Metamorphosis-associated proteolysis in Ceratitis capitata

The induction of several biochemical indicators of larval tissue histolysis in the Medfly, Ceratitis capitata (Diptera: Tephritidae) (Wiedemann) was studied. Using synchronized third instar larvae, we have determined the time of occurence of gut evacuation (12 h before puparium formation, bpf), disappearance of digestive enzymes (10 h bpf), and jumping from the food (8 h bpf). We can also correlate these events temporally with other early landmarks of metamorphosis.The decrease in protein content between 0 hours and 144 hours after puparium formation (0–144 h apf) corresponds to a sharp increase of total acid proteolytic activity measured in vitro from 0 to 44 h apf. This activity appears to be lysosomal, judging by the activation of other lysosomal markers, such as acid phosphatase and -glycosidases. The maximum proteolytic activity occurs during the pre-pupa to pupa transition, i.e. during morphogenesis from the cryptocephalic to the phanerocephalic pupa. The results of endopeptidase inhibitor assays indicate that in dipterans, members of the aspartic and cystein proteinase families are responsible for the degradation of larval tissues.     

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.     

Apical secretion and association of the Drosophila yellow gene product with developing larval cuticle structures during embryogenesis.

Summary The yellow (y) gene of Drosophila melanogaster is required for the pigmentation of larval and adult cuticle structures. The deduced y protein sequence includes two putative N-linked glycosylation sites and a putative signal peptide, suggesting that it might be a secreted molecule. Consistent with the characteristics of a secreted protein, our in vitro translation studies using RNA synthesised from the y cDNA demonstrate that the nascent y polypeptide is a preprotein that cotranslationally translocates into the endoplasmic reticulum (ER) membrane and becomes glycosylated. The N-terminal peptide is cleaved from the preprotein between the two alanine residues at positions 21 and 22, to release the final product into the lumen of the ER. Antibodies raised against the y polypeptide detect the protein starting at 13 h post-fertilization in epidermal cells and in the cuticle structures secreted by them that later become pigmented; in addition, yellow protein is detected in the cuticle structures associated with Keilin's organs. The embryonic -galactosidase staining pattern of a transgene, bearing a construct in which expression of the lacZ gene is driven by the y promoter, is also described and is similar to that of the y protein. Our results indicate that the y gene product is an apically secreted protein which becomes an immobilised structural component of the pigmented cuticle.     

Morphogenesis of <Emphasis Type="Italic">Paragordius varius</Emphasis> (Nematomorpha) during the parasitic phase

The development of Paragordius varius (Nematomorpha), in the parasitic phase of the life cycle, was followed ultrastructurally from 10 days after controlled infection of crickets (Gryllus firmus) to mature animals at 30 days after infection. During this time span, specimens grow from about 1 cm to more than 10 cm and from 93 m to about 400 m in diameter. A thin larval cuticle is replaced at about day 20 by a robust adult cuticle. Epidermis and intestine appear physiologically active during the early stages, but decrease in size and cytological components during further development. This is probably connected to the uptake of nutrients through the larval cuticle, whereas the adult cuticle has only protective function. The longitudinal musculature grows continuously and changes from fewer platymyarian cells to abundant coelomyarian cells. The ventral nerve cord shifts from an intraepidermal to a submuscular position during development. Additionally, basiepidermal nerves are present. Gonads develop from paired dorsolateral compact strands. It seems that in males, cells within these strands separate into epithelial and central cells, which become the gametes, while in females, gametes proliferate from the strands into the primary body cavity and fill it up completely. In males, there is a large quantity of parenchyma, restricting the body cavity to a small periintestinal space, while in females, parenchyma occurs only in the periphery of the gametes.     

Diapausing pupae of the flesh fly Sarcophaga crassipalpis (Diptera: Sarcophagidae) are more resistant to inoculative freezing than non-diapausing pupae

