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Browsing Faculty of Arts & Science by Subject "20-hydroxyecdysone"
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- ItemEffect of 20-hydroxyecdysone and haemolymph on oogenesis in the ixodid tick Amblyomma hebraeum(2008) Seixas, Adriana; Friesen, Kevin; Kaufman, ReubenEarlier 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 (0.45 μg/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 ± 0.5 mg Vt-equivalents/ml) beyond that occurring in vehicle-injected ticks (0.76 ± 0.14 mg Vt-equivalents/ml), and there was a further increase in ticks injected with 20E plus haemolymph from engorged ticks (8.9 ± 1.0 mg Vt-equivalents/ml). On-host injection of 20E alone (6 μg 20E/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 ± 57 μm) compared to vehicle-injected controls (132 ± 11 μm), compared to 20E alone (131 ± 12 μm), or haemolymph alone (124 ± 24 μm). There was a marked stimulation of Vg-synthesis by 31 μg 20E/g bw (6.0 ± 1.5 mg Vt-equivalents/ml) compared to vehicle-injected controls (1.02 ± 33 mg Vt-equivalents/ml). Vt accumulation by ovaries was significantly greater in ticks treated with haemolymph (12 ± 3 μg Vt/mg ovary) or 20E plus haemolymph (56 ± 26 μg Vt/mg ovary) compared to vehicle-injected controls (5.1 ± 1.5 μg Vt/mg ovary). There was also a significant effect of 6 μg 20E/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.
- ItemEffects of 20-hydroxyecdysone and other hormones on egg development, and identification of a vitellin-binding protein in the ovary of the tick, Amblyomma hebraeum(2004) Friesen, Kevin; Kaufman, ReubenPartially fed adult female Amblyomma hebraeum ticks were injected with 20-hydroxyecdysone (20E; up to 43 μg/g body weight (bw)), juvenile hormone III (JH III; up to 100 μg/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.
- ItemQuantification of vitellogenesis and its control by 20-hydroxyecdysone in the ixodid tick, Amblyomma hebraeum(2002) Friesen, Kevin; Kaufman, ReubenOvaries 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.