Pupal-adult Development Research Articles

Polydnavirus DNA of the braconid wasp Chelonus inanitus is integrated in the wasp's genome and excised only in later pupal and adult stages of the female.

Many endoparasitic wasps inject, along with the egg, polydnavirus into their insect hosts, the virus being a prerequisite for successful parasitoid development. The genome of polydnaviruses consists of multiple circular dsDNA molecules of variable size. We show for a 12 kbp segment of the braconid Chelonus inanitus (CiV12) that it is integrated into the wasp genome. This is the first direct demonstration of integration for a bracovirus. PCR data indicated that the integrated form of CiV12 was present in all male and female stages investigated while the excised circular virus DNA only appeared in females after a specific stage in pupal-adult development. The data also indicated that after excision of virus DNA the genomic DNA was rejoined. This has not yet been reported for any polydnavirus. Sequence analyses in the junction regions revealed the presence of an imperfect consensus sequence of 15 nucleotides in CiV12, in each terminus of the integrated virus DNA and in the rejoined genomic DNA. Within these repeats two sequence types (ATA, TAC) were observed in the various virus clones and in the clones encompassing the rejoined genomic DNA; they corresponded to the sequence type in the right and left junction, respectively. To explain this, we propose a model of virus DNA replication in which the genomic DNA is folded to juxtapose the direct repeat of the left with that of the right junction; recombination at specific sites would then yield the two types of virus and rejoined genomic DNA.

A comparative analysis of juvenile hormone metabolyzing enzymes in two species of Drosophila during development

The course of changes in the activities of enzymes degrading juvenile hormone (JH), epoxyde hydrolase (JHEH) and JH-esterase (JHE) was studied in two lines of Drosophila virilis (101 and 147) and in two lines of D. melanogaster (Canton-S and 921283). It was established for D. virilis that changes in the JH titre during pupal-adult development is determined by the activity level of JHE rather than JHEH, while in D. melanogaster developmental changes in JH titre are related to changes in the activity level of both JHE and JHEH. In adults of D. virilis, the high level of JH-hydrolysing activity is determined by JHE and in those of D. melanogaster by JHEH. Differences in the course of changes in the JHE activity level between adults of lines 101 and 147 of D. virilis were found, and also in the JHEH activity level between adults of lines Canton S and 921283 of D. melanogaster. It was shown that attainment of a definite JHE activity level in females of lines 101 and 147 agrees well with the onset of oviposition of fertilized eggs. The possible role of JHE in reproduction of D. virilis is discussed.

水浸漬されたセンチニクバエ成熟幼虫の蛹化および成虫化へのアラキドン酸量の役割

水浸漬されたセンチニクバエ成熟幼虫の蛹化および成虫化へのアラキドン酸量の役割

Influence of development and prothoracicotropic hormone on the ecdysteroids produced in vitro by the prothoracic glands of female gypsy moth ( Lymantria dispar) pupae and pharate adults

Fluctuations in hemolymph ecdysteroid titer are part of a complex mechanism that regulates pupal-adult development. The amount of ecdysteroid produced in vitro by prothoracic glands from female Lymantria dispar (L.) (Lepidoptera: Lymantriidae) pupae and pharate adults, as well as the competency of these glands to respond to a prothoracicotropic hormone (PTTH) stimulus in vitro, each correspond temporally with hemolymph ecdysteroid titers. Based on studies of gland kinetics and dose-responses to brain extract using prothoracic glands from different female pupal and pharate adult ages, an in vitro bioassay for the quantification of PTTH activity was developed using glands from day 2 females incubated without stimulus for 1 h followed by a 3 h incubation with stimulus. Only extracts of brains and corpora allata from pupae and pharate adults possess a PTTH factor. This factor is heat stable and can be separated on high performance size exclusion chromatography into two molecular sizes of 13.75 and 3.2 kDa. Ecdysone and 3-dehydroecdysone are produced in vitro by prothoracic glands from all ages of female L. dispar pupae and pharate adults tested. The amount of ecdysone produced by these glands exceeds that of 3-dehydroecdysone production after 4 h of incubation.

Sterol metabolism in the tobacco hornworm, Manduca sexta--a review.

