Time Of Puparium Formation Research Articles

Smt3 is required forDrosophila melanogastermetamorphosis

Sumoylation, the covalent attachment of the small ubiquitin-related modifier SUMO to target proteins, regulates different cellular processes, although its role in the control of development remains unclear. We studied the role of sumoylation during Drosophila development by using RNAi to reduce smt3 mRNA levels in specific tissues. smt3 knockdown in the prothoracic gland, which controls key developmental processes through the synthesis and release of ecdysteroids, caused a 4-fold prolongation of larval life and completely blocked the transition from larval to pupal stages. The reduced ecdysteroid titer of smt3 knockdown compared with wild-type larvae explains this phenotype. In fact, after dietary administration of exogenous 20-hydroxyecdysone, knockdown larvae formed pupal cases. The phenotype is not due to massive cell death or degeneration of the prothoracic glands at the time when puparium formation should occur. Knockdown cells show alterations in expression levels and/or the subcellular localisation of enzymes and transcription factors involved in the regulation of ecdysteroid synthesis. In addition, they present reduced intracellular channels and a reduced content of lipid droplets and cholesterol, which could explain the deficit in steroidogenesis. In summary, our study indicates that Smt3 is required for the ecdysteroid synthesis pathway at the time of puparium formation.

The dachshund gene is required for the proper guidance and branching of mushroom body axons in Drosophila melanogaster

The dachshund gene encodes a transcription factor required for the proper development of Drosophila eyes, legs, and mushroom bodies. The mushroom bodies of dachshund mutants exhibit a marked reduction in the size of the vertical lobes and disorganization of the horizontal lobes. In mosaic animals, mutant axons fail to contribute significantly to the mushroom body alpha lobe. Here we show that this defect is due to the misrouting of alphabeta axons to the region normally occupied by alpha'beta' axons. This defect is pronounced for clones generated in larval stages but not clones generated after pupariation, indicating that dachshund function is particularly important around the time of puparium formation. In addition, mushroom body axons exhibit excessive branching in dachshund mutant clones. Thus, dachshund is required in mushroom body neurons for proper axon guidance and branching.

Mitochondrial localization and temporal expression of the Drosophila melanogaster DnaJ homologous tumor suppressor Tid50.

The Drosophila melanogaster tumor suppressor gene lethal(2)tumorous imaginal discs (tid) was identified as a homolog of all dnaJ-like genes known to date which have been well preserved in evolution. Homozygous D. melanogaster l(2)tid mutants l(2)tid1, l(2)tid2 and l(2)tid3 are characterized by neoplastic transformation of the adult integumental primordia, the imaginal discs, and the death at the time of puparium formation. The first part of this study is concerned with the identification and subcellular localization of the l(2)tid-encoded protein, Tid50. The second part examines its tissue specific expression during wild-type development and in tumorous imaginal discs. To specify the function(s) of the Tid50 protein polyclonal rabbit antibodies directed against various domains of it were generated and used for staining of Western blots and whole-mounts and paraffin sections of various tissues isolated from wild-type and mutant tumor-developing animals. To identify the mutational events leading in homozygous l(2)tid mutants to abnormal expression level of l(2)tid-encoded RNA and protein, the mutant gene was isolated from homozygous l(2)tid1 and l(2)tid2 animals and sequenced.

4 センチニクバエに見られる囲蛹殻形成時間の地理的変異

4 センチニクバエに見られる囲蛹殻形成時間の地理的変異

Changes of acid phosphatase content and activity in the fat body and the hemolymph of the flesh flyNeobellieria (sarcophaga) bullata during metamorphosis

