical epidermal plasma membrane. It appears to be laid down in Oncopeltus in a fashion similar to that described by Locke (1966) in Calpode s larvae. The tri laminate structure found in Oncopeltus also proves its identity to the similar cuticulin of Locke (1966). These patches of cuticulin grow at their margins and fuse to form a continuous membrane (cf. Filshie & Waterhouse, 1969; Delachambre, 1970). In the newly emerged fourth instar the inner epicuticle contains alternating layers of more and less electron-dense 367 material, but it slowly changes into a uniformly dense layer. Zacharuk (1972) has made similar observations on the transformation of the cuticle of some Elateridae (Coleoptera). Internal to the epicuticle lies a lamellate procuticle, composed of electron-lucent and electron-dense layers. The procuticle of Oncopeltus wings is, therefore, rather similar in general appearance and fine structure to the lamellate day- and night- zone cuticle of the integument of Locusta, Schistocerca and other insects (Neville, 1965a, 1965b). The discrepancy between measurements of the thickness of epicuticle and procuticle as recorded in light and electron microscopy is possibly due to the presence of a transitional zone between the epicuticle and procuticle. In this connection, Zacharuk (1972), working on the integument of some Elateridae, writes: "The first several sub-epicuticular lamellae of cuticle have less distinct cuticular microfibres, are more electron-dense and are thinner than the more medial lamellae that make up the primary thickness of the procuticle. The former appear to be transitional in nature between the dense layer of epicuticle and the typical oriented fibrillar lamellae of procuticle. This transitional zone is about as thick as the dense layer of epicuticle and because these layers were not differentiated by stains used in light microscope preparations, the transitional zone could be easily interpreted as being part of the dense epicuticular layer". This will be permeated during the sclerotisation process by a homogeneous material somewhat similar to that of the dense epicuticle. The innermost layer of cuticle lying close to the epidermis is slightly more electron- dense and probably represents the subcuticle of Schmidt (1956). In all ultrastructural studies reveiwed the subcuticle seems, rather surprisingly, to appear as the first layer in the secretion of new procuticle during the moult period. Schmidt had suggested that the deposition zone forms a subcuticle, whose function was to glue epithelium and cuticle together. Locke (1961), however, concluded that the subcuticle represents the •

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innermost layer of secreted material, continuously in the process of conversion into more normal cuticle. Neville (1975) went further and suggested that the deposition zone may not be simply converted into cuticle, but may exist as a long-term loose matrix through which cuticular macromolecules pass and become oriented on their way to the growing cuticular interface. Taylor & Richards (1965) suggest that this mucilaginous material may function, partly in binding the cuticle to the epidermis during the intermoult period and partly in their separation of apolysis. My observations do not allow one to distinguish between these various possibilities, but the subcuticle and its functional role need further study. (b) Epidermis: In the wing pads and wings of newly emerged instars the integument consists of a single layer of epidermal cells. Junctions along the lateral plasma membrane are distributed in the same apical to basal arrangement at all developmental stages. An intermediate junction (Zonula adherens) is noted typically at the cell apex. The septate junctions characterised by bridge-like septa between cells are found in the area between the intermediate junction and the level of the nucleus (Farquhar & Palade, 1963, 1965; Locke, 1965; Greenstein, 1972a). In the newly emerged larva the wing pad epidermis is actively engaged in the deposition of endocuticle at the apical microvillar plasma membrane. The apical region of the cell seems to be primarily concerned with biosynthesis, for it is filled with ribosomal endoplasmic reticulum, Golgi bodies and secretory vesicles. The dense secretory vesicles are joined to the plasma membrane at the base of plasma membrane infoldings. Their contents seem to be released into the tubular infoldings to add further cuticular substances as described by Locke (1969b) in Calpode s. During procuticular secretion some coated vesicles have been noticed in the apical cytoplasm of Oncopeltus wing pad epidermis which are similar to those described in a number of other •

369 recent studies (Roth & Porter, 1962, 1964; Locke, 1966; Friend & Farquhar, 1967; Filshie & Waterhouse, 1968; Greenstein, 1972a). Although it has generally been thought that coated vesicles are involved in selective cellular uptake of protein (Roth & Porter, 1964) recent studies of the rat vas deferens (Friend & Farquhar, 1967) suggest that some coated vesicles fuse with the apical cell membrane and could serve to convey materials to the apical plasma membrane. A similar type of fusion between the coated vesicles and apical plasma membrane has been noticed in postecdysial wing pad epidermal cells of Oncopeltus. Within 24 hours of ecdysis the flat, thin epidermal cells of the post-ecdysial wing pad has become columnar. There is an increase in the number of mitochondria, polyribosomes, rough- surfaced endoplasmic reticulum and Golgi bodies. As many workers have shown (Karrer, 1960; Palade, 1955), the endoplasmic reticulum is associated with the synthesis of proteins. For example, the increase in endoplasmic reticulum and polyribosomes has been correlated with an increase in their secretory activity in a biochemical study of post diapause development of Hyalophora (Wyatt, 1967b; Ebstein, 1968). An increase in Golgi activity in Oncopeltus wing pad epidermis is indicated by enhanced production of secretory vesicles and an increase in the size and number of the complexes. This rising activity is apparently coincident with a period of increasing protein synthesis (for biochemical data, Wyatt, 1967b). While the increase in number of mitochondria indicates the increase in energy requirements. All these structural changes herald the beginning of epidermal cell-divisions. By 48 hours after the previous ecdysis, the cells increase in number, with the appearance of more numerous mitotic divisions. In the newly moulted insect wing pads, the extracellular space between the basal areas of the cells and between the integumentary layers is more abundant. Soon after ecdysis, the •