A number of intermediates involved in the dealkylation and conversion of the major C28 and C29 phytosterols to cholesterol in insects were first isolated and identified in studies with the tobacco hornworm, Manduca sexta, carried out in our laboratory. We also investigated the effects of a variety of known sterol metabolism inhibitors in Manduca, particularly those affecting the delta 24-sterol reductase enzyme, and synthesized and tested a number of new inhibitors as well. In-depth studies of ecdysteroids in Manduca during embryogenesis and during pupal-adult development provided new information on molting hormone content, biosynthesis, and metabolism. In addition, this insect has been utilized in the study of three specific enzyme systems of ecdysteroid metabolism, namely 20-monooxygenase, 3-epimerase, and phosphotransferase, which are critical to activation and deactivation of molting hormones in insects.

Stage-dependent and Temperature-controlled Expression of the Gene Encoding the Precursor Protein of Diapause Hormone and Pheromone Biosynthesis Activating Neuropeptide in the Silkworm, Bombyx mori

Embryonic diapause and sex pheromone biosynthesis in the silkworm, Bombyx mori, are, respectively, induced by diapause hormone (DH) and pheromone biosynthesis activating neuropeptide (PBAN), which are produced in the subesophageal ganglion from a common polyprotein precursor (DH-PBAN precursor) encoded by a single gene (DH-PBAN gene). Using DH-PBAN cDNA as a probe, we quantitatively measured DH-PBAN mRNA content throughout embryonic and postembryonic development and observed the effects of incubation temperature, which is a key factor for determination of diapause, on DH-PBAN gene expression. The silkworm, which is programmed to lay diapause eggs by being incubated at 25 degrees C, showed peaks of DH-PBAN mRNA content at five different stages throughout the life cycle: at the late embryonic stage, at the middle of the fourth and the fifth larval instars, and at early and late stages of pupal-adult development. In the non-diapause type silkworms programmed by a 15 degrees C incubation, only the last peak of DH-PBAN mRNA in pupal-adult development was found, and the other peaks were absent. Furthermore, interruption of the incubation period at 25 degrees C by incubation at 15 degrees C decreased both DH-PBAN mRNA content in mature embryos and in subesophageal ganglia of day 3 pupae and the incidence of diapause eggs. Thus, there were two types of regulatory mechanisms for DH-PBAN gene expression. One is a temperature-controlled expression that is responsible for diapause induction, and the other is a temperature-independent, stage-dependent expression related to pheromone production.

Developmental changes in urea concentrations in the haemolymph of Daizo (T), an original strain of the silkworm, Bombyx mori reared on an artificial diet and on fresh mulberry leaves

Abstract Urea concentrations in the haemolymph of Daizo (T), an original strain of the silkworm, Bombyx mori , reared on an artificial diet and on fresh mulberry leaves were determined by a urease-indophenol method during the larval-pupal-adult development. Urea concentrations in the fourth and fifth instar larvae reared on an artificial diet (diet I) were between 0.10 and 0.15 mg urea N/ml haemolymph, and increased during the larval-pupal transformation to reach 0.33 mg/ml at the larval-pupal ecdysis. A further increase was observed during pupal-adult development and finally reached 0.48 mg/ml at day 7 pharate adult. In the fourth and fifth instar larvae reared on fresh mulberry leaves, the concentrations were low (0.05 mg/ml). From the larval-pupal ecdysis until day 8 pharate adult, further low urea concentrations (0.04 mg/ml) were observed. By starvation from 72 hr of the fifth instar larvae reared on another artificial diet (diet II), the elevation of urea concentrations (between 1.4- and 3.0-fold against the controls) was observed from just after starvation until day 1 spinning. From day 5 pupae, both the starved and the control insects showed a marked elevation of urea concentrations in the haemolymph, which was never observed on animals reared on diet I.