Changes in the specific and total activity of the lysosomal marker enzyme acid phosphatase (Acph) and in the amount of enzyme protein were examined in the fat body and the hemolymph from the last larval molt to the larval-pupal apolysis. The specific activity showed minor changes during the last larval period. In contrast, the total activity of the enzyme was low during the feeding period and higher during the wandering stage and strikingly increased at the time of puparium formation. We purified a protein having para-nitrophenyl phosphate phosphatase (Acph) activity and raised antisera against it. The amount of Acph protein in the fat body and hemolymph was examined using an ELISA. The specific Acph content showed little variation, but the total amount of the enzyme protein showed a stepwise increase in both organs during last larval stage and was markedly elevated in the pupal stage in the fat body. In contrast, a considerable decrease in the amount of Acph protein was observed in the hemolymph during this period. These data were in agreement with immunohistochemical observations showing an accumulation of the enzyme protein in fat body cells during the prepupal stage with a concomitant disappearance of the enzyme from the hemolymph. Inhibition of ecdysteroid secretion by water stress prevented the changes both in total enzyme activity and in the amount of Acph protein. However, Acph protein content and enzyme activity could be restored when the water stress was followed by a 20-hydroxyecdysone (20-HE) treatment. Taken together, our data show that Acph is secreted by fat body cells into the hemolymph during the larval stage, where it is stored in an inactive form. Increase in the 20-HE titer at the end of last larval stage reverses this process, and the enzyme is taken up by the fat body cells, where it becomes activated and appears in auto- and heterophagic vacuoles. Arch. Insect Biochem. Physiol. 34:369–390, 1997. © 1997 Wiley-Liss, Inc.

Changes of acid phosphatase content and activity in the fat body and the hemolymph of the flesh fly Neobellieria sarcophaga bullata during metamorphosis

Changes in the specific and total activity of the lysosomal marker enzyme acid phosphatase (Acph) and in the amount of enzyme protein were examined in the fat body and the hemolymph from the last larval molt to the larval-pupal apolysis. The specific activity showed minor changes during the last larval period. In contrast, the total activity of the enzyme was low during the feeding period and higher during the wandering stage and strikingly increased at the time of puparium formation. We purified a protein having para-nitrophenyl phosphate phosphatase (Acph) activity and raised antisera against it. The amount of Acph protein in the fat body and hemolymph was examined using an ELISA. The specific Acph content showed little variation, but the total amount of the enzyme protein showed a stepwise increase in both organs during last larval stage and was markedly elevated in the pupal stage in the fat body. In contrast, a considerable decrease in the amount of Acph protein was observed in the hemolymph during this period. These data were in agreement with immunohistochemical observations showing an accumulation of the enzyme protein in fat body cells during the prepupal stage with a concomitant disappearance of the enzyme from the hemolymph. Inhibition of ecdysteroid secretion by water stress prevented the changes both in total enzyme activity and in the amount of Acph protein. However, Acph protein content and enzyme activity could be restored when the water stress was followed by a 20-hydroxyecdysone (20-HE) treatment. Taken together, our data show that Acph is secreted by fat body cells into the hemolymph during the larval stage, where it is stored in an inactive form. Increase in the 20-HE titer at the end of last larval stage reverses this process, and the enzyme is taken up by the fat body cells, where it becomes activated and appears in auto- and heterophagic vacuoles. Arch. Insect Biochem. Physiol. 34:369–390, 1997. © 1997 Wiley-Liss, Inc.

Temporal control of urate oxidase activity during development of the third‐instar larva of Drosophila: The role of 20‐hydroxyecdysone

AbstractThe tissue‐specific enzyme urate oxidase is confined exclusively to the Malpighian tubules of Drosophila melanogaster and expressed only in the third‐instar larva and the adult. Shortly before pupariation urate oxidase activity declines precipitously and is not detectable 24 hours later. That 20‐hydroxyecdysone is the factor that triggers the disappearance of urate oxidase activity in late third‐instar larvae is demonstrated using the temperature sensitive mutant ecd1 which at the nonpermissive temperature of 29°C fails to accumulate a sufficient concentration of 20‐hydroxyecdysone necessary for puparium formation and thus remains a third‐instar larva for 1 to 2 weeks before death. Both the life cycle and the temporal profile of urate oxidase activity in ecd1 larvae at 19°C is identical to that of the wild type. However, at 29°C ecd1 third‐instar larvae retain high urate oxidase activity. A precipitous decline in urate oxidase activity is observed when ecd1 larvae at 29°C are fed 20‐hydroxyecdysone. These data implicate 20‐hydroxyecdysone in the process that controls the rapid decline of urate oxidase activity at the time of puparium formation. In whole homogenates of Malpighian tubules, the urate oxidase polypeptide was identified in SDS‐polyacrylamide gels by its Rf with respect to homogeneously pure Drosophila urate oxidase and also by immunoprecipitation with rabbit anti‐Drosophila urate oxidase IgG. Throughout development the amount of the urate oxidase polypeptide is correlated with the magnitude of urate oxidase activity.

