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

Partially fed adult female Amblyomma hebraeum ticks were injected with 20-hydroxyecdysone (20E; up to 43 microg/g body weight (bw)), juvenile hormone III (JH III; up to 100 microg/g bw), bovine insulin (up to 2000 mU/g bw), or triiodothyronine (up to 200 ng/g bw) in an attempt to stimulate vitellogenesis. Of these, only 20E stimulated synthesis and release of vitellogenin (Vg). Immunoblot analysis revealed that Vg-synthesis occurred in the fat body. However, consistent with earlier observations suggesting that a distinct signal may be required for Vg-uptake, there was no significant Vg-uptake by oocytes of partially fed, 20E-treated ticks. Because Vg-uptake commonly occurs via receptor-mediated endocytosis (i.e., a specific Vg-receptor), we attempted to identify a vitellin (Vt)-binding protein in ovaries of engorged female ticks. A single 86 kDa Vt-binding protein was identified, even under reducing conditions (2-mercaptoethanol), by a ligand-blotting technique. Sodium salt of suramin (5 mM) inhibited binding of Vt to the 86 kDa protein. However, this protein was also detected in ovaries from small partially fed ticks (50-100 mg), suggesting that the inability of 20E to stimulate Vg-uptake in partially fed ticks may not have been due to the absence of a Vg-receptor.     

The impact of RNA interference of the subolesin and voraxin genes in male <Emphasis Type="Italic">Amblyomma hebraeum</Emphasis> (Acari: Ixodidae) on female engorgement and oviposition

Reducing or replacing the use of chemical pesticides for tick control is a desirable goal. The most promising approach would be to develop vaccines that protect hosts against tick infestation. Antigens suitable for the development of anti-tick vaccines will likely be those essential for vital physiological processes, and in particular those directly involved in feeding and reproduction. In this study genes from Amblyomma hebraeum Koch that encode for subolesin and voraxin were studied in male ticks by RNA interference (RNAi). Males (unfed or fed) were injected with dsRNA of (1) subolesin, (2) voraxin, (3) subolesin plus voraxin or (4) injection buffer, after which they were held off-host overnight and then allowed to feed on rabbits together with normal female A. hebraeum. Females that fed together with male ticks injected with subolesin or subolesin + voraxin dsRNA had a higher rate of mortality, weighed substantially less and produced a smaller egg mass than the controls. However, females feeding with males injected with voraxin dsRNA alone were not significantly different from the controls with respect to mortality, engorged weight or fecundity. However, as assessed by semi-quantitative RT-PCR, voraxin was not silenced in this study, the reasons for which remain unknown. The results of this study suggest that A. hebraeum subolesin is worthy of further testing as a candidate tick vaccine antigen.     

Quantification of vitellogenesis and its control by 20-hydroxyecdysone in the ixodid tick,Amblyomma hebraeum

Ovaries of the ixodid tick, Amblyomma hebraeum Koch, grew rapidly after engorgment as a result of yolk uptake. At 26 °C, oviposition began by day 10 post-engorgement, plateaued on days 16-18, and ended by day 38. Vitellin (Vt) was partially purified from ovaries of day 10 engorged ticks by gel filtration and ion exchange chromatography. This Vt comprises seven major and several minor polypeptides. Two polypeptides (211 and 148 kD) from haemolymph of engorged female ticks corresponded to minor polypeptides of similar molecular weight in the ovary. The haemolymph titre of the 211 and 148 kD polypeptides increased up to the onset of oviposition. These polypeptides were absent in males and non-vitellogenic females (day 0 engorged or day 10 partially-fed females), and were thus designated as vitellogenin (Vg). Antibodies raised against haemolymph Vg211 and 148 recognized these polypeptides in partially purified Vt, as well as six of the seven major polypeptides. Using these antibodies we developed an indirect, competitive ELISA to quantify Vg. Rise in haemolymph Vg-concentration lagged slightly behind the rise in haemolymph ecdysteroid (ES)-concentration, and Vg-synthesis was stimulated by injections of 20E into non-vitellogenic females. These observations indicate that an ES is the vitellogenic hormone in A. hebraeum.     

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.     

