Differential hormone regulation of acid DNase activity in the testes and fat body of Spodoptera litura (Lepidoptera-Insecta).

Acid DNase activity in the testes and fat body is high during the early larval instars which may be correlated with the extensive cell division seen in both the tissues during these stages. The increased enzyme activity, observed in the testes of the pupal stage, might be involved in the in vivo degradation of DNA in a large number of degenerating spermatocysts which occur during this stage. Total activity of acid DNase in the fat body is highest in pupal stage. Like acid phosphatase, this enzyme may also be involved in the process of remodelling of the fat body during metamorphosis. 20-Hydroxyecdysone (20-HE) does not have any effect on acid DNase activity in the testes but it alters the enzyme activity in the fat body. Juvenile hormone-I (JH-I) has no effect on the enzyme activity in the fat body.     

Ecdysteroid mediated fat body acid phosphatase activity during larval development of rice moth, Corcyra cephalonica (Lepidoptera)

20-hydroxyecdysone (20-HE) stimulates acid phosphatase activity in the fat body of ligated late-last instar larvae. This effect is time dependent and the specific activity of enzyme increases significantly in hormone treated insects. 20-HE also stimulates general protein synthesis. Cycloheximide treatment either in conjunction with 20-HE or after hormone treatment blocks the increase in enzyme activity as well as increase in protein content. However, actinomycin D treatment does not alter the enzyme activity while it blocks the increase in total RNA as well as increase in protein content.     

Membrane-bound sorbitol 6-phosphatase in fat body cells controls the dynamics of sorbitol 6-phosphate, a major hemolymph sugar in the silkworm

Sorbitol 6-phosphate (S6P) is one of two major sugars (another is trehalose) in the larval hemolymph of Bombyx mori, and its amount dramatically decreases concomitantly with the onset of prepupal period. In the last (fifth) instar larvae, the amount of S6P is approximately 30 micromol/larva at its maximum and decreases to less than 1 micromol at the wandering stage. Incubation of fat bodies of wandering larvae with S6P generates sorbitol in the medium, while S6P in the medium decreases, indicating that fat body possesses sorbitol 6-phosphatase (S6Pase) activity. S6Pase activity in the fat body remains low during the feeding period, abruptly increases at the wandering and decreases to a low level after gut purge. 20-Hydroxyecdysone (20E) increases S6Pase activity in the fat body of feeding larvae, and the activation is dose-dependent. Cell fractionation studies show that S6Pase is mainly associated with the membrane and the optimal pH for membrane-bound S6Pase is 5.5, which is different from that for soluble acid phosphatase (pH 4). Present findings indicate that the S6Pase responsible for a decrease in hemolymph S6P is membrane-bound, and its activity is controlled by a rise of hemolymph ecdysteroid titer at the onset of the wandering stage.     

Bombyx acid cysteine protease (BCP): hormonal regulation of biosynthesis and accumulation in the ovary

We previously reported purification of the cysteine protease from Bombyx eggs (BCP) and the occurrence of the enzyme in various tissues of this insect. In the present paper, we present a detailed analysis of stage-specific changes in activity of BCP between the fourth larval instar and pupal-adult development. A synthetic fluorescent peptide, carbobenzoxy-L-Phenylalanyl-L-Arginine4-methylcoumaryl-7-amide (Z-Phe-Arg-MCA), was used to assay proteolytic activity. When tissue extracts were treated with anti-BCP serum before assay of enzyme activity, most activity towards Z-Phe-Arg-MCA was removed from the extracts. Therefore proteolytic activity in the present experiments is due mainly to BCP. We used Western blot and Northern blot analyses to determine tissue and stage specific expression of the enzyme. In the 5th larval fat body and hemolymph, BCP activity dramatically increased at the time of spinning, returning to the basal level before ecdysis. Northern blot analysis showed that a 1.5 kilobase mRNA which hybridizes to BCPcDNA suddenly appears during this period. Similar results were obtained in 4th instar fat body. In pupal hemolymph and fat body, low basal activity of BCP was detected early (day 0 to day 3 after pupal ecdysis), followed by a pronounced increase to a maximum six days after ecdysis, before returning to the basal level. In ovariectomized female pupae, a significant amount of proteolytic activity accumulated in hemolymph, suggesting that the enzyme is synthesized in the fat body and transferred into the ovary along with vitellogenin. BCP activity increased three days after injection of 20-hydroxyecdysone into ligated pupae. Furthermore, putative BCPmRNA appeared in the fat body within 24 hours after injection. This increase was completely blocked by the administration of cycloheximide. The results suggest that, BCP is synthesized in extraovarian tissues such as fat body and ovarian follicle cells and accumulates in the ovary, thus representing a new class of yolk protein.     