Developmental regulation of phenylalanine hydroxylase activity in Drosophila melanogaster.

Herein we demonstrate that Drosophila larvae possess a synthetic activity capable of converting phenylalanine to tyrosine. This system is readily extractable and displays many characteristics of phenylalanine hydroxylase systems described in other organisms, the most notable being that a tetrahydropteridine is required for full expression of activity. The level of phenylalanine hydroxylase activity present in the organism varies with the stage of development: from an undetected level of activity at the first larval instar, there is a rapid increase in phenylalanine hydroxylase activity which reaches a peak at the time of puparium formation, after which there is a rapid decrease again to an undetected level.

Glucosamine metabolism in Drosophila virilis salivary glands: Ontogenetic changes of enzyme activities and metabolite synthesis

In Drosophila virilis salivary glands the in vitro activities of enzymes involved in the glucosamine pathway were examined during the third larval instar and in the prepupa. While glutamine-fructose-6-phosphate aminotransferase (EC 5.3.1.19) becomes inactive at the time of puparium formation, glucosamine-6-phosphate isomerase (EC 5.3.1.10) and glucosamine-6-phosphate N-acetyltransferase (EC 2.3.1.3) show maximal activities in the prepupal gland. The activity of UDP- N-acetylglucosamine pyrophosphorylase (EC 2.7.7.23) may also decrease prior to puparium formation. Incubation of larval and prepupal glands in medium containing [ 3H]glucose + [ 14C]-uridine or [ 14C]glucosamine and subsequent separation of intermediates of the glucosamine pathway by chromatographic procedures reveal that the capacity of the glands to incorporate the isotopes into these intermediates decreases significantly at the time of puparium formation. The results suggest that in D. virilis salivary glands the formation of aminosugars is mainly controlled by the activities of the two enzymes glutamine-fructose-6-phosphate aminotransferase and UDP- N-acetylglucosamine pyrophosphorylase.

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

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.

Kinetic characteristics and ontogeny of glutamine-fructose-6-phosphate-aminotransferase in Drosophila salivary glands

The first step in the synthesis of mucoproteins in the salivary glands of Drosophila virilis is catalyzed by the enzyme glutamine-fructose-6-phosphate-aminotransferase (E.C.2. 6.1.16). In crude extracts from whole larvae the K m for fructose-6-phosphate is 0.30 mM, for glutamine 0.16 mM. The enzyme is inhibited by excess glutamine and is subject to feed-back control by UDP-N-acetyl-glucosamine. During the second half of the third larval instar most of the aminotransferase activity is found in the salivary glands; it is correlated with the synthesis of secretory granules. The enzyme is inactivated at the time of puparium formation. Correlations with changes in puffing activity are discussed.

Metabolism of 3β,14α-dihydroxy-5β-(3α- 3H)-cholest-7-en-6-one in Calliphora stygia

3β,14α-Dihydroxy-5β-cholest-7-en-6-one (1) is metabolized in Calliphora stygia at the time of puparium formation via the ketotriol (10) and other hydroxylated derivatives to α-ecdysone (6) and β-ecdysone (7). The ketodiol (1) is thus a likely precursor of the ecdysones in Calliphora.

Metabolism of some possible ecdysone precursors in Calliphora stygia

A study has been made, using Calliphora stygia at the time of puparium formation, of the incorporation of a number of labelled sterols into β-ecdysone. [1 α- 3H]-Cholesterol and [4- 14C]-cholesterol are incorporated to a similar extent (0·01-0·02%). [1- 3H]-7-Dehydrocholesterol is better incorporated (0·025%) than cholesterol while [1 α- 3H]-cholesterol sulphate, (22R)-22-hydroxy-[22- 3H]-cholesterol, and 25-hydroxy-[26- 14C]-cholesterol are not incorporated to a significant extent.

Ecdysone levels during metamorphosis of Drosophila melanogaster

The nature of the ecdysones present inDrosophila melanogaster during metamorphosis was investigated by the use of radioimmune assays, gas-liquid chromatography, and high-resolution liquid chromatography. β-Ecdysone was the only ecdysone detected in methanolic extracts of late larvae, prepupae, and early pupae. A small amount of α-ecdysone may be present in mid-third instar larvae. The amount of β-ecdysone found at the time of puparium formation is comparable to the amount required to induce morphogenesis of imaginal discs culturedin vitro. It is concluded that β-ecdysone is the molting hormone responsible for metamorphosis inDrosophila.

