Vitellogenin synthesis,processing and hormonal regulation in the tick,Ornithodoros parkeri (Acari:Argasidae)

This study was undertaken to determine the processing of vitellogenin (Vg) and the role of juvenile hormone (JH) in the regulation of vitellogenesis in the tick Ornithodoros parkeri. Ticks usually require a blood meal to induce vitellogenesis. However, we have shown that a pyrethroid, cypermethrin (CyM), can stimulate Vg synthesis in unfed Ornithodoros moubata females. Vg concentration and synthesis were analyzed by SDS-PAGE spotting-scanning and fluorography using [³⁵S]-methionine. Although unfed females show high titers of Vg in the hemolymph, this is not due to new synthesis. Vg synthesis stimulated by engorgement increases beginning on day 2 after engorgement and reaches a maximum level on day 8. Vg is synthesized in the fat body, secreted into the hemolymph and then processed and incorporated into the ovaries as vitellin. JH I, II and III, methoprene (JHA), and CyM were topically applied to unfed females and Vg synthesis analyzed on day 5 by fluorography. JH and JHA did not stimulate Vg synthesis. CyM stimulated Vg synthesis but not ovarian development. These preliminary results indicate that JH does not function in the regulation of vitellogenin synthesis in this species.     

Cypermethrin induction of vitellogenesis and ovarian development in unfed adult female Ornithodoros moubata (Acari: Argasidae)

Summary

Vitellogenesis in ticks is known to be induced by engorgement and mating. In this paper, the synthetic pyrethroid cypermethrin (CyM) is shown to induce production of yolk protein precursor, vitellogenin (Vg), and ovarian development in unengorged mated adult female Ornithodoros moubata. The levels of Vg found in the hemolymph and ovarian development induced by CyM were dose-dependent. i.e., CyM doses of more than 0.2 and 1.0 μg/tick were needed for significant increase of Vg titer in the hemolymph and yolk deposition in oocytes, respectively. Immunological and electrophoretical analyses of Vg and Vitellin (Vn) induced by CyM were identical with those induced by engorgement. Vg titer induced by CyM in unengorged females followed approximately the same time course as that in the normal engorged females. However, Vg titer induced by CyM continued to increase after day 8 and reached a maximum (95 μg/μ1) on day 10 after treatment, while Vg titer induced by engorgement decreased again after reaching a maximum (60 μg/μ1) on day 6, correlated with yolk Vn deposition in oocytes. Ovarian development induced by even high doses (10 or 20 μg/tick) of CyM was slow compared to normal development stimulated by engorgement. Oviposition was not observed in females treated with CyM.     

Juvenile hormone analog and injection effects on locust hemolymph protein synthesis

In adult female Locusta migratoria, at about day 8 after eclosion, when vitellogenin (Vg) is first produced as a result of induction by juvenile hormone (JH), the intensity of hemolymph protein electrophoretic bands at about 75 kDa and 20 kDa increases sharply, suggesting that JH may induce additional proteins. A major component of the elevated protein is persistent storage protein (PSP; subunit 74 kDa). Administration of the JH analog, methoprene, to precocene-treated adult locusts was followed by a rise in hemolymph levels of PSP but not in apolipophorin III (19 kDa), identified immunochemically and electrophoretically. The synthesis of PSP in adult fat body was confirmed by incorporation of [³H]leucine. At 48 h after treatment with methoprene, Vg synthesis was induced in females (as previously observed) and synthesis of PSP in both sexes was elevated above controls, while synthesis of apolipophorin III was not stimulated. We conclude that in adult locust fat body the synthesis of several proteins responds in different ways to the JH analog: Vg (and a 21 kDa protein described elsewhere) is induced de novo solely in females; PSP (and a 19 kDa protein described elsewhere) is stimulated in both sexes but is not fully JH-dependent; apolipophorin III is not stimulated. In these experiments, methoprene was administered both by injection in mineral oil and topically in acetone. After injection of mineral oil as a vector control, incorporation into secreted proteins was stimulated at 24 h, presumably due to a wound effect; topical application of acetone avoids this effect and is a preferred route for administration of JH analog. © 1992 Wiley-Liss, Inc.     

