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.     

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.     

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.     

Vitellogenin fluctuations in haemolymph and fat body and dynamics of uptake into oöcytes during the reproductive cycle of Diploptera punctata

Haemolymph and fat body soluble protein titres have been examined during the reproductive cycle of Diploptera punctata, with particular emphasis on the occurrence of vitellogenin and its uptake into the developing oöcytes. Vitellogenin was first detected in the haemolymph of mated females 2 days after adult eclosion at about the same time that vitellin deposition in basal oöcytes began. Peak haemolymph titres of vitellogenin occurred on day 6, correlated with the completion of yolk uptake. Thereafter vitellogenin levels declined and were generally undetectable throughout most of gestation, rising again shortly before parturition in association with the second gonotrophic cycle. Total haemolymph protein levels were not correlated with vitellogenesis.Soluble fat body vitellogenin titres of mated females remained low during the first oöcyte growth period but then rose several-fold at its completion and remained high throughout pregnancy and the second gonotrophic cycle. Total fat body soluble proteins decline after adult eclosion in association with oöcyte growth.Vitellin accumulation in basal oöcytes was related linearly to increase in volume until the onset of chorion formation. Thus no post-vitellogenic growth period was detected.     

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.     

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.     

Protein synthesis and secretion by the locust fat body after ovariectomy

The synthesis and secretion of proteins, including vitellogenin, by the locust fat body were investigated in vivo and in vitro at various times after ovariectomy. The rate of overall protein synthesis and secretion by the fat body in vivo is significantly less in ovariectomized animals than in the controls two to three weeks after the operation, the difference in the rates of secretion of vitellin antibody-precipitable protein being even more pronounced and detectable earlier. The significantly greater amount of newly synthesized vitellogenin retained in the fat body of ovariectomized animals is insufficient to account for this difference. The secretion of newly synthesized protein by the fat bodies of ovariectomized locusts in vitro is significantly less than that of control fat bodies, the difference being particularly marked in the case of vitellogenin. The polysome populations of fat bodies of ovariectomized and control females are quantitatively similar and the amounts of total protein and vitellin antibody-precipitable protein synthesized by these polysomes in a cell-free system do not differ significantly.     

Synthesis and release of proteins from cultured larval fat body of the southwestern corn borer,Diatraea grandiosella

The larval fat body of the southwestern corn borer, Diatraea grandiosella, was cultured in vitro to examine the relationship between proteins present in the fat body, those released into the medium, and those present in the haemolymph. While the incorporation of [³H]leucine into fat body proteins was high in last instar pre-diapausing and non-diapausing larvae, it fell in early diapausing larvae to about 11% of that found in prediapausing larvae. Incorporation of [³H]leucine into the diapause-associated protein of the fat body increased gradually in pre-diapausing larvae and reached a maximum in newly-diapaused larvae at a time when the incorporation of [³H]leucine into other proteins of the fat body had declined. The proteins released from the cultured fat body showed identical electrophoretic properties and close immunochemical relationships to most of those present in the haemolymph. Small amounts of the diapause-associated protein were released in vitro from the fat body of larvae of different ages in diapause. Lipophorin was also released in vitro from the fat body of non-diapausing and diapausing larvae, and shown to be immunochemically identical to the lipophorin present in the haemolymph.     

Juvenile hormone-controlled vitellogenin synthesis in Locusta migratoria fat body. Hormonal induction in vivo.

Biosynthetic processes related to the production of vitellogenin (yolk precursor protein) have been examined in the fat body of adult female Locusta migratoria. Vitellogenin-producing capacity was assayed by incubation of fat body with [³H]leucine, followed by precipitation from the medium with specific antiserum. In normal development, vitellogenin synthesis began at about Day 7 after emergence and became maximal at about Day 13, when this protein accounted for 60% of the total fat body protein output. The production of other proteins increased to a lesser extent, becoming maximal at about Day 6. The incorporation of uridine into fat body RNA rose to a maximum at Day 8, which coincided with a marked increase in tissue RNA content. The DNA content in adult female fat body approximately doubled between Days 3 and 8. Vitellogenin synthesis, and the increases in RNA and DNA, were prevented by removal of the corpora allata (the source of juvenile hormone). In allatectomized locusts, vitellogenin synthesis was induced by JH or an analog, ZR-515. Applied topically in acetone, these gave steep dose-response curves, half-maximal at 75 and 150 μg, respectively. After a single treatment with ZR-515, fat body vitellogenin production rose slowly during 48 hr, then steeply to a maximum at 72 hr, but after decay of this effect during 10 days, a second application of ZR-515 induced renewed synthesis with little initial lag. Hormone treatment produced a smaller increase in the output of other proteins, and an increase in incorporation into RNA which preceded the major rise in vitellogenin synthesis. Male fat body produced little or no vitellogenin. These results are consistent with action of JH at the gene level.     