Site of synthesis,tissue distribution,and lipophorin transport of hydrocarbons in Blattella germanica (L.) nymphs

The site of hydrocarbon (HC) synthesis and the amount of HC in various tissues were investigated in relation to developmental stage in the last larval stadium of the German cockroach, Blattella germanica. Abdominal integument linearly incorporated [1-(14)C]propionate into HC for at least 6h in vitro, whereas other body parts synthesized little or no HC. The third through sixth abdominal sternites and tergites were the principal sites of synthesis. High rates of HC synthesis resulted in a fivefold increase in internal HC during the last stadium. We examined the distribution of HC in the hemolymph, fat body, and the developing imaginal cuticle. Hemolymph HC titer was relatively constant at approximately 8&mgr;g/&mgr;l. However, as hemolymph volume increased from 5 to 11&mgr;l in the first 4days of the last stadium, HC content increased and then remained stable the remainder of the stadium. Lipophorin, immunoprecipitated with adult lipophorin polyclonal antibodies, was the only HC carrier protein in nymphal hemolymph and its HC profile was identical to that of hemolymph and similar to that of the epicuticle. The concentration and total amount of hemolymph lipophorin increased until 3days before adult eclosion and declined immediately after ecdysis. The HC content of non-biosynthetic integument (legs, pronotum) doubled during formation of the imaginal cuticle, as did the HC content of sternites, which synthesize HC. HC content of fat body, however, increased threefold during the same period, suggesting that the fat body serves as a storage site for HC during cuticle formation. We conclude that in the last stadium HC is synthesized by abdominal oenocytes, loaded onto hemolymph lipophorin, and transported to fat body and both nymphal and imaginal cuticle. Hydrocarbons associate with the imaginal integument several days before eclosion.     

Hormonal regulation of macromolecular synthesis in testes and accessory reproductive glands of Spodoptera litura during post-embryonic and adult development

In S. litura testicular growth during the last larval instar and early pupal stage is associated with significant increase in DNA, RNA and protein contents. DNA synthesis is stimulated by 20-hydroxyecdysone (20-HE) in the penultimate instar testes. 20-HE injection in ligated late last instars increases the testicular weight and protein content. Accessory reproductive gland (ARG) development takes place during the mid and late pupal stages. Protein synthesis in the pharate adult ARG is stimulated by 20-HE. Juvenile hormone has no effect on ARG protein synthesis.     

Changes in the chemical composition of fat body during the last larval instar of Manduca sexta.

The fat body increases in weight throughout the last larval instar in spite of the loss in total body weight during the wandering and prepupal stages. The protein content of fat body increases dramatically and is greatly responsible for the increase in fat body weight in the wandering and prepupal stages. Lipids do not contribute significantly to the fat body weight gain except during the feeding stage. The amount of total fat body RNA increases until wandering then drops abruptly in the prepupal stage. The total amount of fat body DNA peaks before the onset of wandering and prepupal stages.     

The fate and possible role of arylphorin during the metamorphosis of Mamestra brassicae.

1. Arylphorin, one of the storage proteins has been isolated from the hemolymph of Mamestra brassicae. 2. It has been established that Mamestra arylphorin is the most similar to manducin from among the known storage proteins of other species. 3. A rabbit polyclonal antibody has been developed against arylphorin, and its concentration changes have been determined quantitatively by ELISA in the hemolymph and fat body from the 1st day of the last larval instar to the 3rd day of the imago stage. 4. Histological sections were made on each day during the investigated period and it was shown by immunohistochemical methods that the main quantity of arylphorin was accumulated in the storage protein granules of the fat body and it could be detected even in the imaginal fat body. 5. The uptake of arylphorin by the fat body is induced by 20-hydroxyecdysone. 6. During differentiation of the imaginal cuticle arylphorin is incorporated first in the epidermal cells and it is built in the endocuticular layer of the integument thereafter.     