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basal region of each epidermal cell gives out several cytoplasmic processes from all sides, entangling in all directions with similar processes from other cells, to occupy most of the extracellular space in the wing pad imparting to it a spongy appearance. These processes contain polyribosomes, ribosomal endoplasmic reticulum, mitochondria and microtubules. The presence of similar processes in the normal epidermis of Rhodnius has been described by Wigglesworth (1959, 1977). According to him in the post-feeding insect a mass of processes of varied thickness, developes after feeding, most of them containing numerous microtubules. These processes are in part concerned in securing attachment of the epidermis to air-filled tracheoles in order to ensure their equitable distribution throughout the integument (Wigglesworth, 1959). Wigglesworth considered that these processes are given off by epidermal cells deprived of their oxygen supply, and which thus become attached to air filled tra.cheoles in neighbouring areas, so drawing them into an oxygen- deficient zone. His ultrastructural study (Wigglesworth, 1977) also revealed the fine structure of an epidermal cell deprived of its tracheal supply. The endoplasmic reticulum and mitochondria accumulate proximal to the nucleus (instead of in their normal concentration in the distal part of the cell), and both extend into a conical process, which grows out to form a strand. The presence of similar structures in the pre- and post ecdysial wing pad epidermis of Oncopeltus suggests a response by the divided cells to an oxygen deficiency. The nuclei of the epidermal cells are variously shaped at 48 hours, with regular or irregular profiles and the presence or absence of nucleoli in the dividing cells. These features are presumed to indicate different stages of synthetic activity in the cells, especially in relation to nucleic acid metabolism. In 1967, Krishnakumaran, Berry, Oberlander & Schniderman reported that the presence of smooth nuclear profiles in the epidermis of Cecropia and other Saturniid moths before the onset of moulting •

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coincides with peaks in DNA synthesis. Conversely, Bowers & Williams (1964) showed that irregular nuclear profiles coincide with phases of low DNA sythensis. Greenstein (1972) points out that the compact concentric organisation of nucleoli and the abundance of condensed deeply staining chromosomes during diapause are consistent with low synthesis of RNA and DNA. Further, during development, when nucleic acid synthesis is high, most nucleoli 41 have a looser organisation and chromatin is predominantly diffuse and lightly staining. Mitotic divisions continue in the wing pad epidermis until the cells have increased to a maximum number. The cells resulting from division are smaller and most of their volume is occupied by the oblong nucleus. Each epidermal layer of the wing pad now possesses several layers of cells and forms a stratefied epithelium. Cell boundaries are very prominent in electron micrographs. As these boundaries cannot be identified by light microscope the tissue gives the appearance there of a syncytial stratefied epithelium. As a result of cell divisions most of the former extracellular space between the wing pad integuments becomes occupied by the newly divided epidermal cells. These contain numerous polyribosomes and mitochondria and the nucleoli are very prominent. In the apical cytoplasm there is an extensive development of plasma membrane. The cytoplasm thus gives the appearance of being divided into several "compartments". This extensive development of plasma membranes and construction of cytoplasmic "chambers" represents a major form of histological reorganisation and cellular reorientation during the folding and expansion of wing pad of the pharate larva. It does not seem to have been described adequately in other accounts of wing development and its functional significance is not clear, though the "chambers" are really cross- sections of interdigitating cell-processes. Soon after the completion of the cell divisions, the s 372 epidermal cells again become reorganised into a single layer, and are folded inside the old cuticle. This in turn is followed by an increase in cell size. The basal portion of each cell becomes produced into a long process supported by longitudinally directed bundles of microtubules. From his light-microscopic study Hundertmark (1936) reported the presence of basal cytoplasmic tails containing basiphil miofibrils which he thought supported the wing pads of the newly emerged pupa of Tenebrio and he also observed their retraction after 6 - 8 hours. Microtubules are more abundant in the basal and apical cytoplasm of the late pharate and newly emerged wing pad epidermis of Oncopeltus, and they are also present in tracheal matrix cells, tracheoblasts and glial cells. They appear to be less numerous in the epidermal cells when the wings are covered by a thick sclerotised cuticle. According to Wigglesworth (1977) the microtubules in cytoplasmic process and tracheoles play a major role in resisting tensile forces. The role that microtubules play in cytoplasmic movement and in the determination of final scale shape has been discussed by Locke (1966, 1969b), Lawrence (1966), and Overton (1966, 1967). In Oncopeltus wings the abundance of microtubules in unsupported epidermis, compared with that which is supported by cuticle suggests that they support the epidermal cells mechanically and especially that they strengthen the long cellular processes. Their occurrence in tracheoblasts and glial cells perhaps helps to determine the shape of these cells, to support them and enable them to resist tension. In the late pharate and newly moulted wing pad epidermis, glycogen is very abundant in the apical cytoplasm, concentrated in large masses. On the other hand, during the pre-moult and moulting stages it is accumulated in the basal areas of the epidermal cells. Zacharuk (1972) has brought forward ultrastructural evidence to suggest that during moulting there is a conversion of storage lipids to glycogen in the fat cells, followed •

