Ultrastructure of the “fenestrae” specialized integumentary areas of the desert locust, Schistocerca gregaria forsk. (Orthoptera: Acrididae)

lnt.J. Insect Morphol. & Embryol. 6(5/6): 265-275. 1977, Pergamon Press. Printed in Great Britain.

ULTRASTRUCTURE OF THE "FENESTRAE" SPECIALIZED INTEGUMENTARY AREAS OF THE DESERT LOCUST, SCHISTOCERCA GREGARIA FORSK. (ORTHOPTERA: ACRIDIDAE) G. SBRENNA and M. ANTONELLI Institute of Zoology, University of Ferrara, Via Borsari 46, 44100 Ferrara, Italy

(Accepted 19 May 1977) Abstract--The ultrastructure of the characteristic integumentary areas, called fenestrae on the abdominal terga, was studied in S. gregaria. They exhibit thinner cuticle than the general integument, since the endocuticle is missing. The epicuticle and exocuticle are traversed by pore canals, which are more numerous than those in the exocuticle of the general integument. The fenestrae cells bear a large number of microvilli and rest on a thick basement membrane, which extends far up between them, forming loops, which almost reach the subvillous zone. The cells are tightly packed near the apical surface, where there is a desmosome of "macula adherens" type and a septate junction. In the basal portion of each cell, the plasma membrane infoldings give rise to an intracellular membrane system. As compared with the cells of the general integument, the cytoplasm of the fenestrae shows a greater number of vesicular structures. The probable function of such areas in relation to their ultrastructural appearance is discussed. Index descriptors (in addition to those in title): "Fenestrae", specialized cuticle, locust

integument. INTRODUCTION IN THE acridid integument, certain areas referred to as "fenestrae" (Snodgrass, 1935) or "Slifer's patches" (Uvarov, 1966)are encountered. These zones are easily distinguished from the general integument as they are only slightly sclerotized, almost free o f pigment and b o u n d e d by a distinct bordering line. Snodgrass (1929) and Misra (1947), in their essentially morphological studies, were the first to describe these fenestrae in Schistocerca gregaria and in Dissosteria carolina respectively. Later, Slifer (195 I, 1953a, b) gave a detailed description o f these structures in Locusta migratoria migratorioides, and also enquired into their possible function. On the basis o f her experiments, she assumed these areas to be concerned with thermoreception. Makings (1964, 1968) rejected Slifer's original suggestion, also on the basis o f Weir's (1957) earlier electrophysiological investigation into their thermosensitivity, and showed, using films o f water-sensitive substances, that there was a greater transpiration in these areas than in other parts o f the integument. We have studied the ultrastructure o f the epidermis o f these areas in S. gregaria, and c o m p a r e d them with the general surrounding integument in order to correlate their m o r p h o logy with their probable function. MATERIALS AND METHODS We examined S. gregariaadults from a strain of our laboratory. The insects were reared in crowded cages in which light and heat were provided by 60-W bulbs, with a 12-hr photoperiod, and at 25°C and 65 ~ RH. They were fed on fresh chicory and dry bran. 265

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Newly moulted adults were sacrificed at 16 and 30 days after the last ecdysis. Each individual was injected with cold fixative through the abdomen, and the abdominal terga and sterna were removed under the stereomicroscope. For light microscopy, the integument was fixed in 2 % glutaraldehyde in Hoyle's solution buffered at 6.9. In one group of insectes, the terga and sterna of all the abdominal segments were stained in toto and after dehydration mounted in balsam. The following dyes were used: modified Mallory, Nile Blue sulphate, Oil red, iron hematoxylin. Whole abdominal segments were also fixed in Zenker and Bouin fluids, and embedded in Tissuemat. Sections 7 ~m thick were then stained with Mallory solution. For electron microscopic examination, the terga of the 3rd and 5th segments were fixed either in 1.25% glutaraldehyde in 0.1 Mphosphate buffer at pH 7.0 or in 2% glutaraldehyde in 0.05 Mcacodylate buffer with addition of sucrose, at pH 7.2, or in 2% glutaraldehyde in Hoyle saline solution. After fixation for 2 hr the fragments were post-fixed in 1% osmium tetroxide, dehydrated and embedded in Epon-Araldite. Thin sections were double-stained in uranyl acetate and lead citrate. A Jeol TM 7 electron microscope operated at 60 kV was used to examine the material.

