Role of juvenile hormone in the synthesis and sequestration of vitellogenins in the red cotton stainer, Dysdercus koenigii (Heteroptera: Pyrrhocoridae)

Investigations were carried out to determine the role of juvenile hormone (JH) and 20-hydroxy ecdysone in the synthesis and uptake of vitellogenins, which were earlier identified, purified and characterised, in Dysdercus koenigii. The concentration(s) of vitellogenin(s) in fat body, haemolymph and that of vitellin(s) in ovary were significantly lower after chemical allatectomy at eclosion. In addition, at 70 h after emergence, chemical allatectomy reduced ovarian vitellin concentration, but vitellogenin levels remained normal in the fat body and haemolymph. The haemolymph vitellogenins were not incorporated into oocytes in such insects. Administration of JH-III at 20 h after allatectomy restored vitellogenin levels in the fat body and haemolymph, but the ovary failed to incorporate the available vitellogenins from haemolymph in such insects. However, when JH-III was administered twice, one at 20 h and then at 70 h after allatectomy, vitellogenin concentrations in fat body and haemolymph and also vitellin concentrations in ovary approached control levels. It is suggested that JH has two separate roles, one in vitellogenin synthesis and the other in uptake. 20-hydroxy ecdysone had no apparent role in either vitellogenin synthesis or uptake in D. koenigii.     

Effects of precocene II on female-specific hemolymph polypeptides in Oncopeltus fasciatus

Using polyacrylamide gel electrophoresis (PAGE) under denaturing conditions, two major polypeptides of 200,000 and 170,000 daltons were detected in the hemolymph of mature female Oncopeltus fasciatus, but they were not found in the hemolymph of males or newly emerged females. Those polypeptides constituted the two major bands of early vitellogenic oocytes; however, they were absent from the yolk of mature eggs. The slower-migrating band (200,000 daltons) appears to correspond to a vitellogenic protein already identified in O. fasciatus, whose synthesis has been suggested to be independent of juvenile hormone (JH). Treatment of newly emerged adult females with the corpus allatum cytotoxin precocene II prevented the appearance of the female-specific bands and induced an important accumulation of other proteins in the hemolymph. Yolk deposition was also inhibited in those animals. Topical application of JH to precocene-treated females restored the appearance of the 200,000 and 170,000 dalton polypeptides in the hemolymph. These results suggest that JH is required for the synthesis of female-specific polypeptides in O. fasciatus.     

Significance of haemolymph juvenile hormone titer changes in timing of migration and reproduction in adult Oncopeltus fasciatus

Previous studies had shown that both migratory flight and ovarian maturation in Oncopeltus fasciatus were stimulated by juvenile hormone (JH), yet the two behaviors were mutually exclusive. To understand the relationship of this hormone to these behaviors, haemolymph juvenile hormone titers were determined in both sexes of Oncopeltus throughout the adult stage by the Manduca pigmentation bioassay. Animals reared under long day conditions (17L:7D, 24°C) showed an immediate rise in haemolymph titers of JH after adult emergence whereas those reared in a short day photoperiod (12L:12D, 24°C) had a more gradual increase in hormone titers. Migratory flight behavior occurred during periods of intermediate hormone titers while oviposition did not begin until JH titers had reached their peak. It was concluded that lower JH titers normally stimulate flight in the prereproductive adult whereas higher titers are required for complete ovarian development. The corpus allatum in Oncopeltus thus coordinates migration and reproduction in response to the environmental cues of photoperiod, temperature, and food quality.     

The roles of juvenile hormone and 20-hydroxy-ecdysone during vitellogenesis in isolated abdomens of Drosophila melanogaster

Both juvenile hormone and 20-hydroxy-ecdysone seem to be involved in the regulation of vitellogenesis in Drosophila melanogaster. It is the purpose of this paper to begin to define the functions of these two hormones. Although vitellogenin synthesis does not occur at a high rate in 1-day-old female abdomens isolated from the head and thorax before 0.75 hr after eclosion, both ZR515 (a juvenile hormone analogue) and 20-hydroxy-ecdysone can cause in these preparations vitellogenin synthesis and secretion into the haemolymph. The synthesis and secretion into the haemolymph of all three vitellogenins which are detectable by electrophoresis in sodium dodecyl sulphate-containing gels of polyacrylamide is promoted by both hormones. That result excludes the hypothesis that these two hormones regulate the synthesis of different vitellogenins. A dose-response curve showed that an injection of 0.2 μl of a 10^?6 M 20-hydroxy-ecdysone solution was sufficient to promote vitellogenin synthesis and secretion in isolated abdomens. Ovaries from isolated female abdomens treated with juvenile hormone analogue showed nearly normal amounts of all three vitellogenins and morphologically normal advanced vitellogenic follicles, whereas ovaries from isolated abdomens treated with 20-hydroxy-ecdysone contained little vitellogenin and no vitellogenic follicles. We conclude that under the conditions used, juvenile hormone permits vitellogenin uptake into the oöcyte much more readily than does 20-hydroxy-ecdysone.     