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by a further conversion to monosaccharide and transport through the haemolymph to epidermal cells where it is reconverted into glycogen. In Oncopeltus the wing tissue contributes to the storage of mobile glycogen reserves; which seem to be moved from the basal to the apical surface of the cell during cuticle secretion and are possibly utilised as a substrate in cuticle synthesis. In the late pharate wing pads, where there is more intense metabolism in the growing cells, the cytoplasm contains numerous mitochondria; which are present in varied configurations, ranging from spherical to those with elongate, dumb-bell shaped profiles. In the matrix of some mitochondria are a few dense granules. When the later type of mitochondria were cut transversely, this gives the appearance of a mitochondria isolating cytoplasm; but presumably all that is concerned is a hollow, cup-shaped mitochondrion contained the usual cytoplasmic matrix (cf. Jamieson & Palade, 1968). The fine structure of the epidermal cell of Oncopeltus at the onset of new cuticular secretion resembles that seen in Calpodes larvae (Locke, 1966), Tenebrio (Delachambre, 1970), and Hyalophora (Greenstein, 1972). The rough-surfaced endoplasmic reticulum is swollen and more Golgi bodies are present, with numerous dense vesicles, some coated vesicles and numerous mitochondria. The apical plasma membrane is raised into microvilli with dark tips, over which cuticulin appears as described above. During cuticular secretion vesicles in the apical cytoplasm join the bases of the microvillar projections. These vesicles are interpreted as profiles of secretory material derived from Golgi bodies in the process of transfer to the plasma membrane, and fusing with it, thus contributing to the cuticular material. In Oncopeltus wing pads secretion of the epicuticle and a few lamellae of procuticle is completed prior to ecdysis. (c) Apolysis: In Oncopeltus cell divisions begins very shortly after ecdysis and the process of detachment occurs after a few further •

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days though as pointed out above, Wigglesworth (1973a) had supposed that the epidermis needed to detach before it could undergo mitosis. The approximate time of apolysis is difficult to judge solely from light microscopical observations, since it must then be based on estimates of procuticle thickness and moulting fluid. The cuticle of the wing pads becomes detached artificially at every stage during sectioning (as is also reported by Wigglesworth, 1973a). Locke, • (1979), from work on Calpodes, provides a precise definition of the term 'apolysis'. He found that the epidermal cells usually had small flattened areas or plaques with additional density on the cytoplasmic face. They occur typically at the tips of microvilli. When cuticle deposition ceases the plaques are lost as the area of apical plasma membrane is reduced. They reform again in time to secrete, new cuticulin and fibrous cuticle; and the period when the plaques are absent constitutes, in Locke's view, the time of apolysis. In Oncopeltus wings procuticular secretion continues until the cell number has increased sufficiently. The cuticle then detaches only when the epidermis stops secreting procuticle. The ultrastructure of the apical plasma membrane during procuticular secretion, after its detachment from the cuticle, and during its subsequent reformation into microvilli prior to new cuticle secretion is exactly similar to the events described by Locke (1979). Immediately after the detachment of the epidermis, moulting fluid appears in the exuvial space. In Oncopeltus wing pads, as in many other insects such as Calpodes (Locke, 1970, 1979), Blattella (Kunkel, 1975), and Galle ria (Barbier, 1971), cell division does not require detachment. (d) Exuvial space and moulting fluid: An exuvial space is formed during apolysis and becomes filled with a foam-like secretion similar to the type noted by Noble-Nesbitt (1963b) in Collembola. It is very difficult to give exact times for the formation of the exuvial space and the first appearance of moulting fluid, since they vary with the duration of the instar. However, about 72 hours after ecdysis a foam-like 375 moulting fluid is found in the exuvial space, and appears blue when stained with Mallory's triple stain. This is followed by a stage in which a few red particles are also present in the moulting fluid. Ultrastructurally, the first-formed moulting fluid contains small vesicles surrounded by smooth membranes and enriched with ribosomes. Shortly after the appearance of these vesicles, dense granules are seen in the exuvial space. These aggregate in masses approximately 10 pm in diameter; the granules appear to be released into the exuvial space by the rough-surfaced endoplasmic reticulum of the epidermal cells. They were considered by Noble-Nesbitt as tightly packed arrays of protein-bearing sub- units, the old cuticle being eaten away when in contact with the granule body. Noble-Nesbitt suggests that these contain moulting enzymes, which have become activated to digest the old endocuticle. My ultrastructural observations on the formation and structure of moulting fluid in Oncopeltus wing pad cuticle agree well with the details given by Noble-Nesbitt (1963) for Podura. I have not noticed any ecdysial droplets in the formation of the ecdysial space, as seen in larval Lucilia by Filshie, or by Lock & Krishnan (1973) in Calpo de s . (e) Ecdysial Haemocytes: In the exuvial space of 120-hour fifth instar larval wing pads of Oncopeltus some isolated cells were noticed in addition to the moulting fluid. These were seen under the light microscope as well as in ultrastructural preparations. Barra (1969) noticed similar structures in some Collembola and interpreted them as ecdys-ial haemocytes because of their close resemblance to the granulocytes described by Devauchelle (1971). Zacharuk (1972) also found ecdysial haemocytes present in the exuvial space of the Elateridae, Ctenicera destructor, Limonius californicus and Hypolithus bicolor. The cells are believed to be haemocytes which migrate into the ecdysial space through the epidermal layer from the haemolymph before the new epicuticle layer has become r

