Hormonal control of ovarian development in the silkworm,Bombyx mori

Hormonal control of ovarian development was examined in Bombyx mori. The weight of the ovary increased suddenly by 3 days after pupal ecdysis, and vitellogenin could be immunologically detected in the ovary at that time. The ecdysteroid titers during pupal-adult development, quantified by radioimmunoassay, increased from day 0 to day 2. Ovarian development was arrested for a long period in brainless pupae and isolated pupal abdomens. Injection of 20-hydroxyecdysone into such preparations stimulated development of the ovaries, and vitellogenin could be detected in ovaries 2 days after injection. The results suggest that 20-hydroxyecdysone acts by stimulating the growth of ovary.     

Interactions of ecdysteroid and juvenoid agonists in Plodia interpunctella (Hübner)

The influence of non-steroidal ecdysteroid agonists on Indianmeal moth larvae was assessed by rearing last instar larvae on diet treated with RH-5992 (tebufenozide) or RH-2485 (methoxyfenozide). Larvae were monitored for effects of the ecdysteroid agonists on weight, metamorphosis and mortality. Larvae treated with either of the ecdysteroid agonists at a concentration of 5 ppm or higher gained less weight and had greater mortality than did larvae reared on control diet. For example, the weights of control larvae increased approximately 400% by day 2, compared with only a 50% increase in weight when the larvae were treated with 25 ppm of RH-2485 or RH-5992. Similarly, mortality in control larvae was less than 10%, but was as much as 90–100% in larvae reared on diet treated with one of the ecdysteroid agonists. We also examined the effects of simultaneous treatment with a juvenile hormone (JH) mimic, either methoprene or fenoxycarb. The JH mimics prevented adult emergence, and the larvae continued to feed throughout the month-long observation period. However, larvae treated with a juvenile hormone mimic gained weight despite the presence of an ecdysteroid agonist in the diet. On diets treated with 0.1 ppm of RH-2485 or RH-5992, JH-treated larvae gained even more weight than did untreated controls. Interestingly, although the addition of a JH mimic to ecdysteroid-treated diet resulted in increased weight, it did not lead to reduced mortality. In fact, combinations of a JH mimic with 10 ppm RH 2485 or RH 5992 resulted in nearly 100% mortality compared with 40–70% mortality without the JH compounds. These results indicate that JH mimics overcome the inhibitory effects of ecdysteroid agonists on weight gain; however, they also resulted in increased mortality compared with moderate doses of ecdysteroid agonists alone. One specific action of these compounds at the cellular level was noted in that RH 5992 mimicked ecdysteroids by increasing uptake of ¹⁴C-GlcNAc in a Plodia interpunctella cell line, while fenoxycarb was inhibitory. Arch. Insect Biochem. Physiol. 38:91–99, 1998. Published 1998 Wiley-Liss, Inc.     

Effects of residual chlorfluazuron on haemolymph-borne oviposition-stimulating factors in Spodoptera litura

Acylurea insecticides can have various effects on insect reproduction and may even interfere with haemolymph‐borne oviposition‐stimulating factors (OSFs). This study describes the effects of injecting haemolymph into females of the common cutworm, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), the effects of chlorfluazuron on the activities of OSFs, when sublethal doses (LD₁₀: 1.00 ng larva^?1 or LD₃₀: 3.75 ng larva^?1) were applied topically to newly ecdysed fifth instars under laboratory conditions. The haemolymph of moths of varying age, sex, and mating status resulted in the following increases in oviposition rates within the first 24 h, listed in decreasing order: the haemolymph of mated females injected into mated females (56%), mated females into newly emerged virgin females (53%), virgin males into virgin females (49%), and virgin females into mated females (29%), compared with uninjected mated or virgin females. Two factors are involved in the activation of ovipositon: OSF‐I is found in virgin females, whereas OSF‐II may be formed or received by males during mating. By contrast, the haemolymph taken from chlorfluazuron‐treated adults (treated as fifth instars with sublethal doses) injected into females resulted in the following percentage decreases in oviposition rates within the first 24 h, listed in decreasing order: the haemolymph of LD₁₀‐ and LD₃₀‐treated virgin males injected into untreated virgin females (77 and 84%, respectively), LD₁₀ and LD₃₀‐treated virgin females into untreated mated females (70 and 80%, respectively), LD₁₀ and LD₃₀‐treated mated females into untreated mated females (61 and 69%, respectively), LD₁₀ and LD₃₀‐treated mated females into untreated virgin females (59 or 68%, respectively), compared with untreated ones. Hence, residual chlorfluazuron decreases the activities of OSFs by significantly decreasing the oviposition rates. Moreover, virgin females’ or males’ OSFs are more sensitive to chlorfluazuron than those of mated cutworms. Both OSFs are most likely proteins.     