RESULTS Light microscopy In S. gregaria adults the integumentary fenestrae show a localization similar to that observed in other acridids (Slifer, 1953 a, b). In both males and females, anterior to the base of the antennae, there are 2 small crescentic areas, "antennal crescents" (Slifer, 1951), and a pair of fenestrae in each thoracic segment on either side of the median line. In the abdomen, the fenestrae appear in every segment, but they differ in number and shape according to sex; moreover, their distribution pattern is also different compared with that in the larval stages. In adult males there is a pair of fenestrae in each tergum on either side of the median line, above the chambers of the heart; they are ovoid in shape in the anterior terga and shaped like an inverted L in the posterior ones. A small fenestra is also located near the tracheal stigmata of the second abdominal segment. Fenestrae in adult females also are situated on either side of the dorsal median line, but practically all of them are ovoid in shape. There is also a fenestra close to each tracheal stigma in 2nd-4th terga, exhibiting roughly the same extension of the fenestrae of the dorsal area, except those of tergum 4, which are very small. In the area of the fenestrae, there are setae and sensilla, as well as occasional isolated integumentary glands. The former have a precisely fixed location in each fenestra, while the glands, easily indentifiable by their large secretion-collecting chamber (Sbrenna and De Carli, 1972), become clear only in sexually mature males. The glands are located mainly close to the border of the fenestrae, and are more numerous on the posterior segments. The fenestrae cells have a stellate appearance due to the numerous cytoplasmic interdigitations between apposing cells (Fig. 12). In the cytoplasm of various cells, numerous granules are identifiable by staining with Oil red and Nile Blue sulphate. Similar granules, though greater in number and density, are present in the nearby, epidermal cells, where they are regarded as pigment granules (Weis-Fogh, 1970). In cross sections of the abdominal terga the fenestrae are found above each of the 2 longitudinal tracheal dorsal trunks, at the level of their branching into the transverse branches. Below each area there is a space free of both pericardial cells and fat body, which in vivo is occupied by hemolymph (Fig. 1).

Electron microscopy Electron microscopic observations reported in this study were carried out almost entirely on the fenestrae of the 3rd and 5th abdominal terga of adult males, 16 days after imaginal

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FIGS. 1 and 2. Light micrographs of a transverse section of abdominal fenestra. Cuticle is thinner than that of contigous integument. Note in Fig. 2 characteristic endocuticular enlargement (asterisk) and long infolding of basement lamina (arrow). ex = exocuticle; en = endocuticle; p.s. = perieardial sinus. Fig. 1. x163; Fig. 2. x384 Fins. 3 and 4. Low magnification survey micrographs of cuticle of fenestra (3) and of contiguous general integument (4). Note different thickness of 2 exocuticular lamellae, different density of pore canals, x 6720 FIG. 5. Electron micrograph illustrating appearance of cuticle of a fenestra. Evident are epicuticle (ep), exocuticle and alveolate zone (az) in direct contact with subcuticle (so). Large pore canals (pc) pass almost straight throughout cuticle. × 14,880 Fie. 6. Longitudinal section of a pore canal containing a single filament. ×21,120 FIG. 7. At level of alveolate zone, in lumen of a pore canal is present a translucid vesicle whose significance is unknown (asterisk). my = microvilli. × 13,440 FIG. 8. Oblique section of fenestra cuticle illustrating abundance of pore canals, x 16,800 267

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ecdysis. Ultrastructural differences were not found either in adults of different ages or between males and females.