Acquired immunity to larvae of Amblyomma marmoreum and Ahebraeum by tortoises, guinea-pigs and guinea-fowl

A study of acquired resistance in guinea-pigs, guinea-fowl and tortoises to larvae of the ticks Amblyomma hebraeum Koch and A.marmoreum Koch (Acari: Ixodidae) showed that repeated infestations of the laboratory host resulted in a 29.3-49.4% reduction respectively in weight of engorged larvae. No resistance was induced in guinea-fowl, the natural host for larvae of both species. A small decline in the mean weight of engorged larvae was observed for both species of ticks after three successive infestations on tortoises. The reduction in weight was more pronounced in A.hebraeum (14.5%) which does not naturally feed on tortoises than for A.marmoreum (8.4%), a common tick on this host. The occurrence of acquired resistance in natural tick host relationships as opposed to unnatural tick host relationships is discussed.     

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.     

Molecular characterization of the vitellogenin receptor from the tick,Amblyomma hebraeum (Acari: Ixodidae)

We have identified the full-length cDNA encoding a vitellogenin receptor (VgR) from the African bont tick Amblyomma hebraeum Koch (1844). VgRs are members of the low-density lipoprotein receptor superfamily that promote the uptake of the yolk protein vitellogenin (Vg), from the haemolymph. The AhVgR (GenBank accession No. JX846592) is 5703 bp, and encodes an 1801 aa protein with a 196.5 kDa molecular mass following cleavage of a 22 aa signal peptide. Phylogenetic analysis indicates that AhVgR is highly similar to other tick VgRs. AhVgR is expressed in only the ovary of mated, engorged females, and is absent in all other female tissues and in both fed and unfed males. Unfed, adult females injected with a VgR-dsRNA probe to knock-down VgR expression experienced a significant delay in ovary development and started oviposition significantly later than controls. These results indicate that the expression of AhVgR is important for the uptake of Vg and subsequent maturation of the oocytes.     

17α-ethinylestradiol cometabolism by bacteria degrading estrone, 17β-estradiol and estriol

17alpha-ethinylestradiol (EE2), the active compound of the contraceptive pill, is a recalcitrant estrogen, which is encountered at ng/l levels in wastewater treatment plant (WWTP) effluents and rivers and can cause feminization of aquatic organisms. The aim of this study was to isolate micro-organisms that could remove such low EE2 concentrations. In this study, six bacterial strains were isolated from compost that cometabolize EE2 when metabolizing estrone (E1), 17beta-estradiol (E2) and estriol (E3). The strains belong to the alpha, beta and gamma-Proteobacteria. All six strains metabolize E2 over E1, at mug/l to ng/l concentrations. In 4 days, initial concentrations of 0.5 mug E2/l and 0.6 mug EE2/l were degraded to 1.8 +/- 0.4 ng E2/l and 85 +/- 16 ng EE2/l, respectively. No other metabolites besides E1, E2, E3 or EE2 were detected, suggesting that total degradation and cleavage of the aromatic ring occurred. This is the first study describing that bacteria able to metabolize E2, can subsequently cometabolize EE2 at low mug/l levels.     

γ-Aminobutyric acid (GABA) and other amino acids in tissues of the tick,Amblyomma hebraeum (Acari: Ixodidae) throughout the feeding and reproductive periods

We assayed a variety of tick (Amblyomma hebraeum Koch; Acari, Ixodidae) tissues for a number of amino acids throughout the feeding and early reproductive periods. Our HPLC assay could detect as little as 2–5 pmol per sample of the following: GABA, glycine, serine, glutamine, alanine, taurine, glutamate and aspartate. All of these amino acids could be detected in the salivary gland, synganglion (=total CNS in acarines), haemolymph, Gené's organ, seminal receptacle and ovary. GABA reached high levels in the salivary gland of freshly engorged ticks (685 nmol g^-1) and in the synganglion it exceeded 1000 nmol g^-1 throughout most of the feeding cycle and the first week post-engorgement. GABA also reached a peak titre in the haemolymph of 40 nmol ml^-1. Taurine levels peaked at 1065 nmol g^-1 in the salivary gland from large partially fed ticks. Glutamate and aspartate were likewise found in the salivary gland and synganglion at high concentrations. For most of the amino acids there is insufficient information to correlate these titres (and fluctuations of titres) to neuromodulatory functions. It is possible, however, that the high GABA titre in the salivary gland of engorged ticks is correlated with an augmented level of fluid secretion.Deceased: Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada, T6G 2E9.     