Polydnavirus of the parasitic wasp Chelonus inanitus (Braconidae): characterization, genome organization and time point of replication

Ultrastructural analysis of the polydnavirus of the braconid wasp Chelonus inanitus revealed that virions consist of one cylindrical nucleocapsid enveloped by a single unit membrane. Nucleocapsids have a constant diameter of 33.7 +/- 1.4 nm and a variable length of between 8 and 46 nm. Spreading of viral DNA showed that the genome consists of circular dsDNA molecules of variable sizes and measurement of the contour lengths indicated sizes of between 7 and 31 kbp. When virions were exposed to osmotic shock conditions to release the DNA, only one circular molecule was released per particle suggesting that the various DNA molecules are singly encapsidated in this bracovirus. The viral genome was seen to consist of at least 10 different segments and the aggregate genome size is in the order of 200 kbp. By partial digestion of viral DNA with HindIII or EcoRI in the presence of ethidium bromide and subsequent ligation with HindIII-cut pSP65 or EcoRI-cut pSP64 and transfection into Escherichia coli, libraries of 103 HindIII and 23 EcoRI clones were obtained. Southern blots revealed that complete and unrearranged segments were cloned with this approach, and restriction maps for five segments were obtained. Part of a 16.8 kbp segment was sequenced, found to be AT-rich (73%) and to contain six copies of a 17 bp repeated sequence. The development of the female reproductive tract in the course of pupal-adult development of the wasp was investigated and seen to be strictly correlated with the pigmentation pattern. By the use of a semiquantitative PCR, replication of viral DNA was observed to initiate at a specific stage of pupal-adult development.

Influence of interganglionic interactions on steroid-mediated dendritic reorganization and death of proleg motor neurons during metamorphosis in Manduca sexta.

In Manduca sexta, the larval abdominal prolegs and their muscles degenerate at pupation. The proleg motor neurons undergo a period of dendritic regression, after which a specific subset of them dies. The surviving motor neurons undergo dendritic outgrowth during pupal-adult development, and most die after adult emergence. All of these events are regulated hormonally by ecdysteroids and juvenile hormone, but interactions of the motor neurons with other cells may potentially contribute as well. To investigate the possible influence of interganglionic neural interactions, we chronically isolated individual abdominal ganglia by severing the adjacent rostral and caudal connectives in the larval stage. Subsequent metamorphic changes in proleg motor neurons were examined in the isolated ganglia and ganglia adjacent to the isolated ganglia. Two abnormalities were observed: (1) some imprecision in the timing of motor neuron death, both at pupation and after adult emergence, and (2) the growth of ectopic neurites outside the neuropil boundaries during pupal-adult development (in ganglia with or without neuromas caused by connective transections). Other aspects of proleg motor neuron metamorphosis, including the segment-specific death of motor neurons at pupation, were the same as that in intact and sham-operated insects. Thus, interganglionic interactions appear to play a relatively minor role in the steroid-mediated metamorphic transformation of proleg motor neurons.

Relationships between an Application of KK-42 and Pupal Development in the Silkworm, Bombyx mandarina.

Populations of Chinese origin of the silkworm, Bombyx mandarina, do not show arrested pupal stage, however populations of Japanese origin undergo pupal summer diapause. Hormonal control of this pupal summer diapause in Bombyx mandarina of Japanese origin was investigated using KK-42 treated pupae of Chinese origin. When an imidazole compound (KK-42:1-benzyl-5-[(E) 2, 6-dimethyl-1, 5-heptadienyl] imidazole) was injected into the newly ecdysed pupae of Chinese populations, the pupal-adult development was arrested for as long as 40 days. Resumption of pupal-adult development of the KK-42 treated pupae was induced by injection of 20-hydroxyecdysone (20-HE). Oxygen consumption of the KK-42 treated animals was very low comparing to the control but it increased gradually about 35 to 37 days after treatment. Abrupt increase of oxygen consumption was observed about 12hr after the application of 20-HE in the KK-42 treated animals. These results strongly indicate that the pupal summer diapause in Japanese populations of Bombyx mandarina is due to the low level of ecdysteroids in their body thus preventing them from proceeding to the pupal-adult development.

The Relationships between Pupal Development and Ecdysteroid Levels in Summer Diapausing Silkworm, Bombyx mandarina.