Cycles of protein synthesis during pupal diapause in the flesh fly,Sarcophaga crassipalpis

Protein synthesis is cyclic during pupal diapause in Sarcophaga crassipalpis. These cycles are in phase with infradian MO₂ cycles, which have a periodicity of about 4 days at 25°C. Mean incorporation of [³⁵S]methionine by diapausing pupae was 5.4% during the 2 days of highest MO₂ but dropped to 1.7% during the 2 days of low MO₂. Diapausing pupae treated with a juvenile hormone analog prior to pupariation had a constant high MO₂ similar to peak values observed in untreated pupae, and such pupae consistently incorporated [³⁵S]methionine at a high rate (7.7%). [³⁵S]Methionine incorporation by nondiapausing pupae and pharate adults was eightfold higher than the peak rates observed during diapause. Autoradiography of in vivo labeled proteins indicated quantitative and qualitative changes in the synthesis of proteins by diapausing pupae during different phases of the MO₂ cycle. Brains from diapausing pupae labeled in vitro showed higher incorporation at the peak of the MO₂ cycle than at the nadir of the cycle, but no such differences were detected for integument, fat body, or fat body supernatant. Theses differences in tissue response indicate that control of protein synthesis during diapause is not cell autonomous, but is a function of the metabolism of the intact organism.     

Properties of two follicle proteins and their possible role for vitellogenesis in the African Locust

Summary Insoluble proteins from the maturing follicle ofLocusta migratoria were analyzed by SDS-PAGE. A reproducible pattern of low molecular weight proteins was observed. Five of these proteins did not correspond to yolk or haemolymph proteins. At least two of these show marked quantitative changes during oocyte development. By in vitro incubation of follicles and fat body with a labelled precursor, and by the identification of the labelled polypeptides by SDS-PAGE, we could demonstrate that these two proteins are synthesized only during the time of vitellogenin uptake. This protein is probably a follicle product necessary for yolk formation. The other protein might be necessary for vitelline membrane and/or chorion formation.     

Voltinism and seasonal changes in haemolymph protein pattern of Pyrrhocoris apterus (Heteroptera: Pyrrhocoridae) in relation to diapause

Abstract. The red firebug, Pyrrhocoris apterus (L.), was shown not to be strictly a monovoltine species in Czechoslovakia. A second generation arises from eggs laid by females emerging between June and the beginning of August. Oviposition takes place before diapause is induced in the females by shortening of photophase. Adults that moult later, enter diapause without having laid eggs.
Electrophoretic patterns of haemolymph proteins of adults collected in the field throughout a year showed a characteristic temporal pattern of changes correlated not only with the stages of ovarian development, but also with the progression of diapause and post-diapause quiescence. The changes mainly concerned vitellogenin and polypeptides of molecular weight 78 and 80 kDa.
Vitellogenin was present only in the haemolymph of reproductive females. The polypeptides of 78 and 80 kDa, belonging to the group of storage proteins, accumulated conspicuously in the haemolymph of adults undergoing diapause and post-diapause quiescence. However, the occurrence of these polypeptides seems not to be connected exclusively with cold, because their titres increased with the length of diapause development, even in diapausing adults reared in the laboratory at a constant 26.C     

Vitellogenin titre in haemolymph of Locusta migratoria in normal adults,after ovariectomy,and in response to methoprene