The effect of orally administered Aloe marlothii leaves on Boophilus decoloratus tick burdens on cattle

The effect of a decoction of pulverized aloe leaves (Aloe marlothii) mixed with tap water and administered orally through a gastric tube was determined against ticks on cattle. The decoction was administered to six randomly selected calves seven times at 3-day intervals while the remaining calves received an equivalent amount of tap water only. Five hours after first treatment the calves were infested with Boophilus decoloratus larvae hatched from one gram of eggs. Resultant engorged female ticks were collected, counted and weighed daily. Fertility estimates were determined for 30 engorged female ticks collected from each calf in both the treated and control groups of calves. The treatment had no significant effect on total or daily numbers of engorged females collected per group, nor on their biomasses. Fertility estimates showed the treatment group to yield a marginally higher, yet insignificant, egg laying response (ELR) and reproductive estimate (RE).     

PCR-based detection of Babesia bovis and Babesia bigemina in their natural host Boophilus microplus and cattle

PCR and nested-PCR methods were used to assess the frequency of Babesia bovis and Babesia bigemina infection in Boophilus microplus engorged females and eggs and in cattle reared in an area with endemic babesiosis. Blood and the engorged female ticks were from 27 naturally infested calves and 25 crossbred cows. The frequency of both Babesia species was similar in calves and cows (P>0.05). Babesia bovis was detected in 23 (85.2%) calves and in 25 (100%) cows and B. bigemina was detected in 25 (92.6%) calves and in 21 (84%) cows. Mixed infections with the both Babesia species were identified in 42 animals, 21 in each age category. Of female ticks engorged on calves, 34.9% were negative and single species infection with B. bigemina (56.2%) was significantly more frequent (P<0.01) than with B. bovis (4.7%). Most of the females (60.8%) engorged on cows did not show Babesia spp. infection and the frequency of single B. bovis infection (17.6%) was similar (P>0.05) to the frequency of single B. bigemina infection (15.9%). Mixed Babesia infection was lower (P<0.01) than single species infection in female ticks engorged either in cows (5.7%) or in calves (4.3%). An egg sample from each female was analysed for the presence of Babesia species. Of the egg samples from female ticks infected with B. bovis, 26 (47.3%) were infected while from those from female ticks infected with B. bigemina 141 (76.6%) were infected (P<0.01). The results showed that although the frequency of both species of Babesia was similar in calves and cows, the infectivity of B. bigemina was higher to ticks fed on calves while to those ticks fed on cows the infectivity of both Babesia species was similar.     

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.     

Metabolism of ecdysteroids in the female tickAmblyomma hebraeum (Ixodoidea,Ixodidae): accumulation of free ecdysone and 20-hydroxyecdysone in the eggs

Summary [³H]-20-hydroxyecdysone ([³H]-20E) injected intoAmblyomma hebraeum females 7 days before the beginning of oviposition,viz. at the beginning of vitellogenesis, was converted to 3 polar peaks of unknown nature called 1, 2 and 3, and to apolar conjugates AP1, AP2 and AP3. AP2 have the same retention times as the esters of 20E with long chain fatty acids described inOrnithodoros moubata (Diehl et al. 1985). However, principally unmetabolized 20E was incorporated into the ovaries, and 16% of the injected labelling was recovered in the eggs, 3/4 being free 20E. When 20E was injected during oviposition, it was not converted to the polar products but only to the apolar products. At this time, 76% of the total radiolabel injected accumulated in the egg-batch, principally in the form of free unmetabolized 20E.After injection of [³H]-ecdysone (³H]-E), the three polar metabolites 1, 2 and 3, probably 20-deoxy homologues of 1, 2 and 3 described above were always produced irrespective of the time of injection. In addition, E was metabolized to 20E and to the apolar conjugates AP1, AP2, and AP3. E, 20E and peak 2 were incorporated into the ovary within the first day after injection. These 3 compounds were found in freshly laid eggs in variable proportions, the quantity of E decreasing with time while 20E and peak 2 increased. At the end of oviposition, ca. 60% of the injected radiolabel had been incorporated into the eggs. Apolar products and polar metabolites accumulating in the body were apparently not used as a source of free hormone for the eggs.Our results with tritiated ecdysteroids confirm our data concerning endogenous ecdysteroids of the eggs ofA. hebraeum (Connat et al. 1985). This species, in contrast to 2 other female ticks,Ornithodoros moubata andBoophilus microplus, incorporates free E and 20E instead of ecdysteroid conjugates into its eggs. The role of these free ecdysteroids remains to be elucidated.Abbreviations ES ecdysteroids - E ecdysone - 20E 20-hydroxyecdysone - HPLC high-performance liquid chromatography - RP-18 reyerse phase with C18 aliphatic chains grafted on the silica - RIA radio-immunoassay Part of this work was realized for the PhD thesis of E.M. Dotson     