The cuticle The fenestrae exhibit a cuticle which is approximately 12-15 /~m thick, hence thinner than that of the surrounding general integument, where it is 25-30/zm thick. The cuticle is composed only of the epicuticular and exocuticular layers, the endocuticle being altogether absent, although in its place there is a subcuticular layer 2-3/zm thick (Figs. 3, 5). The epicuticular and exocuticular layers merge without interruption with those of the adjoining integument and appear similar to those both in their lamellate appearance and the number of lamellae (some 40-45) (Fig. 2). However, pronounced differences are observed in the thickness of the cuticular layer and in the number and appearance of the pore canals, which traverse it (Figs. 3, 4). The thickness of the exocuticle in the near general integument of the abdominal terga averages 9/~m, the pore canals are not very numerous and branch into narrower canals. Conversely, in the fenestrae the exocuticle may reach 12-15/zm at its maximum width due to the thickness of the single iamella; the pore canals are about 3 times more numerous, and uniformly distributed (Fig. 8). They follow an almost straight path; their diameter may vary 0.3-0.4 tzm, decreasing slightly only towards the epicuticle, near which they ramify into narrower canals (Fig. 5). At the level of the inferior exocuticular lameUae, they are seen to anastomose by means of irregularly shaped connections, so that the basal portion of the cuticular layer takes on an alveolate appearance (Figs. 5, 7). In longitudinal sections in which the pore canals appear as long tubes, a single, straight filament is visible in their lumen, immersed in a slightly electron-dense matrix (Fig. 6), which resolves into more slender filaments close to the epicuticle. The single filaments inside the pore canals are also evident in the alveolate zone, as well as in the subcuticular one (Fig. 10) where they appear to be in contact with the villi of the cells of the fenestrae. In the subcuticular zone, translucent, roughly ovoid vesicles, are frequently found: their significance is unknown; similar vesicles are also present inside the pore canals of the alveolate cuticle (Fig. 7). As already mentioned, the endocuticle of the integument is absent at the fenestrae levels. The endocuticle of the integument is discontinuous at the edge of the fenestrae and, at this transition point, it thickens, expanding inwardly (Fig. 2). This characteristic endocuticular bulge, produced by the integumentary cells, results in a demarcation line between the general integument and the fenestrae. The cuticular zone of the areas under study also differs from that of the general integument at the level of the subcuticle. In the integument, the subcuticle is heavily electronopaque close to the cuticle and clearer above the very sparse and uneven microvilli (Fig. I0). Conversely, in the fenestrae the subcuticle exhibits a slightly electron-dense matrix throughout its width (Fig. 10). In addition, near the exocuticle, there is a very large number of irregularly arranged fine fibrils, whereas above and between the villi an amorphous material is present, consisting of a fuzzy electron-opaque component (Figs. 7, 10).

The epidermis of thefenestrae The epithelial cells of the fenestrae show the same structure under the electron microscope (Fig. 9). The apical membrane is folded into a series of elongate, thin mierovilli, projecting from the cell surface into the subcuticle; these often appear oriented in different directions (Fig. 10). They contain fine fibrils oriented along their length; in cross-sectioned villi the

Ultrastructure of the "Fenestrae"

FIG. 9. A portion of epithelial layer of a fenestra illustrating organization of cells. Long and irregular intercellular channels are visible (asterisk). n = nucleus; bl = basement lamina, x 7,200 FIG. 10. Apical zone of fenestra epithelium with numerous microvilli. Note filaments of pore canals in subcuticle (arrows) and above tips of microvilli a fine dense granular material, pg = pigment granules; mb = multivesicular bodies; * = intercellular space with fine material. In insert, apical zone of epithelium of general integument, x 19,200 (insert x 13,340) Fie. 11. Specialized junctions between adjacent cells of Fig. 10 at higher magnification. In cytoplasm are evident some vesicles homologous to epidermal granules of pigment (pg). d ~ desmosome; sj ~ septate junction, x 33,600

