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).     

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.     

Study of catheptic and acid phosphatase activities during development and metamorphosis ofDrosophila melanogaster

Summary Developmental stages ofDrosophila melanogaster cultured at 22 ± 1° C were collected and tested for catheptic activity and acid phosphatase activity.It was found that catheptic activity was absent in the egg as well as in the first and second larval instars. The activity first appears in the third instar larva and reaches its peak 24 to 48 hr after puparium formation. It then decreases, at first gradually and at pupal stage 9 (120 to 144 hr after puparium formation) abruptly, reaching zero level just before the emergence of the imago.The pattern of acid phosphatase activity in different developmental stages was found to be broadly similar to that of catheptic activity in the larval and pupal stages. However, the acid phosphatase activity was found to be exceptionally high in the egg in contrast to the catheptic activity.The authors are grateful to Prof. Dr. R. Weber, Zoological Institute of the University of Bern, for constructive criticism of this paper.     

Expression of esterases during ontogenesis of the flour beetle Tribolium castaneum (tenebrionidae; coleoptera)

Two electrophoretically fast-migrating, nonspecific esterases were detected in two strains of the flour beetle Tribolium castaneum and designated F (fast) and S (slow) according to their relative migration distances. Both isozymes are associated with the alimentary canal and display ontogenetic changes. Their activity is very low in the egg stage, increases in the larva, and declines dramatically in the pharate pupa and pupa. The overall activity in the pupal stage is low, yet increases gradually throughout this period. In the adult, the activity of the esterases rises sharply. The larval and adult F and S isozymes were found to hydrolyze - and -naphthylacetate and -naphthylpropionate with almost equal capacity. -Naphthyl laurate was cleaved by the F enzyme of both larvae and adults. The F and S were insensitive to inhibitors of arylesterases and cholinesterases and were markedly inhibited by the organophosphate diisopropylphosphorofluoridate (DFP) and could be classified as carboxylesterases. Differential sensitivities of larval and adult esterases to urea and heat treatment as well as to DFP may indicate the expression of different genes during metamorphosis.     

The development of the sensory organs of the legs in the blowfly,Phormia regina

Summary The development of the sensory neurons of the legs of the blowfly,Phormia regina has been described from the third instar larva to the late pupa using immunohistochemical staining. The leg discs of the third instar larva contain 8 neurons of which 5 come to lie in the fifth tarsomere of the developing leg. Whereas 2 neurons persist at least to the late pupa, the other cells degenerate. The first neurons of gustatory sensilla arise in the fifth tarsomere at about 1.5 h after formation of the puparium. Most of these sensilla, however, appear within a short time period beginning at about 18 h. The femoral chordotonal sensory neurons first appear at the time of formation of the puparium, as a mass of cells situated in the distal femur. During later pupal development 2 groups of these cells come to lie at the femur-trochanter border, where they become the proximal femoral chordotonal organ of the adult; the remaining cells become the distal femoral chordotonal organ. Other scolopidial neurons appear later in development. The nerve pathways of the late pupal leg are established either by the axons of the cells that are present in the larval leg disc or by new outgrowing processes of sensory neurons. In the tibia, the initial direction of new outgrowth differs in different regions of the segment: proximal tibial neurons grow distally, while distal tibial neurons grow initially proximally.     

Puffs and salivary gland function: The fine structure of the larval and prepupal salivary glands ofDrosophila melanogaster

Summary The salivary glands ofDrosophila melanogaster have been examined by electron microscopy for fine structural alterations occurring during larval and prepupal stages. The changes observed in the glands have been correlated with the puffing patterns of the polytene chromosomes at corresponding stages. In early third instar larvae, the lumen of the salivary gland appears empty, and no signs of secretory activity are visible in the glandular cytoplasm. From puff stages 1 to 6 the endoplasmic reticulum becomes reorganized and increases in volume. Electron dense material appears within its cisternae and subsequently within the Golgi saccules. Dense secretory granules then appear to be elaborated from the Golgi by terminal budding; these granules represent the glue for adhering the pupa to its substrate, and gradually increase in size and complexity. By puff stage 6 their contents have been liberated into the glandular lumen. Following puparium formation, those granules which are not extruded coalesce to form larger granules. Other dense bodies and autophagic vacuoles, considered to be lysosomes, appear, and the surplus secretory granules begin to display myelination at their peripheries; ultimately they are reduced to dense residual bodies. At puparium formation, the lumen is depleted of the glue and contains flocculent material. Histolysis commences after puff stage 11, and the cytoplasm becomes vacuolated and opaque; the nucleus becomes reduced in volume and crenated in outline. Nuclear blebbing occurs after puff stage 12, and material seemingly moves from the nucleus into the cytoplasm; the glandular lumen now becomes empty. An attempt has been made to ascertain how the chromosomal puffing activity relates to these cytoplasmic developments.     

