Further Studies of the Third Instar Larval Cuticle of Calliphora erythrocephala. By L. S. WOLFE

Transcription

1 Further Studies of the Third Instar Larval Cuticle of Calliphora erythrocephala By L. S. WOLFE (From the Department of Zoology, Cambridge; now at the Science Service Laboratory, Department of Agriculture, London, Ontario) With one plate (fig. i) SUMMARY The penetration and reduction of ammoniacal silver nitrate solution in the epicuticle of the larva of Calliphora was studied. The epicuticle of the third instar larva is more permeable over the muscle insertions and cuticular sense organs. This finding is related to their development at the previous moult. A surface layer of orientated wax is not present. Proteinaceous and fatty materials from the feeding medium modify the properties of the cuticle surface. Chloroformmethanol extracts a soft light brown acidic lipide from the protein of the epicuticle after contaminants from the medium are removed. The water loss from larvae and puparia of different ages and after various treatments was studied. Young puparia recover from abrasion but larvae do not. An hypothesis that waxy substances are liberated on to the surface of the puparium during hardening and darkening of the cuticle is presented and discussed. The pore canals penetrate the endocuticle until they are cut off from the epidermis by the development of the prepupal cuticle just after the puparial contraction. An inner endocuticle in which pore canals were absent was not found. The structure of the pore canals as shown by phase contrast examination is discussed. The pore canals are three times more concentrated in the lateral regions than in the dorsal or ventral regions. The oenocytes go through a secretory cycle during puparium formation similar to that occurring before moulting of the larva. INTRODUCTION IN the course of a study of the deposition of the third instar larval cuticle of Calliphora erythrocephala Meigen (Wolfe, 1954) some new observations were made on the structure and properties of the cuticle. This paper reports these findings. MATERIALS AND METHODS The rearing of the larvae and the histological methods were the same as described previously (Wolfe, 1954). Ammoniacal silver nitrate solutions were freshly prepared before use. Water loss through the cuticle was studied using the methods of Wigglesworth (1945). The phase contrast microscope was used for studying the structure of the pore canals and the oenocytes. Special techniques are described at the appropriate places in the text. The occurrence and distribution of reducing substances in the cuticle Wigglesworth (1945, 1948) used ammoniacal silver nitrate solution to demonstrate the presence of reducing polyphenols in the insect cuticle and [Quarterly Journal of Microscopical Science, Vol. 96, part 2, pp ,

2 182 Wolfe Further Studies of the also to show the extent of damage to the epicuticle after abrasion. This method was used in this study not only to demonstrate the presence of reducing substances in the larval epicuticle of Calliphora, but also as a qualitative indication of regions in the cuticle more readily penetrated by aqueous solutions. Third instar larvae of different age groups were immersed in a 5 per cent, ammoniacal silver nitrate solution at room temperature for 6 hours and thoroughly washed with distilled water. When examined under a binocular microscope, a series of well-marked deposits of silver were observed in the outer layers of the cuticle. These deposits were localized in feeding larvae in regions of the muscle insertions, cuticular sense organs, and functional and vestigial spiracles. In mature larvae irregularly distributed deposits were found particularly in the spinous regions and were not associated with sense organs or muscle insertions. Sections of the cuticle through these regions revealed that they were produced by small lesions in the outer epicuticle. These lesions were probably produced by the spines tearing and scratching the outer epicuticle during the muscular contortions of the larvae while feeding. Though probably present in young larvae they were not revealed by reduction of silver solutions. Dennell (1947) showed that reducing substances appeared in the inner epicuticle only in mature larvae. Short periods of immersion in ammoniacal silver solution (2-3 hours) resulted in clusters of small deposits of silver at the muscle attachments (fig. 1, A). These deposits were restricted to the outer epicuticle. After longer immersion periods (12 hours) the solution penetrated through the endocuticle and was reduced at the base of the tonofibrillae and the surrounding epi- FIG. 1 (plate). A, reduction of ammoniacal silver nitrate solution by the tips of the tonofibrillae of the muscle attachments in the epicuticle of a mature third instar larva. Immersion time, 2 hours. B, oblique section through a cuticular sense organ of a mature larva after immersion in ammoniacal silver solution for 4 hours. C, surface view of reduction at the cuticular sense organ to show the intensity and extent of the reduction around the sensory peg. D and E, the intense reduction of ammoniacal silver nitrate solution after abrasion of the outer epicuticle of a larva 12 hours before puparium formation. D, abrasion with fine needle; E, abrasion with powdered glass. F, sagittal section through the cuticle of the 'white' puparium to show the pore canals extending into the endocuticle from the epidermis and the absence of an inner endocuticle. The oenocytes are shown immediately beneath the epidermis. Osmic acid / Orcein stained; frozen section. G, the pore canals in the cuticle of a mature third instar larva, 'crop full' stage. The basal and distal portions of the pore canal are easily distinguished. Phase contrast, oil immersion, frozen section. H, reduction of ammoniacal silver nitrate solution by the tips of the pore canals in the inner epicuticle of a mature larva. Immersion time, 24 hours. i-n, phase contrast microphotographs of oenocytes at different stages of their secretory cycle before puparium formation. Mounted in Drosophila ringer. I, 'crop full' stage; j, 30 hours before puparium formation; K, 20 hours before puparium formation; L, quiescent period just before puparial contraction; M, 'white' puparium stage; N, 5-hour puparium just before the separation of the oenocytes from the epidermal cells.