Vitellogenin levels in hemolymph, ovary and hepatopancreas of the freshwater crayfish Cherax quadricarinatus (Decapoda: Parastacidae) during the reproductive cycle

The freshwater crayfish Cherax quadricarinatus is a tropical species of great interest for aquaculture. Vitellogenin (Vg), a lipoprotein precursor of the vitellum accumulated in spawned eggs, can be synthesized in the ovary and/or hepatopancreas of most crustaceans, being the hemolymph the way for transporting Vg throughout the reproductive cycle. Concentration of Vg in hemolymph, ovary and hepatopancreas of Cherax quadricarinatus adult females was measured by means of ELISA, specifically developed after purifying the native Vg. Measurements were made at four periods of the reproductive cycle: pre-reproductive, mid-reproductive, late reproductive and post-reproductive. Besides, both hepatosomatic (HSI) and gonadosomatic (GSI) indexes were determined in each period. Significant variations in Vg levels were detected in both hemolymph and hepatopancreas, being the highest values observed during the mid-reproductive period. Besides, such variations were positively correlated to the HSI. A positive correlation between Vg levels in hepatopancreas and ovary was also seen. These results support previous evidences about the central role of the hepatopancreas as a site of Vg synthesis in the studied species, together with the relevancy of hemolymph for transporting Vg from the hepatopancreas to the ovary. For aquaculture purposes, Vg monitoring in hemolymph could be used as a non-injurious method, to check the reproductive activity of C. quadricarinatus females.     

Ovarian Ecdysteroidogenesis in Both Immature and Mature Stages of an Acari,Ornithodoros moubata

Ecdysteroidogenesis is essential for arthropod development and reproduction. Although the importance of ecdysteroids has been demonstrated, there is little information on the sites and enzymes for synthesis of ecdysteroids from Chelicerates. Ecdysteroid functions have been well studied in the soft tick Ornithodoros moubata, making this species an excellent candidate for elucidating ecdysteroidogenesis in Chelicerates. Results showed that O. moubata has at least two ecdysteroidogenic enzymes, Spook (OmSpo) and Shade (OmShd). RNAi showed both enzymes were required for ecdysteroidogenesis. Enzymatic assays demonstrated OmShd has the conserved functions of ecdysone 20-hydroxylase. OmSpo showed specific expression in the ovaries of final nymphal and adult stages, indicating O. moubata utilizes the ovary as an ecdysteroidogenic tissue instead of specific tissues as seen in other arthropods. On the other hand, OmShd expression was observed in various tissues including the midgut, indicating functional ecdysteroids can be produced in these tissues. In nymphal stages, expression of both OmSpo and OmShd peaked before molting corresponding with high ecdysteroid titers in the hemolymph. In fed adult females, OmSpo expression peaked at 8–10 days after engorgement, while OmShd expression peaked immediately after engorgement. Mated females showed more frequent surges of OmShd than virgin females. These results indicate that the regulation of synthesis of ecdysteroids differs in nymphs and adult females, and mating modifies adult female ecdysteroidogenesis. This is the first report to focus on synthesis of ecdysteroids in ticks and provides essential knowledge for understanding the evolution of ecdysteroidogenesis in arthropods.     

QUANTITATIVE VARIATION OF ECDYSTEROIDS OF IXODID TICKS

Abstract  In order to explore the role of ecdysteroids in development and reproduction of ixodid ticks, we studied the quantitative variation of ecdysteroids in the hemolymph, synganglion, ovary and whole body of the female ixodid ticks, Dermacentor niveus and Haemaphysalis longicornis , before and after engorgement and oviposition by HPLC and RIA. The ecdysteroid content in eggs of these ticks was determined by HPLC. The results indicated that before engorgement the quantitative variation of ecdysteroids in the whole female body was not significant, but their levels increased rapidly after engorgement. The ecdysteroid titer in hemolymph was peaked on the 5th day after engorgement, which was one day prior to oviposition. It may be regarded as a singnal of oviposition. In the synganglion the peak of ecdysteroid level occurred also on the 5th day after engorgement. This is coincident with the secretory activity of neurosecretory cells of synganglion. From the 3rd day after engorgement until oviposition the ecdysteroid level in the ovary increased rapidly. Ecdysteroids were detected in eggs of H. longicornis too. They stem from ovary and accumulated with the process of embryonic development.     

