Modulatory influence of juvenile hormone analogue (JHa) and 20-hydroxyecdysone on lipophorin synthesis in red cotton bug, Dysdercus cingulatus Fabr

Topical supply of methoprene, a juvenile hormone analogue (JHa) caused notable morphological disturbance in insects. Topical supply of methoprene to newly emerged adult female D. cingulatus caused notable disturbance and induced a dramatic reduction in the total haemolymph protein pattern and lipophorin production in tissues like fat body, ovary and haemolymph. Total protein concentration in haemolymph also showed significant reduction in 1 day old insects but increased slightly as age advanced. Application of 20-hydroxyecdysone (20-HE) to 2-day-old adult female stimulated protein synthesis intensively. Lipophorin levels in fat body and ovary also simultaneously increased. Densitometric analysis revealed that methoprene inhibits while 20-HE stimulates lipophorin production in D. cingulatus.     

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.     

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.     

Vitellogenin concentrations in the haemolymph and ovaries of Ixodes scapularis ticks during vitellogenesis

The vitellogenin and vitellin concentrations in the haemolymph and ovaries of Ixodes scapularis females were determined using a double sandwich enzyme-linked immunosorbent assay. The level of vitellogenin in the haemolymph began to increase just prior to tick detachment from the host and continued to increase until 2 days after detachment. There was a slight decrease in the vitellogenin level 4 days after detachment, but a second peak was observed approximately 5 days after oviposition. Subsequent to oviposition, the vitellogenin levels in the haemolymph quickly decreased. The concentration of vitellogenin in the haemolymph ranged from 1.55 to 11.48 g l^-1 during the period after dropping from the host through oviposition. The concentration of vitellin in the ovaries began to increase as the female began rapid engorgement (0.03 mg per female) and declined after oviposition (0.1 mg per female).     

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。     

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.     

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.     

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.     

Effect of 20-hydroxyecdysone and haemolymph on oogenesis in the ixodid tick Amblyomma hebraeum

Earlier work from our laboratory indicated that injection of 20-hydroxyecdysone (20E) into non-vitellogenic female Amblyomma hebraeum ticks stimulates the synthesis of vitellogenin (Vg), but not its uptake into oocytes [Friesen, K., Kaufman, W.R., 2004. Effects of 20-hydroxyecdysone and other hormones on egg development, and identification of a vitellin-binding protein in the ovary of the tick, Amblyomma hebraeum. Journal of Insect Physiology 50, 519-529]. In contrast, Thompson et al. [Thompson, D.M., Khalil, S.M.S., Jeffers, L.A., Ananthapadmanaban, U., Sonenshine, D.E., Mitchell, R.D., Osgood, C.J., Apperson, C.S., Roe, M.R., 2005. In vivo role of 20-hydroxyecdysone in the regulation of the vitellogenin mRNA and egg development in the American dog tick, Dermacentor variabilis (Say). Journal of Insect Physiology 51, 1105-1116] demonstrated that injection of 20E into virgin female Dermacentor variabilis ticks stimulated both vitellogenesis and Vg uptake into oocytes. In addition to the species difference in the two studies there were substantially different methods for injecting 20E. In our earlier work we injected small partially fed ticks after removing them from the host. Thompson et al. injected the females while they remained attached to the host. So in this study we repeated our earlier experiments on A. hebraeum using on-host injection. We also injected 20E into off-host ticks with or without haemolymph collected from engorged ticks (days 2-10 post-engorgement), or from large partially fed mated ticks in the rapid phase of engorgement, to see whether we might detect a 'vitellogenin uptake factor' (VUF) in haemolymph. Off-host injection of 20E (0.45mug/g body weight (bw)) did not induce ovary development beyond that of vehicle-injected controls. But ticks in this study, receiving 20E plus haemolymph from engorged ticks, showed a significant increase in ovary weight beyond that of 20E alone (1.31+/-0.05% bw; 34 for 20E plus haemolymph and 1.03+/-0.05% bw; 25 for 20E alone). However, in normal engorged A. hebraeum, the ovary exceeds 7% bw at the onset of oviposition. As in our earlier work, in this study 20E stimulated Vg-synthesis (3.9+/-0.5mgVt-equivalents/ml) beyond that occurring in vehicle-injected ticks (0.76+/-0.14mgVt-equivalents/ml), and there was a further increase in ticks injected with 20E plus haemolymph from engorged ticks (8.9+/-1.0mgVt-equivalents/ml). On-host injection of 20E alone (6mug20E/g bw) did not produce a statistically significant increase in oocyte length over that of vehicle-injected controls, whereas on-host injection of 20E plus engorged haemolymph resulted in significantly larger oocytes (261+/-57mum) compared to vehicle-injected controls (132+/-11mum), compared to 20E alone (131+/-12mum), or haemolymph alone (124+/-24mum). There was a marked stimulation of Vg-synthesis by 31mug20E/g bw (6.0+/-1.5mgVt-equivalents/ml) compared to vehicle-injected controls (1.02+/-33mgVt-equivalents/ml). Vt accumulation by ovaries was significantly greater in ticks treated with haemolymph (12+/-3mugVt/mg ovary) or 20E plus haemolymph (56+/-26mugVt/mg ovary) compared to vehicle-injected controls (5.1+/-1.5mugVt/mg ovary). There was also a significant effect of 6mug20E/g bw plus engorged haemolymph on ovary weight (1.74+/-0.29% bw) compared to vehicle-injected ticks (0.95+/-0.10% bw), but not compared to ticks injected with 20E alone (1.25+/-0.19% bw). We conclude that at least some of the differences observed between the two laboratories relate to the species difference, and that there is some evidence that the engorged haemolymph of A. hebraeum contains a VUF.     