3 FIG. I L. S. WOLFE

4 Third Instar Larval Cuticle of Calliphora erythrocephala 183 dermal cells. No reduction occurred in the endocuticle. In feeding larvae reduction was localized to the sensory pegs of the sense organs, but in the mature larvae the cuticular depression also showed deposits of silver (fig. 1, B and c). Sections of the cuticle through the sense organs after 12-hour immersion periods showed that the silver solution had penetrated through the epicuticle of the sensory pegs and down the distal processes to the sense cells within the epidermis. The localized reduction of ammoniacal silver solutions by the sense organs and muscle insertions is interpreted as indicating a greater permeability of the epicuticle over these regions. Further confirmation for this conclusion was obtained by immersing larvae for 30 minutes in a saturated solution of cobalt chloride, rinsing in distilled water, and placing them in hydrogen sulphide water. Larvae thus treated showed black patches of cobalt sulphide over the muscle insertions, cuticular sense organs, and spiracles. Addition of detergents (Triton X, Co 9993, Teepol) to the cobalt solution did not increase the size of the sulphide deposits except for a greater depth of penetration down the spiracles. The modification of the epicuticle over the muscle insertions and cuticular sense organs arises during development at the previous moulting cycle (Wolfe, 1954). The tonofibrillae and distal nerve fibres are attached across the exuvial space to the old cuticle until just before ecdysis and the old epicuticle at this time is penetrated by fine fibres of the tonofibrillae at the muscle attachments and the distal nerve fibres at the sense organs. When the cuticle is shed a break occurs at the region where the tonofibrillae of the old cuticle penetrate the newly formed epicuticle. Similarly, a break occurs at a level just below the sense rods where the distal sensory nerve fibre penetrates the epicuticle of the new sensory peg. It is the tips of the cuticularized fibres of the tonofibrillae and the sensory nerve fibres that are thought to give the quick surface reduction of ammoniacal silver solution. Kuhnelt (1949) reported the presence of reducing spots within the cuticle of insects from widely different groups. He also found deposits at the muscle insertions, dermal sense organs, cuticle lesions, and cuticular pores. No openings of ducts or pores were found within the larval cuticle of Calliphora except around the spiracles. The reducing ability of the muscle attachments and cuticular sense organs is not attributed to phenolic substances, The reduction at the cuticle lesions, however, is very probably due to exposure of polyphenolic substances in the inner epicuticle. Feeding larvae whose cuticles had been abraded either by rubbing in finely powdered glass or scratched with a fine needle showed only slight browning at the abraded areas after immersion in ammoniacal silver solution. However, larvae similarly treated at the 'crop full' stage showed an intense reduction (fig. 1, B and E). Powerful reducing substances are added to the inner epicuticle at this stage. Pryor (1940) and Dennell (1947) have conclusively shown that this strong reduction is due to polyphenolic compounds, probably an o-dihydroxyphenol. Mature larvae rubbed in alumina

