In vivo biosynthesis of a stage-specific cuticle glycoprotein during early metamorphosis of the medfly Ceratitis capitata

Cuticle proteins of an insect pest, the Medfly Ceratitis capitata, were resolved in polyacrylamide gels and partially characterized. The pupal cuticle was found to be different from cuticles of other insects since more than 80% w/w of the protein is a single mannose-containing polypeptide (PCG-100). The temporally-regulated in vivo biosynthesis and deposition of cuticle proteins was studied by microinjection of [35S]methionine followed by hand dissection of pupal cuticles. The major pupal glycoprotein, PCG-100, is cuticle- and stage-specific and was the earliest to be labeled and deposited. Its synthesis was maximal at around 46 hours after pupariation and then it decreased. The deposited PCG-100 and other minor pupal cuticle proteins become non-extractable at the end of the instar (7 days after pupariation) probably by sclerotization phenomena. These results provide insight into the temporal control of gene expression programs involved in cuticle deposition during medfly metamorphosis.     

Morphogenesis and cuticular markers during the larval-pupal transformation of the medfly Ceratitis capitata

Changes in morphology during early metamorphosis of the medfly, Ceratitis capitata (Wied.) (Tephritidae) were correlated with biochemical differentiation events. Protein profiles were studied both in the 3rd instar larval cuticle further transformed into puparium and the newly synthesized pupal cuticle. Beta-alanine incorporation into the puparium (0–20 h) correlates with concomitant pigmentation (completed by 16 h) and sclerotization phenomena. This early tannification program seems to be followed by deposition of a layer of substances, probably ecdysial fluid remnants, into the puparium. Their deposition ends approximately at +46 h. Simultaneously, pupal cuticle material starts to be deposited. Synthesis and deposition of the main pupal cuticle protein was detected 48 h after pupariation. At that time, eversion of the pupal head occurs. The definitive profile of pupal cuticle proteins was attained at around +72 h together with the establishment of adult body proportions.     

Cuticle differentiation in the embryo of the amphipod crustacean Parhyale hawaiensis

The arthropod cuticle is a multilayered extracellular matrix produced by the epidermis during embryogenesis and moulting. Molecularly and histologically, cuticle differentiation has been extensively investigated in the embryo of the insect Drosophila melanogaster. To learn about the evolution of cuticle differentiation, we have studied the histology of cuticle differentiation during embryogenesis of the amphipod crustacean Parhyale hawaiensis, which had a common ancestor with Drosophila about 510 million years ago. The establishment of the layers of the Parhyale juvenile cuticle is largely governed by mechanisms observed in Drosophila, e.g. as in Drosophila, the synthesis and arrangement of chitin in the inner procuticle are separate processes. A major difference between the cuticle of Parhyale and Drosophila concerns the restructuring of the Parhyale dorsal epicuticle after deposition. In contrast to the uniform cuticle of the Drosophila larva, the Parhyale cuticle is subdivided into two regions, the ventral and the dorsal cuticles. Remarkably, the boundary between the ventral and dorsal cuticles is sharp suggesting active extracellular regionalisation. The present analysis of Parhyale cuticle differentiation should allow the characterisation of the cuticle-producing and -organising factors of Parhyale (by comparison with the branchiopod crustacean Daphnia pulex) in order to contribute to the elucidation of fundamental questions relevant to extracellular matrix organisation and differentiation. This work was supported by the German Research Foundation (DFG, grant number MO 1714/1-1).     

Effects of exogenous N-acetylhexosaminidase on the structure and mineralization of the post-ecdysial exoskeleton of the blue crab, Callinectes sapidus

A cuticular glycosidase with characteristics of N-acetyl-β- -hexosaminidase (HexNAcase) was identified in post-ecdysial crab cuticle. Its appearance coincided with changes in cuticular glycoproteins and the onset of mineralization. To test if HexNAcase might be the causative agent in the alteration of the glycans and initiation of calcification, newly molted crab cuticle was treated with exogenous HexNAcase. Treating cuticular extracts from crabs at 0 h post-ecdysis with exogenous HexNAcase mimicked those changes observed in vivo. Specifically, the enzyme decreased the concanavalin A affinity of an 83-kDa glycoprotein that binds to calcite crystals in vitro. Treating pieces of 0 h post-ecdysial cuticle with HexNAcase rendered them capable of nucleating calcite in vitro (similar to 5 h post-ecdysial cuticle), while untreated, 0 h controls remained uncalcified. The data imply a role of the cuticular HexNAcase-like enzyme in the initiation of calcite nucleation in the newly formed exoskeleton.     

