<i>OmSpo</i> expression in specific cells of ovaries from nymphs and adult females determined by WISH.

<p>An overall ventral view of tick tissues in a final instar nymph (A). <i>OmSpo</i> localization in dissected reproductive tissues from the nymph in Fig 6A (B). <i>OmSpo</i> localization in small cells of the ovary with a band-like configuration (C, magnification of Fig 6B). Ovary of a final instar nymph stained with the sense probe (D). <i>OmSpo</i> localization in the ovary of a mated female (E). Ovary of mated female stained with the sense probe (F). Arrows indicate staining of <i>OmSpo</i> with the antisense probe. Ticks have a single ovary (Ov) connected to oviducts (Ovd) from both sides. Oviducts open into a uterus (Ut). Developing oocytes (Oo) are attached outside the ovary. Scale bars indicate 1 mm.</p

Effects of dsRNA injection on ecdysis of final instar nymphs.

<p>Statistical significance (p<0.01) between nymphs injected with dsGFP and dsShd are represented with *.</p><p>Effects of dsRNA injection on ecdysis of final instar nymphs.</p

Multiple alignment of OmSpo with orthologs determined for other arthropods.

<p>Location of a P/G rich domain, Helixes (Helix-C and Helix-K), a PERF motif and a Heme binding domain are indicated</p

Effects of 20E injection of nymphs after dsSpo injection.

<p>Effects of 20E injection of nymphs after dsSpo injection.</p

Ovarian Ecdysteroidogenesis in Both Immature and Mature Stages of an Acari, <i>Ornithodoros moubata</i>

<div><p>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 <i>Ornithodoros moubata</i>, making this species an excellent candidate for elucidating ecdysteroidogenesis in Chelicerates. Results showed that <i>O</i>. <i>moubata</i> 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. <i>OmSpo</i> showed specific expression in the ovaries of final nymphal and adult stages, indicating <i>O</i>. <i>moubata</i> utilizes the ovary as an ecdysteroidogenic tissue instead of specific tissues as seen in other arthropods. On the other hand, <i>OmShd</i> expression was observed in various tissues including the midgut, indicating functional ecdysteroids can be produced in these tissues. In nymphal stages, expression of both <i>OmSpo</i> and <i>OmShd</i> peaked before molting corresponding with high ecdysteroid titers in the hemolymph. In fed adult females, <i>OmSpo</i> expression peaked at 8–10 days after engorgement, while <i>OmShd</i> expression peaked immediately after engorgement. Mated females showed more frequent surges of <i>OmShd</i> 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.</p></div

Chromatograms of 20-hydroxyecdysone (20E), produced by enzymatic assay of OmShd, analyzed with LC-MS/MS.

<p>20E chromatograms from the medium of pIZT-OmShd vector (A) or pIZT vector (negative control) (B) were transfected to S2 cells. Intensity was indicated as counts per second (cps). MS/MS spectra of 20E standard (C) and 20E produced by OmShd (D).</p

Primers used in this study.

<p>Primers used in this study.</p

Developmental changes in <i>OmSpo</i> and <i>OmShd</i> expression during nymphal development or egg maturation in adult females.

<p><i>OmSpo</i> (A) and <i>OmShd</i> (B) expression in nymphs was determined for ovaries and midgut, respectively. <i>OmSpo</i> (C) and <i>OmShd</i> (D) expression in adult females determined for ovaries and midgut, respectively. (n = 3–4, mean±se)</p

Multiple alignment of OmShd with orthologs determined for other arthropods.

<p>Location of a P/G rich domain, Helixes (Helix-C, Helix-I and Helix-K), a PEFR motif and a Heme binding domain are indicated.</p