Induction of dauer pupae by fenoxycarb in the silkworm,Bombyx mori

Topical application of fenoxycarb (1 μg per animal) at 129 or 132 h of the fifth instar larvae of the silkworm, Bombyx mori, did not induce morphological abnormalities in the pupal stage, but these animals became dauer (permanent) pupae. This condition of B. mori and the endocrine events leading to permanent pupae are discussed in this work. Application of fenoxycarb at 132 h of the fifth instar elicited a high ecdysteroid titre in the pharate pupal stage and a steadily high ecdysteroid titre in the pupal stage. The fenoxycarb-induced permanent pupae had non-degenerating prothoracic glands that secreted low amounts of ecdysteroid and did not respond to recombinant prothoracicotropic hormone (rPTTH) late in the pupal stage. The Bombyx PTTH titre in the haemolymph, determined by a time-resolved fluoroimmunoassay, was lower than that of controls at the time of pupal ecdysis, but higher than controls later in the pupal stage in fenoxycarb-treated animals. After application of fenoxycarb, its haemolymph level, measured by ELISA, reached a peak at pupal ecdysis, then remained low. These results suggest that the fenoxycarb-mediated induction of permanent pupae is only partially a brain-centred phenomenon. It also involves alterations in the hormonal interplay that govern both the initiation of pupal-adult differentiation and changes in the steroidogenic pathway of the prothoracic glands of B. mori.     

RH-5992--an ecdysone agonist on model system of the silkworm Bombyx mori

RH-5992 is a novel synthetic non-steroidal ecdysteroid agonist with a high selectivity towards Lepidopteran species. The effect of ecdysone agonist RH-5992 on larval period, larval weight, silk gland weight and haemolymph protein profile were examined in the model organism, the larvae of silkworm, Bombyx mori. The LD50 values were found to be 16.21 and 12.01 micrograms/larva for 72 and 96 hr respectively. In the present study, three sublethal concentrations of 1/5th, 1/10th and 1/20th of LD50 at 72 hr were selected and applied on the mid-dorsal line of the silkworm B. mori. The maximum mortality of 35% was observed in the group which received the highest (3.2 micrograms/larva) concentration of RH-5992. The mortality rate was found to be dose dependent as well as time dependent. Interesting results were observed in haemolymph profile of the RH-5992 treated larvae as staining intensity of 30 kDa protein decreased significantly whereas the effect was not marked on other major proteins like storage proteins and vitellogenin polypeptides. From the results, it is confirmed that RH-5992 causes changes in larval characters and protein profile of silkworm B. mori. It is proposed that RH-5992 may cause negative effect specifically on reproductive characters like development of ovary and egg production due to decrease in 30 kDa protein.     

Hormonal regulation of phase polymorphism and storage-protein fluctuation in the common cutworm, Spodoptera litura

Three storage proteins are synthesised by Spodoptera litura last-instar larvae as detected by an antiserum against pupal fat body proteins. The putative pupal storage proteins 1 and 2, appear in the haemolymph of the last-instar larvae 36 h after ecdysis under crowded rearing conditions: they appear 1 day later in isolated conditions. The appearance of these proteins in the haemolymph is prevented by juvenile hormone treatment and enhanced by allatectomy. Injection of 20-hydroxyecdysone into ligatured larvae does not induce appearance of these 2 proteins. Accumulation of protein 3 that reacts with Bombyx mori arylphorin antiserum is not blocked by juvenile hormone and is similar in both phases. It also accumulates to a small extent in the haemolymph during the moult to the final-larval instar and then disappears at ecdysis. One-hundred ng/ml ecdysteroid caused the sequestration of these proteins by the fat body, but a higher concentration of ecdysteroid (200 ng/ml) produced pupal cuticle in the isolated abdomens, suggesting that different ecdysteroid concentrations are necessary for these two events.     