蜕皮激素在银盾革蜱生殖滞育中的作用(英文)

用高效液相色谱法和放射免疫测定法测定了银盾革蜱滞育雌虫的合神经节、血淋巴、卵巢和整体中蜕皮激素的含量,并与正常发育的雌虫做比较。结果表明,滞育雌虫血淋巴和卵巢中蜕皮激素的含量在饱血后前10天与正常发育雌虫基本上相同。饱血10天以后,滞育雌虫蜕皮激素的含量下降,较正常发育雌虫少得多。蜕皮激素的缺乏影响了卵母细胞的发育。为阐明蜕皮激素对滞育雌虫的影响,在不同时间向银盾革蜱滞育雌虫体内注入不同剂量的β-蜕皮素。刚饱血者注入大剂量(1.375和10000ng/蜱)的β-蜕皮素引起雌虫死亡。饱血后20—30天,注入一定剂量(50,70,100ng/蜱)的β-蜕皮素可以促进卵黄形成,并解除了雌虫的生殖滞育,但其所产的卵量明显少于正常发育的雌虫。应用外源蜕皮激素解除硬蜱雌虫的生殖滞育现象尚属首次报道。     

Cinnamaldehyde—A potential antidiabetic agent

Cinnamonum zeylanicum (cinnamon) is widely used in traditional system of medicine to treat diabetes in India. The present study was carried out to isolate and identify the putative antidiabetic compounds based on bioassay-guided fractionation; the compound identified decreased the plasma glucose levels. The active compound was purified by repeat column and structure of cinnamaldehyde was determined on the basis of chemical and physiochemical evidence. The LD(50) value of cinnamaldehyde was determined as 1850+/-37 mg/kg bw. Cinnamaldehyde was administered at different doses (5, 10 and 20 mg/kg bw) for 45 days to streptozotocin (STZ) (60 mg/kg bw)-induced male diabetic wistar rats. It was found that plasma glucose concentration was significantly (p<0.05) decreased in a dose-dependent manner (63.29%) compared to the control. In addition, oral administration of cinnamaldehyde (20 mg/kg bw) significantly decreased glycosylated hemoglobin (HbA(1C)), serum total cholesterol, triglyceride levels and at the same time markedly increased plasma insulin, hepatic glycogen and high-density lipoprotein-cholesterol levels. Also cinnamaldehyde restored the altered plasma enzyme (aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase and acid phosphatase) levels to near normal. Administration of glibenclamide, a reference drug (0.6 mg/kg bw) also produced a significant (p<0.05) reduction in blood glucose concentration in STZ-induced diabetic rats. The results of this experimental study indicate that cinnamaldehyde possesses hypoglycemic and hypolipidemic effects in STZ-induced diabetic rats.     