Structure,haemolymph titre,and regulatory effects of juvenile homone during ovarian maturation in Leucophaea maderae

Juvenile hormone (JH) synthesized and secreted in vitro by the corpora allata of mated adult Leucophaea maderae females was determined to be JH III (methyl-10,11-epoxy-3,7,11-trimethyl-2,6-dodecadienoate).The haemolymph titre of JH was determined during maturation of the terminal oöcytes in the first reproductive cycle of L. maderae. In virgin females, JH is not detectable in the haemolymph during the first eight days following adult emergence; however, by 10 days after emergence, trace quantities of JH are apparent. Mating stimuli induce a dramatic increase in the concentration of haemolymph JH, with a peak occurring approximately 12 days after mating; thereafter, the JH concentration declines until it has reached an undetectable level 19 days after mating, at the time of chorion deposition.During ovarian maturation, changes in the rates of synthesis of vitellogenin by the fat body and DNA by the ovary correlate closely with the haemolymph titre of JH. However, no such correlation exists between the JH titre and the extensive ovarian protein synthesis that occurs in L. maderae coincident with chorion formation.The effects of JH I and JH III on both vitellogenin synthesis and ovarain DNA synthesis are statistically similar.     

Haemolymph vitellogenin,juvenile hormone,and oöcyte growth in the adult cockroach Nauphoeta cinerea during first pre-oviposition period

In the cockroach Nauphoeta cinerea the incorporation of a protein of low solubility into the oöcytes begins at day 5 of its adult life. An immunologically identical protein appears in the haemolymph two days earlier. The concentration of this protein, i.e. ‘vitellogenin’ in the haemolymph increases up to the onset of yolk incorporation into the oöcytes. During ovarian development no correlation could be detected between vitellogenin titre and several other parameters (ovary dry weight, length of the basal oöcytes, haemolymph protein concentration, body weight and age when ovulation occurred). In young females vitellogenin titre depends on the age, i.e. the volume of the corpora allata and hence on the presence and the titre of JH. During the period of egg maturation the total haemolymph protein concentration generally tends to drop while materials not precipitable by trichloracetic acid circulate at higher concentration after ecdysis and before ovulation.Early decapitation prevents vitellogenin synthesis and oöcyte growth, but when JH is applied to decapitated females, the normal vitellogenin titre is re-established, ovarian development, however, cannot be fully resumed. A dose-response curve shows that serial application of the hormone is much more effective than single large doses. Farnesylmethylester, a JH mimic, is about a hundred times less active, but more persistent than JH. Copulation seems to enhance the synthesis and release of endogenous JH, while food and water uptake are necessary to guarantee and optimal ovarian development. JH and high vitellogenin titre never restore ovarian development in females deprived of food and/or water or in those decapitated shortly after ecdysis.     

Synthesis of vitellogenins and diapause proteins by the fat body of Leptinotarsa, as a function of photoperiod

ABSTRACT. During maturation of adult female Leptinotarsa decemlineata , synthesis of specific proteins by the fat body was investigated with the aid of in vitro incubations. Both vitellogenins and diapause proteins were present in all females. Under long days high rates of vitellogenin synthesis were observed, which were not reflected in the amount of vitellogenin present in the haemolymph. Under short days synthesis of three diapause proteins predominated. The highest rates of synthesis of these proteins occurred during the second half of the prediapause period. At the same time storage of two diapause proteins by the fat body was observed. Comparison of the synthesis of specific proteins in vivo and in vitro revealed the fat body as the main producer of haemolymph proteins.     

Synthesis of 5 S and transfer RNA during embryogenesis in the milkweed bug, Oncopeltus fasciatus

Synthesis of 5 S RNA and transfer RNA has been shown to begin at the onset of gastrulation in eggs of the milkweed bug, Oncopeltus fasciatus. 5 S RNA synthesis parallels that of ribosomal RNA, but transfer RNA synthesis is somewhat different.     

The relationship between nutrition,vitellogenin, vitellin and ovarian development in the housefly, Musca domestica L.