The resistance of diapausing (overwintering) and non‐diapausing (summer) Sarcophaga crassipalpis (Diptera: Sarcophagidae) pupae to inoculative freezing was examined. Although both types of pupae resisted inoculative freezing after 24‐h submergence in water, diapausing pupae were overall significantly more resistant than non‐diapausing pupae. Exposing the thin pupal cuticle by removing the ends of the puparial case eliminated the capacity of both pupal types to resist inoculative freezing, indicating that resistance to inoculative freezing resides with the puparium. Pupae submerged in surfactant solution were significantly less resistant to inoculative freezing than those submerged in water. Furthermore, the puparial water content of pupae submerged in surfactant solution was significantly greater than that of puparia from pupae submerged in water. Surfactant may have promoted inoculative freezing by facilitating the spread of water over the surface of and into the puparium, thereby creating bridges between external ice and pupal body fluids. Extracting puparial surface lipids with chloroform/methanol (2 : 1, v:v) decreased the resistance of non‐diapausing pupae to inoculative freezing but did not significantly affect that of diapausing pupae. This finding indicates that the puparium of diapausing pupae contains protection against inoculative freezing separate from its surface lipids. This barrier may be important in protecting the freezing‐intolerant overwintering pupae against inoculative freezing within their soil hibernaculum.     

Periclinal penetration of potassium permanganate into mature cuticular membranes ofAgave andClivia leaves: new implications for plant cuticle development

Staining cuticular membranes ofAgave americana andClivia miniata en bloc with potassium permanganate results in a strong contrast in the interior cuticular layer while the exterior part remains unstained. This is not caused by a selective chemical reaction with the interior part but by the unidirectional penetration of the reagent from the interior side, the outside being protected by the cuticle proper. In transverse cryosections of the cuticular membrane, permanganate penetrates nearly as easily into the exterior cuticular layer as into the interior one giving the same contrast. However, compared with the periclinal penetration into the cuticle proper this penetration is accelerated five-to tenfold by the polysaccharide network within the cuticular layer which serves as a distribution-channel system. Periclinal penetration into the cuticle proper occurs independently in each cutin penetration unit included between two obvious lucent lamellae and further divided into subunits.     

A light and electron microscopic study of stigmas inAneilema andCommelina species (Commelinaceae)

Summary The stigmas of species inAneilema andCommelina are trifid and comprise elongate papillae. Progressive degeneration of papular cells is observed in stigmas from open flowers and at anthesis papillae may be moribund and collapsed. Fluid emanating from the hollow style flows onto the surface through ruptures in the cuticle at the interpapillar junctions into the interstices at maturity. This secretion stains positively for protein. Stigmas are of the wet type.The cuticle overlying the papillar cells is ridged and at the final stages prior to flowering this cuticle becomes detached from the underlying cellulosic wall. The sub-cuticular space so formed is filled with secretion. InAneilema species detachment of cuticle is at the papillar tip and along the lateral walls. InCommelina species the anticlinal walls of adjacent papillae are strongly attached for much of their length and thus detachment of cuticle is restricted to the papillar tip. The cell wall at the tip in both genera may proliferate forming a rudimentary transfer-cell type wall. The secretion is considered to be produced by the papillar cells. It is PAS positive but fails to stain for protein and in both the light and electron microscopes appears heterogenous.Pollen attachment, hydration, germination and early tube growth are very rapid following self-pollination, the pollen tubes entering the neck of the style within ten minutes of attachment.A unique character combination involving pollen and stigmas in these genera indicates a monophyletic origin.     

Control of mannose/galactose ratio during galactomannan formation in developing legume seeds

Galactomannan deposition was investigated in developing endosperms of three leguminous species representative of taxonomic groups which have galactomannans with high, medium and low galactose content. These were fenugreek (Trigonella foenum-graecum L.; mannose/galactose (Man/Gal) = 1.1), guar (Cyamopsis tetragonoloba (L.) Taub.; Man/Gal = 1.6) and Senna occidentalis (L.) Link. (Man/Gal = 3.3), respectively. Endosperms were analysed at different stages of seed development for galactomannan content and the levels, in cell-free extracts, of a mannosyltransferase and a galactosyltransferase which have been shown to catalyse galactomannan biosynthesis in vitro (M. Edwards et al., 1989, Planta 178, 41–51). There was a close correlation in each case between the levels of the biosynthetic mannosyl- and galactosyltransferases and the deposition of galactomannan. The relative in vitro activities of the mannosyl- and galactosyltransferases in fenugreek and guar were similar, and almost constant throughout the period of galactomannan deposition. In Senna the ratio mannosyltransferase/galactosyltransferase was always higher than in the other two species, and it increased substantially throughout the period of galactomannan deposition. In fenugreek and guar the galactomannans present in the endosperms of seeds at different stages of development had the Man/Gal ratios characteristic of the mature seeds. By contrast the galactomannan present in Senna endosperms at the earliest stages of deposition had a Man/Gal ratio of about 2.3. During late deposition this ratio increased rapidly, stabilising at about 3.3, the ratio characteristic of the mature seed. The levels of -galactosidase in the developing endosperms of fenugreek and guar were low and remained fairly constant throughout the deposition of the galactomannan. In Senna, -galactosidase activity in the endosperm was low during early galactomannan deposition, but increased subsequently, peaking during late galactomannan deposition. The developmental patterns of the -galactosidase activity and of the increase in Man/Gal ratio of the Senna galactomannan were closely similar, indicating a cause-and-effect relationship. The endosperm -galactosidase activity in Senna was capable, in vitro, of removing galactose from guar galactomannan without prior depolymerisation of the molecule. In fenugreek and in guar the genetic control of the Man/Gal ratio in galactomannan is not the result of a post-depositional modification, and must reside in the biosynthetic process. In Senna, the Man/Gal ratio of the primary biosynthetic galactomannan product is controlled by the biosynthetic process. Yet the final Man/Gal ratio of the galactomannan in the mature seed is, to an appreciable extent, the result of galactose removal from the primary biosynthetic product by an -galactosidase activity which is present in the endosperm during late galactomannan deposition.Abbreviations al galactose - Man mannose This work was carried out with the aid of a Cooperative Research Grant (No. CRG 1) awarded by the Agricultural and Food Research Council, UK.     