Hormonal control of growth and differentiation of insect tissues cultured in vitro

Summary This paper reviews the effects of insect hormones on lepidopteran imaginal discs cultured in vitro.β-ecdysone stimulated both evagination and cuticle deposition of wing discs ofPlodia interpunctella (Hübner). However, evagination required a shorter exposure to ecdysone than did cuticle deposition. Cuticle deposition was obtained under the following conditions: (a) a 24-hr pulse ofβ-ecdysone (0.5–5.0μg/ml); (b) continuous treatment with 0.2μg/mlβ-ecdysone; or (c) continuous treatment with 0.5 to 50.0μg/mlβ-ecdysone in medium conditioned with larval fat body. Investigations of some biochemical effects of ecdysone showed that RNA and protein synthesis was required for evagination and cuticle deposition. In particular, studies with actinomycin D and cycloheximide (at nontoxic levels) showed that RNA and protein synthesis during the ecdysone-dependent period was essential for subsequent development. These findings support the hypothesis that stimulation of macromolecular synthesis is fundamental to the action of ecdysone on imaginal discs. The influence of beta-ecdysone on chitin synthesis was also examined.β-ecdysone stimulated uptake and incorporation of tritiated-glucosamine by culturedP. interpunctella wing discs. Addition of hexosamines to the culture medium had no influence on ecdysone-induced cuticle deposition, but inhibition of glucose-uptake by cytochalasin B prevented the formation of cuticle. The action of ecdysone on particular enzymes in the chitin pathway remains to be elucidated. Presented in the formal symposium on Information Transfer in Eukaryotic Cells, at the 26th Annual Meeting of the Tissue Culture Association, Montreal, Quebec, June 2–5, 1975.     

Variation in thickness and protein content of the cuticle of the female of Glossina austeni

The thickness and total protein content of the ventral abdominal cuticle of the female tsetse, Glossina austeni, increase during the early part of each pregnancy cycle, reaching a maximum at approximately 2 days after ovulation. They decrease thereafter, and reach a minimum value just before larviposition. Virgin females do not exhibit a cycle of protein content or thickness in the cuticle. Preliminary data on the incorporation of [³H]tyrosine or [³H]leucine into the water-soluble proteins of the ventral abdominal cuticle at the time of the second larviposition suggest that there is rapid turnover of protein in the cuticle at this time. These observations are consistent with the net storage of protein in the cuticle during the early part of pregnancy cycle followed by a net depletion of that store as the nutritional demands of the rapidly growing larva in utero exceed the capacity of the ingested blood meals to supply them.     

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.     

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.     

Glycosidase activity in the post-ecdysial cuticle of the blue crab, Callinectes sapidus

We have previously demonstrated a marked change in sugar moieties of glycoproteins of the cuticle of the blue crab, Callinectes sapidus, between 0.5 and 3 h post-ecdysis. The present study has identified a glycosidase that appears in the cuticle during the early post-ecdysial hours. The enzyme has affinities for p-nitrophenyl derivatives of both N-acetylglucosamine and N-acetylgalactosamine. Both activities are competitively inhibited by chitobiose, suggesting that the enzyme could be a N-acetylhexosaminidase (EC 3.2.1.52). Atypical of N-acetylhexosaminidases described to date, this enzyme has a pH optimum of 7.0. The enzyme activity is high during the post-ecdysial period coincident with the changes in glycoprotein profiles observed in vivo. Partial purification of the enzyme has been accomplished by Sephacryl size-exclusion chromatography followed by concanavalin A affinity chromatography.     

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.     