Developmental and sex-dependent regulation of storage protein synthesis in the silkworm,Bombyx mori

The mechanism of sex-dependent expression of a major plasma protein, referred to as storage protein 1 (SP-1) was studied during development of the silkworm, Bombyx mori. SP-1 occurred in the hemolymph of the female as well as in the male larvae until the end of the fourth larval instar. In the last instar larvae, the amount of SP-1 in the hemolymph greatly increased in females, but markedly declined in males. The level of fat body mRNA for SP-1 reflected the developmental and sex-dependent changes in the hemolymph concentration of SP-1. The developmental patterns of hemolymph proteins in the third and the fourth instar larvae of sex-mosaic individuals were quite analogous to those observed in normal larvae at the same developmental stages. The hemolymph concentration of SP-1 at the last larval instar of the sex mosaics varied among individuals irrespective of the gonad compositions. In vitro culture of the fat body cells dissected from several locations of a sex-mosaic larva provided evidence that each fat body cell in a common hemolymph milieu synthesizes a high (female type) or a low (male type) level of SP-1 depending on the sex chromosome composition. The amount of vitellogenin in the hemolymph of the sex-mosaic pupae was in proportion to that of SP-1 at the last larval instar. From these results, it is suggested that the sex-dependent expression of SP-1 and vitellogenin in B. mori is genetically determined and developmentally regulated without participation of the reproductive organs or any sex-specific humoral factors.     

Age-dependent changes of fat body stores and the regulation of fat body lipid synthesis and mobilisation by adipokinetic hormone in the last larval instar of the cricket, Gryllus bimaculatus

Data on the hormonal regulation of the formation and mobilisation of fat body stores are presented and discussed in relation to general parameters of last instar larval development such as growth, food intake, and moulting. Crickets feed voraciously during the first half of the last larval stage. With the onset of feeding, fat body lipid synthesis increases, leading to increasing lipid stores in the fat body with a maximum reached on day 5. Lipid (42% of fat body fresh mass) is the main constituent of the fat body stores, followed by protein (6%) and glycogen (2%). During the second half of the last larval stage, feeding activity dramatically decreases, the glycogen reserves are depleted but lipid and protein reserves in the fat body remain at a high level except for the last day of the last larval stage when lipid and protein in the fat body are also largely depleted. The process of moulting consumes almost three quarters of the caloric equivalents that were acquired during the last larval stage. Adipokinetic hormone (AKH) inhibits effectively the synthesis of lipids in the larval fat body. Furthermore, AKH stimulates lipid mobilisation by activating fat body triacylglycerol lipase (TGL) in last larval and adult crickets. Both effects of AKH are weaker in larvae than in adults. This is the first report on the age-dependent basal activity of TGL in larval and adult insects. In addition, for the first time, an activation of TGL by AKH in a larval insect is shown.     

Molecular cloning of silkworm paralytic peptide and its developmental regulation

The silkworm paralytic peptide (PP) is a member of the ENF peptide family that exerts multiple biological activities involved in defense reaction and growth regulation. We isolated its cDNA and examined mRNA expression profiles. cDNA encoded 131 amino acids from which the 23-residue PP sequence was found at the C-terminal portion. Immunoblot analysis and paralytic activity assay indicated that inactive pro-protein in larval hemolymph was processed into active peptide immediately after bleeding. In the last larval instar, 0.6-kb PP mRNA was expressed in various tissues, of which the fat body was predominant. Its expression in the fat body decreased during the feeding period and then increased during metamorphic process. Juvenile hormone and 20-hydroxyecdysone upregulated its expression. At the embryonic stage, 1.5-kb mRNA, in addition to 0.6-kb mRNA, was expressed from 1 day after oviposition to hatching. PP was thus expressed stage-specifically under hormonal control.     

