In vitro effects of RH-0345 and KK-42 on ecdysteroid levels and cuticle synthesis by pupal integument of mealworms

In this study, the effects of two insect growth regulators (KK-42 and RH-0345), applied either alone or in combination, were evaluated on the ecdysteroid production in vitro. Integument explants from the abdominal sternites of newly ecdysed pupae of mealworms, Tenebrio molitor, were cultured and the amounts of ecdysteroids, released into the culture medium, were determined by an enzyme immunoassay (EIA) after various intervals of incubation. In combined treatments, explants were cultured for the first two days in a medium added with the first compound and then transferred in new medium containing the second compound. EIA measurements showed that RH-0345, either alone or followed by KK-42, resulted in higher amounts of ecdysteroids as compared to controls. In contrast, KK-42 alone caused a significant reduction. But, when KK-42 was followed by RH-0345, the ecdysteroid amounts were equal to controls. In another series of experiments, the effects on cuticle secretion were examined. Only in the case that explants were treated with RH-0345, either alone or followed by KK-42, new cuticle synthesis was observed.     

Effects of ecdysteroid agonist RH-2485 reveal interactions between ecdysteroids and juvenile hormones in the development of Sesamia nonagrioides

Larvae of Sesamia nonagrioides developing under long day (LD) conditions pupate in the 5th or 6th instar, whereas under the short day (SD) conditions, they undergo several supernumerary larval molts and are regarded as diapausing. The development in early larval instars occurs in the LD larvae at a moderate and in the SD larvae at a high juvenile hormone (JH) titer; ecdysteroid titer cycles similarly under both conditions. The transformation to pupa is initiated by a burst of ecdysteroids at undetectable JH levels, whereas extra larval molts in the diapausing larvae are associated with moderate JH titer and irregular rises of ecdysteroids. Application of 0.2 ppm RH-2485 to the diet of the 6th instar larvae promotes hormonal changes supporting metamorphosis in the LD larvae and slightly accelerates larval molts in the diapausing SD larvae. The 0.5- and 1-ppm doses revert these patterns of endocrine regulations to a mode typical for early larval instars. Particularly dramatic is a JH titer increase provoked within 24 h in the LD larvae. After the treatment, both the LD and SD larvae undergo a series of larval molts, suggesting that hormonal programming of the larval development has been stabilized. A few insects receiving 1 ppm RH-2485, and a high proportion of those fed with 5 ppm RH-2485, deposit two cuticles within a single apolysis and die.     

A juvenile hormone analogue,methoprene as a circadian and developmental modulator in Diatraea grandiosella (Pyralidae)

A juvenile hormone analogue (methoprene) affected the circadian system controlling the adult eclosion rhythm in Diatraea grandiosella. The effect of methroprene was compared with those of photoperiod, sex and temperature. The phase difference (ψ̃rl) between the rhythm and the light-dark cycle and its variance increased as the photoperiod increased. Males emerged earlier than females. Higher temperatures induced earlier eclosion and reduced the variance. A parallel phase advance was observed in papae treated topically with methoprene. At the higher dose tested, methoprene induced earlier eclosion. The effect of methoprene was both dose and age dependent, with earlier application more effective. A dosage of 3.0 μg was lethal, however, if applied in the first 2 days of pupal stage. Synthetic juvenile hormone I had less effect than methoprene. Methoprene accelerated adult differentiation and emergence in both sexes.     

Activity of kk-42 in combinated treatment with RH-0345 or 20-hydroxyecdysone on morphometric measurements and free ecdysteroid in eggs of mealworms

RH-0345 (halofenozide), a bisacylhydrazine derivative, is a nonsteroidal ecdysteroid agonist that mimics the action of the moulting hormones, while KK-42, an imidazole compound, is a potent inhibitor of ecdysteroid biosynthesis. Recent experiments with Tenebrio molitor suggested that the reduction of ecdysteroid titer, leading to a reduction of reproductive capacity, is due to a direct and rapid action of KK-42 on ecdysterold biosynthesis. Moreover, RH-0345 could partly restore the effects on reproductive events induced by KK-42. On the other hand, RH-0345 was found to affect growth and development of ovaries in a manner similar to that 20-hydroxyecdysone (20-E) and increased the ecdysteroid production. Therefore, the present study evaluates KK-42 applied topically (10 microg/insect) in combination with RH-0345 or 20-E in order to obtain more information on the mode of action of this compound on reproduction in T. molitor. In a first series of experiments, the compounds were assayed on morphometric measurements of freshly laid eggs. Results showed that both the weight and the volume of eggs increase significantly in series treated by KK-42 followed by 20-hydroxyecdysone as compared to controls and treated series by KK-42 followed by RH-0345. Data from enzyme immunoassay measurements revealed that KK-42 applied before 20-E was found to reduce significantly the amounts of free ecdysteroids in eggs comparatively to the others series.     