Association of a fungal endophyte in perennial ryegrass with antibiosis to larvae of the southern armyworm, Spodoptera eridania

Larvae of the Southern armyworm Spodoptera eridania Cramer (Lepidoptera:Noctuidae), feeding on perennial ryegrasses (Lolium perenne L.) infected with an endophytic fungus (Acremonium loliae Latch, Christensen and Samuels), had a much lower survival rate (7–13%) than larvae feeding on endophyte-free ryegrasses (82–90%). Death of the larvae on endophyte-infected entries occurred rapidly between 144 h and 168 h of feeding. This corresponded with armyworms feeding on the base of the plant, where endophyte concentration is highest. Twenty-four hours after the start of the bioassay the larval mass and rate of larval development were significantly higher on endophytic entries. From 48–144 h no differences were seen, but after 144 h the mass of larvae on endophyte-infected grasses sharply declined. Observations from this bioassay further substantiate the association between A. loliae-infected ryegrass and antibiosis to several lepidopterous and coleopterous insect pests.

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Résumé Le ray-grass vivace (Lolium perenne), contaminé par le champignon endophyte Acremonium loliae Latch, Christensen & Samuels, a présenté une augmentation de la resistance à de nombreux coléoptères et lépidoptères nuisibles. Cette note examine les réactions de Spodoptera eridania Cramer (Lépido., Noctuidae) alimenté sur trois lignées de ray-grass contaminées par le champignon et trois lignées saines. Après 168 h d'alimentation sur ray-grass contaminé, les chenilles presentent une très forte mortalité; la survie n'est que de 7 à 13% contre 82 à 90% pour le ray-grass sain. Le décès brutal des chenilles correspond à leur alimentation sur la base de la plante ou la concentration du champignon est la plus forte. Les chenilles consomment constamment, broutant plus des 2/3 du feuillage du ray-grass; les broutements des six séries ne différaient pas significativement.Au bout de 24 h, la nombre de chenilles passées du 3ème au 4ème stade, et l'augmentation de poids sont plus élevés pour les séries sur plante contaminée, ce qui suggère un avantage initial pour les chenilles en présence de champignon endophyte, l'analyse en poids sec a montré que l'augmentation de poids initial est réel. Entre 48 et 144 h, cependant, le nombre de 4ème stade et le poids des chenilles sont les mêmes dans les deux séries. Après 144 h, le poids des chenilles sur ray-grass contaminé diminue significativement; aucune n'était parvenue au 5ème stade, contre 11% sur ray-grass sain. Nous n'avons pas observé de signes apparents de neurotoxicité. Au lieu de cela, il ya a eu interaction avec un processus physiologique fondamental, ce qui a provoqué une forte perte de poids larvaire et la mort, indiquant l'intervention d'inhibiteurs métaboliques.

     

Enhanced rates of P700<Superscript>+</Superscript> dark-reduction in leaves of<Emphasis Type="Italic"> Cucumis sativus</Emphasis> L. photoinhibited at chilling temperature

The changes in electron transport within photosystem I (PSI) were studied in detached leaves of Cucumis sativus L. during the course of irradiation with moderate white light (300 mol photons m^–2 s^–1) at 4°C. When intact leaves were exposed to the combination of moderate light and low temperature, the amplitude of far-red light-induced P700 absorbance changes at 820 nm (A₈₂₀), a relative measure of PSI, progressively decreased as the light treatment time increased. Almost no oxidation of P700 was noticeable after 5 h. Methyl viologen accelerated the oxidation of P700 to a steady-state level and also increased the magnitudes of A₈₂₀ changes in photoinhibited leaves, reflecting the rapid removal of electrons from native carriers. Photoinhibition under moderate light and chilling temperature also accelerated the rate of P700⁺ reduction after far-red light excitation as the half-times of the two exponential components of P700⁺ decay curves decreased relative to the control ones. A detailed analysis of the kinetics of P700⁺ reduction using diuron alone or the combination of diuron and methyl viologen strongly favours an increased rate of electron donation from stromal reductants to PSI through the plastoquinone pool following photoinhibitory treatment. Importantly, the marked acceleration of P700⁺ re-reduction is the consequence of the irradiation of leaf segments at low temperature and not caused by chilling stress alone.Abbreviations A ₀ and A ₁ Primary acceptor chlorophyll and secondary electron acceptor phylloquinone - FR Far-red light - F _X , F _A , and F _B Iron–sulfur centers - MT Multiple-turnover flash - MV Methyl viologen - Ndh NAD(P)H-dehydrogenase - PQ Plastoquinone - PS Photosystem - P700 Reaction-center chlorophyll of PSI - ST Single-turnover flash     