Vitellogenin synthesis in blood-fed Aedes aegypti in the absence of the head,thorax and ovaries

A blood meal initiates oöcyte maturation in Aedes aegypti, and we have used rocket immunoelectrophoresis to investigate the function of midgut, ovaries, and head in the onset of vitellogenin synthesis. Non-blood-fed females and those fed blood (by enema) containing soybean trypsin inhibitor never contained vitellogenin. This demonstrates that the pressure of an undigested blood meal on stretch receptors of the midgut plays no role in the induction of vitellogenin synthesis, rather the stimulus is a digestion product of blood.When females were ovariectomized or decapitated and then fed blood, the haemolymph contained newly synthesized vitellogenin 24 h later. This was also demonstrated in isolated ovariectomized abdomens. Apparently, induction of vitellogenin synthesis does not require factors from either the head, thorax, or ovaries. When ovariectomy or decapitation was postponed after a blood meal, the level of vitellogenin in the haemolymph rose. Therefore, interaction of factors from the head and ovaries maintain the synthesis needed for oöcyte maturation.     

Vitellogenins and v itellins of the Mediterranean field cricket, Gryllus bimaculatus: isolation, characterization and quantification

ABSTRACT In the Mediterranean field cricket, Gryllus bimaculatus de Geer, two immunochemically distinct female specific proteins were identified in haemolymph (Vg I and II) and in vitellogenic oocytes (Vn I and II). The corresponding Vgs and Vns seem to be immunochemically identical. On polyacrylamide gels Vg I and II as well as Vn I and II could not be clearly separated because Vg I and Vn I produced broad bands. The M_rs of both Vgs and Vns are approximately 525 kD. Upon dissociation by sodium dodecyl sulphate the Vgs and Vns each yielded at least nine polypeptides in the range of 50–215 kD. Haemolymph Vg titres were determined by rocket immunoelectrophoresis. The time of first appearance of Vg is temperature-dependent and correlates with the onset of Juvenile Hormone synthesis by the corpora allata. Peak values of Vg were 17 μg μl^-1 haemolymph in normal vitellogenic females. After ovariectomy, Vg appeared at the normal time, but then increased to about 55 μg μl^-1 haemolymph. Ovariectomy also resulted in an accumulation of Vg in the fat bodies. Enforced virginity did not affect the haemolymph Vg titre. In allatectomized or head-ligated females no Vg/Vn was detectable. Topical application of 100 μg Methoprene onto head-ligated females induced Vg synthesis, whereas injection of 20-hydroxyecdysone did not induce Vg synthesis.     

Hormonal control of vitellogenin synthesis in Oncopeltus fasciatus

Previous work indicated the existence of two vitellogenins (A and B) in the haemolymph of Oncopeltus fasciatus, and that vitellogenin B was juvenile hormone (JH)-dependent whereas A was not (Kelly and Telfer, 1977). We have extended these results using several electrophoretic techniques in combination with limited proteolysis of key proteins to show that (1) vitellogenin B is present in eggs in a modified form while vitellogenin A cannot be detected in eggs. (2) Vitellogenin A may be a precursor of B since it has a molecular weight of 200,000D, approximately three times that of vitellogenin B (68,000D) and analysis by limited proteolysis shows that two proteins to be nearly identical. (3) Neither ovariectomy nor treatment with the anti-allatotropin, precocene II prohibits the appearance of vitellogenins A and B in the haemolymph. (4) Injection of ecdysone or 20-hydroxyecdysone into adult, male Oncopeltus fasciatus induces the appearance of both vitellogenin A and B in the haemolymph, suggesting the possible involvement of ecdysteroids in the control of vitellogenin synthesis in this species. (5) We have no evidence for JH control of the synthesis of vitellogenin, however, the ratio of vitellogenin A to B in the haemolymph is higher in the precocene-treated females.     

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.     

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.     