Growth of the Thermophilic Bacterium Geobacillus uralicus as a Function of Temperature and pH: An SCM-Based Kinetic Analysis

The synthetic chemostat model (SCM), originally developed to describe nonstationary growth under widely varying concentrations of the limiting substrate, was modified to account for the effects of nontrophic factors such as temperature and pH. The bacterium Geobacillus uralicus, isolated from an ultradeep well (4680 m), was grown at temperatures ranging from 40 to 75°C and at pH varying from 5 to 9. The biomass kinetics was reasonably well described by the SCM, including the phase of growth deceleration observed in the first hours after a change in the cultivation temperature. At an early stage of batch growth in a neutral or alkalescent medium, bacterial cells showed reversible attachment to the glass surface of the fermentation vessel. The temperature dependence of the maximum specific growth rate (_m) was fitted using the equation _m = Aexp(T)/{1 + expB[1 – C/(T + 273)]}, where A, , B, and C are constants. The maximum specific growth rate of 2.7 h^–1 (generation time, 15.4 min) was attained on a complex nutrient medium (peptone and yeast extract) at 66.5°C and pH 7.5. On a synthetic mineral medium with glucose, the specific growth rate declined to 1.2 h^–1, and the optimal temperature for growth decreased to 62.3°C.     

Integument and sensory nerve differentiation ofDrosophila leg and wing imaginal discs in vitro

Summary An ultrastructural analysis is presented of the cuticular and neural structures formed by the prothoracic leg and wing imaginal discs of maleDrosophila melanogaster larvae during culture in vitro with 0.2 g/ml of -ecdysone. A pupal cuticle, and subsequently an imaginal cuticle with a well-defined epicuticle and a laminated endocuticle is formed. The ultrastructure of the epidermis and of cuticular structures such as bristles, trichomes, apodemes, and tracheoles is very similar to that found in situ. Dendrites and nerve cell bodies are formed in vitro, and sensory axons form nerve bundles similar to those of normal appendages in situ, despite their isolation from the central nervous system. It is concluded that at the ultrastructural level, differentiation in vitro closely parallels the normal course of development.     

Phase transitions in plant cuticles

The effect of temperature on wet plant cuticles has been investigated with the following techniques: Calorimetry, densitometry, spin-label electron-spin-resonance-(ESR)-spectroscopy, photo bleaching, and light and electron microscopy. At low temperatures cuticles ofCitrus aurantium L. andHedera helix show, at 16.3°C, a sharp transition (T0.5°C) with a latent heat of 4.7±0.5 J g^-1-cuticle. Below transition: The main orientation of the polymer matrix is parallel to the normal of the cuticle and the main orientation of the layer with soluble lipids is perpendicular to the normal. The cuticle is in a rigid state. Above transition (between 16.3°C and 38°C): Only the orientation of the polymer matrix has changed (tilted in parts). There exist several very sharp (T0.1°C) transitions (38°C, 41°C, 45°C, 49°C, ...) with a latent heat in the order of 0.4 J g^-1-cuticle. Above 38°C: The lamella of the soluble lipids is in a fluid state. Above 45°C there is a change in the molecular orientation of the soluble lipids as well as in the polymer matrix. The soluble lipids are mainly oriented parallel to the normal. The dry cuticles show no phase transition between 0°C and 200°C. At room temperature a dry/wet transition can be observed.Abbreviations ESR-spectroscopy electron-spin-resonance-spectroscopy     

