The pupal cuticle of Drosophila: biphasic synthesis of pupal cuticle proteins in vivo and in vitro in response to 20-hydroxyecdysone

We investigated the synthesis and localization of Drosophila pupal cuticle proteins by immunochemical techniques using both a complex antiserum and monoclonal antibodies. A set of low molecular weight (15,000-25,000) pupal cuticle proteins are synthesized by the imaginal disk epithelium before pupation. After pupation, synthesis of the low molecular weight proteins ceases and a set of unrelated high molecular weight proteins (40,000-82,000) are synthesized and incorporated into the pupal cuticle. Ultrastructural changes in the cuticle deposited before and after pupation correlate with the switch in cuticle protein synthesis. A similar biphasic accumulation of low and high molecular weight pupal cuticle proteins is also seen in imaginal discs cultured in vitro. The low molecular weight pupal cuticle proteins accumulate in response to a pulse of the insect steroid hormone 20-hydroxyecdysone and begin to appear 6 h after the withdrawal of the hormone from the culture medium. The high molecular weight pupal cuticle proteins accumulate later in culture; a second pulse of hormone appears to be necessary for the accumulation of two of these proteins.     

The role of nutrition in creation of the eye imaginal disc and initiation of metamorphosis in Manduca sexta

With the exception of the wing imaginal discs, the imaginal discs of Manduca sexta are not formed until early in the final larval instar. An early step in the development of these late-forming imaginal discs from the imaginal primordia appears to be an irreversible commitment to form pupal cuticle at the next molt. Similar to pupal commitment in other tissues at later stages, activation of broad expression is correlated with pupal commitment in the adult eye primordia. Feeding is required during the final larval instar for activation of broad expression in the eye primordia, and dietary sugar is the specific nutritional cue required. Dietary protein is also necessary during this time to initiate the proliferative program and growth of the eye imaginal disc. Although the hemolymph titer of juvenile hormone normally decreases to low levels early in the final larval instar, eye disc development begins even if the juvenile hormone titer is artificially maintained at high levels. Instead, creation of the late-forming imaginal discs in Manduca appears to be controlled by unidentified endocrine factors whose activation is regulated by the nutritional state of the animal.     

The hormonal coordination of cuticulin deposition and morphogenesis in Drosophila imaginal discs in vivo and in vitro

Cuticulin is the first layer of the insect cuticle to be deposited and is laid down as a continuous inelastic sheet over the apical surface of cuticle-secreting cells. During metamorphosis in Drosophila melanogaster, imaginal discs deposit the cuticulin layer of the pupal cuticle between 3 and 7 hr after puparium formation. This is a period of rapid morphogenesis involving cell shape changes and cell rearrangements. We have examined cuticulin deposition in vivo and in vitro with a view to understanding the coordination of cuticulin deposition with morphogenesis. We find that the optimum hormonal regimen (of the steroid hormone, 20-hydroxyecdysone) for the completion of both morphogenesis and cuticulin deposition in vitro parallels the changes in hormone titer observed in vivo. We also find that cuticulin is deposited last over cell boundaries, thereby allowing cell rearrangements to occur as cuticulin is laid down. We have identified in vitro conditions under which cuticulin deposition is completed precociously, inhibiting further morphogenesis. Cytochalasin B and colchicine do not inhibit cuticulin deposition and we therefore conclude that an intact cytoskeleton is not necessary for secretion of this extracellular structure. Finally, we present a preliminary protocol for the partial purification of cuticulin synthesized in vitro by mass isolated discs.     

The cell biology of Drosophila wing metamorphosis in vitro

Summary We have examined the metamorphosis of the wing imaginal disc of Drosophila during culture in vitro in the continuous presence of 20-hydroxy ecdysone (0.1 g/ ml). We find that the sequence of cellular changes in the wing blade during culture closely match those occurring in situ, involving two periods at which the dorsal and ventral surfaces are joined only by cell processes containing trans-alar microtubule arrays. Good pupal and imaginal cuticle secretion is found in this system.     

Tissue-specific effects of the juvenile hormone analogue ZR 515 during metamorphosis in Drosophila cell cultures

The juvenile hormone analogue ZR 515 has specific effects on ecdysone-induced metamorphic differentation of Drosophila cells cultured in vitro. The number of vesicles containing imaginal cuticular structures is reduced to 10% of control levels. Similarly, the differentiation of adult fat body is partly inhibited by ZR 515. The differentiation of adult tubular and fibrillar muscles, however, is not affected. ZR 515 does not inhibit cuticle secretion by tracheal cells and larval epidermal cells.     

