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

Abstract

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.

Sequence and the developmental and tissue-specific regulation of the first complete vitellogenin messenger RNA from ticks responsible for heme sequestration

“…If Dv Vg exists as a dimer in hemolymph, the predicted molecular weight would be 412 kD (2 times 206 K). This is in reasonable agreement with that for Vg and Vn previously reported for this species (Rosell and Coons, 1991;Sullivan et al, 1999;Gudderra et al, 2001Gudderra et al, , 2002aThompson et al, 2005). Although we cannot confirm the exact oligomeric form of Vg and Vn in D. variabilis, Vg is known to exist in oligomeric forms in its native state in other Arthropods (Sappington and Raikhel, 1998).…”

“…1. The molecular weight of Vg from D. variabilis determined by native PAGE and gel permeation chromatography was reported previously in the range of 320-486 kD (Sullivan et al, 1999;Gudderra et al, 2001Gudderra et al, , 2002aThompson et al, 2005). The size of vitellin (Vn) from Dv by electrophoresis was in the range of 370-480 kD as determined by Rosell and Coons (1991) and also shown in Fig.…”

“…The initial Vg cDNA fragment from D. variabilis was obtained from a cDNA library made from the fat body of replete (mated, vitellogenic) females as described in Thompson et al (2005). To obtain the remainder of the 5 0 region of the Vg cDNA, 5 0 RACE was performed using SMART RACE (Clontech, Palo Alto, CA).…”

“…A partial Vg cDNA from D. variabilis was previously cloned in our laboratories (Thompson et al 2005). A reverse primer designed from the 5 0 end of the partial cDNA and 5 0 RACE were used in the current study to obtain an additional upstream fragment of the Vg message.…”

“…In ticks, incomplete sequences have been reported from B. microplus (GenBank accession number U49934; Tellam et al, 2002), Amblyomma americanum (GenBank accession number BI27356; Bior et al, 2002) and Dermacentor variabilis (Dv, GenBank accession number AY885250; Thompson et al, 2005). The site of synthesis of Vg in ticks is also in question.…”