Acid phosphatase activity in the larval salivary glands of developing Drosophila melanogaster.

Both the biochemical profile and the optical and fine structural localization of acid phosphatase activity in the larval salivary glands of developing Drosophila melanogaster is described. Biochemically, acid phosphatase shows peak activity in the glands of feeding larvae, followed by a marked decline. Directly preceding the onset of cell histolysis however, enzyme activity increases 1.5 fold and is maintained at this level. Histochemically, acid phosphatase activity initially appears as discrete point or lysosomal sources. As development proceeds, an intense and diffuse form of enzyme is seen, accompanying an extremely vacuolated cytoplasm. Ultrastructurally, the enzyme is located in lysosomes, Golgi elements, multivesicular bodies and both within, and on the extracisternal surface of the rough endoplasmic reticulum. This extracisternal or cytosolic form appears directly preceding cell lysis and eventually shows a comprehensive cellular distribution. Large numbers of acid phosphatase positive haemocytes are attached to the basal glandular surface at all developmental stages. In morphologically intact gland cells, discrete extracisternal enzyme activity appears associated with local areas of degradation.     

Ultrastructural changes of Drosophila larval and prepupal salivary glands cultured in vitro with ecdysone

Summary Alterations in the ultrastructure of in vitro cultured larval salivary glands of Drosophila melanogaster in response to the steroid hormone ecdysone were studied in relation to complex changes in puffing patterns. We found that the changes in the fine structure of cultured glands reflected progression of the puffing pattern, and they paralleled those seen in vivo. We observed that glue secretion by exocytosis, the main function of salivary glands, took place between puff stage 5 (PS5) and PS7. Glue could not be expectorated under culture conditions but was slowly released from the lumen through a duct into the medium. After the cultured glands reached PS13/PS14, further progress of puffing and fine structural alterations required that the ecdysteroid titer be transiently extremely low or absent. Under in vitro conditions we did not observe the putative new secretory program(s) described for glands in vivo after PS12. However, ultrastructural changes which unambiguously indicated that an autohistolytic process had begun in vitro started to appear after PS17. Many salivary gland cells developed numerous features of progressive self-degradation between PS18 and PS21. Actual degradation of salivary glands in vivo seemed to be rapid, but in vitro degradation was never completed, probably due to a lack of exogenous factors from the hemolymph. Manipulations of ecdysone titer in vitro in the culture medium, known during the larval puffing cycle to cause premature induction of developmentally specific puffing patterns, did not affect the normal development of ultrastructural features of the cytoplasm and nucleus.     

Polarized secretion of an ectopic protein in Drosophila salivary glands in vivo.

Epithelial cells can secrete specific proteins in a polarized manner, either from the apical or basolateral surface. Intracellular protein sorting which results in polarized secretion has previously been studied using epithelial tissue culture cells. We describe here the use of Drosophila larval salivary glands for the study of polarized secretion by epithelia in vivo, and address whether an ectopically synthesized secretory protein can be sorted and targeted to the correct cell surface for secretion. Larval cuticle proteins (LCPs) and salivary gland secretion (Sgs) proteins of Drosophila melanogaster are apically secreted proteins that are produced respectively by the epidermis and salivary glands. We have transformed Drosophila with a hybrid gene consisting of the sgs-4 promoter sequence and the coding sequence for a variant (LCP-f2) of LCP-2. We have found that transgenic late third instar larvae produce LCP-f2 only in the salivary glands and that LCP-f2 is properly secreted in vivo in a polarized manner from only the apical surface of the cells into the gland lumen. The results indicate that apical secretion does not depend on a tissue-specific targeting signal contained within the protein.     

Respiration of larval salivary glands of Drosophila in relation to the activity of specific genome loci

Oxygen consumption of intact larval salivary glands of Drosophila hydei was measured after the addition of intermediates of the citric acid cycle or amino acids to the incubation medium. The effect of these substances on respiration of glands previously submitted to anaerobiosis in vivo was compared with that of glands of control larvae. Only isocitrate and tyrosine stimulated respiration of anaerobically treated glands to a much higher extent than glands of control larvae. This stimulatory effect was abolished when RNA or protein synthesis was inhibited. It is suggested that some of the specific puffs occurring as a consequence of anaerobiosis reflect gene activity required for an increase in utilization of isocitrate and tyrosine for respiration under conditions of stress.     

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).     