Fat body is the site of vitellogenin synthesis in the soft tick,Ornithodoros moubata

Summary Vitellogenin (Vg) synthesis in cultured tissues was analysed biochemically in a soft tick,Ornithodoros moubata. Nine tissue fractions dissected from reproductive females were incubated in vitro in a specially designed Ringer containing³⁵S-methionine. The synthesis of total protein and Vg was assayed by the radioactivity incorporated into precipitates with trichloroacetic acid and antivitellin (Vn)-serum, respectively. Fat body was the most active tissue in Vg synthesis, which comprised 46% of the Vg synthesis by all tissues and 42% of total protein synthesis by fat body. Protein synthesized by the fat body and precipitated with anti-Vn-serum was shown by electrophoresis and fluorography, to consist of six radioactive polypeptides corresponding to the components of Vg. Vg synthesized in cultured fat body was first accumulated in the tissue and secreted into the medium during incubation. Some tissues other than fat body showed low Vg synthesis (in each, less than 12% of total protein synthesis) which, however, may be due to contamination by fat body cells as seen with the scanning electron microscope (SEM). SEM also showed that fat body cells in the active stage of Vg synthesis expanded about 10-fold in length. Immunohistochemical analysis showed a very strong reaction with anti-Vn-IgG in the cytoplasm of fat body from reproductive females. Fat body from unfed females and other tissues including midgut, did not show any specific fluorescence. A positive reaction was obtained with developing oocytes. These results indicate that the fat body is the only site of Vg synthesis in this tick.Abbreviations Vg vitellogenin - Vn vitellin - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - SEM scanning electron microscopy - TCA trichloroacetic acid     

硬蜱中蜕皮激素含量的变化(英语)

为阐明蜕皮激素在硬蜱发育和生殖中的作用,用高效液相色谱法(HPLC)和放射免疫测定法测定了银盾革蜱和长角血蜱中饱血和产卵前后雌虫整体、血淋巴、合神经节及卵巢中蜕皮激素含量的变化.此外,还用HPLC测定了长角血蜱的卵和幼虫中蜕皮激素的含量.结果表明:在饱血前雌虫整体中蜕皮激素含量的变化不大,饱血后迅速增加.血淋巴中的蜕皮激素在饱血后第5天(即产卵前1天)达到高峰,可做为产卵的信号.合神经节中蜕皮激素含量的高峰在饱血后第5天,与血淋巴中高峰出现的时间一致,这与神经分泌细胞的分泌活性变化相吻合.从饱血后第3天起,卵巢中蜕皮激素含量增加很快,直到产卵时止,在长角血蜱的卵中也测到α-蜕皮素,来源于卵巢,随胚胎发育进程其含量逐渐增加.卵产出后第4天到第12天,蜕皮激素的含量增加十几倍.     

Quantification of vitellogenin-mRNA during maturation and breeding of a burying beetle

Burying beetles (Nicrophorus orbicollis) are unusual in that to breed they require an unpredictable and valuable resource, a small carcass. Thus the timing of reproduction is unpredictable and beetles' physiological response must be fast. We hypothesized that their pattern of vitellogenin (Vg) synthesis might reflect these requirements. We examined the expression of two Vg genes (sequenced for this study) during sexual maturation and through a reproductive bout. Vg-mRNA, juvenile hormone (JH) titers, ovarian development, and hemolymph concentrations of Vg were quantified in the same individuals. All four variables gradually increased during maturation to peak 15-20 days after eclosion. Twelve hours after the discovery of a carcass, a few hours before oviposition, mRNA was high, hemolymph Vg had decreased, JH and ovarian weight had increased. After oviposition, mRNA was low, hemolymph Vg concentrations and JH were high. This is consistent with our hypothesis that beetles produce and store Vg in the hemolymph prior to the discovery of a breeding resource and replace it quickly. Partial regression of these variables (with the effect of time removed) indicated that JH was not correlated with mRNA, hemolymph Vg, or ovarian weight at any time. Thus the role of JH as a gonadotropin remains unclear.     