The effect of adult nutrition on oögenesis during the first gonotropic cycle was studied in three strains of the housefly, Musca domestica. Two of the strains were anautogenous and the third was autogenous. In these strains, three subunits (51, 43 and 42 kdaltons) of vitellogenin and vitellin were electrophoretically identical using SDS-PAGE electrophoresis for haemolymph proteins of vitellogenic females and for egg extracts. Each developmental stage of the ovary in individual females flies of both autogenous and anautogenous strains fed on either sugar or protein clearly reflected the appearance of electrophoretic bands for vitellogenin and vitellin. Using immunological analysis, a very small amount of vitellogenin was detectable in the haemolymph of previtellogenic flies. The highest level of vitellogenin appeared in the haemolymph at the middle of vitellogenic phase and reached about 25% of the total haemolymph protein. There were differences in vitellogenin concentration in females with mature eggs between the two anautogenous strains: vitellogenin was not detectable in one strain, and the other showed 30% of the maximal level.     

Vitellogenins in the firebrat,Thermobia domestica (Packard): Immunological quantification during reproductive cycles and in relation to insemination (Thysanura: Lepismatidae)

An antiserum raised against the major fractions (240 + 300 Kd) of the vitellins extracted from maturing oöcytes of Thermobia domestica, allowed the immunological relationship between these proteins and their haemolymph precursors (vitellogenins) to be established, as well as the female specificity of these protein fractions. Using rocket immunoelectrophoresis, the vitellogenin concentration in the female haemolymph was measured on each day of a reproductive cycle. The changes in vitellogenin titre appear to be correlated with ovarian maturation: very low levels during the previtellogenic phase and increasing levels during the vitellogenic growth phase of the basal oöcytes. Also, these changes are synchronized with the moulting cycles which persist in the adults of these primitive insects. An insemination at the beginning of the interecdysial period causes a large increase in the vitellogenin titre synchronized with accelerated oöcyte growth.     

Vitellogenin accumulation in the fat body and haemolymph of Locusta migratoria in relation to egg maturation

Vitellogenins first appear in the fat body of Locusta migratoria during subphase I of vitellogenesis and increase to a constant level during subphase II. A second increase occurs shortly before the oöcyte attains maximal size. Vitellogenin content of fat body subsequently returns to that of subphase I, appropriate to the size of the subterminal oöcyte. The absolute amount of vitellogenin in the fat body is low compared to that found in the haemolymph. Fat body and haemolymph vitellogenins have immunological properties similar to oöcyte yolk proteins—when challenged with oöcyte protein antiserum. They exhibit similar electrophoretic mobility in polyacrylamide gel electrophoresis and are complex glyco-lipoproteins.     

Phase polymorphism in Schistocerca gregaria: Assessment of juvenile hormone synthesis in relation to vitellogenesis

Juvenile hormone (JH) synthesis by the corpora allata of gregarious and solitarious phase females of Schistocerca gregaria was determined in vitro during the penultimate and last stadia as well as during the first gonotrophic period of adults. Generally, the corpora allata of solitarious females showed higher rates of JH synthetic activity. In addition, in adult females there was a temporal difference between the corpora allata activities of gregarious and solitarious locusts, the latter exhibiting relatively higher rates of JH synthesis early in the first gonotrophic period. The corpus allatum volumes of solitarious females were also generally larger than those of their gregarious counterparts; there was no synchrony between fluctuations in JH synthetic activity and changes in corpus allatum volume in either phase.The early onset of relatively high JH synthetic rates in solitarious females was correlated with the early detection, by rocket immunoelectrophoresis, of vitellogenin in the haemolymph and vitellin in the oöcytes. Vitellogenin appeared in the haemolymph on day 4 in solitarious females and on day 6 in gregarious females and vitellin appeared in the oöcytes on days 6 and 8 respectively. Oöcyte length at which vitellogenesis was first detected was 1.8 mm for gregarious and 1.3 mm for solitarious females. However, despite the accelerated onset of both vitellogenin synthesis and uptake, oöcyte maturation time of solitarious females was longer. In both gregarious and solitarious females, vitellogenin titres increased until oöcytes reached a length of about 4 mm and declined thereafter. Vitellin content of ovaries increased proportionately to oöcyte growth until they attained a length of 5.0 mm. The subsequent increase in length of oöcytes to maturity is attributed to postvitellogenic growth, possibly by hydration.     