5 184 Wolfe Further Studies of the dust showed no increase in reduction within the cuticle when immersed in ammoniacal silver solution. The outer epicuticle is very resistant and must be deeply abraded to expose the inner epicuticle. The epicuticle The staining reactions of the epicuticle of Calliphora larvae and the changes in these reactions at puparium formation differed little from the closely related species Sarcophaga falculata (Dennell, 1946), and Rhagoletis cerasis (Wiesmann, 1938). However, the outer epicuticle stained red in Mallory's stain and black in Heidenhain's haematoxylin. In young larvae the inner epicuticle stained pink in Mallory's stain but in mature larvae became a deep blue and also gave stronger Millon's and ninhydrin reactions. The appearance of phenolic substances and oxidase in the inner epicuticle was found by Malek (1952) to coincide with the presence of more protein. The protein-lipide association in the inner epicuticle before puparium formation contains all the requisite materials for the formation of the sclerotin of the exocuticle (Pryor, 1940, 1947). Sclerotin formation commences in the inner epicuticle at puparium formation and spreads to the outer endocuticular layers. The term 'exocuticle' should be applied only to sclerotinized cuticle whether it is of epicuticular or endocuticular origin or both. The exocuticle of the puparium consequently includes both endocuticle and epicuticle which become indistinguishable. The outer epicuticle remains distinct, but its pale amber colour suggests that it also contains sclerotin. The surface of the larval epicuticle is hydrophil. An orientated superficial wax layer on the epicuticle of the type described by Beament (1945) and Wigglesworth (1945) is absent from the Calliphora larva. However, lipides are incorporated in the epicuticle. The epicuticle breaks down into oily droplets when treated with concentrated nitric acid and potassium chlorate (cuticulin reaction). Chloroform extracts small quantities of a soft, almost liquid, waxy material of indefinite melting-point from the epicuticle. Beament (1945)' calculated a wax thickness of 0-27/0. on the washed puparium and i-i/x on the unwashed larval cuticle. He attributed this difference of wax thicknessto the presence of contaminants from the larval environment on the unwashed cuticle. Experiments were performed to investigate the nature of the hydrophil cuticle and the extent the larval environment affects cuticle wettability. Two hundred unwashed mature Calliphora larvae were rolled in alumina dust for 15 minutes, removed from the dust, and washed quickly with a jet of alcohol. The alumina dust was then extracted with 2:1 chloroformmethanol, a solvent mixture that extracts little non-lipide material. This procedure yielded 17-5 mg. of a strongly smelling acidic grease with indefinite melting-point. The yield, after repeating the above procedure with larvae previously washed in distilled water, was only 0-7 mg. An approximate calculation gives a wax thickness of 1 JJ, on the unwashed and /" on tne washed larva] cuticle. The latter value is so small that the presence of a superficial wax layer on the epicuticle appears unlikely. The waxy materials.