Hemolymph proteins in ticks

In comparison to insects and Crustacea, our knowledge of the predominant hemolymph proteins in ticks is minimal. The hemolymph protein most studied in ticks has been vitellogenin (Vg). Vg is synthesized by the tick fat body after female adults obtain a blood meal, is released into the hemolymph and is absorbed by developing oocytes as vitellin (Vn). Much of what we know about Vg is from studies of Vn. In general, the carbohydrate, lipid and amino acid composition is similar to insects except that in the tick, Vg contains heme, most likely from the digestion of host hemoglobin. In the American dog tick, Dermacentor variabilis, Vg is comprised of two native proteins and seven subunits on SDS-PAGE. Vg has been characterized in five tick species but the amino acid sequence is not yet available. Another predominant hemolymph protein, apparently a carrier protein (CP), has recently been studied in two tick species. This protein is found in the hemolymph of both male and females adults, in adult tissues outside of the hemolymph in some tick species, in coxal fluid of soft ticks and in whole body homogenates from eggs, larvae and nymphs. CP from the hard tick, D. variabilis, contains cholesterol, phospholipids, monoacylglycerides, triacylglycerides, free fatty acids, carbohydrate and heme. Under identical assay conditions, the analogous protein in the soft tick, Ornithodoros parkeri, did not contain heme. CP in the American dog tick consists of two subunits, one of which has 61% identity to the biliprotein, artemocyanin, from the fairy shrimp. CP is identical to a heme-lipoprotein (HeLp) from Boophilus microplus. The exact roles of CP and HeLp have not yet been fully determined, but they apparently are important in heme sequestration and as a storage depot for protein and lipid. Macroglobulin, lectin, antimicrobial, JH binding, JH esterase, and other tick hemolymph proteins are also discussed.     

Dynamics of vitellogenin mRNA expression and changes in hemolymph vitellogenin levels during ovarian maturation in the giant freshwater prawn Macrobrachium rosenbergii

The dynamics of vitellogenin mRNA expression during ovarian maturation in Macrobrachium rosenbergii were examined by measuring hemolymph vitellogenin (Vg) levels and Vg mRNA expression in the hepatopancreas and ovary at differing reproductive stages in both intact and eyestalk ablated animals. Vg mRNA was quantified using real-time RT-PCR and hemolymph Vg was measured by enzyme immunoassay. In intact animals, Vg mRNA levels in the hepatopancreas and hemolymph Vg levels showed a gradual increase during the molt cycle concomitant with increasing gonadosomatic index (GSI), with Vg levels decreasing prior to ecdysis although GSI continued to increase. Eyestalk ablation was seen to accelerate Vg synthesis as well as ovarian maturation, although it did not alter the overall pattern of Vg expression. Vg mRNA expression was negligible in the ovary of both intact and eyestalk ablated animals, confirming that the hepatopancreas is the principal site of Vg synthesis in M. rosenbergii with the ovary being only a minor contributor. This study has shown that Vg synthesis is correlated to ovarian maturation and the molt cycle in M. rosenbergii.     

In vivo role of 20-hydroxyecdysone in the regulation of the vitellogenin mRNA and egg development in the American dog tick, Dermacentor variabilis (Say)

Injection of the hormone 20-hydroxyecdysone (20-E) into partially fed (virgin) female adults of the American dog tick, Dermacentor variabilis, while they are attached and feeding on the rabbit host, initiated the expression of the vitellogenin (Vg) gene, and Vg protein secretion and uptake by the ovary. The induction of egg production by 20-E in this bioassay was dose dependent in the range of 1-50 times the concentration normally found in a replete, vitellogenic female. Ticks examined 4 d after the 50 x treatment were still attached to the host, had numerous enlarged vitellin-filled (brown) oocytes in their ovaries, but had not engorged to repletion. The ovaries reached weights similar to those found in untreated, replete (mated) females (pre-oviposition) while solvent-injected controls demonstrated no increase in oocyte size or increase in ovary weight. An increase in the levels of a putative Vg protein was observed in hemolymph samples collected 1, 2 and 3d post-20-E injection but was not observed in the corresponding solvent controls as determined by native PAGE. Analysis of the ecdysteroid-induced protein by tryptic digestion-mass fingerprinting and BLASTP found that the putative Vg had the strongest match to GP80 (U49934), the partial sequence for the vitellogenin protein from Boophilus microplus. A partial Vg cDNA was cloned and sequenced from replete females of D. variabilis with a high similarity to GP80. Using this message as a probe, Northern blots conducted with RNA collected from partially fed, virgin females 1, 2 and 3d post-20-E injection showed upregulation of the Vg mRNA on all 3 days. Controls injected with solvent only showed no Vg mRNA. Injections with juvenile hormone III did not stimulate Vg expression, oocyte growth or full engorgement. These studies indicate that ecdysteroids and not JH can initiate expression of the Vg gene, Vg protein synthesis and release into hemolymph, and Vg uptake into developing oocytes under bioassay conditions mimicking normal feeding on the host.     