Venom of ectoparasitoid,Euplectrus sp near plathypenae (Hymenoptera: Eulophidae) regulates the physiological state of Pseudaletia separata (Lepidoptera: Noctuidae) host as a food resource

Euplectrus sp. near plathypenae is an ectoparasitoid that can parasitize from 3rd to day 0-6th instar Pseudaletia separata. The developmental period of the parasitoid from the egg to the pupal stage is about 13 days. Parasitized hosts are developmentally arrested and never molt to the next stadium. The injection of venom fluid results in similar effects on P. separata larvae as does parasitization. The inhibitory effect of the venom on molting was dose dependent. Injection of 0.3 female equivalents of venom into day 0-5th host instar resulted in a similar developmental arrest as seen in parasitized hosts. The amount of total lipid in the hemolymph of the host increased as a function of the amount of venom injected, while the lipid content of the fat body was similar to lipid levels in the fat body of parasitized larvae. The amount of total protein in the hemolymph also increased when venom was injected, whereas the protein level of the fat body did not increase. The lipid concentration within the parasitoid larva was maintained at the same level throughout larval development, but increased before pupation. We conclude that the injected venom increased the hemolymph content of lipid and protein to support the growth and development of the ectoparasitoid larva.     

Changes in the titer of the female-predominant storage protein (81K) during larval and pupal development of the waxmoth,galleria mellonella

The levels of an 81K storage protein in the waxmoth, Galleria mellonella, were monitored during the course of development using rocket immunoelectrophoresis. During the fifth and sixth larval stadia, 81K protein levels increased during feeding and growth but sharply declined at each larval molt. During the fifth and sixth stadia hemolymph levels of the 81K protein increased to about 1 and 2.5 mg/ml, respectively, with no discernible differences between levels in males and females. Neither the fat body nor the remainder of the carcass contained the 81K protein, indicating that the accumulation of this protein during the intermolt period was exclusively in the hemolymph and redistribution of the 81K protein into other tissues does not occur at the final two larval molts. During the seventh (final) larval stadium the absolute quantities of the 81K protein increased from 23 μg per insect to over 1,600 μg in females and to 300 μg in males. The hemolymph concentration of the 81K protein reached 28 mg/ml in females and 6 mg/ml in males with only low levels found in the remaining tissues. Shortly after pupal apolysis, marked by eyespot retraction, the fat body in both sexes rapidly and quantitatively sequestered the 81K protein from the hemolymph. The 81K protein in the hemolymph of both males and females rapidly dropped to nearly zero concentration by pupation. The 81K storage protein remained localized in the fat body cells after uptake occurred, even though the fat body cells disaggregate and reaggregate during metamorphosis. During pharate adult development the 81K storage protein disappeared from the fat body without entering the hemolymph. At adult eclosion 81K was virtually absent from the tissues of both males and females.     

Clathrin‐dependent endocytosis predominantly mediates protein absorption by fat body from the hemolymph in Bombyx mori

During insect larval–pupal metamorphosis, proteins in the hemolymph are absorbed by the fat body for the maintenance of intracellular homeostasis; however, the type of proteins and how these proteins are internalized into the fat body are unclear. In Bombyx mori, the developmental profiles of total proteins in the hemolymph and fat body showed that hemolymph‐decreased protein bands (55–100 kDa) were in accordance with those protein bands that increased in the fat body. Inhibition of clathrin‐dependent endocytosis predominantly blocked the transportation of 55–100 kDa proteins from the hemolymph into the fat body, which was further verified by RNA interference treatment of Bmclathrin. Six hexamerins were shown to comprise ～90% of the total identified proteins in both the hemolymph and fat body by mass spectrum (MS) analysis. In addition, hemolymph‐specific proteins were mainly involved in material transportation, while fat body‐specific proteins particularly participated in metabolism. In this paper, four hexamerins were found for the first time, and potential proteins absorbed by the fat body from the hemolymph through clathrin‐dependent endocytosis were identified. This study sheds light on the protein absorption mechanism during insect metamorphosis.     

Increase in acid phosphatase activity in the fat body during larval and pharate pupal development in Calliphora erythrocephala

Biochemical evidence was obtained for an increase in acid phosphatase activity in the larval fat body of Calliphora erythrocephala during larval and pharate pupal instars. This observation is in conflict with published data indicating a decreasing enzyme activity in late third stage larvae. Centrifugation and filtration studies showed that the pH of the homogenisation medium has a strong influence on the solubilisation of acid phosphatase and its distribution in homogenate components. Differences in biochemical techniques including the pH value may explain the discrepancy between the published results and the present findings.The observed increase in acid phosphatase activity is related to the activity of the lysosomal system in the period immediately preceding pupal-adult apolysis.     