Circadian synchronization and rhythmicity in larval photoperception-defective mutants of Drosophila

A single light episode during the first larval stage can set the phase of adult Drosophila activity rhythms, showing that a light-sensitive circadian clock is functional in larvae and is capable of keeping time throughout development. These behavioral data are supported by the finding that neurons expressing clock proteins already exist in the larval brain and appear to be connected to the larval visual system. To define the photoreceptive pathways of the larval clock, the authors investigated circadian synchronization during larval stages in various visual systems and/or cryptochrome-defective strains. They show that adult activity rhythms cannot be entrained by light applied to larvae lacking both cryptochrome and the visual system, although such rhythms were entrained by larval stage-restricted temperature cycles. Larvae lacking either pathway alone were light entrainable, but the phase of the resulting adult rhythm was advanced relative to wild-type flies. Unexpectedly, adult behavioral rhythms of the glass60j and norpAP24 visual system mutants that were entrained in the same conditions were found to be severely impaired, in contrast to those of the wild type. Extension of the entrainment until the adult stage restored close to wild-type behavioral rhythms in the mutants. The results show that both cryptochrome and the larval visual system participate to circadian photoreception in larvae and that mutations affecting the visual system can impair behavioral rhythmicity.     

Programmed cell death in salivary glands of Drosophila arizonae and Drosophila mulleri

Programmed cell death (PCD) in insect metamorphosis assumes a great diversity of morphology and controlling processes that are still not well understood. With the objective of obtaining information about the PCD process, salivary glands of Drosophila arizonae and D. mulleri were studied during larval-pupal development. From the results, it can be concluded that the type of the PCD that occurs in these organs is morphologically typical of apoptosis (formation of apoptotic nuclei, followed by fragmentation into apoptotic bodies). Histolysis happens in both species, between 22 and 23 h after pupation. There were no significant differences between the species studied. Apoptosis does not occur simultaneously in all cells. Cytoplasmic acid phosphatase activity gradually increases during development, suggesting the existence of acid phosphatases that are only expressed during the apoptotic stage. Twenty hours after pupation, salivary glands already show biochemical alterations relative to nuclear permeability such as acidification, possibly due to the fusion of lysosomes with the nucleus a few hours before apoptosis. Autophagy seems to act together with apoptosis and has a secondary role in cell death.     

Differential Replication of DNA Sequences in Drosophila Chromosomes

During the DNA replications involved in polyploidization orpolytenization in Drosophila cells, not all DNA sequences arereplicated to the same extent. Cytological studies have demonstratedthat certain chromosome regions, such as the a-heterochromatin,are in some cells under-replicated and in other cells not replicatedat all. Similarly, such DNA fractions as the highly repeatedsatellite DNAs are also under- or non-replicated in polyploidand polytene cells. The genes for rRNA in polytene cells replicateone to three rounds less than the euchromatic DNA and are capableof differential synthesis to compensate for deficiencies. Thedifferential replication of DNA sequences indicates that thereis regulation of DNA replication at a level intermediate betweenthe replicon and the entire genome. Chromosomes of terminallydifferentiated polyploid or polytene cells have several domainsof DNA sequences, which in replication are controlled as units.This paper, which reviews the literature on differential replicationof DNA in Drosophila, discusses possible controls over thisprocess.     

Rickettsiae-like structures in the larval salivary gland cells of Drosophila auraria

Rickettsiae-like structures were found in the salivary gland cells of Drosophila auraria during different larval and prepupal developmental stages, from the early 3rd instar up to 14 hr after spiracle inversion. These microorganisms are surrounded by a membrane, are constantly intracellular, and occur singly or in groups. Their widespread occurrence in various tissues of other Drosophila species indicates that they can be considered as symbionts, but their actual functional significance (if any) is unknown.     

DNA synthesis in larval brains of Drosophila nasutoides

The incorporation of H³-TdR and H³-TTP into embryos, embryo homogenates, and larval brains and imaginal discs of Drosophila nasutoides was examined. The highest incorporation was observed with brains and brain-associated imaginal discs incubated in Schneider's medium containing H³-TdR. A specific activity of 50,000 cpm/µg DNA or higher was obtained. By use of this method, the specific activities of the main DNA and the satellite DNAs of D. nasutoides were determined. It was observed that the specific activities were nearly the same regardless of labeling period, indicating that the main DNA and the satellite DNAs have similar extents of H³-TdR incorporation.     

Activities of the nuclear envelope in the salivary glands of Drosophila

Morphological data are presented concerning the single-membrane-bound vesicles ("oval bodies") associated with the nuclear envelopes of larval salivary gland cells of Drosophila. Data are also presented concerning the existence of cytoplasmic annulate lamellae in these same cells. The mode of formation of these structures, as well as the relationships between them and with other cytoplasmic organelles are described. The possible functional significance of these phenomena is discussed.