Vitellogenin in the haemolymph of Locusta migratoria was assayed by rocket immunoelectrophoresis to elucidate aspects of its regulation. In many normal adult females, vitellogenin first appeared on days 5–9, rose quickly to peak levels, and declined before a second vitellogenic cycle; in others, it appeared later and built up more slowly. The timing of first appearance of vitellogenin, and proportions of early and late-developing individuals, differed markedly in groups from the same colony assayed in different years, suggesting effects of both genetic and environmental variation. Average peak levels of vitellogenin were 25–30 mg/ml. After ovariectomy, vitellogenin appeared near the normal time and increased for several weeks to about 300 mg/ml; haemolymph volume also increased greatly, so that the total haemolymph-vitellogenin pool reached about 300 mg/individual, or 100 times the normal amount. After ovariectomy, no cyclicity of vitellogenin accumulation was apparent. These results show that the ovary is not required for stimulation of vitellogenin synthesis, and suggest that normal cycling may depend on inhibition by the mature ovary. Females treated with ethoxyprecocene on day 1 of adult life to inactivate the corpora allata did not produce vitellogenin, but were induced to do so with the juvenile hormone analogue, methoprene. After injection of 150 μg of methoprene in mineral oil, there was one day lag, then vitellogenin increased in the haemolymph to the normal peak level and declined slowly to zero during 5 weeks; after a second injection of methoprene, vitellogenin re-appeared more rapidly, with less lag, reflecting accelerated secondary hormonal stimulation of vitellogenin synthesis in the fat body. Adult males showed no detectable haemolymph vitellogenin even after injection of large doses of methoprene.     

蝗虫微孢子虫对东亚飞蝗卵黄原蛋白含量的影响

采用免疫学方法,对东亚飞蝗Locusta migratoria manilensis感染蝗虫微孢子虫Nosema locustae后体内卵黄蛋白含量的变化进行了研究。结果表明,感病蝗虫与对照健虫相比,卵黄发生有严重障碍,脂肪体和卵巢中卵黄原蛋白或卵黄蛋白含量极低,导致感病雌虫丧失产卵能力。脂肪体中卵黄原蛋白含量最高峰健虫为18.7 mg/mL,而病虫只有4.7 mg/mL;血淋巴中卵黄原蛋白含量最高峰健虫为7.6 mg/mL,而病虫只有2.6 mg/mL;卵巢中卵黄蛋白含量最高峰健虫为73.4 mg/mL,而病虫只有4.9 mg/mL。     

Control of vitellogenin synthesis in the Monarch butterfly by juvenile hormone.

Yolk formation in the Monarch butterfly, as in many other insects, entails juvenile hormone-induced synthesis in the fat body, transport in the hemolymph, and uptake into the oocytes of specific sex-limited protein, the vitellogenin. In the Monarch, vitellogenin first appears in the hemolymph 1 day after emergence of the adult butterfly and then rises rapidly in concentration for at least 3 days. The synthesis of vitellogenin in adult females was shown by the incorporation of [³H]leucine. Active labeling was observed at the same time as the presence of vitellogenin in the hemolymph was first detectable, and the radioactivity in vitellogenin expressed as a percentage of radioactivity in total blood proteins reached 50–60% 3 days after adult emergence. The appearance of vitellogenin was prevented by ligature of freshly emerged butterflies at the neck and restored by injection of juvenile hormone. A low yet significant stimulation of vitellogenin synthesis could be detected as early as 10 hr after administration of the hormone into neck-ligated butterflies, and after 30 to 50 hr about 40% of the total blood protein label was found in the specific protein. With C₁₈-juvenile hormone (JH-I), 0.1 μg per animal was effective in inducing a low level of vitellogenin synthesis, and between 0.01 and 1 μg, a linear relationship between synthesis and log dose was observed. The synthetic analog ZR-512 was also active. In a preliminary experiment, actinomycin D effectively blocked the induction of vitellogenin synthesis.     

Presence and function of ecdysteroids in adult insects

1. Ecdysteroids have been found in both male and female adults of many insect species.2. In Diptera vitellogenin synthesis is primarily controlled by ecdysteroids. In male flies vitellogenin synthesis can easily be induced by ecdysone and 20-OH ecdysone. In most other insect orders ecdysteroids do not directly control vitellogenin synthesis in the fat body.3.In vivo the ovary readily takes up [³H]ecdysone from the haemolymph.In vitro, at least, the ovary releases ecdysteroids.4. A high ecdysteroid peak was found in non-reproducing prediapausing adult Colorado potato beetles.5. Ecdysteroids do occur in adult males but the titre in their haemolymph is usually much lower than that found in females.6. The exact role of ecdysteroids in processes other than vitellogenin synthesis in Diptera remains to be elucidated.     