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continuous, though none were seen within the epidermis in any of the above authors' preparations or in the present study. They are thought to disintegrate to release lysosomes containing lytic enzymes able to digest lamellate endocuticle. It is obviously difficult or impossible to verify such an hypothesis ultrastructurally. (f) Gland cells:

0 The presence of unicellular glands and their structure was discussed when dealing with the light microscopy of the developing wing pads. The ultrastructure of 96-hour and 120-hour fourth instar wing pads showed the presence of unicellular gland cells among normal epidermal cells and in association with dividing bristle- forming cells. Gland cells are larger than ordinary epidermal cells and possess a large central lumen, surrounded by long finger-like microvilli. Towards the basal side, the cytoplasm is traversed by apposed plasma membranes indicating that a form of cellular invagination has taken place. These membranes are joined by intermediate junctions and septate desmosomes. The abundance of rough-surfaced endoplasmic vesicles in the cytoplasm surrounding the lumen suggests that the whole area is involved in secretory activity. Secretions (perhaps proteins) appear in the microvilli as small vesicles, and the tips of the microvilli are cut off into the lumen. Neither a duct nor the discharge of the secretions were noted during the present study. In the early stages of development the gland cells lie close to the adjacent normal epidermal cells, with their plasma membranes eaprated by a narrow intercellular space, but the mature cell shows a wider intercellular space and the entire cell perhaps detaches and enters the exuvial space to liberate its contents, which may contribute to the moulting fluid. Philiptschenko (1907) noted similar unicellular glands in the epidermis, which change their appearance at the onset of ecdysis, when the cytoplasm becomes foam-like. Hoop (1933) described moulting glands somewhat similar to those of Oncopeltus in the larval epidermis of Limnophila, Nematus and others, though they are not found in all s

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insects. As in Oncopeltus they appear to lack a duct and are not surmounted by cuticle through which the secretions are eliminated (the two major types of epidermal glands discussed by Nairot & Quennedy (1974). Hoop considers that these glands which show cytological changes in relation to the moulting cycle are not responsible for secreting the moulting fluid, which he believes to be a product of the normal epidermal cells. Their ultrastructure and function seem to need further study. (f) Chromatin droplets: While the epidermal cells are actively dividing, large flask shaped cells, with a small cytoplasmic volume and large chromatin masses in the nucleus were found among them. The nuclear envelope disappears and the chromatin is liberated as droplets. Wigglesworth (1942) and Laurence (1966b) have described similar forms of cell degeneration during the moult (see

also Wigglesworth, 1954). - (g) Extracellular structures: During feeding and growing stages, the extra-cellular space in the wing pad encloses some relatively large, highly vacuolated structures enclosed by fibrillar membranes and measuring about 5 atm in diameter. They are filled with ribosomes and small vesicles and some times they even possess a little ribosomal endoplasmic reticulum and a few Golgi bodies. During cell division the contents are lost and only empty profiles are left. There is no clear evidence to show their nature. They are without nuclei and therefore cannot be some sort of haemocyte. They are not cut portions of basal cytoplasmic tails since they do not include mitochondria or microtubules. It is possible that during the feeding and growing stages, unwanted ribosomes or ribosomal endoplasmic reticulum may be eliminated from the epidermal cells in the form of small vesicles such as are known to occur in the exuvial space, and that these then aggregate to form a large structure which can provide reserves that are reutilised by •