Integration between Steinernema feltiae and some of environmental friendly compounds to control Spodoptera littoralis (Lepidoptera: Noctuidae)

The efficacy of different concentrations of the commercial neem-based insecticide, Nimbicidine^® and the ecdysone agonist compound Methoxyfenozide (RH-2485), was evaluated against larvae of Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae). RH-2485 and Nimbicidine^® significantly (p?<?0.001) reduced adult longevity by 2.7 and 1.9 d at the higher concentrations tested, respectively, but no significant differences were observed at low concentration. The tested compounds strongly affected the reproduction of S. littoralis by producing a high percentage of sterility. Fecundity and fertility were significantly affected by both insecticides. No egg laying has been recorded with the higher concentration 0.5% of Nimbicidine^® and 0.1?ppm of RH-2485, while no significant (p?>?0.05) difference was noticed on total number of eggs laid by the female when lower concentration 0.0001?ppm of RH-2485 was applied in the same stage as compared to the control. In the second part of this study, the invasion rate of Steinernema feltiae was affected by the addition of both Nimbicidine and RH-2485 to the diet of experimental host. At higher concentration of both compounds, the invasion rate was decreased despite the infection rate, while the percentage of invading nematodes increase to 56.7% with the combination treatment indicating a considerable improvement in the efficacy of S. feltiae nematode applied in combination with the lower concentration of Nimbicidine^® over that of nematode alone. On the other hand, the invasion of S. feltiae to the insects that were fed on the diet with the addition of ecdyson compound was strongly decreased with increasing concentration of RH-2485 in host diet.     

Haemolymph ecdysteroid in the housefly,Musca domestica,during oögenesis and its relationship with vitellogenin levels

Ecdysteroid titre in the haemolymph of the housefly, Musca domestica, cycled during oögenesis and peaked at ～50 pg/μl during stages 5, 6 and 7. Levels of 10–20 pg/μl were found in houseflies with pre- and post-vitellogenic ovaries. Removal of the corpus allatum and corpus cardiacum complex resulted in low ecdysteroid levels (10 pg/μl). Ovariectomized flies also had lower ecdysteroid levels than the controls at 2 days (5 pg/μl) after emergence but not at 6 days (22 pg/μl). It is possible that the ecdysteroid peak that occurred during stages 5, 6 and 7 was produced by the ovaries because ovaries secreted and synthesized ecdysteroid in vitro. Endogenous haemolymph ecdysteroid levels had a linear correlation with the amount of vitellogenin that held for hormone concentrations of 5–43 pg/μl. Furthermore, the injection of 20-hydroxyecdysone at doses of 10 ng?1.0 μg/fly increased the amount of vitellogenin from 6 h to 12 h after injection; by 24 h, the vitellogenin returned to control levels. When 20-hydroxyecdysone was injected into ovariectomized flies, it was rapidly degraded and 96% was cleared from the haemolymph within 1 h.     

Gene-controlled incorporation of haemolymph protein into the ovaries of Bombyx mori

The haemolymph protein concentration in Bombyx mori decreases normally by about one-fourth during pharate adult development. In females homozygous for the small egg gene, the concentration of haemolymph protein remained constant throughout the pupal and pharate adult stages. The sm gene does not influence the synthesis of vitellogenic female protein of pupal and pharate adult haemolymph (FP). Normal ovaries transferred to the haemocoele of sm females undergo normal vitellogenesis. In the absence of normal alleles of sm, the ovaries encounter difficulties in the incorporation of FP into their oöcytes from pharate adult haemolymph. These results suggest that an active translocation mechanism is involved in the transfer of haemolymph protein into the ovaries.     