Control of pupal commitment in the imaginal disks of Precis coenia (Lepidoptera: Nymphalidae)

When final (5th) instar larvae of Precis coenia were treated with the juvenile hormone analog (JHA) methoprene, they underwent a supernumerary larval molt, except for certain regions of their imaginal disks, which deposited a normal pupal cuticle. Evidently those regions had already become irreversibly committed to pupal development at the time JHA was applied. By applying JHA at successively later times in the instar, the progression of pupal commitment could be studied. Pupal commitment in the proboscis, antenna, eye, leg and wing imaginal disks occurred in disk-specific patterns. In each imaginal disk there were distinct initiation sites where pupal commitment began during the first few hours of the final larval instar, and from which commitment spread across the remainder of the disk over a 2- to 3-day period. The initiation sites were not always located in homologous regions of the various disks. As a rule, pupal commitment also spread from imaginal disk tissue to surrounding epidermal tissue. The regions of pupal commitment in all disks except those of the wings, coincided with the regions of growth of the disk. Only portions of the disk that had undergone cell division and growth underwent pupal commitment. Shortening the growth period did not prevent pupal commitment in the wing imaginal disk, indicating that, in this disk at least, a normal number of cell divisions was not crucial in reprogramming of disk cells for pupal cuticle synthesis. The apparent growth spurt of imaginal disks that occurs during the last part of the final larval instar is merely the final stage of normal and constant exponential growth. Juvenile hormone (JH) and ecdysteroids appeared to play little role in the regulation of normal imaginal disk growth. Instead, growth of the disks may be under intrinsic control. Interestingly, even though endogenous fluctuation in JH titers do not affect imaginal disk growth, exogenous JHA proved able to inhibit both pupal commitment, cell movement, and growth of the disks during the last larval instar. This function of JH could be important under certain adverse conditions, such as when metamorphosis is delayed in favor of a supernumerary larval molt.     

The pupal instar of Stomoxys calcitrans: Cuticle deposition and chitin synthesis

Incorporation of radioactive N-acetyl-d-glucosamine peaked at 1 and also at 4 days post-pupation. Histological studies indicated that these peaks were related to the production of the ecdysial membrane and underlying imaginal cuticulin layer on the first day and the production of imaginal cuticle on the fourth day. The formation and/or secretion of the larval exocuticle, pre-pupal cuticle and ecdysial membranes were studied.     

Site of synthesis,tissue distribution,and lipophorin transport of hydrocarbons in Blattella germanica (L.) nymphs

The site of hydrocarbon (HC) synthesis and the amount of HC in various tissues were investigated in relation to developmental stage in the last larval stadium of the German cockroach, Blattella germanica. Abdominal integument linearly incorporated [1-(14)C]propionate into HC for at least 6h in vitro, whereas other body parts synthesized little or no HC. The third through sixth abdominal sternites and tergites were the principal sites of synthesis. High rates of HC synthesis resulted in a fivefold increase in internal HC during the last stadium. We examined the distribution of HC in the hemolymph, fat body, and the developing imaginal cuticle. Hemolymph HC titer was relatively constant at approximately 8&mgr;g/&mgr;l. However, as hemolymph volume increased from 5 to 11&mgr;l in the first 4days of the last stadium, HC content increased and then remained stable the remainder of the stadium. Lipophorin, immunoprecipitated with adult lipophorin polyclonal antibodies, was the only HC carrier protein in nymphal hemolymph and its HC profile was identical to that of hemolymph and similar to that of the epicuticle. The concentration and total amount of hemolymph lipophorin increased until 3days before adult eclosion and declined immediately after ecdysis. The HC content of non-biosynthetic integument (legs, pronotum) doubled during formation of the imaginal cuticle, as did the HC content of sternites, which synthesize HC. HC content of fat body, however, increased threefold during the same period, suggesting that the fat body serves as a storage site for HC during cuticle formation. We conclude that in the last stadium HC is synthesized by abdominal oenocytes, loaded onto hemolymph lipophorin, and transported to fat body and both nymphal and imaginal cuticle. Hydrocarbons associate with the imaginal integument several days before eclosion.     