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fibrils display a circular arrangement. The lateral plasma membranes of 2 adjacent cells are about 0.05-0.15 t~m apart from each other, giving rise to an irregularly winding slit-like space reaching the basement lamina (Figs. 9, 10). At the upper part of these intercellular spaces, near the apical surface of the cells, there is a short, well-defined zone of membrane junctions, which involves the sequence of a desmosome "zonula adherens" type and a septate junction (Fig. 11). The connections between the cells of the fenestrae are different from those observed between the epidermal cells of the integument, in which the plasma membranes are tightly joined by long septate junctions, and the intercellular spaces are very rare. The basal plasma membrane of every cell lines the interdigitating cytoplasmic processes of adjoining cells above the basement membrane, forming a characteristic intracellular system of membranes, in Whose lumen a very fine granular material is evident (Fig. 12). In electron micrographs small cytoplasmic compartments are frequently observed at the basal border of the epithelium (Fig. 13). These irregular enclaves of cytoplasm are bounded by a continuous plasma membrane. They are interpreted as cross sections of the lateral processes that extend radially from the basal portion of the adjacent fenestrae cells. Evidently, they correspond to the cellular processes, referred to as interdigitations, visible in whole mounts (Fig. 12). A reconstruction showing the interdigitating cytoplasmic processes of adjoining cells, is shown in Fig. 19. A number of cytoplasmic organelles characterize these cells, thus sharply differentiating their cytoplasm from that of the cells of the normal integument. Numerous mitoehondria, are found throughout the cell, although they sometimes appear less numerous near the membranes which border the basal infoldings, and in greater numbers below the villous surface (Figs. 14, 16). Single cisternae of the endoplasmic reticulum, with which polyribosomal configurations are associated, are arranged into long strands parallel to the membranes bordering the basal channels (Fig. 12). Golgi bodies comprising smoothsurfaced cisternae (Figs. 12, 14) and slightly electron-dense vesicles are located in the proximity of the subvillous cytoplasmic zone, where multivesicular bodies are also present (Fig. 15). Small electron-dense coated vesicles (Fig. 14) are unevenly distributed, being more numerous in the vicinity of the membranes bordering the basal channels, where small pits are visible; these are pinched off into the cell as pinocytotic invaginations (Figs. 13, 18). In addition, 2 types of clear vesicles are present. The first type consists of vesicles about 0.5 /~m in diameter (Fig. 10), which are few in number, and lie in the upper area of each cell, and are homologous to pigment granules (Weis-Fogh, 1970). Numerous vesicles of this type are found in the subvillous cytoplasmic zone of epidermal cells (Fig. 10). The second type consists of variably distributed vacuole-like vesicles averaging 1-1.5/~m, which contain a finely granular material (Fig. 17). Lastly, in every cell, in the cytoplasmic compartments between the basal infolds, irregularly shaped lysosome-like dense granules are found (Figs. 9, 12, 13). The cells composing the epithelium of the fenestrae are separated from the hemolymph of the underlying sinus by a basement membrane on which they rest, being fastened to it by hemidesmosomes (Figs. 12, 13). This lamina is approximately 0.5-1.5 /~m thick, i.e. nearly twice the general integument, and is composed of amorphous material in which collagen fibres are embedded. This basement lamina is not evenly straight, but is frequently inflected between the cells, forming loops which extend far up towards the subvillous zone, almost contacting it (Fig. 18). A similar invagination of the basement lamina encloses the entire area of the fenestrae at the level of the integumentary endocuticular expansion mentioned above (Fig. 2). By intervening between the peripheral cells of the fenestra and

Ultrastructure of the "Fenestrae"

FIG. 12. Two large cytoplasmic compartments at level of basal zone of epithelium with long isolated cisternae of endoplasmic reticulum disposed parallel to cellular membranes. In insert, whole mount of abdominal integument showing cells of fenestra with a stellate appearance. G b = Golgi bodies. x 15,000 (insert x240) FIG. 13. A region of basal epithelium. Above basement lamina small cytoplasmic compartments are present (asterisk) and at level of plasma membranes small vesicles pinching off into cytoplasm are evident (arrows). x 17,280 FIGS. 14 and 15. Cytoplasmic areas near upper cellular edge illustrating numerous organelles sparsely distributed, arrows = coated vesicles; asterisk = polysome configurations, x22,080

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Fios. 16 and 17. Multivesicular bodies (mb), vesicles(v), microtubules (mt) in cytoplasm of fenestrae cells. Fig. 16. x24,000; Fig. 17. x18,240 Fio. 18. Long infolding of basement lamina between 2 adjacent cells of fenestra. Plasma membrane is joined to basement lamina by hemidesmosomes (ed). arrows = coated vesicles, x 27,360 the adjacent ones of the general integument, the basement lamina approaches the overlying cuticular layer, thereby forming a kind of barrier between the 2 epithelia. DISCUSSION At the ultrastructural level, the fenestrae of the abdominal tergites of S. gregaria display a cuticle and an epithelium, both differing in structure from those of the general integument (Fig. 19). The cuticle overlying the cell is devoid of the endocuticular layer and appears thinner than that of the integument, but the exocuticular layer, because of the greater thickness of the lamellae of which it is made up, appears thicker than that of the corresponding general integument. This might be due to partial sclerotization, or lack of it, at the level of the fenestrae, as already suggested by Slifer (1951). The predominantly characteristic features of this cuticular area are undoubtedly the pore canals, on account of their high number and the wide diameter. A cuticular layer structurally similar to that of the fenestrae is found in the hydropyle of the chorion of eggs of Melanoplus differentialis (Slifer and Sekhon, 1963). These authors suggest that this limited chorial region is concerned in water uptake, and this process seems to take place osmotically. The epithelium of the fenestrae, is formed by a thin cell layer separated from the hemolymph of the underlying pericardiac sinus by a basement lamina of considerable thickness with extensive intercellular loops. In addition, the surface extension of the cell membranes is strikingly enlarged both at the level of the basement lamina, where the characteristic intercellular system of membranes is present and at the apical zone, which is rich in thin