天蚕卵黄发生的激素调控

报告了蜕皮激素和保幼激素对天蚕Antheraea yamamai卵黄发生的调控作用。当单独以20－羟基蜕皮酮或保幼激素类似物methoprene处理,以及同时用这两种激素处理天蚕蛹时,蛹期脂肪体和血淋巴中卵黄原蛋白（Vg)含量明显高于对照,即二对Vg的合成起促进作用。然而,卵巢中卵黄蛋白（Vt)含量则因激素种类而异,以保幼激素处理时明显低于对照,以20－羟基蜕皮酮处理则反之,即前抑制卵巢对Vg的摄取,而后则起促进作用。离体培养脂肪体并以激素处理的结果表明,20－羟基蜕皮酮和methoprene均能促进Vg合成,但前作用更。综合考虑上述结果可以认为蜕皮激素对该蚕的卵黄发生起主要调控作用。     

HORMONAL CONTROL OF THE VITELLOGENESIS IN THE JAPANESE OAK SILKWORM, ANTHERAEA YAMAMAZ (LEPIDOPTERA: SATURNIIDAE)

Abstract Effects of ecdysteroid and juvenile hormone (JH) on vitellogenesis of the Japanese oak silkworm, Antheraea yamami are reported in this article. After topical treatment with 20-hydroxyecdysone alone or JH analog (i.e. methoprene) alone and combined treatment with these two chemicals, vitellogenin (Vg) titers in the fat body and haemolymph at the pupal stage were mostly higher than those of the control, indicating that both ecdysteroid and JH exerted a promoting effect on the synthesis of Vg. In contrast, the Vg uptake was markedly inhibited by JH while stimulating effect of the ecdysteroid could be shown that vitellin (Vt) titer in the ovary was lower after methoprene treatments, but higher after 20-hydroxyecdyson treatments. Meanwhile, effects of these two hormones on Vg synthesis in the fat body were also tested with the incubation in vitro with Grace medium containing H-leucine and the hormones. The results demonstrated that Vg synthesis was stimulated after treating with methoprene alone or 20-hydroxyecdysone alone and combined treating with these two chemicals, and particularly ecdysteroid had more marked positive effect. To comprehensively concluded our results, it could be regarded that ecdysteroid play the main role in the regulation of vitellogenesis for the Japanese oak silkworm.     

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.     

Sublethal effects of Bacillus thuringiensis on the spruce budworm, Choristoneura fumiferana

Exposing larvae of the spruce budworm, Choristoneura fumiferana (Clemens), to sublethal ( 50% lethal dose) levels of Bacillus thuringiensis subsp. kurstaki at various stages of their development significantly increased development time to the pupal stage and reduced pupal size and number of eggs laid per female, but did not affect the proportion of embryonated eggs. The changes in larval development time, pupal weight and fecundity depended on the larval stage that was treated. Exposure of fourth instars delayed larval development and reduced only male pupal weights with no effects on fecundity. Exposure of sixth instars delayed larval development to a lesser extent than exposure of fourth instars but had a pronounced effect on weight of both male and female pupae. The effect on pupal weight was sex dependent, as males tended to be more affected than females. The reduction in male pupal weight did not appear to influence fecundity, because the effect of exposure was explained by the change in female pupal weight. Effects on larval growth and pupal weight were proportional to the dose that was ingested during exposure, and were observed at doses as low as one-tenth of the LD₅₀. Ingestion of an LD₅₀ caused a 29 or 45% delay in development of, respectively, female or male larvae when exposed as fourth instars and a 30% reduction in female pupal weight when larvae were exposed as sixth instars.     

Comparative analysis of proteome maps of silkworm hemolymph during different developmental stages

Background  

The silkworm Bombyx mori is a lepidopteran insect with four developmental stages: egg, larva (caterpillar), pupa, and adult. The hemolymph of the silkworm is in an open system that circulates among all organs, and functions in nutrient and hormone transport, injury, and immunity. To understand the intricate developmental mechanisms of metamorphosis, silkworm hemolymph from different developmental stages, including the 3^rd day of fifth instar, the 6^th day of fifth instar, the 3^rd day of pupation, the 8^th day of pupal stage and the first day of the moth stage, was investigated by two-dimensional electrophoresis and mass spectrometry.     