6 Third Instar Larval Cuticle of Calliphora erythrocephala 185 extracted from the unwashed larvae originate as suggested by Beament (1945) from substances in the feeding medium adsorbed on to the epicuticle. Three separate batches of fifty washed, isolated, and dried cuticles of mature Calliphora larvae were weighed and extracted for 1 hour with chloroform-methanol. After removal of solvent, 3 < 2±o-3 mg. of a soft light brown wax of indefinite melting-point remained representing I-I per cent, of the dry cuticle weight. This wax is the true epicuticular lipide of the cuticle. A mature Tenebrio larva floats when dropped into a Petri dish containing distilled water and the epicuticle surface remains perfectly dry. If an unwashed Calliphora larva was added to a dish containing a floating Tenebrio larva it immediately sank and within 2 minutes the Tenebrio larva also sank with its epicuticle completely wetted. Three washed and blotted Calliphora larvae placed in a fresh dish containing a floating Tenebrio larva sank, but in this case the Tenebrio larva remained floating for minutes. A Tenebrio larva previously placed among unwashed Calliphora larvae sank immediately when dropped into a fresh dish of distilled water. These experiments indicate that the contaminants on the unwashed Calliphora cuticle can affect the wettability of the Tenebrio cuticle. This may result from the formation of a hydrophil film on the cuticle surface, the alteration of the surface tension of the water, or a genuine disruption of the wax layer. Hurst (1941, 1948) observed that Calliphora larvae in contact with Tenebrio larvae caused the latter to die from desiccation. He stated that the hydrophil free lipides of the Calliphora epicuticle disrupted the hydrophobe wax layer of the Tenebrio epicuticle. Richards, Clausen, and Smith (1953) discredited this interpretation and concluded that the Tenebrio larvae used by Hurst were damaged. The hydrophil free lipides of Hurst are nothing but contaminants from the larval environment and these can alter the surface properties of the Tenebrio epicuticle. Water loss through the cuticle The transpirational loss of water through the cuticle of larvae and puparia of different ages and after various treatments with their spiracles sealed with Celamel was determined by the methods of Wigglesworth (1945). The results are summarized in table 1. Water loss from the larva decreased with age (table 1, A). There is a parallelism between this observation and observations of the penetration of aqueous poisons through the cuticle. Second instar larvae were immobilized within 30 seconds when immersed in o-i per cent. KCN or HgCl a. Mature third instar larvae were immobilized in minutes. Ammoniacal silver nitrate solution immediately penetrated the first and second instar cuticles and was reduced at the epidermis. A greater permeability of the early instars to arsenite was shown by Lennox (1940) for Lucilia cuprina and by Ricks and Hoskins (1948) for Sarcophaga securifera. Speyer (1925) found a 200 per cent, increase in the resistance of Lucilia sericata to penetration by poisons after the first 24 hours of the larval period. The increased resistance to water loss and to penetration of aqueous poisons by third instar larvae may be correlated

7 186 Wolfe Further Studies of the with epicuticle thickness. In the first and second instars the epicuticle thickness is less than iju. whereas in the third instar it varies from 3 to 7^, in TABLE I Percentage loss of weight of Calliphora erythrocephala larvae and puparia after various treatments and exposure to dry air over P for 4 hotirs at 25 0 C. Object of treatment A. Second instar larva untreated Third instar larva feeding,, Mature larva White puparium,, Puparium 4 hours Puparium 40 hours,, B. Larva 'crop full', control immersed cold CHC1 3 3 minutes immersed hot CHC1 3 3 minutes,, rubbed with alumina dust; dust left on,, heated first to 6o C. smeared with Co 9993 C. Puparium 2 hours, control immersed in CHCL 3 for 3 minutes immersed in benzol for 3 minutes immersed in ale.-ether (3 :1) immersed in acetone for 3 minutes immersed in abs. ale. for 3 minutes D. Puparium 2 hours, control,, surface scraped to damage epicuticle surface scraped; left 12 hours surface scraped; left 12 hours; smeared Co9993 surface smeared Co 9993,, rubbed with alumina dust,, rubbed with alumina dust; rinsed dist. water; left 12 hours E. Puparium 10 hours, control,, surface scraped puparium removed in section to level of prepupal cuticle F. Puparium 35 hours, control,, puparium removed post half puparium removed post half; pupal cuticle smeared with Co 9993 ditto; pupal cuticle immersed in CHC1 3 for 3 minutes,, immersed with puparium intact in CHC1 3 for 3 minutes Per cent, loss of weight i-8 I- S 16 I-I 21 2I-O 44'O "3 S2' S 229 5" 1 1 "4 54' " O 3O-4 i-o thickness. Also an inner epicuticle cannot be seen in the epicuticle of first and second instar larvae. It may be that the presence of an inner epicuticle is essential for the control of water loss as well as water penetration. Richards, Clausen, and Smith (1953) have recently shown that the inner epicuticle of Sarcophaga bullata is essential for the ph-enomenon of asymmetrical penetra-