Different pathways of DNA synthesis are operative in the fat body of Spodoptera litura (insecta) during larval and pupal development

In Spodoptera litura, the DNA content of the fat body increases during the last larval instar. Radiolabelling studies reveal high degree of H3-thymidine incorporation in the fat cell DNA, during last larval stage of development. At prepupal stage the DNA synthesised during larval development is degraded, at least partially and the resultant nucleotides are most likely used as building blocks for the fat cell DNA synthesis during pupal development and pupal-adult metamorphosis, because very little H3 -thymidine injected into pupae is incorporated in DNA, though in fact, significant amount of DNA is synthesised during this period.     

Correlation of hemocyte counts with different developmental parameters during the last larval instar of the tobacco hornworm, Manduca sexta

We determined the changes in hemocyte titer and in the abundance of hemocyte types of the tobacco hornworm Manduca sexta during the fourth and fifth larval stadium and the beginning of the pupal stadium. As we analyzed the samples of individual insects at daily intervals, we were able to correlate phenotypical features, body weight, as well as total protein content and lysozyme activity in the hemolymph with the observations on hemocytes. In the course of the fifth larval stadium, the hemocyte titer decreased slightly and declined further after pupation. Using calculated values for total hemocyte numbers, females had about five times and males three times more hemocytes in the circulating population at the beginning of the wandering stage (in the middle of the fifth larval stadium) than immediately after the last larval--larval molt (from the fourth to the fifth larval stadium). This sexual difference was mainly due to an increase in the number of plasmatocytes, which was more prominent in females than in males. Granular cells were dominant in early fifth larval stadium while plasmatocytes were the most abundant cells in pupae. Oenocytoids and spherule cells disappeared during the wandering stage. Lysozyme activity in the hemolymph rose to a maximum during the wandering stage, with females having lysozyme values twice as high as those for males. These changes in lysozyme activity, however, did not correlate with the increase of total hemolymph protein titer which occurred already at the beginning of the wandering stage. We postulate that changes in hemocyte titers are under direct hormonal control, which has to be proven in future experiments.     

Purification and characterization of epidermis-origin hemolymph protein in Galleria mellonella

Epidermis-origin hemolymph protein (EOHP) was identified and purified from the last instar larval hemolymph of Galleria mellonella by anion exchange chromatography, chromatofocusing chromatography, and Sephadex G-100. The EOHP has a native molecular mass of 47 kDa and is composed of one subunit. The isoelectric point of the EOHP was determined to be 5.3. The amino acid composition of the EOHP was rich in aspartic acid, glutamic acid and lysine, but poor in tyrosine, methionine, and tryptophan. EOHP is present in hemolymph over the period from the 4th instar larvae to the adult stage examined. Concentration of EOHP is high during the larval stage but gradually decreased during the developmental stage from pupal to adult stage. EOHP is present in the cuticle, fat bodies and trachea but not in hemocytes, fore gut, mid gut and hind gut.     

Effects of the steroid hormone, 20-hydroxyecdysone, on the growth of neurites by identified insect motoneurons in vitro.

During metamorphosis in the hawkmoth, Manduca sexta, identified larval leg motoneurons survive the degeneration of their larval targets to innervate new muscles of the adult legs. The dendrites and axon terminals of these motoneurons regress at the end of the larval stage and then regrow during adult development. Previous studies have implicated the insect steroid, 20-hydroxyecdysone (20-HE), in similar examples of dendritic reorganization during metamorphosis. The present studies were undertaken to test whether 20-HE acts directly on the leg motoneurons to regulate dendritic growth. Larval leg motoneurons were labeled with a fluorescent dye to permit their identification in culture following the dissociation of thoracic ganglia at later stages of development. Leg motoneurons isolated from early pupal stage animals (just before the normal onset of dendritic regrowth) survived in vitro and grew processes regardless of whether 20-HE was added to the culture medium. The extent of process outgrowth, however, as measured by the total length of all processes and the number of branches, was significantly greater for motoneurons maintained in the presence of 20-HE. The enhancement could be blocked by the addition of a juvenile hormone analog. By contrast, larval leg motoneurons that were isolated just before the normal period of dendritic regression did not show enhanced growth of neurites in the presence of 20-HE. The results suggest that 20-HE acts directly on the leg motoneurons to regulate the growth of processes during metamorphosis.