Sequence alignment of the conserved regions of <i>Tribolium</i> Vvl and the vertebrate POU3F4 proteins.

<p>Sequences were downloaded from NCBI and aligned using the Geneious 6 software. The POU domains and homeodomains are highlighted.</p

Proposed mechanism of Vvl action.

<p>Vvl influences JH production via <i>jhamt</i> expression and hence the timing of metamorphosis. Vvl also regulates the biosynthesis of ecdysteroids and the molting pathway.</p

Vvl removal leads to decreased ecdysteroid titer.

<p>Ecdysone titers in <i>amp^r</i> and <i>vvl</i> dsRNA-injected fifth instar larvae on day 4. A 10 µl sample represents two larval equivalents of extracted ecdysteroids.</p

Effects of 20E or RH-2485 treatments on larvae injected with dsRNA as fifth instars.

<p>Effects of 20E or RH-2485 treatments on larvae injected with dsRNA as fifth instars.</p

Expression profile of <i>vvl</i> and knockdown verification for RNA interference.

<p>(A) Expression profile of <i>vvl</i> during the late larval and prepupal stages of <i>Tribolium.</i> Expression profile was determined by qPCR of <i>vvl</i> from whole body sixth and seventh instars and prepupae. <i>Ribosomal protein 49 (rp49)</i> was used as the internal control. For all of the treatments, each sample consisted of RNA pooled from five sixth instars, three seventh instars and three prepupae. Three biological replicates were used per treatment, and each sample was run in triplicates. (B) Expression of <i>vvl</i> in the CNS, epidermis, fat body and gut of day 0 seventh instar larvae. mRNA was isolated from tissues pooled from 20 individuals. (C) Knockdown verification of <i>vvl</i> and <i>Met</i> in early prepupae. Expression profiles for <i>vvl</i>, <i>Met</i>, and <i>rp49</i> (control) in <i>amp^r</i>, <i>vvl</i>, and <i>Met</i> dsRNA-injected animals. Cycle numbers for <i>vvl</i>, <i>Met</i> and <i>rp49</i> were 34, 35, and 28, respectively. (D) Quantitative real-time PCR data showing the expression of <i>Met</i> (left) and <i>vvl</i> (right) in <i>vvl</i> and <i>Met</i> knockdown prepupae, respectively.</p

<i>vvl</i> knockdown leads to decreased expression of ecdysone biosynthesis genes.

<p>(A–E) Ecdysone response gene expression in <i>amp^r</i> and <i>vvl</i> dsRNA-injected fifth instar larvae on day 4. Anterior represents expression in the head and thorax. Posterior represents expression in the abdomen. Three biological replicates were used per treatment, and each sample was run in triplicates. Each biological sample consisted of pooled RNA from five larvae. Error bards represent standard error. (F) Relative expression of <i>spo</i> expression in the oenocytes of day 4 <i>amp^r</i> and <i>vvl</i> dsRNA-injected fifth instar KT817 larvae. Oenocytes from 15 larvae were pooled per treatment.</p

Effects of ectopic application of methoprene on developmental timing in larvae injected with dsRNA as fifth instars.

<p>Effects of ectopic application of methoprene on developmental timing in larvae injected with dsRNA as fifth instars.</p

The timing of metamorphosis initiation in larvae treated with <i>vvl</i> dsRNA and 5 ng methoprene.

<p>(A) Timing of prepupa formation in dsRNA injected fifth instars. (B–D) Timing of prepupa formation in dsRNA-injected fifth instars treated with acetone and methoprene. <i>amp^r</i> RNAi (B), <i>Met</i> RNAi (C) and <i>vvl</i> RNAi (D) indicate animals injected with <i>amp^r</i> dsRNA, <i>Met</i> dsRNA and <i>vvl</i> dsRNA, respectively. The time to the onset of prepupal period was recorded. All animals were maintained at 29°C and 50% humidity.</p

Phenotypic effects of dsRNA injection into fifth instar larvae.

<p>*includes animals that eclosed as abnormal adults without pupating.</p

Effect of <i>vvl</i> knockdown on genes involved in JH and ecdysone signaling.

<p>(A–C) Effect of knockdown of <i>vvl</i> on the expression of <i>jhamt3</i> (A), <i>Met</i> (B) and the JH response gene, <i>kr-h1</i> (C), in fifth instar larvae. Three biological replicates were used per treatment, and each sample was run in triplicates. (D) Effect of methoprene application on the expression of <i>kr-h1</i> in <i>vvl</i> knockdown larvae. <i>vvl</i> dsRNA-injected larvae were treated with either acetone (control) or methoprene (15 µg) on day 0 of the fifth instar stage. <i>amp^r</i> dsRNA-injected animals treated similarly were used as a comparison. Four biological replicates were used per treatment, and each sample was run in triplicates. Means not sharing the same letter are significantly different (p<0.05, ANOVA with Tukey HSD test). (E, F) Effect of knockdown of <i>vvl</i> on the expression of <i>E75</i> (E) and <i>HR3</i> (F). (G) Effect of 20E injection on the expression of <i>HR3</i> in <i>vvl</i> knockdown larvae. Three biological replicates were used per treatment, and each sample was run in triplicates. For (A)–(F), each sample consisted of RNA pooled from five larvae. For (G), each biological replicate consisted of RNA pooled from three larvae. Data are represented as mean +/− SEM.</p