Effects of insect growth regulators on the hairy rose beetle, Tropinota squalida (Col., Scarabeidae)

Abstract:  Saponin extract from alfalfa roots, azadirachtin from the neem seed oil, synthetic ecdysteroid agonist RH-2485, and the juvenoid hydroprene disturb the development and reproduction of Tropinota squalida . Feeding beetles on diets containing 750 p.p.m. saponins, 7.5 p.p.m. RH-2485, and 1.13 p.p.m. azadirachtin reduces their progeny from 51 second instar larvae per female to 24, 15, and 15 larvae, respectively. When the larvae of untreated adults are fed for 1 week on dung with 75 p.p.m. saponins, 50 p.p.m. RH-2485, and 0.45 p.p.m. azadirachtin, the rate of adult emergence drops from 80% (controls) to 20, 0 and 13%, respectively. No adults emerge when the treatment is continued through the second and third larval instars. Two topical treatments of larvae with 0.2  μ g hydroprene decrease the rate of adult emergence from 90 to 11%, and treatments with 2  μ g prevent adult development in all insects. The observed effects warrant testing of azadirachtin, RH-2485, and hydroprene in the field. Several types of their application for the control of T. squalida are suggested.     

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.     

The effect of juvenile hormone on prothoracic gland function during the larval-pupal development of Manduca sexta: An in situ and in vitro analysis

The application of juvenile hormone I or ZR 512 to neck-ligated, day-5 fifth instar (V₅) larvae reduced the time to pupation in a dose-dependent manner when compared to neck-ligated controls treated with methyl epoxy stearate. Haemolymph ecdysteroid titres determined by radioimmunoassay (RIA) reflected the ability of juvenile hormone I and ZR 512 to stimulate larval-pupal development, i.e. the ecdysteroid titres were similar to those of normally developing larvae although the ecdysteroid peak elicited by ZR 512 lagged that in the normal titre by 1 day, while that elicited by juvenile hormone I lagged the ecdysteroid peak in normal larvae by 2 days. Neck-ligated V₅ larvae that were untreated ultimately pupated and the haemolymph ecdysteroid peak eliciting pupation in these animals was 7 μg/ml haemolymph, almost double that of normal animals and ZR 512- and juvenile hormone I-treated, ligated larvae. The data indicated that juvenile hormone I does stimulate the prothoracic glands but to determine whether this stimulation was direct or indirect, an in vitro approach was taken. Prothoracic glands from V₅, V₆ and V₇ larvae were incubated in vitro under conditions in which they could be stimulated by prothoracicotropic hormone, and were exposed to concentration of free juvenile hormones I, II, III or ZR 512 ranging from 10^?5M to 10^?10M. In no case were the prothoracic glands stimulated in a dose-dependent manner that would be indicative of hormone activation. Similar results were obtained when juvenile hormone bound to binding protein was incubated with the prothoracic glands. Studies with the acids of the three juvenile hormone homologues revealed them to be ineffective in activating prothoracic glands, although juvenile hormone III acid does appear to inhibit the synthesis of ecdysone by day-0 pupal prothoracic glands. The significance of the latter effect is unknown. It is concluded from these data that juvenile hormone can, indeed, activate late larval prothoracic glands in situ, but does so indirectly.     

Haemolymph ecdysteroid titre and epidermal cell commitment of the female southwestern corn borer larvae