Eucalyptus stomata with occluded anterior chambers

Summary This paper describes variations of a mode of stomatal development already described in a species (E. orbifolia) ofEucalyptus L'Herit. (Carr andCarr, Protoplasma 96, 127, 1978) in which the outer part of the stomatal pore (ostiole) is formed by the creation of a break in the thin layer of cuticle lying over the stomatal chamber. In a number of species with a thick cuticle (e.g., E. cooperana) the process of breakthrough is different: additions to the guard cell upper thickenings extend from them as ridges, pressing the leaf cuticle outwards. Breakthrough of the cuticle occurs above the tips of these extensions. The anterior chamber is lined throughout by the extensions, which become heavily cutinized. This mode of stomatal development is typical of many other species of eucalypts, including those dealt with in this paper.In addition, inE. halophila thickenings develop on the end walls of the anterior chamber above the unusual upturned poles of the guard cells. Cutinized thickenings, pseudo-outer stomatal ledges are also formed on the upper guard cell walls. All these wall thickenings occlude the anterior chamber, leaving only a narrow passage in the form of a letter H.Similar occlusions are found inE. balladoniensis but here the thickenings are developed into the chamber from its lateral walls. InE. gracilis, and the related speciesE. celastroides andE. calycogona, less regular occluding thickenings develop principally from the lateral walls of the chamber. In addition, large pseudo-outer stomatal ledges may be formed.These phenomena are discussed in terms of the mechanism underlying the formation of the occluding thickenings and the possibility of their adaptive significance.     

Thermodynamic analysis of nonelectrolyte sorption in plant cuticles: The effects of concentration and temperature on sorption of 4-nitrophenol

The sorption of 4-nitrophenol (4-NP) in enzymatically isolated cuticles ofLycopersicon esculentum fruits andFicus elastica leaves was studied as a function of temperature and solute concentration. Plots of the concentrations of 4-NP sorbed in the cuticle versus the equilibrium concentrations in the aqueous phase gave linear isotherms at low concentrations that tended to approach plateaus at higher sorbate concentrations ( 10 mmol·kg^-1). At low concentrations of sorbed 4-NP, cuticles have sorptive properties similar to those of organic solvents which are able to form intermolecular hydrogen bonds, while at higher concentrations their solid nature becomes apparent. During sorption of 4-NP the cutin matrix swells and new sorption sites are successively formed. The partition coefficients of 4-NP in the system cuticle/buffer are functions of temperature and concentration. At high sorbate concentrations (approx. 1 mol·kg^-1) they approach a value of 1. Different sorptive properties were observed for the cutin regions normally encrusted with soluble cuticular lipids (SCL) and those without SCL. Increasing temperature augmented the number of sorption sites in the cutin ofLycopersicon while no effect was observed withFicus. The changes of partial molar free energy (G ^o _tr), enthalpy (H ^o _tr), and entropy (S ^o _tr) for the phase transfer of 4-NP also depended on sorbate concentration: H ^o _tr and S ^o _tr were negative and steeply decreased at high sorbate concentrations. This is due to solute-solute interactions replacing solute-cutin interactions at high concentrations resulting in solid precipitates of solute within the cutin matrix. This formation of ordered solid domaines starting from a small number of nonelectrolyte molecules interacting with the cutin is proposed as a model for the intracuticular deposition of SCL.Abbreviations CM cuticular membrane - MX polymer matrix membrane - 4-NP 4-nitrophenol - SCL soluble cuticular lipids     

Roles of dopa decarboxylase and phenoloxidase in the melanization of the tobacco hornworm and their control by 20-hydroxyecdysone