In the silkworm, Bombyx mandarina, duration of pupal-adult development was mainly divided into three different types among the Japanese population: the first one was 2 weeks duration, the second was about 3 weeks and in the third type more than 30 to 40 days were necessary for the adult development, although these were dependent on the rearing seasons. However, the average number of days required for adult development was 14 in the majority of the Chinese population. In the third type of pupal-adult development of Japanese population, the development of pupal stage was arrested. Oxygen consumption of the animals of which adult eclosion occurred at 34 days after pupation was very low during the pupal development. Ovarian development of these animals was also suppressed considerably. The fluctuation patterns of ecdysteroid titer in haemolymph of Japanese population were quite different from that of the Chinese population. Ecdysteroid titer was undetectable at 4 to 13 days after pupation in the Japanese population, but the titer increased by day-15 and reached a maximum level of 4.5 μg at day-20. The reinitiation of ovarian development was coincident to the increase of ecdysteroids in haemolymph of these animals. It was suggested that the delay of pupal-adult development may be due to the low levels of ecdysteroids in the Japanese population of Bombyx mandarina.

The Relationships between Oxygen Consumption and Duration of Pupal-Adult Development in the Silkworm Bombyx mandarina.

Duration of pupal stage of Bombyx mandarina is 2 to 4 times longer than that of Bombyx mori. We attempted to investigate whether this represents a state of diapause or not. Based on the pupal duration and oxygen consumption, developmental pattern of pupae in B. mandarina was mainly classified to three types, A, B and C, though these were dependent on the rearing seasons. In the spring season, typical type-A pupae had the shortest duration the same as in B. mori. Type-B pupae were intermediate of type-A and C. Pupae that had the longest duration were classified as type-C. Oxygen consumption decreased quickly soon after pupation and each type showed a typical U-shape respiration curve during pupal stage. Oxygen consumption in type-C animals was lowest of all 3 types. It reached extremely low levels of 5μl/hr/g, which went on for more than 2 weeks. When the type-A pupae were maintained in low temperature, pupal duration was elongated in proportion to the temperature decrease. However, in this experiment the respiratory activity curve in the lowest temperature of 20°C was clearly different from that of type-C pupae. In another experiment, type-A and B pupae showed increased respiration, proportionally to the temperature increase, while there was no increase in respiration of the C-type animals when subjected to the different temperatures. Finally, we can conclude that the low level of oxygen consumption in the C-type animals is characteristic to diapausing insects and this is in good accordance with the insensitivity of these animals to different temperatures. It was consequently determined that in B. mandarina there is a kind of facultative diapause in pupal stage.

Changes in the Titer of Bombyxin-Immunoreactive Material in Hemolymph during the Postembryonic Development of the Silkmoth Bombyx mori: (Bombyx moril/bombyxin/monoclonal antibody/radioimmunoassay/hemolymph).

Monoclonal antibodies were raised against pure, native bombyxin-II (bombyxin-II antibody) and against a synthetic nonapeptide corresponding to the amino-terminus of the C-peptide of the bombyxin precursor protein (C-peptide antibody). The bombyxin-II antibody recognized both bombyxin and probombyxin. A radioimmunoassay for bombyxin using the bombyxin-II antibody was developed, and developmental change in the titer of bombyxin immunoreactivity in the Bombyx hemolymph was investigated. The titer was low and almost constant during the fourth and early fifth instars. In the male, the titer rose abruptly 3 days after the beginning of wandering. One day after pupation it rose again steeply to reach the maximal level which lasted until the middle of the developing adult stage. The titer decreased thereafter and increased again at adult emergence. In the female, the pattern of titer fluctuation was similar to that in the male, but the female titers during pupal-adult development were 2-3 times higher than the male titers.

Proteolytic activity in the fat body during the pupal-adult metamorphosis of the silkworm,Bombyx mori

Proteolytic activity with an optimum pH of 4.0 was found in fat bodies of 5 to 6 day-old female pupae of the silkworm, Bombyx mori, using azocoll as a synthetic substrate. The activity was strongly inhibited by p-chloromercuric benzoate at 0.5 mM, although inhibitory effect of several kinds of compounds on proteolytic activity was examined. Developmental changes in the activity were associated with two peaks of proteolytic activity related to the larval-pupal and pupal-adult transformation, the activity in the fat body of females was clearly higher than that of males. The profile of the proteolytic activity after chromatography on a column of Sephacryl S-300 showed that, in the female fat body, the highest activity was in the fraction with a molecular weight of about 232,000 daltons while, in male fat body, the highest activity was in the fraction with a molecular weight of about 105,000 daltons. The storage proteins were digested when extracts from fat bodies, which contain storage proteins and proteases, were incubated and only when the pH of the extract was adjusted to 4.0. In the case of female fat body, storage protein 1 was degraded earlier than storage protein 2. Furthermore, the administration of 20-hydroxyecdysone (5 μg/animal) to the isolated abdomens of female pupae (dauer-pupae) induced the proteolytic activity in the fat body in parallel with ovarian development. These results suggest that the proteases in female and male fat bodies are responsible for the utilization of storage proteins during pupal-adult development.