Physiological characterization and stress-induced metabolic responses of Dunaliella salina isolated from salt pan

A Dunaliella strain was isolated from salt crystals obtained from experimental salt farm of the institute (latitude 21.46 N, longitude 72.11 degrees E). The comparative homology study of amplified molecular signature 18S rRNA, proves the isolated strain as D. salina. The growth pattern and metabolic responses such as proline, glycine betaine, glycerol, total protein and total sugar content to different salinity (from 0.5 to 5.5 M NaCl) were studied. The optimum growth was observed at 1.0 M NaCl and thereafter it started to decline. Maximum growth was obtained on 17th day of inoculation in all salt concentrations except 0.5 M NaCl, whereas maximum growth was observed on 13th day. There were no significant differences (P < 0.01) in chlorophyll a/b contents (1.0-1.16 +/- 0.05 mug chl. a and 0.2-0.29 +/- 0.01 mug chl. b per 10(6) cells) up to 2.0 M NaCl, however at 3.0 M NaCl a significant increase (2.5 +/- 0.12 mug chl. a and 0.84 +/- 0.4 mug chl. b per 10(6) cells) was observed which declined again at 5.5 M NaCl concentration (2.0 +/- 0.1 mug chl. a and 0.52 +/- 0.03 mug chl. b per 10(6) cells). Stress metabolites such as proline, glycine betaine, glycerol and total sugar content increased concomitantly with salt concentration. Maximum increase in proline (1.4 +/- 0.07 mug), glycine betaine (5.7 +/- 0.28 mug), glycerol (3.7 +/- 0.18 ml) and total sugar (250 +/- 12.5 mug) per 10(5) cells was observed in 5.5 M NaCl. A decrease in total protein with reference to 0.5 M NaCl was observed up to 3.0 M NaCl, however, a significant increase (P < 0.01) was observed at 5.5 M NaCl (0.19 +/- 0.01 mug per 10(5) cells). Inductive coupled plasma (ICP) analysis shows that intracellular Na(+) remained unchanged up to 2.0 M NaCl concentration and thereafter a significant increase was observed. No relevant increase in the intracellular level of K(+) and Mg(++) was observed with increasing salt concentration. Evaluation of physiological and metabolic attributes of Dunaliella salina can be used to explore its biotechnological and industrial potential.     

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.     

Ovarian and haemolymph titres of ecdysteroid during the gonadotrophic cycle in Diploptera punctata

The free (non-conjugated) ecdysteroid in the ovaries during the first gonadotrophic cycle of Diploptera punctata was identified as 20-hydroxyecdysone. The hormone, quantified by radioimmunoassay and by ultraviolet absorbance, was detectable in the ovary toward the end of vitellogenesis; the quantity increased rapidly during chorion formation. Ovaries with chorionated eggs contained 67 μg of 20-hydroxyecdysone per g fresh weight. The haemolymph free-ecdysteroid, not identified physicochemically, was quantified by radioimmunoassays. The highest concentration was observed at adult emergence; the titre declined between days 1–3 and then remained at a relatively constant level through oviposition (which occurs between day 7 and 8); titres in pregnant females were higher. Ovariectomized females exhibited the same pattern of ecdysteroid titres in the haemolymph as the sham operated controls throughout the period corresponding to the first gonadotrophic cycle. Thus the ovary may not be the only source of haemolymph ecdysteroid related to reproduction in adult females.     

Beauveria bassiana: Synergistic effect with acaricides against the tick Hyalomma anatolicum anatolicum (Acari: Ixodidae)

Owing to the need to combat the spread of chemical acaricide resistance in ticks, we evaluated the efficacy of a mixture of the entomopathogenic fungus Beauveria bassiana AT17 and acaricides for the control of Hyalomma anatolicum anatolicum in China. A mixture of B. bassiana AT17 at the concentration of 10⁸ conidia/mL and the synthetic pyrethroid deltamethrin at concentrations of 2500, 250, 25, 5, 2.5, 0.5 and 0.25 ppm was tested in vitro. The germination capability, vegetative growth, conidia production, and viability of B. bassiana AT17 were assessed and the efficacy of the mixture in killing engorged H. anatolicum anatolicum females was measured. High mortality rates were achieved when the entomopathogen was combined with different concentrations of deltamethrin. Neither B. bassiana AT17 nor deltamethrin alone at the same concentrations could cause the higher mortality rates seen with the combination. In addition the combination killed the ticks more rapidly than did either agent alone (3–5 days more rapidly). Our results indicate that the mixture of B. bassiana AT17 and deltamethrin has potential as a new type of reagent for integrated control of H. anatolicum anatolicum.