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the cells, leaving only the empty profiles behind. (h) Basement membrane: The ultrastructure of the third instar wing pad shows that there is a thick continuous basement membrane between two layers of integument. This separates into two layers near the lacuna and encircles it from both sides. At every stage the lacunae are encircled by a basement membrane but there may be variations in • its thickness depending upon the stage. As was previously noted in the wing epidermis of Hyalophora cecropia (Greenstein, 1972), the basement membrane is sometimes absent between most of the epidermal cells (of the fourth and fifth instar wing pads in Oncopeltus). The light microscopical appearance of the basement membrane may be due to the presence of long, thin processes of haemocytes and the elongate, bent basal processes of epidermal cell. In the past, various light microscopic studies of wings have reported the presence of a thick, compound basement membrane (middle membrane) between the integuments. Marshall (1915), for example, says the basement membranes of two epithelia comes together in certain areas and fuse more or less completely to give rise to a conspicuous but incomplete layer called the middle membrane of the wing. A middle membrane has been reported to be a true membrane (Richards, 1951), but it has also been described as very thin or absent in various insects (Jannone, 1939; Waddington, 1940). Its absence may perhaps facilitate the exchange of substances between the two layers of integuments. The light-microscopic and ultrastructural studies of Wigglesworth (1933, 1955, 1956, 1959b, 1972 and 1979) have fully demonstrated the formation of a basement membrane by haemocytes. Early in the moulting cycle of Oncopeltus (as Wigglesworth also describes for other species) the plasmocytes settle in large numbers below the basement membrane and discharge inclusions which merge with the substance of the original membrane. When the plasmocytes are more conspicuous the basement membrane increases in thickness (Wigglesworth, 1979). The thickness is 379 variable even in a single specimen and Wiggle sworth showed that it depends partly upon nutrition. In Oncopeltus the presence of a basement membrane around the lacuna and in early instars is associated with the presence of a blood circulation. In the areas where their blood circulation is apparently absent, the basement membrane is also absent. It has been well established in the present study that plasmocytes can give off long processes, from which secretions are added to the basement membrane. (i) Lacunae: Both light and ultrastructural observations show that there are five prominent lacunae in the third instar wing pads of Oncopeltus, all lined by a basement membrane. Micrographs from the tip of a wing pad show empty lacunae containing neither nerves or tracheae. The micrographs from the base of the wing pad, on the other hand, show well developed tracheae enclosed within the lacunae. This and other evidences discussed above shows that the formation of lacunae takes place in the developing wings earlier than the entrance of tracheae (Holdsworth, 1941). (j) Tracheae: Each lacuna in the wing pad encloses a single trachea, which is circular in cross-section, composed of tracheal intima, tracheal epithelia and basement membrane. The lumen is lined by a dense cuticulin and is helically folded into prominent taenidia. The intra-taenidial and taenidial portions of epicuticle show micropapillate processes except over the inner surface of the taenidia (Locke, 1957). Internal to the cuticulin is a less dense cuticle staining blue with Mallory's triple stain; which confirms that a procuticle is present in the trachea of Oncopeltus. According to Locke (1964) and Smith (1968), there is no procuticle between the taenidia and epithelia, but Miller (1964), Richards (1951), Wigglesworth (1965), and Whitten (1969, 1972) all showed the presence of procuticle in the trachea. Locke has concluded that there may be a procuticle in the larger tracheae only. The tracheal 380 epithelium is single layered, with flat cells and cytoplasmic organelles similar to those of the general epidermis. The abundance of cytoplasmic organelles is related to the growth and secretion of cuticular intima by the cells. The tracheal matrix cells contain microtubules (Smith, 1968), arranged parallel to the length of the tracheae. These may have a supporting function or play a role in tracheal formation. Cell boundaries are very prominent and similar to the description given by Beaulation (1964). The outer and inner plasma membranes of tracheal epithelia are connected by folds

"mestracheon" (see Edwards et al. , 1958). As the wing pad integument, so also in the tracheal epithelium, cell division and apolysis take place successively from one end of the trachea to the other, rather than simultaneously at all places. Apolysis occurs earlier in the tracheae than in ordinary wing epidermis, but the secretion of new cuticle appears later. As early as 24 hours after ecdysis, cell divisions and degeneration of some cells were noticed in the wing pad trachea. The phenomena of growth and decay are responsible for cellular reorganisation and increase in diameter of tracheal tube (Wigglesworth, 1954a). When the epithelium retracts from the tracheal intima, an exuvial space appears between them and becomes filled by an electron-dense fibrous material to form a continuous membrane. Whitten (1969, 1972) suggests that an ecdysial membrane helps to determine surface patterns by supporting cytoplasmic extensions. Hinton (1974) suggested that the function of a moulting membrane is to protect the epidermal cells mechanically while they are naked after apolysis. The presence of dense fibrous material between the old intima and the epithelium of the developing trachea has also been reported by Locke (1966, 1969). The epithelium slowly detaches from it, just before the secretion of new cuticulin. In the third instar cuticular patches appear over small dense areas of apical plasma membrane in the tracheal epithelia. In the fourth and fifth instars, however, they appear on long narrow •

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stacks of microvilli and fuse to form a continuous membrane (Locke, 1966). Locke (1966) and Whitten (1972) found that in small tracheae and tracheoles the cuticulin arises in small patches directly above the non microvillate plasma membrane (or sometimes over slightly raised portions) whereas in large tracheae it appears, over long stacks of microvilli. In Oncopeltus the apical plasma membrane accompanies the cuticulin as it buckles to form taenidia. When the • taenidial form is fully established, the plasma membrane retracts towards its original form (cf. Locke, 1966). The secretions from the dark tips of the microvilli enter the taenidial fold and a solid taenidium is thus formed. Ultimately a thin layer of procuticle is formed around the trachea between the cuticulin layer and the epithelial ring. In the present study the formation of new tracheal branches was not specially studied, but in the third instar a tracheal tube at the base of the wing pad was seen to be connected with a chain of epidermal cells lying inside the wing pad. This is evidently the beginning of new tracheal growth from an existing trachea, as described by Wigglesworth (1954); the new tracheae and tracheoles arise by outgrowth of columns of cells from the sides or endings of existing tracheae. Tracheae are encircled by a basement membrane which separates the epithelium from haemolymph. At some places the trachea lie close to one side of the integument without any intervening basement membrane. The basement membrane lining the lacuna is, however, closely applied to the remaining portion of the tracheae matrix cells. In the same way, wherever the trachea and nerve come close together the basement membrane of the trachea disappears or becomes fused with that of the nerve. These direct contacts between epidermis and trachea or trachea and nerve perhaps facilitate exchange of metabolite between the cell layers in contact. (k) Tracheoles: The inner lining of the tracheoles is formed from a smooth, dense epicuticle (cuticulin of Locke, 1957, 1966). It is helically •