The permeability of the midgut of three insects to cardiac glycosides

The uptake of a polar and nonpolar cardiac glycoside by three insects, Oncopeltus fasciatus, Schistocerca gregaria and Periplaneta americana was investigated. Of these insects, the midgut of only O. fasciatus was found to be permeable to cardiac glycosides. Ouabain was not metabolized by this insect and crossed the midgut slowly and passively. It was sequestered from the haemolymph into the dorsolateral spaces against a concentration gradient and at relatively fast rates suggesting that uptake from the gut is rate limiting. Digitoxin was metabolized at the level of the midgut but not in the isolated haemolymph or dorsolateral space fluids. Twenty-four hours after feeding O. fasciatus labelled digioxin, digitoxin metabolites but no unchanged digitoxin could be detected in the haemolymph while both metabolites and a small amount of unchanged digitoxin could be detected in the dorsolateral space fluids.     

Effect of JH and PII on the protein content of haemolymph and ovaries of Spilostethus pandurus

• 1.1. An SDS-PAGE study of the qualitative and quantitative differences in protein bands from haemolymph and ovaries of Spilostethus pandurus females treated with JH or chemically allatectomized with precocene II, has been done.
• 2.2. The SDS-PAGE study of haemolymph revealed the occurrence of three female-specific proteins. In the ovary appeared three protein fractions (A, Band C) with mol. wts similar to those from haemolymph.
• 3.3. The three female-specific proteins from the haemolymph, and the ovary bands B,C and D were absent in the samples from PII-treated females.
• 4.4. JH accelerates ovary growth and the relative amounts of bands B, C and D were in relation to the physiological stage of the considered ovaries.

     

Influence of a juvenile hormone analogue on vitellogenin synthesis and oögenesis in larvae of Nauphoeta cinerea

In experiments on the synthesis of the vitellogenic protein, farnesylmethylester, a juvenile hormone (JH) analogue, was injected into female Nauphoeta cinerea larvae at various stages during their development. Two and 4 days after injection, 2 μl of haemolymph were assayed in a vitellogenin immunodiffusion test. In second last and last instar larvae less than 6 days before adult ecdysis, high doses (100 μg) of farnesylmethylester are necessary to induce vitellogenin synthesis, whereas older last stage larvae and decapitated adults respond to small doses (1 μg) with the synthesis of vitellogenin. It seems that the competence to synthesize the vitellogenic protein changes at the time of induction of the moulting process. If farnesylmethylester is injected into last instar larvae with a supposedly high titre of ecdysone, the vitellogenic protein can be detected in the haemolymph of a small percentage of animals only.Oöcyte maturation can be observed in last instar larvae injected after the fifth to ninth day with farnesylmethylester. The observed volume changes of the corpora allata suggest that an absence of JH for a short time is necessary for the oöcytes to become competent to grow. Last instar larvae treated with farnesylmethylester become larval-adult intermediates with partly developed oöcytes, demonstrating a simultaneous juvenilizing and gonadotropic influence of the JH analogue. In last instar larvae injected with farnesylmethylester a partial degeneration of already maturing oöcytes is induced at the time when the ecdysone titre is supposedly high and the possible reasons for this are discussed.     

Inhibition of vitellogenin synthesis in Apis mellifera workers by a juvenile hormone analogue,pyriproxyfen

Insect juvenile hormone (JH) has been related to modulation of vitellogenin (Vg) synthesis, a protein produced by fat body cells, secreted in haemolymph and sequestered by developing oocytes. A stimulatory JH action has been described for the majority of species studied thus far. In some insects, however, Vg synthesis has been inhibited or unaffected by JH. The aim of this study was to re-examine the action of JH on Vg synthesis in Apis mellifera workers, since contrasting effects of this hormone were described. Newly emerged worker bees were treated with different doses of pyriproxyfen (PPN), a potent JH analogue. Vg and total protein were quantified in haemolymph samples of newly emerged up to 6-day-old worker bees. Protein synthesis activity of fat body cultured in vitro and ultrastructure of fat body cells were also examined. High doses (1.25, 2.5, 5 and 10 &mgr;g) of PPN inhibited the onset and accumulation of Vg in the haemolymph of young worker bees in a dose-dependent fashion. This inhibition was not a result of fat body cell degeneration or death, as illustrated by fat body cells ultrastructure analysis, but by impairing Vg synthesis, as demonstrated by in vitro culture of fat body cells. Low doses (0.001, 0.01 and 0.1 &mgr;g) neither affected the normal synthesis and secretion of Vg into the haemolymph nor caused an early onset of Vg in treated bees (which could be interpreted as a JH-activating effect), as shown by Vg quantification at 24-h intervals. The results suggest that a low JH titre in honey bee workers permits the onset and accumulation of Vg in haemolymph, whereas high JH levels turn off Vg synthesis.     