Metamorphosis of the corpus allatum and degeneration of the prothoracic glands during the larval-pupal-adult transformation of Drosophila melanogaster: A cytophysiological analysis of the ring gland

The degeneration of the prothoracic glands of Drosophila melanogaster during pupal-adult metamorphosis was analyzed by light microscopy, scanning, and transmission electron microscopy. The ultrastructural observations were correlated with the ability of the ring gland to synthesize ecdysteroids in vitro. The ring gland is prominent during larval life and is identifiable until just before adult eclosion but undergoes dramatic changes in location, shape, size, ultrastructure, and function during pupal-adult development. Prothoracic gland degeneration is characterized by: a gradual decrease in its ability to synthesize ecdysteroids; a decreasing quantity of smooth endoplasmic reticulum (SER) and mitochondria; the absence of intercellular channels; cytoplasmic fragmentation; and the separation of the prothoracic gland from the corpus allatum and corpus cardiacum. An ultrastructural analysis of the corpus allatum during larval-pupal-adult metamorphosis and adult life was also correlated with function, i.e., juvenile hormone biosynthesis, using a radiochemical assay of ring glands and adult corpora allata in vitro. A relatively high concentration of SER, mitochondria, and mitochondrion-scalariform junction complexes are typical features of an active corpus allatum cell. The migration of the corpus allatum from the ring gland to its position as a separate gland in the adult fly was studied in detail. The capacity of the corpus allatum to synthesize juvenile hormone is at its peak in the ring gland of the early wandering third instar larva, whereas the corpus allatum of 2-day-old female adults displayed the greatest synthetic activity during adult life. The physiological significance of the alterations in gland activity is discussed.

Phosphorylation of endogenous proteins by a cyclic amp-dependent protein kinase in the wing epidermis of Manduca sexta

The wing epidermis of Manduca sexta has 8 prominent proteins of molecular weights ranging from 18,000 to 55,000, the in vitro phosphorylation of which is enhanced significantly by cAMP. The level of protein phosphorylation during pupal-adult development can be correlated with the changing hemolymph ecdysteroid titer. These protein substrates are not limited to the wing epidermis, being present in the pupal brain, fat body, prothoracic gland and gut, as well as larval integumented epidermis, muscle and the wing imaginal discs. Most of the phosphoproteins stimulated by cAMP were localized in the microsomal fraction of tissue homogenates. The 31/32 kDa doublet phosphoproteins were further localized to a ribosome enriched microsomal fraction and have properties similar to those of mammalian ribosomal protein S6.

Precursors of pattern specific ommatin in red wing scales of the polyphenic butterfly Araschnia levana L.: Haemolymph tryptophan and 3-hydroxykynurenine

In the seasonally diphenic butterfly Araschnia levana 14C-labelled tryptophan and 3-hydroxykynurenine, the principal precursors of ommochromes, injected into young pupae caused a pattern specific radiolabel of mature red scales. [ 14C]glucose and [ 35S]methionine also labelled red scales but only when injected shortly before or during the time of pigment synthesis in the wing. In developing non-diapause pupae contents of 3-hydroxykynurenine increased until an abrupt decrease when pigments appeared in the wings. In diapausing pupae 3-hydroxykynurenine remained low but increased after injection of 20-hydroxyecdysone which induced pupal-adult development. Supply of wing scale cells with ommochrome precursors via the haemolymph was analysed after injection of [ 3H]tryptophan. In developing pupae haemolymph contents of [ 3H]tryptophan and [ 3H]3-hydroxykynurenine increased at the time of wing pigment formation and decreased shortly before adult emergence. In diapausing pupae haemolymph contents of [ 3H]tryptophan and [ 3H]3-hydroxykynurenine were low compared to non-diapause pupae but increased at the time of wing pigment formation after injection of 20-hydroxyecdysone. Isolated wings incubated in Grace's medium containing [ 14C]tryptophan and [ 14C]3-hydroxykynurenine incorporated radiolabel specifically into red portions of the wing colour pattern due to labelling of ommatin. Incorporation into red wing areas occurred specifically depending on different colour patterns of the spring- and the summer-morph. The results demonstrate that both tryptophan as well as 3-hydroxykynurenine are delivered via the haemolymph, and both can serve as precursors of ommatin formation in the scale cells. Therefore, the complete set of enzymes for the tryptophan-ommatin pathway is present in scale-forming cells.