Developmental changes in genome activity in Drosophila lebanonensis casteeli pipkin

Puparium formation in Drosophila lebanonensis casteeli is obviously restricted to a certain phase in circadian oscillation. The question whether or not the release of molting hormone is the actual process which is controlled by the circadian oscillation could be approached by using molting hormone-specific changes in genome activity as indication for changes in hormone titer. The identification of hormone specific changes in the puffing pattern of polytene chromosomes should provide a basis for this study.—To this end, a chromosome map of the 7 polytene chromosome arms (1 acrocentric and 3 metacentric chromosomes) of the species was made. Changes in the puffing pattern associated with puparium formation are described and compared with those occurring in response to experimental administration of -ecdysone.—89 puffs were regularly observed in midthird instar larvae. Prior to puparium formation 5 new puffs arise, one at an early stage and 4 attaining their maximum size immediately before puparium formation. Concomitantly, 5 puffs increase considerably in size. These changes in the puffing pattern can be reproduced by injection of ecdysone.—Upon injection of the hormone a clear differentiation between fast reacting loci (within 30–60 min) and slow reacting loci (after 3–4 hours) can be found. As in other Drosophila species the immediate response (within 30–60 min) comprises more than one (5) locus.In memory of Professor Dr. J. Schultz.     

Ultrastructural enzyme-cytochemical study of the intrinsic glandular cells in the corpus cardiacum of Locusta migratoria: relation to the secretory and endocytotic pathways,and to the lysosomal system

Summary Ultrastructural aspects of the secretory and the endocytotic pathways and the lysosomal system of corpus cardiacum glandular cells (CCG cells) of migratory locusts were studied using morphological, marker enzyme, immunocytochemical and tracer techniques. It is concluded that (1) the distribution of marker enzymes of trans Golgi cisternae and trans Golgi network (TGN) in locust CCG cells corresponds to that in most non-stimulated vertebrate secretory cell types; (2) the acid phosphatase-positive TGN in CCG cells is involved in sorting and packaging of secretory material and lysosomal enzymes; (3) these latter substances are produced continuously; (4) at the same time, superfluous secretory granules and other old cell organelles are degraded; (5) the remarkable endocytotic activity in the cell bodies and the minor endocytotic activity in cell processes are coupled mainly to constitutive uptake of nutritional and/or regulatory (macro)molecules, rather than to exocytosis; (6) plasma membrane recycling occurs mainly by direct fusion of tubular endosomal structures with the plasma membrane and little traffic passes the Golgi/TGN; and (7) so-called cytosomes arise mainly from autophagocytotic vacuoles and represent a special kind of complex secondary lysosomes involved in the final degradation of endogenous (cell organelles) and exogenous material.     

Ecdysone-induced changes in glycoprotein synthesis and puff activities in Drosophila virilis salivary glands

During five hours after the injection of -ecdysone into the hemolymph of D. virilis late third instar larvae the formation of larval glycoproteins in the salivary glands is terminated and the synthesis of a different set of glycoproteins which is characteristic for the prepupal gland is initiated. The data presented suggest that products from early puffs inhibit the formation of larval glycoproteins while the induction of late puffs may be responsible for the appearance of prepupal glycoproteins.     

Preparation of Single Cell Detritus from <Emphasis Type="Italic">Laminaria saccharina</Emphasis> as a Hatchery Diet for Bivalve Mollusks

A high-yield technique is described for the elaboration of single cell detritus (SCD) from Laminaria saccharina, based on the sequential action of C1H, enzymes (endoglucanases and cellulases) and 2 bacteria showing a high degree of cellobiotic, proteolytic, and alginolytic activity (CECT 5255 and CECT 5256). Over 85% of dried particles of L. saccharina were transformed into a suspension of free cell and bacterial and detrital particles after 24 hours of bacterial activity with this technique. These particles were less than 20 m in diameter, constituting a suitable diet for bivalve mollusks. After 72 hours 99% of the total particulate volume consisted of particles less than 20 m in diameter. Tests of hatchery diets for the seed of clam Ruditapes decussatus revealed increases of 54% and 68% for live weight and length, respectively, when SCD from L. saccharina was used as the sole dietary component compared with a live phytoplankton diet. However, SCD from L. saccharina is not a suitable food for the larvae of R. decussatus.     