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folded into taenidia. In tracheoles the taenidia are regularly arranged, and the inner walls are smooth, without any micropapilla. The tracheoles are included within the main body of the tracheoblast or in its prolonged cytoplasmic extensions which lie close to the tracheolated epidermis. Tracheole formation, associated with invaginated plasma membranes, in the form of "vacuolated" intercellular space, was noticed in Oncopeltus wing pads. The close S association of Golgi bodies and numerous secretory vesicles with these vacuolated spaces and with the formation of cuticulin inside the vacuole suggests that the plasma membranes of tracheal end-cells develop vacuoles as described by Wigglesworth (1973), in the unmodified epidermis. The vacuoles from Golgi areas fuse with these plasma membranes and in this way a new intima may be formed. The mechanism of tracheole formation in insects has never been satisfactorily described. According to some, vacuoles arise intra- cellularly on whose walls the new intima is deposited (Keister, 1948; Wigglesworth, 1954), while Edwards (1958), considers that there may be tubular infolding of plasma membrane to form cavities which would be linked to the cell surface by a "mestracheoin" analogus to the nerve "mesaxon". Locke (1966) suggests that in some tracheoles the lumen arises by the fusion of vacuoles from the Golgi bodies, or that it might arise entirely by growth of plasma membrane facing the tracheal lumen. (1) Nerve: The nerve in the wing pad resembles any other multiaxonal peripheral nerve branch of insects. Larger axons are surrounded individually by glial cytoplasm and minute nerve axons form a bundle and lie in a common glial envelope (Edwards, 1960; Smith & Treherne, 1963). The nerve lying in the sub-costal lacuna is placed in the centre of the lacuna and is surrounded by a basement membrane. Other nerves lie inside the extracellular space of the dorsal wing integument, closely applied to the basal portions of the epidermal cells towards one side but separated by a narrow inter- 383

cellular space. The ventral side is bounded by a lacuna. (m) Adult wings: The ultrastructure of the mesothoracic wings or hemelytra of Oncopeltus was studied in this investigation. The hemelytra of Oncopeltus show special ultrastructural changes associated with the development of trabeculae, the corio-membranic border and veins and the peculiar process of tracheal retraction in the membrane. S In the newly emerged wings the epidermis is in an apparently normal living condition. The tracheae are somewhat oval in cross-section. The epidermal cells are more or less conical in structure. In the membrane portion of the wing most of the basal surfaces of the cells are free inside the wing, supported by microtubules (miofibrils of Hundertmark, 1936; or tonofibrils of Holdsworth, 1942). Some of the basal processes from apposed cells come in contact with each other and are joined by a desmosomal attachment. The formation of desmosomes in the epidermis of Rhodnius has been reported by Wigglesworth (1977) when the epidermal processes that he refers to as "strands" are in contact with the basement membrane or where they come in contact with similar strands. The fenestrated middle membrane in the corium is partly formed from processes of the haemocytes, and is present between the epidermal layers. The desmosome attachments, the long thin processes of haemocytes and the curved basal processes of epidermal cells all seem to be responsible for the light microscopic appearance of a middle membrane in the wings. The cuticle covering the dorsal surface of the corium is much thicker than that of the ventral surface and from the tip of the membrane. The epidermis is still actively engaged in the deposition of endocuticle at the apically microvillar plasma membrane and the adjacent associated vesicles. The increase in Golgi bodies, dense vesicles, and coated vesicles coincides with the period of increasing protein synthesis. The basal portions of some cells are produced into long thin processes, running towards the 384 lacunae and sometimes joined to epithelial cell process of the trachea. The ultrastructure of these processes is similar to that of the cytoplasmic strands described by Wigglesworth (1977), and given off by epidermal cells when deprived of oxygen to run in the direction of air filled tracheae. The presence of similar processes in the late pharate and post ecdysial adult wings of Oncopeltus suggests a response by the dividing cells to a possible oxygen deficiency. A few hours after ecdysis the basal extremeities of epidermal cells from both dorsal and ventral surfaces of the corium extend into the interior compartment of the wing and join by desmosomal attachments. Because of these structures the interior compartment of the wing gives the impression in transverse section of being divided into small chambers. These spaces are filled with haemocytes and cytoplasmic fragments. Whitten (1964) reported that the haemolymph of newly emerged Sarcophaga bullata adults contained cellular fragments derived from the degenerating haemocytes, and found that the haemocytes are actively engaged in phagocytosis of these fragments. Similar phagocytotic blood cells have already been described in Calliphora (Crossley, 1965, 1968, 1975). In the corium the epidermis contains variously shaped nuclei and the structure of the chromatin shows that the cells are undergoing further division. The cytoplasm of these epidermal cells contain a greatly increased rough-surfaced endoplasmic reticulum with numerous Golgi bodies and mitcohondria, thus showing a phase of high secretory activity at the beginning of the cell divisions. The accumulation of abundant glycogen deposits and some lipid droplets in the epidermis (mainly in the cells lying around the lacunae) suggests that haemolymph circulation continues in the corium of Oncopeltus wings at least for some time after emergence. At 24 hours after ecdysis there is little change in the epidermal cells in the membrane of Oncopeltus except for an increase in the amount of rough-surfaced endoplasmic •