Summary WhenManduca sexta larvae are allatectomized 5 h before head capsule slippage (HCS) in the final larval molt, the new larval cuticle contains granules that melanize 3 h before ecdysis when the ecdysteroid titer falls (Curtis et al. 1984). In both the epidermis and hemolymph of these allatectomized larvae dopamine was higher than dopa prior to and at the time of melanization. Dopamine also increased in the new cuticle as melanization began. Dopa decarboxylase (DDC) activity increased in the epidermis, cuticle, and fat body beginning 16 h after HCS, with a two-fold greater increase in the epidermis of allatectomized larvae. Both -MDH and -fluoromethyl-dopa inhibited epidermal DDC activity and inhibited melanization in vitro when dopa was used as a precursor. Addition of dopamine to the medium allowed melanization in the presence of the inhibitors. All these results indicate that dopamine is likely the primary precursor of cuticular melanin. The diphenoloxidase in the premelanin granules was activated in vivo between 19 and 21 h after HCS and was found to prefer dopamine to dopa and not to convert tyrosine to melanin. The activation of the prophenoloxidase was inhibited by 20-hydroxyecdysone (20-HE), both in vivo and in vitro, if hormone was given by 16 h after HCS. Infusion of 1.2 g/ml 20-HE into allatectomized larvae for 24 h from HCS prevented both the increase in DDC activity and the activation of the premelanin granules. Although the larvae ecdysed after a 15 h delay, melanization never occurred.Abbreviations -MDH L-3-(3,4 dihydroxyphenyl)-2-hydrazine-methylpropionic acid - -FM-dopa R-S--fluoromethyl-dopa - DCC dopa decarboxylase - 20-HE 20-hydroxyecdysone - JH juvenile hormone - HCS head capsule slippage     

Experimental study of the influence of errors of genetic correlation estimates on unrestricted,optimum, and desired gains selection indices

Summary Effects of errors in estimates of the genetic correlation on the accuracy of unrestricted, optimum, and desired gains selection indices were examined experimentally in Tribolium castaneum. Three lines were selected for three generations for pupal weight at 21 days and adult weight at 31 days, using unrestricted (I9), optimum (O9), and desired gains (G9) index selection methods. The genetic correlation between pupal and adult weights in the base population was 0.95. The optimum index was designed to set the response of pupal weight by a fixed amount, while in the desired gains index the responses of pupal and adult weights were specified as being equal to 31. Three other indices were constructed using a deliberately incorrect genetic correlation (0.25), i.e., unrestricted (I2), optimum (O2), and desired gains (G2). Responses observed in unrestricted index lines (I9 versus I2) and optimum index lines (O9 versus O2) did not differ significantly, even though lines I9 and I2 differed in a practical sense. Responses in desired gains index lines (G9 versus G2) differed significantly. Responses obtained for aggregate genotype (pupal weight + adult weight) and for the component traits were greater in line I9 than those obtained in line I2. Responses obtained in the O9 and O2 lines for pupal and adult weights were similar, while those obtained in the G9 and G2 lines were similar for pupal weight but not (P<0.05) for adult weight. Therefore, underestimation of the genetic correlation seems to affect the efficiency of a desired gains index more than that of unrestricted or optimum indices.     

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.     

Interspecific hybridization of cultivated rice, Oryza sativa L. with the wild rice, O. minuta Presl.

Crosses were made between four varieties (Mahsuri, Setanjung, MR84 and MR103) of Oryza sativa L. (2n=24, AA) and one accession of O. minuta (2n= 8, BBCC). The seed set obtained ranged between 9.5% and 25.1% depending on the rice variety used. By rescuing 14-day-old embryos and culturing them on 25%-strength MS medium we obtained a total of 414 F₁ hybrids. The F₁s were vigorous, tillered profusely, were perennial and male-sterile. The hybrids were triploid (ABC) with 36 chromosomes and showed irregular meiosis. The average frequency and range of chromosome associations at metaphase I or early anaphase I pollen mother cells of F₁ plants were 29.31(16–36) Is +3.32(0–10) IIs+0.016(0–1) IIIs+0.002(0–1) IVs. Upon backcrossing the original triploid hybrids and colchicine-treated hybrids to their respective recurrent parents, and further embryo rescue, 17 backcross-1 (BC₁) plants were obtained. Of all the crosses using MR84, no BC₁ plant was obtained even after pollinating 13 894 spikelets of the triploid hybrid. The BC₁s were similar in appearence to the F₁s and were male-sterile, their chromosome number ranged from 44 to 48. By backcrossing these BC₁s and nurturing them through embryo rescue, we obtained 32 BC₂ plants. Of these, however, only 18 plants grew vigorously. One of these plants has 24 chromosomes and the other 17 have chromosome numbers ranging between 30 and 37. The 24-chromosome plant was morphologically similar to the O. sativa parent and was partially fertile with a pollen and spikelet fertility of 58.8% and 12.5% respectively. All of the F₁ and BC₁ plants were found to be resistant to five Malaysian isolates (XO66, XO99, XO100, XO257 and XO319) of Xanthomonas campestris pv oryzae. Amongst the BC₂s, the reaction varied from resistant to moderately susceptible. The 24-chromosome BC₂ plant was resistant to the four isolates and moderately resistant to isolate XO100 to which the O. sativa parent was susceptible.Part of PhD thesis submitted by first author to Universiti Kebangsaan Malaysia, Bangi     