Enhancing the egg's natural defence against bacterial penetration by increasing cuticle deposition

The cuticle is a proteinaceous layer covering the avian egg and is believed to form a defence to microorganism ingress. In birds that lay eggs in challenging environments, the cuticle is thicker, suggesting evolutionary pressure; however, in poultry, selection pressure for this trait has been removed because of artificial incubation. This study aimed to quantify cuticle deposition and to estimate its genetic parameters and its role on trans‐shell penetration of bacteria. Additionally, cuticle proteins were characterised to establish whether alleles for these genes explained variation in deposition. A novel and reliable quantification was achieved using the difference in reflectance of the egg at 650 nm before and after staining with a specific dye. The heritability of this novel measurement was moderate (0.27), and bacteria penetration was dependent on the natural variation in cuticle deposition. Eggs with the best cuticle were never penetrated by bacteria (P < 0.001). The cuticle proteome consisted of six major proteins. A significant association was found between alleles of one of these protein genes, ovocleidin‐116 (MEPE), and cuticle deposition (P = 0.015) and also between alleles of estrogen receptor 1 (ESR1) gene and cuticle deposition (P = 0.008). With the heritability observed, genetic selection should be possible to increase cuticle deposition in commercial poultry, so reducing trans‐generational transmission of microorganisms and reversing the lack of selection pressure for this trait during recent domestication.     

Elicitor induction of the synthesis of a novel lectin-like arabinosylated hydroxyproline-rich glycoprotein in suspension cultures of Phaseolus vulgaris L.

A novel lectin-like glycoprotein which accumulates in response to fungal elicitor action has been characterised in endomembranes from suspension cultures of French bean (Phaseolus vulgaris L.). The lectin, which has specificity towards N-acetylglucosamine oligomers, consists of a polypeptide of apparent molecular weight (M_r) 31 000 which is rich in glycine and contains 6.7% hydroxyproline O-linked to arabinose-containing oligosaccharides to give a glycoprotein of M_r 42500. A dual-labelling technique has been used to identify changes in the synthesis of the glycoprotein in cells exposed to fungal elicitor molecules. Thus, incorporation of [¹⁴C]proline into membranes in vivo and of [1^-3H]arabinose from uridine 5-diphosphate [1^-3H]arabinose in vitro and analysis by isoelectric focussing-polyacrylamide gel electrophoresis gave absolute correspondence of the labelled isoform of the glycoprotein. Having established the absence of contaminating polypeptides, subsequent analysis of microsomal fractions bysodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the peak of sythesis of the M_r-42500 glycoprotein occurred 4 h after the addition of fungal elicitor. The changes in the level of incorporation into the glycoprotein monomers were concomitant with increases in the activity of prolyl hydroxylase (EC 1.14.11.2)Incorporation of [¹⁴C]proline and its subsequent post-translational modification to hydroxyproline in microsomal polypeptides was followed by rapid transfer into the wall with an average t _1/2 of about 7 min. The M_r-42500 glycoprotein was rapidly transferred out of the endomembrane fraction with a t _1/2 of 2 min and could be detected in wall fractions where it became progressively less extractable. The glycoprotein, which clearly differs from bean extensin, accounts for up to 40% of the hydroxyproline newly exported in response to elicitor action. The lectin, which resembles those found in the Solanaceae and which is coinduced with enzymes of phytoalexin synthesis, may play some role in disease resistance.Abbreviations HRGP hydroxyproline-rich glycoprotein - IEF isoelectric focussing - M_r apparent molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate     

DNA replication in homogenates of Physarum polycephalum

Homogenates of Physarum polycephalum incorporate [³H] dATP into nuclear DNA at an initial rate of approximately 15% of the in vivo rate. To attain this level of synthesis, cultures are homogenized in a medium containing Mg⁺⁺, EGTA, glucose and spermine. Incorporation is strongly stimulated by the addition of ATP and all four deoxyribonucleoside triphosphates to homogenates prior to incubation. Various inorganic cations other than Mg⁺⁺ either do not affect synthesis or are inhibitory. Incorporation is inhibited by a nonionic detergent, Triton X-100. DNA synthesis in this cell-free nuclear system is similar in several respects to that which occurs in vivo: (1) The rate of DNA synthesis in the intact organism at a given time in the mitotic cycle is reflected by the level of synthesis in homogenates prepared from cultures at that time of the cycle; (2) DNA strands labeled in vitro exhibit alkaline sucrose density gradient sedimentation properties similar to those of daughterstrand DNA pulse-labeled in vivo; and (3) Homogenates of cultures which were pre-treated with cycloheximide incorporate [³H]dATP at about 60% of the level observed in homogenates of untreated controls.     