8 Third Instar Larval Cuticle of Calliphora erythrocephala 187 tion to occur. The suggestion by Bonnemaison and Cayrol (1951) that the endocuticle thickness is a factor in resistance to penetration of insecticides seems less likely. Treatment of the larva and early puparium with organic solvents greatly increased the water loss through the cuticle (table 1, B and C). This is almost certainly a result of extraction of waxy substances and disorganization of the epicuticular protein-lipide complex. Larvae rubbed in alumina dust or smeared with the powerful detergent Co 9993 (cetyl ether of polyethylene glycol) showed no increase in water loss. This is further evidence for the absence of an orientated surface wax layer controlling water loss on the larval cuticle of Calliphora. A curious difference was found between the mature larva and the early puparium. Rubbing the early puparium with alumina dust led to a significant water loss, but if the puparium was left for 12 hours impermeability was completely restored (table 1, D). Recovery also occurred after light scraping of the epicuticle of the puparium. The reasons for this recovery reaction are not clear. A possible explanation is that wax is continuously secreted during the darkening and tanning of the puparium. The puparium progressively darkens and hardens during the first hours, and this is precisely the period before pupation when recovery from abrasion was observed. However, wax could not have been secreted continuously by the epidermis or from gland cells during this period because they are separated from the puparial cuticle 2 hours after the puparial contraction by the formation of a very thin prepupal cuticle. Recovery of the larval cuticle from abrasion was not observed. The proteinaceous and waxy materials on the surface of the larval epicuticle derived from the feeding medium are also present as a solidified and oxidized layer on the surface of the puparium. The puparium is not wetted as readily as the larva and also shows a higher resistance to water loss. Rubbing the puparium in alumina dust led to a much greater increase in water loss than in the larva (table i, D). This indicates that the surface waxy materials on the puparium do control water loss and suggests that besides the contaminants carried over to the puparium from the feeding medium there may be a wax layer formed on the puparium. Pryor (1940) concluded that sclerotin formation made the cuticle 'lipophil'. He regarded the epicuticle as a simple protein later tanned and impregnated with lipides. It is suggested that the formation of sclerotin within the protein-lipide epicuticle of the larva of Calliphora during puparium formation leads to the exclusion of lipide on to the lipophil surface forming a distinct waxy layer. Abrasion of this layer by alumina dust or its disruption by detergents might be expected to result in an increase in water loss. This process of exclusion of waxy substances from within the epicuticle on to its surface would continue as long as the process of hardening and darkening occurs. The puparium is not fully hardened until pupation. The prepupal cuticle does not control water loss in the early puparium (table 1, E). At pupation, occurring 25 hours after puparium formation, water

9 188 Wolfe Further Studies of the loss is efficiently controlled by the waxy layer of the delicate pupal cuticle (table i, F). This has been extensively studied by Beament (1945). The pore canals The pore canals in newly moulted third instar Calliphora larvae appear as cytoplasmic filaments extending as far as the inner epicuticle. The deposition of endocuticle during the third instar results in the retraction of the cytoplasmic part of the filament; the outer non-cytoplasmic portion then becomes extremely difficult to distinguish from the surrounding endocuticle by usual staining procedures. Sections of cuticle, however, treated with 2 per cent, osmic acid show the pore canals very clearly. This observation suggests that during the retraction of the cytoplasmic filaments from the inner epicuticle a little lipidal material is left in the pore canals. Sudanophil material is also present particularly in the branching filaments just beneath the inner epicuticle. Pore canals branching fan-like within the inner epicuticle have been observed by Plotnikow (1904) in Bombyx, Dennell (1946) in Sarcophaga, and Way (1950) in Diataraxia. Fresh sections of mature larval cuticle when examined either with transmitted light or under phase contrast show the pore canals clearly differentiated from the endocuticle. Phase contrast examination has revealed several interesting points about their structure in the mature cuticle (fig. 1, G). TWO distinct regions of the canal are shown. The basal portion contains epidermal cytoplasm extending approximately one-third of the way through the endocuticle (25-30 fi). The distal portion shows what appear to be numerous fine granules within the laminae of the endocuticle and ends in the inner epicuticle. The distal portion of the canals does not show any lining and certainly does not look like a duct. The canal is not helicoidal but runs an almost straight course through the endocuticle. However, in young growing larvae, the pore canals are very irregular in their course through the endocuticle and appear as irregular wavy lines crossing the laminae of the endocuticle. A spiral or helicoidal course of the pore canals through the endocuticle sufficiently regular to ascribe a pitch to the helix as recorded by Dennell (1946) for Sarcophaga and Richards and Anderson (1942) for Periplaneta has not been observed. One of the functions of the pore canals is the secretion of the inner epicuticle (Wolfe, 1954). Dennell (1946) reported the presence in Sarcophaga falculata of an endocuticular layer, the inner endocuticle, which contained no pore canals and was secreted in the mature larva just before puparium formation. This inner endocuticle was not found in Calliphora. Pore canals were found in osmic acid Orcein stained preparations connected to the epidermis right up to the commencement of browning of the puparium (fig. 1, F). Larvae at the 'crop full' stage when immersed in ammoniacal silver nitrate solution for long periods (25-30 hours) showed series of distinct spots of silver within the epicuticle (fig. 1, H). These deposits corresponded to the pore canals in the inner epicuticle. They are not shown in larvae immersed