The epidermal cell commitment (to pupation or formation of immaculate larvae) and related haemolymph ecdysteroid titres of the southwestern corn borer, Diatraea grandiosella were studied in both nondiapause-bound and diapause-bound last-instar female larvae. Cell commitment was estimated by examining the characteristics of new cuticle secreted in response to an injection of 20-hydroxyecdysone. Haemolymph ecdysteroid titres were determined by radioimmunoassay. Juvenile hormone effect on epidermal cell commitment was studied by applying a juvenile hormone mimic (ZR-515) to last-instar non-diapause-bound larvae and examining the resulting cuticle.In non-diapause-bound larvae, the epidermis of different body regions was committed to pupal development at different times. When pupal cuticular characteristics were evaluated by a scoring system, it appeared that the development of normal pupal cuticle is discontinuous. Three sudden increases in pupal characteristics were observed at 1.67, 2.67 and 3.67 days into the last-larval instar. Haemolymph ecdysteroid titre changes were correlated with the sudden increases in pupal characteristics. Peak ecdysteroid titres were found at 1.67, 2.33, and 3.33 days into the final instar. A fourth ecdysteroid peak (138.8 ng/ml of haemolymph) occurred in pharate pupae. In contrast, the commitment of diapause-bound larvae to produce immaculate integument was made in a fast and continuous fashion. Full commitment was made by 50% of the individuals 4 days (ca. first quarter) into the stadium. Haemolymph ecdysteroid titres fluctuated during the first 2 weeks of the stadium but no significant peaks were observed prior to pharate stage. An ecdysteroid peak (29.8 ng/ml of haemolymph) was identified in pharate immaculate larvae.Pupal development could be completely prevented in 26.7% of nondiapause-bound larvae as late as 4 days into the last instar by topical application of ZR-515. This indicates that the commitment to pupation as revealed by 20-hydroxyecdysone injection is reversible.     

Temporal events of gypsy moth vitellogenesis and ovarian development

Abstract The vitellogenic period of gypsy moth ovarian development starts on day 3 of the pupal stage and continues through adulthood. During this period, rapid increases occur in follicle size, protein content, and wet weight of the ovary. Patency is observed on day 3 of the pupal stage.
Pre-vitellogenic follicles are formed in the last larval stadium. Newly formed follicles detach from the germarium on day 4, and increase rapidly to 140 per ovariole at the end of the last larval stadium. The pre-vitellogenic follicles are uniformly around 50 um in diameter. No vitellogenin is incorporated into the oocytes until the pupal stage.
Polyacrylamide gel electrophosesis (PAGE) in the presence of sodium dodecylsulphate (SDS) analysis of male and female haemolymph samples and vitellogenic ovaries demonstrates the presence of two female-specific subunits of vitellogenin of 180 kD and 160 kD. These proteins are detected only in haemolymph and ovarian extracts of vitellogenic females. The molecular weight of the native protein determined by size exclusion chromatography is approximately 400–420 kD.
A highly sensitive double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed to monitor the temporal changes in vitellogenin titre in haemolymph. Vitellogenin production starts on day 2 of the last larval stadium, reaching a maximum level by day 6 of the last larval stadium, and decreasing in the late pupal stage as vitellogenin was internalized into the oocytes. This is the first report of vitellogenin production occurring in the larval stage of a holometabolous insect. The fact that vitellogenin production and uptake occur during different stages of development in the gypsy moth, opens up some interesting questions concerning the underlying regulatory mechanisms controlling each process.     

Azadirachtin potentiates the action of ecdysteroid agonist RH-2485 in Spodoptera littoralis

Edysteroid agonist RH-2485 induces an immediate and fatal molt in Spodoptera littoralis when added to the diet of the 2nd and 4th instar larvae at 1 ppm, and to that of the 6th instar larvae at 0.001 ppm concentration. Ten times lower doses fed to the larvae continuously allow an apparently normal larval development that is terminated by a supernumerary larval molt. The other effects of RH-2485 include death during metamorphosis and impaired fertility of emerged adults. The number of progeny is reduced even with low RH-2485 doses that do not interfere with moltings; e.g., insects fed 0.0001 ppm since the 2nd, 4th, and 6th instar produce 72%, 62%, and 22%, respectively, less progeny than the controls. Feeding larvae with 10-1000 ppm Suneem oil (containing about 0.1-10 ppm azadirachtin) causes, in a stage- and dose-dependent manner, a cessation or reduction of feeding, delay of molts, death of larvae and pupae, and sterility of emerged adults; with 10 ppm Suneem oil, the number of progeny is reduced by 20-32%. Presence of Suneem oil in the diet does not influence the potential of RH-2485 to induce a prompt molt, but it increases ten times the potency to elicit a supernumerary larval molt. Certain combinations of RH-2485 with Suneem oil provoke up to 3 extra larval molts. Lethal developmental derangements and sterility are more frequent, and the response of larvae of different age is more uniform, when Suneem oil and RH-2485 are combined than when each of these agents is administered alone.