The role of pressure in controlling the entry of water into the developing eggs of the Australian plague locust Chortoicetes terminifera (Walker)

The normal internal hydrostatic pressure and the additional pressure necessary to rupture the egg shell was measured in the eggs of Chortoicetes terminifera, Newly laid white eggs burst at c 0.15 kg cm^-2, but after external tanning the chorion withstands c 0.5 kg cm^-2 when removed from its tanned foam ‘corset’ and 1.0 kg cm^-2 if left embedded in the egg pod material. Older eggs with formed cuticles often withstand 2.0 kg cm^-2 but yield at rather lower pressures if they develop ‘pin-holes’. As the OP of the egg contents always exceeds 7.7 kg cm^-2 the rigidity of the wall is clearly insufficient to permit the generation of high hydrostatic pressures capable of preventing water entry during the non-absorbing phases of development. Real hydrostatic pressures are lower than 0.06 kg cm^-2 in the young intact egg and reach only c 0.5 and 0.3 kg cm^-2, respectively, during the absorptive and post-absorptive phases of development. Several events contribute to the sigmoid form of the water uptake curve. Water is at first excluded by a permeability barrier associated with the chorion. Absorption is delayed until the yolk is completely enclosed by the serosal cell layer. After undergoing cleavage, the yolk is then rapidly mobilized to furnish precursors for cuticle synthesis; in consequence, the internal OP rises from δ 0.76d?K to 0.93d?K despite the massive inflow of water which is governed by the osmotic gradient. At blastokinesis the serosa becomes detached from the cuticle; cuticle deposition and yolk mobilization are halted, the OP falling rapidly to cδT 0.53d?K. The bulk entry of water then ceases. Any excessive hydrostatic pressures which develop later are relieved by the formation of self-sealing ‘pin-holes’.     

Temporal programming of epidermal cell protein synthesis during the larval-pupal transformation ofManduca sexta

Summary During the final larval instar the epidermis of the tobacco hornworm,Manduca sexta, synthesizes the larval cuticular proteins and the pigment insecticyanin. Then at the onset of metamorphosis the cells first become pupally-committed, then later produce the pupal cuticle. The changes in the pattern of epidermal protein synthesis during this period were followed by incubating the integument in vitro with either³H-leucine or³⁵S-methionine, then analyzing the proteins by 2-dimensional gel electrophoresis. Precipitation by larval and pupal cuticular antisera and by insecticyanin antibody identified these proteins. Three distinct changes in epidermal protein synthesis were noted: 1) Stage-specific proteins, some of which are larval cuticular proteins, appear just before and during the change of commitment on day 3. (2) By late the following day (wandering stage), synthesis of these and many other proteins including all the identified larval cuticular proteins and insecticyanin was undetectable. Several noncuticular proteins were transiently synthesized by this pupally committed cell during wandering and sometimes the following day. (3) During the production of pupal cuticle a new set of pupal-specific cuticular proteins as well as some common cuticular proteins (precipitated by both antisera) were synthesized. Some of the latter were also synthesized during the period between pupal commitment and pupal cuticle deposition.In spite of an apparent absence of methionine in both larval and pupal cuticle, many cuticular proteins incorporated³⁵S-methionine. Thus they may be synthesized as proproteins.Insecticyanin was shown to have two forms differing in isoelectric point, the cellular form being more acidic than the hemolymph form. Synthesis of the cellular form ceased before that of the hemolymph form.     