Vitellogenin and egg production in the moth,Heliothis virescens

The characteristics of vitellogenin (Vg) and the relationship between Vg production and egg production in the tobacco budworm, Heliothis virescens, were studied. The relationship between Vg production and juvenile hormone (JH) and the impact of mating on Vg and egg production were also investigated. Vg appears in the hemolymph of H. virescens about 6 h after moth eclosion. Vg may be separated into two apoproteins (ApoVg-I and ApoVg-II) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights were calculated to be 156,065 ± 800 for ApoVg-I and 39,887 ± 323 for ApoVg-II. SDS-PAGE analysis revealed that the female hemolymph Vg polypeptides appear to be identical to those from eggs but are absent in male hemolymph. Vg concentration was significantly higher in mated females than in virgin females of the same age at 48 h after emergence. Rates of egg production increased as Vg production increased; rates of egg production in mated females were significantly higher than those of virgin females at 48, 72, 96, and 120 h postemergence. Vg production is dependent on JH, because hemolymph from decapitated females lacked Vg while that of decapitated females treated with synthetic JH had Vg at levels comparable to similarly aged, normal H. virescens females. Hemolymph JH titers in mated females were significantly higher compared with those in virgin females at all sampling periods. The high JH level in mated females may explain the high Vg and egg production in mated H. virescens. Arch. Insect Biochem. Physiol. 34:287–300, 1997. © 1997 Wiley-Liss, Inc.     

Molecular characterization,expression, and function of Vitellogenin genes in Phytoseiulus persimilis

Vitellogenin (Vg) is an important factor that impacts oocyte maturation, egg formation and embryonic development in Arthropoda. Two orthologs of Vg gene were obtained from the genome of Phytoseiulus persimilis and termed as PpVg1 and PpVg2. Both orthologs belong to the large lipid transfer protein superfamily. The expression of PpVg1 and PpVg2 was low in immatures and male adults, and increased rapidly in female adults after mating, and reached a peak before the first egg was laid (168×? and 20.5×? the level in virgin females, respectively). When PpVg1 and PpVg2 were interfered with dsRNA, the relative expression decreased by 81.0 and 30.9%, respectively, and 7.8 and 31.4% interfered individuals died within 24 h. Among surviving individuals, ca. 51.1 and 44.8% are infertile. Factors that might be related to expression of Vg genes are also discussed.

     

Tebufenozide disrupts ovarian development and function in silkmoths

Adult development and production of up to 400 eggs within the pupal case of female silkmoths are both dependent on 20-hydroxyecdysone (20E), the steroid hormone of insects. When adult development was initiated with tebufenozide, the non-steroidal ecdysteroid agonist, instead of 20E, full development of all epidermal tissues like the wing was witnessed, but ovarian growth and egg formation was minimal. Administration of tebufenozide to female pharate adults caused disruption of the follicular epithelium, produced nurse cell damage, and inhibited oogenesis. Reduced ability to synthesize RNA and protein accompanied these tebufenozide induced morphological disturbances of the follicles. In vivo accumulation of vitellogenin (Vg) from the hemolymph was reduced in tebufenozide treated female ovaries as well as their ability to accumulate Vg in vitro. Determination of protein staining intensity and antibody reactivity of Vg pointed out that hemolymph Vg level remained fairly constant all through adult development whether induced by 20E or tebufenozide. Measurement of hemolymph volumes and hemolymph Vg levels of control and experimental animals allowed us to conclude that egg development involves the uptake of all the hemolymph proteins and not Vg alone. The loss of hemolymph that accompanies egg maturation was considerably reduced in tebufenozide initiated female pharate adults. 20E could not overcome ovarian growth inhibitory effects of tebufenozide. Dual mechanisms, one involving ecdysteroid antagonist action at the beginning of development, and the other unrelated to that function during heightened egg formation, are needed explain the biphasic inhibitory actions of tebufenozide on silkmoth ovaries.     