Ultrastructure of the "Fenestrae"

273

RE IAL

~A~AL

LAMINA

FIG. 19. Diagrammatic reconstruction of organization of cuticle and cells involved in construction of integumental fenestra.

microvilli. On the whole, this ultrastructural picture reminds one of water and/or iontransporting epithelial structures, and suggests that the areas investigated here may be concerned in solute and water movements (Maddrell, 1971; Kiimmel, 1973; Wall and Oschman, 1975). It is now widely accepted that in some secretory epithelia active transport of solute into intercellular spaces can establish local osmotic gradients that generate water absorption (Diamond and Bossert, 1968; Berridge and Oschman, 1969). At this point, however, it should be remembered that in the cells of the integumentary fenestrae mitochondria are less numerous than in the cells of the above mentioned epithelia. In insects, special structures, such as rectal pads, rectal papillae, are responsible for ion and water absorption: these are composed of cuticular and cellular layers. In our opinion, however, these structures differ structurally from the fenestrae, having just a few features in common. The cuticle of the rectal region, though appearing lamellate, is very thin, and pore canals are rarely found (Noirot and Noirot-Timoth6e, 1969). With the exception of the rectal epithelium of mosquito larvae (Meredith and Phillips, 1973), tracheae are always present; those in the apical region of the epithelium branching into fine tracheoles that enter large intercellular spaces. The cells are characterized by the presence of a number of mitochondria lecated in close contact with the highly folded lateral and apical plasma membranes (Gupta and