Fluoride in tissues of Krill Euphausia superba Dana and Meganyctiphanes norvegica M. Sars in relation to the moult cycle

Summary The fluoride content of whole animals and different tissues of the euphausiid species Euphausia superba and Meganyctiphanes norvegica was analyzed by two different and improved methods of isolation and determination. In contrast to other authors our findings show that the internal organs (muscle, hepatopancreas and hemolymph) contain less than 6 ppm d.w. fluoride this being the same order of magnitude as for vertebrates. The high concentrations reported by other authors must be mainly due to contamination of the soft tissue during storage (post-mortem migration of fluoride from shell) and/or contamination caused by minute fractions of cuticle during dissection. Over 99% of the total fluoride content is located in the cuticle (i.e. integument) of the euphausiids (2600 ppm/d.w. in E. superba and 3300 ppm/d.w. in M. norvegica in pleon cuticle). Analysis of F^- levels in relation to the moulting cycle showed that the uptake in both euphausiids occurs at a comparable and fast rate during the same physiological phase shortly after moult, parallel to the general construction of the cuticle. The internal organs show homeostasis in respect to fluoride. Accordingly, no internal deposition takes place, and F^- is reaccumulated from the external medium at each moult.This work was supported by grants from the DFG No. Ad 24/9 and Bu 548/1     

Cell proliferation in the subependymal layer of the adult mouse in vivo and in vitro

Pulse labelling experiments with [³H] thymidine (dT) and double labelling experiments with [³H]dT and bromodeoxyuridine (BrdUrd) were carried out on cells of the subependymal layer in the brain of adult normal mice in vivo, in vivo/in vitro and in vitro. The results should (i) lead to information about cell cycle parameters of these cells in the brain of adult mice, since these cells have been studied mostly in the rat brain up to now and (ii) answer the question whether results concerning cell proliferation obtained in vivo correspond with those from brain slices incubated in vitro with or without prelabelling in vivo. In vivo an LI of 20.2 ± 2.7% (x?± SEM) and T_s= 7.2 ± 0.7h were found. Furthermore, grain count halving experiments led to a surprisingly short cycle time (T_c) of 11.2–14.2 h. The longer T_c values (18–20 h) reported in the literature for subependymal cells in the rat brain seem to be due to evaluations of different areas around the lateral ventricle without considering the migrating behaviour of these cells which is quite different regionally. The in vitro studies (with or without prelabelling in vivo) showed a significantly reduced LI due to the fact that about 20% of the S phase cells, possibly lying in the middle of S, stopped further DNA synthesis after transfer to culture. This was shown by comparing the cell fluxes at the G₁/S and S/G₂ borders of in vivo vs. in vitro studies.     

Autoradiographic and fine structural study of chitin deposition in the cuticle of a barnacle using [3H]-D-glucosamine incorporation

[³H)-D-Glucosamine was injected into the rostral sinus of Balanus eburneus (barnacle) and the distribution of labelled chitin in the cuticle was studied with autoradiography and electron microscopy. When the pattern of labelling was examined in different body regions of the same organism where thickness of fully formed cuticle varied, it was observed that the rate of chitin deposition varied, being greater in thick than in thin regions. The density of Ag grains overlying cuticle was also greater in the thick regions. When the pattern of labelling was examined in regions of cuticle, comparable in thickness, taken from a series of organisms sacrificed at different time points a comparable value for the rate of chitin deposition was obtained. In addition, asynchrony in deposition of cuticle in different body regions of the same organism as well as uptake of the label by substances other than chitin, i.e. glycogen and glycoprotcins were described.     

The formation of the pupal cuticle by Drosophila imaginal discs in vitro

Mass-isolated imaginal discs of Drosophila melanogaster form a chitin-containing pupal procuticle In vitro. Optimal procuticle deposition occurs when the discs are incubated for 4–6 hr with 0.5–1.0 μg/ml of 20-hydroxyecdysone and then with less than 0.05 μg/ml of 20-hydroxyecdysone. The formation of the chitin-containing procuticle is demonstrated using three independent assays: with fluorescene-conjugated cuticle proteins that bind to chitin; by electron microscopy; by incorporation of [³H]glucosamine into a chitin fraction. Synthesis and deposition of pupal cuticle proteins are also demonstrated. Incorporation of [³H]glucosamine into chitin is sensitive to inhibitors of protein, RNA and chitin synthesis, but has little sensitivity to inhibitors of DNA synthesis, and dolichol-dependent glycosylation.