保幼激素类似物对斜纹夜蛾核型多角体病毒在宿主血淋巴中增殖动态的影响

通过研究保幼激素类似物(juvenile hormone analogues, JHA) methoprene对斜纹夜蛾核型多角体病毒（Spodoptera litura nucleopolyhedrovirus, SpltNPV）在宿主血淋巴中增殖的影响,以探明JHA促进 SpltNPV增殖的初步机制,为阐明JHA促进病毒增殖提供更全面的理论依据。应用SDS PAGE及Western blot法,分析了methoprene对SpltNPV多角体蛋白(polyhedron, POLH)在宿主斜纹夜蛾6龄幼虫血淋巴中合成的影响。结果表明：经methoprene处理后2～3天可明显促进幼虫血淋巴液中POLH的合成。在此基础上,通过荧光定量PCR检测,发现methoprene对SpltNPV在幼虫血淋巴液复制的影响主要发生在处理后的第4和第5天,该期间polh基因的拷贝数比对照显著增加,拷贝数的峰值达1.22×10¹⁰/mL。     

The effect of a juvenile hormone analogue on ecdysteroid titre during development and HnRNA formation in the moth,Ephestia cautella

The juvenile hormone analogue, methoprene was found to interfere with normal development of Ephestia in a manner dependent on age. Young embryos, prior to the stage of blastokinesis, and animals, shortly before and after pupation, were found to be the most sensitive to the compound. The JHA inhibited metamorphosis and produced giant larvae when it was given to immature larvae, however, when it was given to larvae 2–3 days prior to pupation or to young pupae it did not affect metamorphosis but prevented adult emergence. Comparison of the ecdysteroid titre determined in control and treated animals in the various developmental stages showed that JHA depressed the ecdysteroid titre totally only when it was given to young larvae and partially when it was applied shortly before pupation. It seems that the action of methoprene on ecdysone regulated systems and/or ecdysone producing systems in Ephestia appears to be mainly during the larval stage prior to the appearance of the small ecdysteroid peak and the formation of HnRNA in the transition period from larvae to pupae.     

The ovicidal effects of a juvenile hormone and a juvenile hormone analogue on the fleshfly, Sarcophaga bullata

Egg fertility was reduced in adult females of Sarcophaga bullata treated topically with a juvenile hormone (Cecropia JH-1) or a juvenile hormone analogue, ZR-515 (isopropyl-11-methoxy-3,7,11-trimethyl-dodeca-2,4-dienoate), the effect was most noticeable when the flies were treated on the day of ovulation. Depending on the time of treatment, JH and JHA affected different stages of development. Treatment on the day before ovulation produced a high percentage of embryonic arrest; when the flies were treated on the day of ovulation a major proportion of fully developed embryos failed to hatch. Treatment at the early stages of embryogenesis caused mortality of larvae before molting to third instar. There was no delayed mortality after the third instar.

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Résumé Les effets ovicides d'une hormone juvénile (Cecropia JH-1) et d'un analogue d'hormone juvénile (ZR-515) ont été examinés par application locale sur des femelles adultes de Sarcophaga bullata à différents stades du dévelopement des ufs.Le traitement des mouches le jour de l'ovulation est très efficace et empèche les embryons totalement développés d'éclore et de donner des larves de premier stade.L'application de ces produits un jour avant l'ovulation provoque l'arrêt de l'embryogenèse; quand les mouches sont traitées un jour après l'ovulation, la mortalité est plus tardive et une proportion importante de larves meurt avant la mue donnant le troisième stade.