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reticulum. The presence of multivesicular bodies, cytolysosomes, autophagic vesicles and large electron-lucent vacuoles all indicate the beginning of cell degeneration by autolysis. Seligman & Filshie (1975) report that the first ultrastructural indication of epidermal degeneration in the developing wings of Lucilia cuprina is the appearance of large vacuoles and extracellular channels penetrating these cells. • (n) Cell degeneration and retraction of tracheae in the wing membrane: Twenty four hours after adult emergence, the epidermis near the tip of the membrane has started to retract from the cuticle, and undergoes degeneration through autophagy accompanied by phagocytotic attack, apparently by other epidermal cells. As Goldsmith (1966) has pointed out, the death of the cell is accomplished economically with the digestion and reutilisation of its old components. The degenerating cells in the wings of Oncopeltus undergo a sequence of events similar to those in degenerating cells from the wings of Drosophila melanogaster (Fristrom, 1968). Ultimately the entire membrane epidermis disappears. The cuticles from the dorsal and ventral sides thus come to be pressed closer together. Snodgrass (1935) and others have pointed out that when wing development is completed the epidermis has largely disappeared; the mature wings are almost entirely cuticular structures. Bland & Nutting (1969) agreed that this generalisation applies in the Endopterygota, but claimed that living epidermal cells persist in Melanoplus lakinus and, apparently, in other Exopterygota. In Oncopeltus, however, Snodgrass's view appears to be correct. In the newly emerged wings the tracheal intima is oval in cross-section, but this oval profile is lost through the collapse of the tracheae within a few hours after emergence of the adult. The collapsed tracheae from the membrane move up to the border with the corium accompanied by the retracted epidermis. 386

The degeneration of tracheae in this region takes place as the last major event in wing development. The intima of the tracheal tube remains coiled, adjacent to corio-membranec border. (o) Trabeculae: In the areas where the two surfaces of the wing are joined by epidermal cell-process, the cuticular thickening from the dorsal integument grows by addition of cuticle by the trabecular epidermis, which contains long tubules of rough-surfaced endoplasmic reticulum and Golgi bodies. The cuticular column reaches the ventral side of the wing, and here the epidermis from which it has developed is attenuated where it meets (though never fuses with) the ventral cuticle. The two surfaces of the corium are separated by a blood space across which run the longitudinal rows of trabeculae. (p) Formation of veins and intervein areas: The epidermal cells of the corium lining the ventral surface of the lacunae start to secrete cuticulin about twenty four hours after the final moult, so forming the ventral walls of the tubular vein. During this period the degeneration of collapsed tracheal epithelia takes place in the corium. By the third day of adult life, larger portions of cytoplasm and the associated organelles from epidermal cells lying around the lacuna and tracheal epithelia have been replaced by intracellular cuticulin deposits. By fifty hours the formation of veins due to the deposition of sclerotised cuticle around the lacuna has been completed. The epidermal cells lying in the intervein areas of the wing still contain major zones of cytoplasmic organelles, though cuticle deposition is also taking place there. Eventually the fully formed adult wings contain highly cuticularised wing membranes crossed by strongly slcerotised veins, each enclosing the intima of a collapsed trachea and their detached epithelium, who is also sclerotised. The dorsal wall of the vein contains a nerve and apparently remains alive, perhaps throughout the life of the adult insect. 387

(1) A morphological account of the adult wings of Oncopeltu.s faciatus, including their venation, basal attachment and axillary sclerites, is given. Based on these observations, an appropriate system of homologies and notation has been proposed for the wing veins which are denoted as sub-costa, radius, radial sector, media, cubitus, first and second anal. (a) The development. of a tracheal supply to the larval wing pad is described in relation to the venation. Tracheae from anterior and posterior sides of the wing pad enter the lacunae in the second instar. A pattern of adult wing tracheation is well established in the third instar. Sc, R and M arise from the costo- radial trunk, while Cu, lA and ZA arise from the cubito-anal trunk. Both groups are connected by a transverse basal connection. (3) In the fifth instar, the radial trachea of the fore wing gives off a small anterior branch near the corio-membranic margin. This branch, observed here for the first time, is identified as R and thus confirms the presence of a radial sector in the membrane. (4) Some variations have been reported in the cubital trachea. (5) Within 24 hours after the emergence of the adult, tracheae from the posterior-half of the wing retract slowly, coil up and lie between the medial and cubital veins adjacent to the corio- membranic border. Ultrastructurally it has been established that they are the coiled tracheal intimae left after the complete degeneration of the tracheal epithelia. (6) Light microscopic histology and ultrastructural study provided a detailed account of the differentiation of the wing pad at selected intervals throughout the moulting cycle and intermoult period. The newly moulted wing pads show a single layer of epidermal cells containing numerous polyribosomes and mitochondria. Ribosomal endoplasmic reticulum, Golgi bodies and •