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Berridge, 1966; O s c h m a n a n d Wall, 1969). W e t h i n k t h a t greater similarities can be d e m o n strated between the cells o f S. gregaria fenestrae a n d those o f the cephalic kidneys o f a p t e r y g o t e s ( F a i n - M a u r e l a n d CaNNier, 1971), which function as excretory organs, a n d also those o f the m a l p i g h i a n tubules which have excretory a n d o s m o r e g u l a t o r y functions (Eichelberg a n d Wessing, 1975). In the latter, b o t h experimentally a n d by electron-microscope histochemistry, several different m e c h a n i s m s o f transepithelial passage o f substances can be shown, including passage by means o f vesiculation (cytopempsis) a n d passage t h r o u g h the g r o u n d c y t o p l a s m a n d the e n d o p l a s m i c reticulum (see review by Wessing a n d Eichelberg, 1975). U n d e r the electron microscope, the cells o f b o t h the fenestrae a n d o f the above-cited excretory organs, besides a n u m b e r o f intercellular channels a n d microvilli, display a c y t o p l a s m rich in c o a t e d vesicles, multivesicular bodies, a n d p o l y s o m i c f o r m a tions. All these organelles c o u l d be related to exo- a n d e n d o c y t o t i c processes as well as to a r a p i d m e m b r a n e turnover. The mere u l t r a s t r u c t u r a l aspect, howe.ver, is insufficient to establish or clarify the function o f these specialized areas. O u r investigation suggests that they m a y be c o n c e r n e d in the passage o f fluids, the wide p o r e canals serving in this case an excretory f u n c t i o n ; their presence in such c o n s p i c u o u s n u m b e r s m u s t evidently relate to the specialized f u n c t i o n o f these areas. This w o u l d fit in with M a k i n g s (1968) research, a c c o r d i n g to which the f u n c t i o n o f S. gregaria fenestrae m a y be the t r a n s p i r a t i o n o f b o d y water to the outside. REFERENCES BERRIDGE,M. J. and J. L. OSCHMAN. 1969. A structural basis for fluid secretion by Malpighian tubules. Tissue Cell 1 : 274-72. DIAMOND,J. M. and W. H. BOSSERT.1968. Functional consequences of ultrastructural geometry in "backwards" fluid-transporting epithelia. J. Cell Biol. 37: 694-702. EICHELBERG,D. and A. W~SSING.1975. Morphology of the Malpighian tubules of insects. Fortschr. Zool. 23: 124-47. FAIN-MAUREL,M. A. and P. CASSIER.1971. Diff6renciations cytoplasmiques en relation avec la fonction excr6trice darts les reins c6phaliques de Petrobins maritimus Leach (Insecte, Apt6rigote). J. Microsc. (Paris) 10: 163-78. GUPTA,B. L. and M. J. BERRIDGE.1966. Fine structural organization of the rectum in the blowfly Calliphora erythrocephala (Meig.) with special reference to connective tissue, tracheae and neurosecretory innervation in the rectal papillae. J. Morphol. 120: 23-81. KOMMEL, G. 1973. Filtration structures in excretory systems: a comparison, pp. 221-40. In L. BOLLS,K. SCHMIDT-NIELSEN and S. H. P. MADDRELL (eds.) Comparative Physiology. North-Holland, Amsterdam. MADDRELL,S. H. P. 1971. The mechanisms of insect excretory systems. Adv. Insect Physiol. 6: 199-331. MAKINGS,P. 1964. "Slifer's Patches" and the thermal sense in Acrididae (Orthoptera). J. Exp. Biol. 41: 473-97. MAKINGS,P. 1968. Transpiration through the Slifer's patches of Acrididae (Orthoptera). J. Exp. Biol. 48: 247-63. MEREDITH,J. and J. E. PHILLIPS. 1973. Rectal ultrastructure in salt- and fresh-water mosquito larvae in relation to physiological state. Z. Zellforsch. 138: 1-22. MISRA, S. D. 1947. Studies on the somatic musculature of the desert locust Schistocerca gregaria (Forskal) phasegregaria. Part III. The pterothorax. IndianJ. Entomol. 9: 19-72. NOIROT, Ch. and C. NOIROT-TIMoTHI~E.1969. La cuticule proctod6ale des insectes--I: Ultrastructure compar6e. Z. Zellforsch. 101: 477-509. OSCHMAN, J. L. and B. J. WALL. 1969. The structure of the rectal pads of Periplaneta americana L. with regard to fluid transport. J. Morphol. 127: 475-510. SBRENNA, G. and G, DE CARLI. 1972. Aspetti ultrastrutturali delle ghiandole a feromoni dell'epidermide dei maschi adulti di Schistocerca gregaria (Orthoptera). 9th Congr. Naz. Entomol. 9:119-24. SLIFER, E. H. 1951. Some unusual structures in Locusta migratoria migratorioides and their probables function as thermoreceptors. Proc. R. Soc. B. 138: 414-37. St.lrER. E. H. 1953a. The pattern of specialized heat-sensitive areas on the surface of the body of Acrididae (Orthoptera). Part I. The males. Trans. Amer. Entomol. Soc. 79: 37-68.

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SLIFER,E. H. 1953b. The pattern of specialized heat-sensitive areas on the surface of the body of Acridadae (Orthoptera). Part II. The females. Trans. Amer. EntomoL Soc. 79: 69-97. SLIFER, E. H. and S. S. SEKHON. 1963. The fine structure of the membranes which cover the egg of the grasshopper Melanoplus differentialis, with special reference to the hydropyle. Q. J. Miscrosc. Sci. 104: 321-34. SNODGRASS, R. E. 1929. The thoracic mechanism of a grasshopper, and its antecedents. Smithson. Misc. Collect. 82: 1-111. SNODGRASS,R. E. 1935. Principles of Insect Morphology. McGraw-Hill, New York. UVAROV, B. P. 1966. Grasshoppers and Locusts. Anti-Locust Res. Centre, Univ. Press, Cambridge. WALL, B. J. and J. L. OSCnMAN. 1975. Structure and function of the rectum in insects. Fortschr. ZooL 23: 193-222. WEIr, J. S. 1957. Preliminary investigation of thermoreception on the thin regions of the cuticle of Locusta migratoria migratorioides. Report to Anti-Locust Res. Centre, London. WEIS-FOGH, T. 1970. Structure and formation of insect cuticle, pp. 165-85. In A. C. NEVILLE (ed.) Insect Ultrastructure, Blackwells, Oxford. WESSiNG, A. and D, EICHELBERG.1975. Ultrastructural aspects of transport and accumulation of substances in the Malpighian tubules. Fortschr. Zool. 23: 148-72.