10 Third Instar Larval Cuticle of Calliphora erythrocephala 189 for short periods. The solution must penetrate through the outer epicuticle to the tips of the pore canals before being reduced. The outer epicuticle is slightly permeable to aqueous solutions in the mature larva. The van Wisselingh chitin test was performed on isolated pieces of cuticle which were mounted and examined in surface view. The pore canals showed up as dark purple dots on a paler purple background, and were found to be more concentrated in the lateral than the dorsal or ventral regions; approximately 17,400/sq. mm. in the lateral regions and 5,600/sq. mm. in the mid-dorsal and ventral regions. Fresh, unstained, transverse sections of the cuticle showed the endocuticle laminae crossed by many more lines in the lateral regions than elsewhere. The endocuticle increases in thickness during the growth of the third instar larva, reaching a maximum of /n. The laminae of the endocuticle are at all stages of growth penetrated by cytoplasmic extensions of the epidermis which continue distally as chitinized filaments into the inner epicuticle (fig. 1, G). An examination of the pore canals of fresh sections of the mature cuticle under phase contrast did not show any space between pore canal contents and surrounding endocuticle that might suggest plugs or cords of chitin within the pore canal lumen. Chitinous filaments were not found projecting from teased laminae of the outer region of the endoculicle. However, in young larvae, filaments were found projecting from endocuticular laminae in certain sections that had become teased apart during section cutting. These filaments were pieces of the cytoplasmic part of the pore canals which in the newly moulted larva extend up to the inner epicuticle (Wolfe, 1954). As the larva grows and the endocuticle thickness rapidly increases these cytoplasmic filaments are retracted. A marked differentiation still remains in the newly secreted endocuticle connecting the cytoplasmic portion of the pore canals to the inner epicuticle. It is this distal portion of the pore canals that gives a strong chitin reaction as shown by Dennell (1946). The cytoplasmic portion did not give a chitin reaction. This may be the reason why Dennell was unable to find pore canals in the inner region of the endocuticle in the mature larva. The observations made above indicate that the pore canals remain connected to the epidermis throughout the third stadium. Way (1950) has shown in Diataraxia that in the soft cuticle the pore canals function only during the early stages of development and are then cut off from the epidermis by the development of an inner endocuticle. In areas of hard cuticle there was a thick heavily tanned exocuticle that continued to develop throughout the stadium and required the maintenance of the pore canal system from the epidermis. In Calliphora, however, the darkening and hardening processes occur at the end of the third stadium when the cuticle has reached its maximum thickness. During the period immediately before puparium formation polyphenols, basic protein, and enzymes accumulate in the inner epicuticle. It appears necessary that the pore canal conducting system be maintained between epidermis and inner epicuticle until puparium formation.