Drosophila Syntaxin Is Required for Cell Viability and May Function in Membrane Formation and Stabilization

The role of the Drosophila homologue of syntaxin-1A (syx) in neurotransmission has been extensively studied. However, developmental Northern analyses and in situ hybridization experiments show that SYX mRNA is expressed during all stages and in many tissues. We have isolated new mutations in syx that reveal roles for syx outside the nervous system. In the ovary, SYX is present in the germarium, but it is predominantly localized to nurse cell membranes. Mitotic recombination experiments in the germ-line show SYX is essential for oogenesis and may participate in membrane biogenesis in the nurse cells. In the early embryo, a large contribution of maternally deposited RNA is present, and the protein is localized at cell membranes during cellularization. After the maternal contribution is depleted, zygotically produced SYX assists secretion events occurring late in embryogenesis, such as cuticle deposition and neurotransmitter release. However, SYX is also required in larval imaginal discs, as certain hypomorphic mutant combinations exhibit rough eyes and wing notch defects indicative of cell death. Furthermore, recombinant clones that lack syx cause cell lethality in the developing eye. We propose that, similar to its roles in cuticle secretion and neurotransmitter release, SYX may mediate membrane assembly events throughout Drosophila development.     

Interaction Between Ecdysteroid, Eclosion Hormone, and Bursicon Titers inManduca sexta

SYNOPSIS. The end of the molting process in the tobacco hornwormincludes the rapid digestion of the old cuticle, molting fluidresorption, ecdysis of the old cuticle, and expansion and hardeningof the new cuticle. The coordination of these processes is accomplishedby three hormones. Each ecdysis during the life of Manduca appearsto be triggered by eclosion hormone. Depending on developmentalstage, the hormone comes either from the brain-corpora cardiacacomplex or from the chain of ventral ganglia. The neural programstriggered by eclosion hormone include a neuroendocrine event,the release of the tanning hormone, bursicon, thereby ensuringthat tanning of the new cuticle must follow ecdysis. Ecdysis,itself, appears to be controlled by the ecdysteroid levels sinceecdysteroid injections delay ecdysis at physiological concentrationsand in a dose dependent fashion. This delay is due to inhibitionof eclosion hormone secretion and to the retardation of theterminal phases of the molt including the digestion of the oldcuticle and the onset of sensitivity to eclosion hormone. Thus,eclosion hormone secretion and the ecdysis it triggers are coordinatedwith the end of development because both are influenced by thesame endocrine signal—the decline in the ecdysteroid titer.     

Inhibition of cuticle production in imaginal discs of Plodia interpunctella (cultured in vitro): Effects of colcemid and vinblastine

The relationship of the microtubular system to the production of cuticle was evaluated by culturing wing imaginal discs from Plodia interpunctella in medium that contained 20-hydroxyecdysone and either colcemid or vinblastine. Examination of the treated tissue with both a dissection stereomicroscope and an electron microscope showed that the microtubule inhibitors prevented the formation of cuticle. The inhibitors also prevented the synthesis of chitin, but did not reduce protein synthesis. These results support the hypothesis that the secretion of cuticular components by insect cells requires the integrity of the microtubular system.     

Diflubenzuron-Induced alterations during in vitro development of Tenebrio molitor pupal integument

The effects of diflubenzuron (DFB) in Tenebrio molitor pupae were first investigated on cuticle secretion induced by 20-hydroxyecdysone in vitro. The sternal integuments were treated by DFB either 3 days before culture or during culture. DFB, when applied before culture, did not prevent the molting hormone from inducing a new cuticle deposition by integument explants in vitro. However, this cuticle showed several architectural alterations and a thickness reduction. When applied during the culture in the presence of 20-hydroxyecdysone, DFB at high dose (≥ 20 μg/ml) was able to inhibit cuticle secretion, but lower doses (? 10 μg/ml) resulted in epicuticle deposition. These observations confirm in vivo studies showing antagonistic effects of DFB and ecdysteroids at the level of epidermal cells. In another series of experiments, the DFB effects were analyzed without addition of exogenous molting hormone in vitro. Because it had been observed in previous studies that pupal epidermal explants of Tenebrio secrete low but significant amounts of ecdysteroids in the culture medium, this in vitro secretion was measured by radioimmunoassay after DFB treatment. It was observed that DFB, when applied either before or during culture, significantly reduced the hormonal secretion in vitro. This reduction, observed at the level of epidermal cells, could be homologous with the diminution of the endogenous ecdysteroid peak previously described after in vivo DFB treatment in Tenebrio pupae.     