     

The effects of nutrition and methoprene treatment on ovarian ecdysteroid synthesis in Drosophila melanogaster

Radioimmunoassay of in vitro culture medium from ovaries of Drosophila melanogaster indicates that detectable ovarian ecdysteroid synthesis begins between 6 and 12 h after eclosion and reaches a peak between 24 and 30 h, when animals are reared at 25°C, 12 h photophase. Analysis of 24 and 72 h medium by a combination of high-performance liquid chromatography and radioimmunoassay demonstrates three ecdysteroid regions, two comigrating with known standards of ecdysone and 20-hydroxyecdysone and a third highly polar region containing one or more unidentified radioimmunoassay-active ecdysteroids. In 72 h medium the polar region comprises the majority of radioimmunoassay-active material while in 24 h medium the majority is in the ecdysone region. Provision of a nutritionally deficient diet to females at adult eclosion prevents the normal increase in vitellogenic-stage follicles and ovarian ecdysteroid synthesis. Methoprene treatment of such females stimulates a transient burst of ovarian ecdysteroid synthesis and the production of near normal numbers of vitellogenic oöcytes by 24 h, although by 48 h the number of vitellogenic oöcytes is less than normal.     

Effects of methoprene,a juvenile hormone analogue,on the larval and pupal stages of the yellow fever mosquito, Aedes aegypti

Exposure of early fourth-instar larvae of Aedes aegypti to the juvenile hormone analogue Altosid ZR15^® (methoprene) significantly increased the concentration of carbohydrates in the haemolymph of late fourth-instar larvae and reduced the haemolymph carbohydrate concentration of 24-h-old pupae relative to controls. Such treatment also effected a decline in haemolymph amino nitrogen levels of the pupal stage and a depletion of haemolymph proteins in late fourth-instar larvae as well as pupae. Two of nine protein fractions in the haemolymph of larvae were significantly depleted following methoprene treatment. Fourteen soluble protein fractions were present in the haemolymph of control pupae; two of these were missing from the pupae which were treated as larvae with methoprene. A further protein fraction, common to the haemolymph of both treated and control pupae, was significantly reduced in concentration as a consequence of exposure to methoprene. The juvenile hormone analogue impaired the capacity of the fat bodies of late fourth-instar larvae and pupae to synthesise proteins, resulting in a lowered concentration of fat body proteins. Glycogen levels in the fat bodies of treated larvae were significantly lower than in controls and glycogenolysis was suppressed due to an overall depletion of glycogen phosphorylase and, in pupae, a lowered ratio of active: inactive enzyme. The data are consistent with the proposition that the juvenile hormone analogue elicits neuroendocrinological changes in the target insect.     

Regulation of vitellogenin synthesis by juvenile hormone analogue in Coccinella septempunctata

Vitellogenesis in the lady beetle, Coccinella septempunctata, is controlled by juvenile hormone (JH). When immature females were reared on an artificial diet, they were characterized by hypertrophy of the fat bodies and slow development of the ovaries. Vitellogenin (Vg) synthesis in the fat body remained at a very low level throughout adult development. RNA synthesis also stayed at a relatively low level. Treatment with hydroprene induced vitellogenesis in these non-fecund females. Stimulation in Vg synthesis in hormone-treated females was demonstrated both in vivo and in fat body culture. Synthesis of fat body RNA also increased after hormone treatment. Fat body RNA from hormone-treated females directed the synthesis of Vg polypeptides both in Xenopus oocytes and in a reticulocyte lysate. Thus the induction of Vg synthesis by JH involves an increase in the level of translatable Vg mRNA.     

Vitellogenin induced in adult male Diploptera punctata by juvenile hormone and juvenile hormone analogue: Identification and quantitative aspects

The effect of the juvenile hormone analogue hydroprene on the appearance in the haemolymph of the yolk-protein precursor vitellogenin in male Diploptera punctata has been assessed using rocket immuno-electrophoresis. Vitellogenin was induced in a dose-dependent fashion, with a minimum dose of 10 μg hydroprene required for its appearance. Implantation of male corpora allata into male and female hosts also resulted in the appearance of vitellogenin in the haemolymph of the hosts, with females showing apparent greater sensitivity to the implantation.The identity of vitellogenin in male haemolymph was further confirmed using Ouchterlony double immunodiffusion and SDS polyacrylamide gel electrophoresis of immunoprecipitates. The electrophoretic pattern of immunoprecipitated haemolymph from females and hydroprene-treated males was similar, further confirming that the immunoprecipitable product was in fact vitellogenin.     