Milk-clotting protease production by <Emphasis Type="Italic">Nocardiopsis</Emphasis> sp. in an inexpensive medium

The milk-clotting and proteolytic activities of extracellular enzyme preparations from Nocardiopsis sp. were investigated under different culture conditions. A soybean flour medium was used, with concentrations of soybean flour and of glucose varying from 0.25 to 1% w/v and from 0 to 1% w/v, respectively. Growth was monitored with 2ml samples withdrawn from the culture medium at 8-h intervals, for determination of total protein, proteolytic activity, milk-clotting activity and sugar reduction. The best milk-clotting protease production, with a specific activity of 24.49U/mg at 40h, was obtained in the glucose-free medium containing soybean flour 1% w/v.     

Dual localization of acid hydrolases in endoplasmic reticulum and in lysosomes

Summary Dissimilar enzyme locations obtained on occasion by the post- and simultaneous-coupling techniques employing the substrate naphthol AS-BI -glucosiduronic acid were attributed to the inadequate incorporation of substrate into lysosomal membranes in the post-coupling technique on the one hand, as well as to the inhibition of cytoplasmic enzyme by diazotate in the simultaneous coupling technique on the other hand. The use of a fixative solvent mixture prior to the enzyme staining reaction appeared to labilize lysosomal membranes, to improve fixation and to eliminate fiber artefacts. In male mice which have been androgenized by the injection of gonadotrophin, kidney homogenates, subsequently prepared, exhibited an immediate increase in the specific activity of microsomal -glucuronidase while lysosomal -glucuronidase was unchanged for the first 36 hours.This event at 36 hours corresponded with enhanced cytoplasmic but not lysosomal staining. Diffuse reactions in enzyme morphology are discussed as well as the origin of lysosomal -glucuronidase in mouse kidney and the dual localization of hydrolases in endoplasmic reticulum and lysosomes.     

A comparative ultrastructural study of ‘glue’ production and secretion of the salivary glands in different species of theDrosophila melanogaster group

Summary Salivary gland cells of members of theDrosophila melanogaster group (from four different subgroups) were examined electron microscopically and histochemically during the late larval period of development. The secretory product, which is supposed to be utilized as glue at the time of puparium formation, appears, by analogy to Palade and Jamieson's results, to be synthesized partially in the rough endoplasmic reticulum (RER) and partially in the Golgi complex. The latter is also the usual site of the packaging of the product into secretory granules, except in the case of one of the secretory granule components ofD. lucipennis. The phylogenetic relationships among the subgroups, implied by the morphological appearance of the secretory granules, fit well with the existing phylogenetic relationships within the group. The secretory granules of each species have their own morphological features; granules of species of the same subgroup share some of these features. Secretion occurs from the cells via exocytosis during which the morphology of the secretory granules changes. Light microscope examination of PAS (Periodic Acid-Schiff reaction) stained glands shows a strong positive reaction in most species, with the exception of the species of thesuzukii subgroup which show a weak, or a negative reaction (D. rajasekari). Electron histochemical localization of polysaccharides in the secretory granules was possible inD. melanogaster and the species of theananassae subgroup.     

Developmental morphology of the cyprinid fish Horadandia atukorali

Embryonic, larval, and juvenile development of a small Indian cyprinid, Horadandia atukorali, is described from laboratory-reared specimens. The eggs, measuring 0.7–0.8mm in diameter, were demersal, almost spherical in shape, transparent and unpigmented, with a pale yellow yolk without oil globules. Hatching occurred 47–54h after fertilization at 26.3–27.5°C. The newly hatched larvae, measuring 2.3–2.6mm in body length (BL) with 16+13=29 myomeres, had no melanophores, except on the eye, a single melanophore occurring on the lower margin, and xanthophores surrounding the pupil. The yolk was completely absorbed at 3.0mm BL. Notochord flexion was initiated at 4.0mm BL and finished at 4.4mm BL. Aggregate numbers of all fin rays were completed at 8.0mm BL. Squamation was initiated at 6.4mm BL and completed at 9.5mm BL. Although the eggs of Horadandia atukorali resembled those of other small danionin species, including Aphyocypris chinensis, Chela dadiburjori, Danio rerio, Devario malabaricus, and Hemigrammocypris rasborella, they differed from those of Danio rerio and Devario malabaricus in having a narrower perivitelline space. The larvae and juveniles of Horadandia atukorali were also similar to those of the latter five species in general morphology, especially in the presence of a melanophore on the lower margin of the eye at hatching, as in C. dadiburjori. However, the early life stage morphology of Horadandia atukorali differed from the other danionin species in having a conical yolk sac at hatching, no cement organ on the forehead in the yolk-sac larval stage, a divided gas bladder in the flexion larval stage, two dark lateral streaks on the head and chevron-like melanophores on the ventral body surface from the preflexion to postflexion larval stages, and xanthophores on the eyes at hatching.     