Vitellogenin mRNA in locust fat body: coordinate induction of two genes by a juvenile hormone analog

Levels of vitellogenin (Vg) mRNA in Locusta migratoria fat body were determined as indicators of gene expression induced by the juvenile hormone analog methoprene. After injection of methoprene into juvenile hormone-deprived locusts, excised fat bodies were cultured with [3H]leucine for immunochemical assay of Vg synthesis, and RNA was assayed for Vg mRNA content by hybridization with probes from the previously cloned locust Vg genes A and B. In general, the rise in Vg mRNA paralleled the rise in Vg synthesis. During the primary response to methoprene (in female locusts in which the corpora allata had been destroyed immediately after emergence), Vg mRNA was first detected after 18-24 hr and accumulated rapidly between 36 and 48 hr. The secondary response (in locusts allatectomized during vitellogenesis and kept until Vg disappeared) was accelerated, as Vg mRNA was detectable at 12 hr and titers rose steeply after 18 hr. When Vg synthesis was prematurely induced by injection of methoprene into fifth-stage female larvae, the kinetics of mRNA accumulation were similar to those of primary stimulation in the adult. After allatectomy of vitellogenic females, fat body Vg mRNA decayed with a half-life of about 24 hr, roughly paralleling the decline in Vg synthesis. Assays with the two Vg probes showed coordinate accumulation of gene A and gene B messages under all conditions tested: during primary and secondary stimulation in adult females and in the low-level response obtained by treating male larvae with methoprene.     

保幼激素类似物对德国小蠊卵黄发生及繁殖的影响

应用ELISA、单向免疫扩散法,研究了保幼激素类似物（灭幼宝）对德国小蠊Blattella germanica卵黄发生的影响;同时,观察了灭幼宝对德国小蠊繁殖的影响。灭幼宝促进了德国小蠊脂肪体Vg的合成,使血淋巴Vg的浓度明显增高,出现时间早于对照组;且变化规律不同于对照组,呈双峰模式。灭幼宝处理组昆虫卵巢中Vt的含量在第5天、第7天明显高于对照组,而在第9天明显下降,仅为对照组的50%左右,而此时对照组卵巢的Vt含量则上升到处理组第7天水平。灭幼宝处理新羽化的雌虫,导致不育。     

Nutritional and hormonal effects on storage proteins in the silkworm,Bombyx mori L.

Starvation of 48 h old fifth instar larvae depressed storage protein titres initially for 48 h but retained the levels comparable to control thereafter, possibly due to nutrients obtained during the 48 h feeding after fourth ecdysis. After an initial decline ligated larvae accumulated maximum storage proteins in haemolymph. This is because of inhibitory juvenile hormone titre at the basal level besides the appropriate release of 20-hydroxyecdysone from the ectopic source(s). Injection of methoprene (10 Μg/larva) repressed accumulation of storage proteins while 20-hydroxyecdysone (10 Μg/larva) increased the same. P-soyatose injection to starved and ligated larvae accelerated storage protein accumulation in haemolymph, signalling nutrient indispensability for initiation of storage protein synthesis at the appropriate time of last instar development inBombyx mori.     

The effects of allatectomy and ovariectomy on haemolymph proteins and lipid mobilization in Locusta

After ovariectomy the concentrations of diacylglycerol and protein in the haemolymph increase markedly. The increased diacylglycerol is associated with increased quantities of the ‘heparin-precipitable’ protein (lipoprotein A) that carries diacylglycerol in the blood of normal resting locusts. After the injection of adipokinetic hormone (AKH), the blood of ovariectomized locusts contains only slight quantities of the ‘heparin-soluble’ lipoprotein A⁺ whereas this forms in large amounts in the blood of sham-operated locusts after AKH injection. After allatectomy, the increase in the adipokinetic response is slower and the full level of responsiveness observed in sham-operated locusts is never attained. Nevertheless, allatectomized locusts develop a marked adipokinetic response which tends to stabilize as they age; it does not deteriorate as it does in aged sham-operated locusts.The effects of ovariectomy on blood metabolites can be prevented completely by allatectomy, but only partially by cautery of the cerebral neurosecretory cells. Treatment with a juvenile hormone analogue (JHA R-20458) counteracts the effects of allatectomy in ovariectomized locusts.     

龟纹瓢虫卵黄蛋白的分子特性及发生动态

研究了龟纹瓢虫Propylea japonica (Thunberg) 卵黄蛋白的基本特性以及卵黄发生过程中卵黄蛋白的动态变化。PAGE和SDS-PAGE实验表明,龟纹瓢虫卵黄蛋白分子量为294.81±40.70 kD,并由分子量分别为144.68±0.03 kD和51.23±0.27 kD的两种亚基组成。对卵黄蛋白的氨基酸组成和含量分析发现,其必需氨基酸总量占57.48％,略高于非必需氨基酸,其中谷氨酸（Glu）含量最高,为15.26％;色氨酸（Trp）和蛋氨酸（Met）含量较低,分别为0.50％和0.11％。采用间接竞争ELISA法,系统测定了龟纹瓢虫成虫期脂肪体、血淋巴和卵巢中卵黄蛋白的动态变化,结果表明：脂肪体是卵黄原蛋白合成的场所,卵黄原蛋白的合成始于羽化后第2天;脂肪体、血淋巴和卵巢中卵黄原蛋白的滴度在羽化后第4天开始迅速上升,至成虫期的第8天左右达到高峰期。