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mitochondria increase in number prior to the cell division, nuclei round up, and cells divide before the epidermis becomes detached from the cuticle. The wing pads of the pharate phase are provided with stratified epithelium. The cell boundaries are very clear in ultramicrographs. Before secreting the new cuticle the epidermal cells become re-organised into a single layer. (7) The basal cytoplasm of the pharate wing pad epidermis t becomes produced into elongate processes, supported by longitudinally directed bundles of microtubules. (8) The basal cytoplasmic processes of the pharate wing pads meet those of the opposite surface or the basement membrane, but some processes merge with the ends of neighbouring cells to leave intercellular spaces, which fuse to form the lacunae. When first formed, these do not contain tracheae. Tracheae grow into these channels only when their pattern has been established. (9) A continuous basement membrane between two layers of wing pad integument is shown in the ultra-micrographs of third instar wing pads, but in the fourth, fifth and adult instar wings it is absent in most areas of the wing except around the lacunae. (10) During the moulting cycle, dividing bristle cells, degenerating cells and unicellular glands were found among the normal epidermal cells. The unicellular glands enclose a large central lumen, surrounded by microvilli. They are not provided with a duct, or lined by any cuticular investment. They disappear before the new cuticle is secreted again. They are not present in the adult wings nor in the tracheal epithelia. (11) Paranotal regions of cell proliferation have been discovered in the prothroax and are perhaps serially homologus with the wings, but they regress later in post embryonic life, perhaps through lack of nutrition and oxygen supply, since lacunae and tracheae never seem to enter these prothoracic expansions. (12) In the newly emerged adult wings, the epidermis, tracheal epithelia and nerves are in a living condition. The lacunae contain 389

numerous blood cells. The epidermis, which is actively engaged in cuticular secretion, is provided with mitochondria, glycogen granules, ribosomal endoplasmic reticulum, Golgi bodies, dense secretory vesicles and coated vesicles. (13) A few hours after its emergence, the tracheae of the adult wing collapse, and this is followed by the degeneration of the tracheal epithelia in the corium. • (14) 24 hours after emergence the epidermis from the membrane of the adult wing starts to degenerate through autophagy, accompanied by phagocytotic attack by other epidermal cells. Within 48 hours the entire tissue from the wing membrane has degenerated completely and, the cuticular layers are pressed closely together. (15) The epidermis in the corium undergoes mitotic divisions to give rise to columns of cells where the trabeculae later appear. The secretion of the trabeculae takes place from the dorsal to the ventral surface, and they do not fuse with the ventral cuticle. (16) In the corium of the adult wing, the epidermal cells lining the lacunae secrete the cuticular substance from which the walls of the vein will be formed, and the epidermis degenerates completely. The epidermal cells from the interlacuna spaces also secrete cuticular substance and degenerate. The fully formed adult corium of the hemelytron contain highly cuticularised wing membranes crossed by strongly sclerotised veins and enclosing the intima of collapsed tracheae. (17) The dorsal wall of each vein contains a nerve, which remains alive for the rest of the insect's life. •

PART VI

s LIST OF ABBREVIATIONS USED IN ILLUSTRATIONS •

390

VI LIST OF ABBREVIATIONS USED IN ILLUSTRATIONS

anal vein (or trachea) a axon of anal furrow anp anterior notal process • atr - anterior trachea av autophagic vesicle axi, ax2, ax3, ax4 - axillary scierites axc - axillary cord axm axillary membrane bas - basalar membrane btr basal trachea ch chromatin cl clavus cm costal margin cmb - corio-membranic border cor corium cp cytoplasmic process c-r costo- radial crg - costo-radial group cs claval suture c. s campaniform sensilla Cu cubital vein (or trachea) cu-a cubito-anal cuag cubito-anal group cuf cuticular fold cum - cuticular material cut cuticle (old, cutl ; new, cut2 ) cv coated vesicle d - de smo some dc dead cell dep electron-dense epidermal cell s

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dg electron-dense granule dgc - degenerating cell dmp distal median plate dsv electron-dense secretory vesicle dv dense vesicle ecdm ecdysial membrane ecsp extracellular space e. cut epicuticle elv - electron lucent vacuole ep epidermis er - endoplasmic reticulum exsp - exuvial space f furrow G - Golgi body g glycogen gl - gland cell glc glial cell gls glial sheath h humeral angle haem - haemocyte hp - humeral plate ib isolation body ic - intercellular junction (normal) icsp intercellular space ij - intermediate junction 1 lumen lac lacuna lc - light cell ld - lipid droplet ler - lamellae of rough-surfaced endoplasmic reticulum ly - lysosome-like body M - medial vein (or trachea) m - mitochondria r

392

ma - mesaxon mac macrotrichium mb - microbody mc bristle mother cell mdsv - moderately dense secretory vesicle me - membrane met - mestracheon • mfl moulting fluid mic - microtrichium mit - mitotic division mm middle membrane mt microtubule my - microvillus mvb - multivesicular body n - nucleus ncl - nucleolus ne nuclear envelope np nuclear pore nt neurotubules nv - nerve oen oenocyte p micropapillae Pi - plasma membrane infolding pm plasma membrane pmp proximal median plate pnp posterior notal process

po. m - posterior margin pr polyribosome s pro-cut procuticle pw p - pleural wing process R radial vein (or trachea) rer rough-surfaced endoplasmic reticulum Rs - radial sector 393 s suture Sc sub-costal vein (or trachea) scp scutellar process sd septate desmosomes sub sub alar sclerite sub- cut subcuticle sv secretory vesicle t trachea tbt - transverse basal trachea tec tracheal end cell tep - tracheal epithelia thl - prothorax th2 mesothorax th3 - metathorax ti - tracheal intima tl tracheole to - taenidia to - tormogen cell ton tormogen nucleus tr - trichogen cell trn trichogen nucleus trab trabeculae u. r. upturned ridge V - vein v vacuole ve r - vesicle of rough-surfaced endoplasmic reticulum vf ventral fold W r - wing rudiment •

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