A dual function of the Notch gene in Drosophila sensillum development

We have investigated the function of the neurogenic gene Notch (N) during development of the adult sensilla of Drosophila. Heat pulses were applied to flies carrying the temperature-sensitive Notch allele Nts1 at different larval and pupal stages. We can show that the reduction of Notch+ function during a short interval prior to the onset of sensillum precursor division, resulting from a heat pulse between 0 and 14 hr after puparium formation (apf), leads to an increase in microchaete precursors at the expense of epidermal cells. The structure and cellular composition of the sensilla produced by these supernumerary precursors are normal. Later heat pulses which include the interval immediately after sensillum precursor division (14-20 hr apf) lead, among the progeny of the sensillum precursors, to a hyperplasia of sensory neurons, at the expense of accessory cells. The resulting "sensilla" consist of neurons only and lack the external cuticular structures (i.e., shaft, socket). These results demonstrate that similar mechanisms both of which involve the function of the Notch gene may be operating to sort out (premitotic) sensillum precursors from epidermal precursors and (postmitotic) sensory neurons from accessory cells. They further show that in postmitotic sensillum cells the differentiative fate is not yet irreversibly fixed, but presumably requires cell-cell interaction to become established.     

Induction of apoptosis by a cachectic-factor in murine myotubes and inhibition by eicosapentaenoic acid

Treatment of C₂C₁₂ myotubes with a tumour-derived proteolysis-inducing factor (PIF) at concentrations between 1 and 10 nM was shown to stimulate the activity of the apoptotic initiator caspases-8 and -9 and the apoptotic effector caspases-2, -3 and -6. This increased caspase activity was attenuated in myotubes pretreated with 50 M eicosapentaenoic acid (EPA). At least part of the increase in caspase activity may be related to the increased proteasome proteolytic activity, since a caspase-3 inhibitor completely attenuated the PIF-induced increase in chymotrypsin-like enzyme activity, the predominant proteolytic activity of the proteasome. However, Western blot analysis showed that PIF induced an increase in expression of the active form of caspase-3, which was also attenuated by EPA.Further Western blot analysis showed PIF increased the cytosolic content of cytochrome c, as well as expression of the pro-apoptotic protein bax but not the anti-apoptotic protein bcl-2, which were both attenuated by 50 M EPA. Induction of apoptosis by PIF in murine myotubes was confirmed by an increase in free nucleasomes formation and increased DNA fragmentation evidenced by a nucleasomal ladder typical of apoptotic cells. This process was again inhibited by pre-incubation with EPA. These results suggest that in addition to activating the proteasome, PIF induces apoptosis in C₂C₁₂ myotubes, possibly through the common intermediate arachidonic acid. Both of these processes would contribute to the loss of skeletal muscle in cancer cachexia.     

Development of adult sensilla on the wing and notum of Drosophila melanogaster

We have investigated the temporal pattern of appearance, cell lineage, and cytodifferentiation of selected sensory organs (sensilla) of adult Drosophila. This analysis was facilitated by the discovery that the monoclonal antibody 22C10 labels not only the neuron of the developing sensillum organ, but the accessory cells as well. The precursors of the macrochaetes and the recurved (chemosensory) bristles of the wing margin divide around and shortly after puparium formation, while those of the microchaetes and the stout and slender (mechanosensory) bristles of the wing margin divide between 9 h and 18 h after puparium formation (apf). The onset of sensillum differentiation follows the terminal precursor division within a few hours. Four of the cells in an individual microchaete organ are clonally related: A single first-order precursor cell divides to produce two second-order precursors; one of these divides into the neuron and thecogen cell, the other into the trichogen cell and tormogen cell. Along the anterior wing margin, two rounds of division generate the cells of the mechanosensory sensilla; here, no strict clonal relationship seems to exist between the cells of an individual sensillum. At the time of sensillum precursor division, many other, non-sensillum-producing cells within the notum and wing proliferate as well. This mitotic activity follows a spatially non-random pattern.