Effects of the ecdysteroid agonists RH 5849 an RH 5992, alone and in combination with a juvenile hormone analogue, pyriproxyfen, on larvae ofSpodoptera exigua

Biological activity assays with RH 5849 and RH 5992 indicated that both compounds affected growth and development of last-instar larvae ofSpodoptera exigua (Hübner) (Lepidoptera: Noctuidae) in a dose-dependent manner. Within the first 24 h after treatment by continuously offering leaves dipped in a water solution of ≥50 mg/l RH 5849 and ≥0.5 mg/l RH 5992, symptoms of a prematurely induced larval moult and head capsule apolysis were visible. Intoxicated larvae died shortly afterwards, showing signs of unsuccessful ecdysis. LC₅₀-values of RH 5849 and RH 5992 for fifth-instarS. exigua larvae were 110 and 2.5 mg/l, respectively. Pyriproxyfen alone affected the larval stage and disturbed normal metamorphosis. One supernumerary larval instar occurred occasionally. LC₅₀-value for pyriproxyfen was 1.7 mg/l. Larvae simultaneously treated with RH 5849 or RH 5992 and pyriproxyfen, continued to grow until they attained a size and weight about 2–3 times that of the controls. This growth was accompanied by at least one and sometimes two supernumerary moults. Concerning thein vivo imaginal wing disc growth and development, only in larvae treated with 10 and 50 mg/l RH 5849 or 0.5 mg/l RH 5992, tracheole migration was observed earlier than in the controls. When applying 300 mg/l RH 5849 or 3–7 mg/l RH 5992, the discs remained small and no signs of tracheole migration were observed. In larvae simultaneously treated with RH 5849 or RH 5992 and pyriproxyfen, tracheole migration was not prematurely induced and a pupal cuticle was produced in the discs of larvae, undergoing a supernumerary moult. No clear signs of evagination were observed.     

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.     

Changes in ecdysteroid and juvenile hormone levels in developing eggs of Bombyx mori

It is well-known that insect eggs can contain very high concentrations of ecdysteroids, which undergo drastic changes during embryogenesis. We found that this is equally valid for juvenile hormones. Three juvenile hormone-immunoreactive compounds were observed in developing Bombyx mori eggs. They were assumed to be juvenile hormones 1, 2 and 3 according to their retention time in HPLC. These hormones underwent drastic and sudden changes. In the space of one day their concentration was seen to rise rapidly from an undetectable level up to as high as 4 × 10^?6 micromoles per mg of eggs. Their presence was detected as early as the first day of embryonic development, as well as during the blastokinesis period (day 5 to day 9) and in late embryos (day 12 to day 14). Their relative concentrations varied greatly. On two occasions, day 1 and day 8, all three hormones were simultaneously present. Moreover, juvenile hormone 3 was present during the blastokinesis period, either alone or in combination with hormone 2. The latter was the only hormone present in late embryos, before hatching. Thus, with regard to both ecdysteroids (ecdysone and 20-hydroxyecdysone) and juvenile hormones, each day of embryonic development displayed a different hormonal pattern. These patterns undoubtedly constitute a “hormonal code” of embryogenesis control. While 20-hydroxyecdysone can be assumed to trigger cuticulogenesis in embryos as it does in larvae, the effects of the other hormones as well as their possible interactions are questionable.     

The effects of mating,exogenous juvenile hormone and a juvenile hormone analogue on pheromone titre,calling and oviposition in the omnivorous leafroller moth (Platynota stultana)

Mating in Platynota stultana resulted in the termination of calling, the gradual reduction of pheromone in the pheromone glands to non-detectable levels (<0.1 ng/♀) within 14 h, and oviposition of the first batch of eggs 20–24 h after copulation. Decapitation of virgin females resulted in a similar decline in pheromone titre, and also eliminated oviposition and calling. Pheromone production appears to be controlled via the head. Mating probably terminates neural or hormonal input required for pheromone production and/or removes neural or hormonal inhibition of pheromone degradation. A juvenile hormone analogue (ZR-512) and juvenile hormones I, II and III applied exogenously to virgin females elicited oviposition comparable to mated females and terminated calling within 48 h. The juvenile hormone analogue also appeared to block pheromone production in virgin females. These results suggest that juvenile hormone may be involved in the switch from virgin to mated behaviour in this species.     