Influence of neem kernel extract on morphogenesis and vitellogenic oocyte development inTrogoderma granarium everts

Neem seed kernel extracts adversely affect the growth and morphogenesis of insects. InTrogoderma granarium, neem seed kernel extract was found to inhibit normal pupal-adult development. The malformed adults comprised a heterogenous group of transitory forms between the pupal and adult types. Older pupae were less sensitive to the compound. In the seemingly normal surviving adults ofTrogoderma granarium a reduction in the number of vitellogenic oocytes was found. The active compound(s) in the neem seed kernel extracts is proposed to produce the observed effects by regulating morphogenetic hormone titres, though the precise mode of action cannot be safely defined.

Identification of virus-specific polypeptides and translatable mRNAs in the isolated pupal abdomens of the silkworm, Bombyx mori, infected with nuclear polyhedrosis virus

Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that infection of the isolated pupal abdomens of the silkworm, Bombyx mori, with B. mori nuclear polyhedrosis virus (BmNPV) caused generation of a number of polypeptides with a concomitant decrease of cellular polypeptides. These generated polypeptides were not identified as viral structural polypeptides, but were characterized as the degradation products of cellular polypeptides, as evidenced by the reaction with specific antiserum against storage proteins 1 and 2. Immunoblot analysis using anti-BmNPV serum identified at least 14 virus-specific polypeptides in the BmNPV-infected isolated pupal abdomens and showed that 12 of these polypeptides represented authentic structural polypeptides of the virus. In vitro translation and subsequent immunoprecipitation with anti-BmNPV serum showed that translation yielded at least 15 polypeptides at the expense of cellular polypeptides. Time-course experiments showed that the viral structural polypeptides and virus-specific translatable mRNAs were not detectable until 3 days postinoculation. On the basis of the fact that the isolated pupal abdomens are in an arrested state of development, the delayed onset of virus-specific macromolecule production in the infected isolated pupal abdomens, as compared with that in the developing intact pupae, implies that some cellular function associated with the pupal-adult development is an important prerequisite for the efficient commencement of BmNPV replication.

Characterization of a calcium and diacylglycerol-activated and phospholipid-dependent protein kinase in the pupal brain of the tobacco hornworm, Manduca sexta: The particulate associated enzyme

A particulate associated, calcium and diacylglycerol-activated, phospholipid-dependent protein kinase (protein kinase C; PKC) has been demonstrated in the pupal brain of the tobacco hornworm, Manduca sexta. The particulate enzyme could be measured accurately only after its solubilization by non-ionic detergents, Triton X-100 being the most effective. The basic enzymatic properties of the particulate associated PKC are similar to those of the cytosolic brain enzyme. Maximal enzyme activity was obtained in the presence of 0.75 mM Ca 2+ and 200 μg/ml phosphatidylserine. Diacylglycerol (1,2 diolein; 50 μg/ml) further enhanced both PKC activity and Ca 2+ sensitivity in the presence of an optimal concentration of phosphatidylserine. Particulate PKC is inhibited by trifluoperazine in a dose-dependent manner with an IC 50 of approx. 30 μM. Subcellular distribution studies revealed that 86% of the pupal brain PKC activity is present in the soluble cellular fraction. A study of PKC activity during pupal-adult development revealed that the specific activity of day 9 pharate adult brain PKC was 6-fold higher than that observed for day 0 pupal brains. SDS-polyacrylamide gel electrophoresis and autoradiography demonstrated that a microsomal enriched 34 kDa peptide was phosphorylated by PKC in the presence of Ca 2+ and phosphatidylserine.