THE ROLE OF ECDYSTEROIDS IN THE REPRODUCTIVE DIAPAUSE OF DERMACENTOR NIVEUS NEUMANN

Abstract  The ecdysteroid levels in hemolymph, ovary, synganglion and whole body of diapausing female Dermacentor niveus were detected by HPLC, and compared with the results of nondiapausing female. It is revealed that the ecdysteroid levels in hemolymph and ovary of diapausing female are similar basically to that of nondiapausing female in the first few days after engorgement. From the 10th day after engorgement, the ecdysteroid levels of diapausing female decreased and even became distinctly lower than that of nondiapausing female. The paucity of ecdysteroids in these individuals would influence the normal development of oocytes. In order to explore the effect of ecdyateroids on the diapausing female, we injected 20-hydroxyecdysone with different dosages at different time into the ticks, and found that after just complete engorgement the injection with large dosages (10000 and 1375 ng/tick) caused death of the ticks. From 10th to 20th day after engorgement the ecdysteroid levels of diapausing female are lower than that of nondiapausing one before oviposition, the injection with certain dosages 50, 70 and 100 ng/tick> of 20E can accelerate vitellogenesis and terminate reproductive diapause, but the amount of eggs produced by them is less than that produced by nondiapausing female. The termination of diapause in female of ixcdid tick by exogenous ecdysteroids is reported for the first time.     

Biochemical and physiological studies of certain ticks (Ixodoidea): specific gravity of hemolymph and coxal and gut fluids of Argas (Persicargas) persicus and Argas (Persicargas) arboreus (Argasidae)

Specific gravity (sp. gr.) of cell-free hemolymph and gut and coxal fluids was determined at different states of the gonotrophic cycle (unfed + 15 days, engorgement day before and after coxal fluid emission, engorgement + 1 day, oviposition day, and oviposition completion + 1 day) of female Argas (Persicargas) persicus and A. (P.) aboreus (Argasidae). The patterns of hemolymph and gut fluid sp. gr. change differed from each other during the gonotrophic cycle, but both patterns were similar in the 2 Argas species. Hemolymph sp. gr. decreased to a minimum one day after feeding (1.0085 and 1.0081 for persicus and arboreus, respectively), and increased through oviposition to a maximum on oviposition + 1 day (1.0187 and 1.0221). Minimum gut fluid sp. gr. occurred on engorgement day before coxal fluid emission (1.0565 and 1.0697). Afterward, gut fluid sp. gr. increased to a maximum on engorgement day + 1 for persicus (1.1089) and on oviposition day for arboreus (1.0973), and then decreased during oviposition in both species. In each tested state of each species, the sp. gr. was consistently higher in gut fluid than in hemolymph. In each species, coxal fluid and hemolymph sp. gr. were the same on engorgement day.     

Purification,characterization, and synthesis of vitellin from the cabbage butterfly Pieris rapae L.

Vitellin from the cabbage butterfly Pieris rapae L. was purified and characterized by electrophoresis. Vitellin from P. rapae is a phosphorylated glycolipoprotein of 380,000 ± 10,000 molecular weight as determined by nondenaturing polyacrylamide gel electrophoresis. Two subunits with an M_r of 150,000 and 40,000 were obtained from vitellin. The native molecule is thought to be a tetramer composed of two molecules of each of these subunits. The isoelectric point, as determined by isoelectric focusing on polyacrylamide gels, is 6.10. Vitellin and vitellogenin were indistinguishable by immunological methods such as double diffusion and tandem-crossed immunoelectrophoresis. Vitellogenin from the hemolymph and vitellin from the ovary were quantified by rocket immunoelectrophoresis. Vitellogenin and vitellin were first detected in 6-day-old pupae, and their levels increased continuously during ovarian development. Vitellogenin synthesis by the fat body in 4-day-old female pupae could be induced by juvenile hormone I.