Effect of bisacylhydrazine ecdysteroid mimics (RH-5849 and RH-5992) on chromosomal puffing, imaginal disc proliferation and pupariation in larvae of Drosophila melanogaster

Two bisacylhydrazine insecticides with ecdysone-mimetic action, RH-5849 and RH-5992, have been subjected to several bioassay procedures that are prerequisites for ecdysone action in Drosophila larvae: (a) induction of early ecdysone-specific puffs on the polytene chromosomes of the larval salivary glands; (b) secretion of glycoprotein glue into the lumen of the salivary glands; (c) evagination of imaginal discs of adult wings and legs; and (d) partial rescue of wild-type phenotypic expression in ecdysone-deficient mutants (ecdysoneless1 (ecd1) and suppressor of forkedts67g (su(f)ts67g). In all these bioassays on Drosophila larvae, the two purely synthetic hydrazines exhibited similar dose-response relationships as did the natural steroid hormone, 20-hydroxyecdysone. In assays involving induction of early chromosomal puffs (74EF, 75B) or regression of the preexisting puffs (25AC, 68C), the dosages required for induction of standard ED-50 effects were one order of magnitude larger for the hydrazines in comparison with 20-hydroxyecdysone. In the assays related to glycoprotein glue secretion, evagination of imaginal discs, or rescue of phenotypic expression in ecdysone-deficient mutants, 20-hydroxyecdysone was two orders of magnitude more active than RH-5849 and RH-5992. We conclude that, in spite of these quantitative differences, the two hydrazine compounds studied are able to duplicate in Drosophila larvae the complex of qualitative biological effects that are a prerequisite for ecdysteroid hormones. The hormonomimetic stimulus of RH-compounds has been given at very low, intracellular, chromosomal level.     

Effects of the juvenile hormone analogue methoprene and dietary protein on male melon fly Bactrocera cucurbitae (Diptera: Tephritidae) mating success

The effect of access to dietary protein (P) and the topical application of a juvenile hormone analogue (methoprene (M)) on mating behaviour of male melon fly Bactrocera cucurbitae was assessed in the laboratory and in field cages. Age, dietary protein and methoprene application increased the mating success and influenced the mating behaviour. Treatment with methoprene (M+) to protein-deprived (P−) males had only a modest effect on the acceleration of sexual maturity, but application of methoprene (M+) to protein-fed (P+) males greatly accelerated sexual maturity. Protein diet (P+) increased mating success of males in comparison to protein-deprived (P−) males. Protein and methoprene have a synergistic effect on mating behaviour, since M + P+ treated males exhibit reduced mating latency and achieved higher mating in younger ages than methoprene and/or protein-deprived males. Copulation duration was correlated with nutritional status and M + P+ males copulated longer at the age of advanced sexual maturity than M − P+ males. Our results suggest that in this species with a lek mating system, females discriminate between the males based on their sexual signals, which were influenced by protein in the adult diet, methoprene application and age. The results are discussed in the light of mating competitiveness of precocious treated young males and their relevance to Sterile Insect Technique application against this pest species.     

Binding of juvenile hormone,methoprene and hydroprene to haemolymph proteins of larvae of the southwestern corn borer,Diatraea grandiosella

The binding of tritiated juvenile hormone I, and the juvenile hormone mimics, methoprene and hydroprene (alkyl-3,7,11-trimethyl-2,4-dodecadienoates), to proteins of the haemolymph of larvae of the southwestern corn borer, Diatraea grandiosella, was examined in vitro. The techniques of analytical isoelectric focusing, disc electrophoresis and gel filtration were employed. Juvenile hormone was shown to bind to a high affinity low molecular weight protein in the haemolymph of diapausing larvae. This protein has an apparent dissociation constant of about $\text{3.1 × 10}{}^{\mathrm{-7}}$ M at 4°C, an isoelectric point of about 4.85, and a molecular weight of about $\text{3 × 10}{}^4$. Juvenile hormone was also bound to at least one low affinity high molecular weight protein fraction. High juvenile hormone binding activity was found in the haemolymph of newly diapaused larvae. Methoprene and hydroprene bound selectively to a different protein fraction present in the haemolymph of pre-diapausing larvae. This protein fraction had a low relative mobility when subjected to electrophoresis at pH 8.9.A preliminary study of the binding of tritiated juvenile hormone I to proteins of the haemolymph of pre-diapausing larvae of the southern cornstalk borer, Diatraea crambidoides, was also conducted in vitro. A low molecular weight binding protein with identical electrophoretic properties to that of D. grandiosella was present. The high molecular weight binding protein fraction of the two species, however, had different electrophoretic properties.