11 190 Wolfe Further Studies of the The pore canals of Calliphora, it is suggested, should not he regarded as distinct ducts or canals in the cuticle. Rather, they are thought to be differentiated regions through the laminae of the endocuticle possessing a greater porosity and able to transport materials necessary for the formation of the puparium quickly and selectively to the inner epicuticle. The oenocytes at puparium formation The oencytes exhibit a secretory cycle at moulting and at puparium formation. During the period when the larvae are migrating away from the feeding medium, the amount of secretory granules within the oenocyte cytoplasm increases rapidly and reaches a maximum at the time of contraction of the cuticle to form the white puparium and then decreases during the first 5 hours of the puparium. The secretory cycle was followed by examining, under phase contrast, fresh oenocyte groups dissected from the larvae and puparium at different times during the third stadium (fig. 1, I-N). The staining properties of the oenocyte secretion elaborated before moulting and puparium formation are those of an acidic, unsaturated lipide in association with protein. The larval oenocytes are completely histolysed by the 25th hour after the white puparium stage. The role of the oenocytes in puparium formation is very obscure. None of the changes leading to the formation of the puparial cuticle seem to be correlated with oenocyte activity. It might be suggested that the oenocytes are associated with the production of a component necessary for the darkening and hardening of the puparium. But the phenolic precursors for this process are present in the inner epicuticle before secretion appears in the oenocyte cytoplasm. Moreover, histochemical tests on isolated oenocytes for phenols, oxidases, and dehydrogenases were negative. However, a suggestive correlation exists between the secretory activity of the oenocytes and the secretion of the prepupal cuticle during the first 6 hours after the white puparium stage. At moulting oenocyte secretive activity follows strikingly the deposition of the protein-lipide epicuticle (Wigglesworth, 1948; Wolfe, 1954). The secretion granules within the oenocyte cytoplasm as well as the prepupal cuticle are stained by Sudan black. The peak in the secretive activity of the oenocytes occurs just before the prepupal cuticle is formed. Immediately after the secretion of the prepupal cuticle histolysis commences in the epidermal cells and oenocytes, which separate from the cuticle and become replaced by bands of actively dividing imaginal epidermal cells spreading over the larval epidermal cells displacing them into the interior of the puparium. I wish to thank Professor V. B. Wigglesworth for his excellent supervision and criticism, and Dr. J. W. L. Beament and Dr. M. G. M. Pryor for their valuable comments throughout the course of this work. This research was carried out during the tenure of an 1851 Exhibition Scholarship at the University of Cambridge.

12 Third Instar Larval Cuticle of Calliphora erythrocephala 191 REFERENCES BEAMENT, J. W. L., J. exp. Biol., 21, 115. BOHM, O., Pflanzenschutzber., 7, 33. BONNEMAISON, L., and CAYROL, R., C. R. Acad. Agric. Fr., 37, 112. DENNELL, R., Proc. Roy. Soc, B, 133, Ibid., 134, 79. and MALEK, S. R. A., Nature, 171, 298. HURST, H., Ibid., 147, Disc. Faraday Soc., 3, 193. KCHNELT, W., Osterr. Zool. Z., 2, 223. LENNOX, F. G., Council Sci. Ind. Res. Australia, Pamph., 101, 67. MALEK, S. R. A., Nature, 170, 850. PLOTNIKOW, N., Z. wiss. Zool., 76, 333. PRYOR, M. G. M., Proc. Roy. Soc, B, 128, Nature, 159, 399. RICHARDS, A. G., CLAUSEN, M. B., and SMITH, M. N., J. cell. comp. Physiol., 42, 395. and ANDERSON, T. F., J. Morph., 71, 135. RICKS, M., and HOSKINS, W. M., Physiol. Zool., 21, 258. SPEYER, W., Z. angew. Ent., n, 395. WAY, M. J., Quart. J. micr. Sci., 91, 145. WIESMANN, R., Vjschr. naturf. Ges. Zurich, 83, 127. WIGGLESWORTH, V. B., J. exp. Biol., 21, Quart. J. micr. Sci., 89, 197. WOLFE, L. S., Ibid., 95, 49.