Temporal variation of vitellogenin synthesis in Ectatomma tuberculatum (Formicidae: Ectatomminae) workers

Workers of the ant species Ectatomma tuberculatum (Ectatomminae) have active ovaries and lay eggs that are eaten by the queen and larvae (trophic eggs). Vitellogenins are the main proteins found in the eggs of insects and are a source of nutrients. The aim of this study was to characterize the period of vitellogenin production in workers of E. tuberculatum. The vitellogenin was identified from queen and worker eggs by SDS-PAGE. Anti-vitellogenin antibodies were obtained and used to detect this protein in the fat body and haemolymph of workers at different ages. Vitellogenin from E. tuberculatum consists of two polypeptides of 31 and 156 kDa. In the eggs of queens, the 156 kDa polypeptide is cleaved into two subunits of 36 and 123 kDa. The analysis of the haemolymph of workers showed that the secretion of vitellogenin varies with age. The secretion is initiated around the fifth day after emergence, with peak production from days 20 to 60, and stops around day 100. The variation in production is related to the different activities performed by the workers within the colony, suggesting that vitellogenin may have an important role in maintaining age polyethism.     

Diapause phenomena in non-diapausing last instar larvae,pupae, and pharate adults of the Colorado beetle

From the first day of the last (fourth) larval instar no trace of juvenile hormone (JH) can be detected in the haemolymph by Galleria bioassay. Three specific diapause proteins, which are also found in diapausing adults, appear in the haemolymph. These proteins disappear towards the end of the pupal stage. Study of the ultrastructure of the fat body revealed the formation from lysosomes of proteinaceous bodies which are also characteristic for adult diapause. The behaviour of last instar larvae and pupae resembles that of prediapausing and diapausing adults respectively. Injection of synthetic JH delays the appearance of the diapause proteins in the haemolymph and of proteinaceous bodies in the fat body for 2 to 3 days. The absence of JH seems to trigger off these diapause phenomena.     

Proteins of the fat body of non-diapausing and diapausing larvae of the southwestern corn borer, Diatraea grandiosella: Effect of juvenile hormone

The proteins of the fat body of non-diapausing, pre-diapausing, and newly-diapaused larvae of the southwestern corn borer, Diatraea grandiosella, were examined. Since a low titre of juvenile hormone (JH) is present in the haemolymph throughout the final instar of non-diapausing larvae, the hormone does not appear to stimulate the pre-metamorphic synthesis of proteins. In contrast, the high titre of JH in the haemolymph during the final instar of pre-diapausing larvae appears to stimulate the synthesis of selected proteins. For example, pre-diapausing larvae store in their fat body a low molecular weight protein which has been named the ‘diapause-associated protein’. When non-diapausing larvae were treated topically with C₁₇-JH or a JH mimic, from 50 to 70% entered a diapause-like state as fully grown larvae. These hormone-treated larvae accumulated the diapause-associated protein and a high molecular weight protein in their fat bodies. Both of these proteins were shown to be released from the fat body of newly-diapaused larvae in vitro, and may function in the haemolymph during diapause. The high molecular weight protein, isolated from the haemolymph, was shown to contain neutral and polar lipids, including biochromes. Its storage in the fat body and release into the haemolymph may be essential for the transport of lipids during diapause. The fat body proteins of newly-diapaused larvae of the southern cornstalk borer, Diatraea crambidiodes, were also examined electrophoretically. They were found to contain a similar protein pattern to that of D. grandiosella, including the presence of a diapause-associated protein.     

Vitellogenesis inhibition in Oncopeltus fasciatus females (Heteroptera: Lygaeidae) exposed to cadmium

Newly moulted females of the insect Oncopeltus fasciatus were exposed to cadmium (Cd) dissolved in the drinking water (50-400 mg l(-1) Cd) for 5 days. Cd exposure delayed ovarian maturation and inhibited egg production. Exposure to Cd, moreover, decreased hemolymph levels of the two major vitellogenin polypeptides of O. fasciatus, VG1 and VG2, in a concentration-dependent way, probably by a reduction in their synthesis. The ovarian levels of VG1 and VG2 were also decreased in Cd-exposed females. It was next investigated whether Cd effects might be a consequence of the endocrine disruption of vitellogenin synthesis, which is controlled by juvenile hormone (JH). JH replacement therapy did not restore VG1 or VG2 levels in Cd-exposed females, but did so in starved females. Our results do not therefore support a disturbance of JH production or a reduction in feeding as the cause of the reduced vitellogenin polypeptide levels, but rather point to the site of action of JH, the JH receptor, as the target of Cd effects.