Ecdysone levels during metamorphosis of Drosophila melanogaster

The nature of the ecdysones present in Drosophila melanogaster during metamorphosis was investigated by the use of radioimmune assays, gas-liquid chromatography, and high-resolution liquid chromatography. β -Ecdysone was the only ecdysone detected in methanolic extracts of late larvae, prepupae, and early pupae. A small amount of α -ecdysone may be present in mid-third instar larvae. The amount of β -ecdysone found at the time of puparium formation is comparable to the amount required to induce morphogenesis of imaginal discs cultured in vitro . It is concluded that β -ecdysone is the molting hormone responsible for metamorphosis in Drosophila .

Ecdysone biosynthesis in the blowfly Calliphora stygia

3β,14α-Dihydroxy-5β-[3α-3H]cholest-7-en-6-one (1) is metabolised in Calliphora stygia at the time of puparium formation via hydroxylated derivatives to α-ecdysone (2) and β-ecdysone (3) and thus may be a precursor of ecdysones in this insect.

Developmental studies in Drosophila: VI. Ultrastructural analysis of the salivary glands of Drosophila pseudoobscura during the late larval period

Salivary gland cells of Drosophila pseudoobscura were examined electron microscopically and cytochemically during the late larval stage of development. The data indicate that several synthetic processes are occurring in the glands during this period. The first, a presumably digestive secretion, is produced throughout the gland in the earliest stages studied, and only in the most proximal cells with increasing time. Mucoprotein granules, which are secreted and utilized as a “glue” at the time of puparium formation, are produced in the most distal cells of the gland beginning at about 100–106 hours after hatching, and are eventually found throughout the medial region as well, but never in the proximal portion of the gland. Such granules are produced in the Golgi regions and have nonmucoprotein “caps” at their periphery. Several types of material are also found within the cisternae of the rough endoplasmic reticulum of the cells; the function and significance of these crystals, granules, and aggregations of granules is unknown. Lysosomal activity is present in the cells from the earliest stages, and increases with time.

Ontogeny and tissue distribution of alcohol dehydrogenase inDrosophila melanogaster.

Alcohol dehydrogenase (ADH) activity inDrosophila larvae and adults is localized primarily in fat body, intestine, and Malpighian tubules. In adult males, it occurs furthermore in derivatives of the genital disk.When expressed as ADH activity/whole organism, increasing values are found throughout the larval stages. Around the time of puparium formation, the activity decreases, to increase again around hatching of the adult.As the genital disk undergoes metamorphosis, it synthesizes (or activates) ADH rather than acquiring the enzyme from elsewhere in the organism.

A study of a melanic mutant of the blowfly Lucilia cuprina

The melanic mutant of the blowfly Lucilia cuprina (Wied.) is homozygous for three recessive characters, yellow eyes, rusty body, and a black puparium whereas the normal wild strain has red eyes, black body, and a chestnut-brown puparium. In the mutant the three pigment characters appear to be independent. The puparium of the mutant has a layer of melanin just inside the epicuticular layer. A study of the cuticular o-diphenoloxidases, the dopa-decarboxylases and the utilization of radioactive phenolic compounds shows the same process of sclerotization to occur in both strains at the time of puparium formation. The larval haemolymph of the mutant contains a prophenoloxidase which, when activated by an extract of the cuticle, has activity on tyrosine and dopa. This activity increases greatly as the larva approaches the time for puparium (and melanin) formation and is presumably responsible for the formation of melanin. In the mutant, sclerotization and melanization are independent processes. The same prophenoloxidase appears to be in the normal wild strain larval haemolymph but the necessary activator is not in the cuticle; the activator in the cuticle causes the pro-enzyme to become highly active only on substrates such as dopamine and 4-methylcatechol. Cystine is present in hydrolysates of puparia of both strains and, at least in the normal wild strain, accounts for all the sulphur present.

The fine structure of the male Drosophila genital disk during late larval and early pupal development.

The fine structure of male genital disks of Drosophila between the late second larval instar and early pupal stage is described. Two major developmental changes were observed. One, the incidence of rough endoplasmic reticulum increases around the time of puparium formation. Two, the incidence of microvilli increases around the time of the moult from the second to the third larval instar.