Ultrastructure des disques de patte de drosophile cultivésin vitro. Evagination,sécrétion de la cuticule nymphale et apolysis

Summary Ultrastructural observations were made on imaginal leg discs ofDrosophila cultured in vitro in Mandaron's medium M in the presence of - or -ecdysone.During evagination of discs in vitro the tight epidermal folds of the disc flatten out progressively and the cells change their shape.As the discs evaginate, the pupal cuticle is secreted at the apex of the cells: it comprises a three-layered cuticulin, an epicuticle and a thick endocuticle. Secretion of pupal cuticle always starts by the appearance of dense deposits at the apex of microvilli. Only the latter apparently participate in the construction of the three successive layers of the pupal cuticle.During disc development an abundant rough endoplasmic reticulum is formed. Nuclei and nucleoli become progressively enlarged. At the same time, the nucleus, initially located near the base of the cell, moves closer to the apical cell surface. Microtubules become fragmented as soon as evagination begins and are reorientated parallel to the cell surface (i.e. parallel to the new long axis of the cell) at the end of evagination.

---

---

Ce travail a été réalisé avec l'aide du CNRS (Action thématique programmée) «Différenciation cellulaire», contrat n^o A 6554324.

---

Ce mémoire représente une partie de la thèse qui sera soutenue par l'auteur devant l'Université Scientifique et Médicale de Grenoble.     

Synthesis and deposition of PCG-100, the main pupal cuticle glycoprotein of the medfly Ceratitis capitata

We have studied the developmental expression of the main pupal cuticle glycoprotein, PCG-100, in the Medfly Ceratitis capitata. A polyclonal antiserum was raised against this protein. Western blotting analysis showed that this glycoprotein is integument- and stage-specific. No PCG-100 or immunologically related polypeptides were detected in other tissues or instars. As studied by microinjection of [³⁵S]methionine in individual flies, in vivo synthesis and deposition of PCG-100 begins approximately 48 h after the onset of pupariation, shortly after the time of head eversion. Synthesis is maximal at 54–64 h, decreases at 72 h, and practically ceases in fully shaped 4-day-old pupae. The time required for PCG-100 deposition into the cuticle was found to be less than 10 min after its synthesis. This is the first time such in vivo analysis has been performed. © 1994 Wiley-Liss, Inc.     

Substrate-histochemical investigations and ultrahistochemical demonstrations of acid phosphatase in larval and prepupal salivary glands of Drosophila melanogaster

Summary A major function of the larval salivary glands of Drosophila melanogaster is known to be the production of a mucopolysaccharide that serves as an adhesive during puparium formation. In order to localize the mucosubstances during development substrate histochemical methods were used, and the site of acid phosphatase was demonstrated by the ultrahistochemical lead-salt method. It could be shown that the glue-granules in the corpus cells of larval salivary glands as well as the large secretion vacuoles in the prepupal corpus cells give a positive -amylase-resistent PAS-reaction, which indicates neutral mucosubstances. Granular PAS-positive deposits in the larval and prepupal collum cells were reduced after preincubation with -amylase and may represent glycogen, which has also been seen in electron micrographs of these cells. The Hale-reaction gave a weak indication that acid mucosubstances are present in the larval glue granules and in the large prepupal secretory vacuoles. After digestion of sialic acid with -neuraminidase the weak indication was absent showing that the acid mucosubstances had been sialomucines. Ultrahistochemical demonstration of acid phosphatase indicated the presence of this enzyme in Golgi fields and lysosomal structures. Acid phosphatase seems to be missing in the large secretion vacuoles of the prepupal salivary gland.It is concluded, that the large vacuoles in the corpus cells of prepupal salivary glands represent a secretion product, obviously a mucosubstance. The lysosomal structures, containing acid phosphatase, may be accumulated in preparation for the autolysis of the gland which begins about two hours after the pupal moult, i.e. 15 hours after puparium formation.This investigation was supported by grants from the Deutsche Forschungsgemeinschaft (Ga 97/6).