In vivo and in vitro effects of the nonsteroidal ecdysteroid agonist tebufenozide on cuticle formation in Spodoptera exigua: An ultrastructural approach

To evaluate the ecdysteroid-like mode of action of tebufenozide (RH-5992), the effects on the fine structure of the integument in last- and third-instar larvae of the beet armyworm, Spodoptera exigua, and on cuticle formation in cultured imaginal wing discs, were studied. After 3 h of treatment with tebufenozide, the first signs of a normal moult were observed in treated larvae. A few hours later, ecdysial space formation and secretion of a new epicuticle were started. Furthermore, the new cuticle was incomplete in treated larvae; the new procuticle was absent or contained only a very low number of lamellae. In addition, epidermal cells showed many vacuoles and symptoms of degeneration with increase in time. Only a few lamellae of the old procuticle were digested, and normal ecdysis was inhibited which led to the presence of a double cuticle within 24–48 h after treatment. Similarly, cultured discs were stimulated to deposit a new cuticle within 12 h after cultivation in a medium containing tebufenozide. Our observations in treated S. exigua larvae on the one hand and in imaginal discs cultured with tebufenozide on the other hand are indicative of a hyperecdysteroid action, and confirm that the moult accelerating mode of action of tebufenozide resulted in a forced, untimely synthesis of cuticle by activation of epidermal or epithelial cells, and that its ecdysis inhibitory activity is mediated by its effect on post-apolysis processes. © 1996 Wiley-Liss, Inc.     

Action of juvenoids on metamorphosis of the honey-bee, Apis mellifera

The morphogenetic action of several structurally unrelated bioanalogues of juvenile hormone resulted in two different types of effects: (1) Topical application of these compounds on worker bee larvae prior to the cessation of feeding induced development of imaginal structures characteristic for queen individuals and (2) administration of the compounds at later stages caused inhibition of differentiation of imaginal characters at various phases of adult development. The moment of application appeared to be crucial for the extent and quality of these effects. Effects on pupal morphogenesis were not proved unequivocally, since the formation of a second pupal cuticle was not observed. The juvenile hormone activities of 33 chemicals representing several types of hormonally active agents have been compared and some principal structure-activity relationships have been outlined.     

Distribution of cuticular protein mRNAs in silk moth integument and imaginal discs

The distributions of mRNAs for two cuticular proteins of Hyalophora cecropia were examined with RT-PCR and in situ hybridization. For major regions of larval and pupal cuticle, there was a strong correspondence between the type of cuticle and the predominant cuticular protein message found. Epidermal cells underlying soft cuticle had mRNA for HCCP12, with a RR-1 consensus attributed to soft cuticle, while the epidermal cells associated with hard cuticle had predominantly mRNA for HCCP66, a protein with the RR-2 consensus attributed to hard cuticle. Both messages were found in all areas of the pupal fore- and hind-wings, with modest area-specific difference in concentration being much less than differences in the relative abundance of these cuticular proteins.

mRNA for HCCP12 was present in imaginal discs of feeding larvae of H cecropia. Data from Bombyx mori available at SilkBase (http://www.ab.a.u-tokyo.ac.jp/silkbase/) revealed that imaginal discs from feeding larvae had abundant mRNA for RR-1 cuticular proteins, representing six distinct gene products. Only discs from spinning larvae had mRNAs that coded for RR-2 proteins arising from 10 distinct genes. Thus, lepidopteran wing imaginal discs can no longer be regarded as inactive in larval cuticle production.     

Histogenesis of the cuticle of the adult flies,Sarcophaga bullata and S. argyrostoma

Sequential patterns of cuticle deposition and “melanization” in the imaginal cuticle of Sarcophaga argyrostoma in parts of the body darkening before or after emergence are examined on a histological basis. The patterns in the cuticles examined range from a simple absence of “melanization” to a complex of histological changes involving “melanization” and deposition. Ultrastructural changes in the post-emergent cuticle of Sarcophaga bullata during the hardening and darkening process and cuticle deposition are described.