An immunochemical study of the diapause-associated protein of the southwestern corn borer,Diatraea grandiosella

The distribution and titre of the diapause-associated protein (DAP), which accumulates in the fat body of diapausing larvae of the southwestern corn borer, Diatraea grandiosella, were determined using an antibody raised to the purified protein. Immunoblotting and immunodiffusion showed that the highest concentration of DAP was present in the fat body. Small amounts of the protein were present in the haemolymph and even lower levels were detected in other tissues. Rocket immunoelectrophoresis showed that DAP accumulated in the fat body at the beginning of diapause, reached a plateau, and gradually declined towards the end of diapause. Similarly, the titre of DAP in last instar non-diapausing larvae treated with a JH analogue increased, reached a plateau and then declined. Double immunodiffusion and immunoblotting using the DAP-antibody and extracts of the fat body of D. grandiosella and the southern cornstalk borer, Diatraea crambidoides, and the sugarcane borer, Diatraea saccharalis, revealed the partial immunochemical identity of the DAP of D. grandiosella and a protein present in these two species. Denaturing electrophoresis with immunoblotting showed that the DAP-related proteins of D. crambidoides and D. saccharalis have apparent molecular weights of 33,000 and 36,000, respectively, as compared to a molecular weight of 35,000 for DAP of D. grandiosella. A dot blot analysis showed that no cross reaction occurred between the “diapause proteins” present in the haemolymph of adults of the Colorado potato beetle, Leptinotarsa decemlineata, and the DAP of D. grandiosella.     

Termination of vitellogenin synthesis by mosquito fat body, a programmed response to ecdysterone

ABSTRACT. In the blood-fed mosquito, peak vitellogenin synthesis occurs 24–32 h after the meal, dropping to resting levels by 40 h. Challenging fat body with ecdysterone in vitro at various times after a blood meal demonstrated a refractory period at about 50 h, when there was also a drastic decrease in mitochondria, rough endoplasmic reticulum, ribosomes, and glycogen in fat body cells. When fat bodies from sugar-fed females were incubated with continuous ecdysterone in vitro , vitellogenin synthesis reached a peak at 30 h, but then declined even in the presence of ecdysteroné. This was not due to the in vitro conditions since fat bodies were responsive, even if first exposed to ecdysterone, after 80 h in vitro. If ecdysterone was removed, vitellogenin synthesis ceased. If it was replaced, the fat body responded again only if the initial removal was done during the first 30 h. It is proposed that the falling ecdysterone titre is the major cause of cessation of vitellogenin synthesis, but that synthesis is programmed to decline even if exposure to ecdysterone is abnormally prolonged.     

An electrophoretic study of proteins of the ovary,fat body,and haemolymph in the migratory grasshopper, Melanoplus sanguinipes

Electrophoretic separation of saline extracts from the ovary revealed 14 proteins. Twelve proteins were detected in the fat body, of which seven had electrophoretic mobilities identical to those in the ovary. Similarly, eight of 16 proteins in the haemolymph of vitellogenic females ahad electrophoretically identical counterparts in the ovary. As these proteins accumulate in the haemolymph of ovariectomized females, the findings suggest that most yolk proteins are synthesized in the fat body. Although most female haemolymph proteins are present in males, two of the predominant yolk protiens are absent and represent female-specific proteins.Although certain proteins accumulate in the haemolymph of allatectomized females, the major ovarian proteins are absent or present in low concentrations. However, 48 hr after allatectomized females are treated with a juvenile hormone analogue, the haemolymph protein pattern resembles that of a normal female. This suggests that the corpora allata stimulate the synthesis of female-specific and other vitellogenic proteins. The median neurosecretory cells (mNSC) are also necessary for synthesis of female-specific proteins. Furthermore, proteins which are present in allatectomized females are absent in mNSC-cauterized insects suggesting that the mNSC stimulate general protein synthesis.     

Inhibition of the formation of protein storage granules by glutaurine in the larval fat body cells of Mamestra brassicae (Insecta, Lepidoptera)

Correlative changes in the protein contents of haemolymph and fat body and the accumulation of protein storage granules in the fat body cells of Mamestra brassicae were investigated during the last larval stage in normally developing larvae and following administration of glutaurine (1 X 10(-4) mg/g body weight). The protein content of the haemolymph of untreated larvae increased up to the 4th day of the stage, declined during days 5 and 6, and increased again before pupation. In the glutaurine-treated larvae the amount of proteins in the haemolymph was as high as in the controls during the first four days but continued to rise up to the end of the stage. The protein content of the fat body started to increase from the 3rd day and heavy accumulation of protein storage granules in the cells of fat body was observed on the 5th and following days. The protein content of the fat body of glutaurine-treated larvae remained at a low level and the protein storage granules were absent in the cells. The inhibition of the selective uptake of haemolymphatic storage proteins by fat body following glutaurine treatment is suggested.