Effect of <i>TcKnk3</i> dsRNA-treatment on the development of <i>T. castaneum</i>.

<p> (A) dsRNAs specific for different exons and intron of <i>TcKnk3</i> (exon 6, exon 8, exon 9 and exon 8a) were injected into penultimate instar larvae, last instar larvae and pharate pupae. Approximately 60–100% of the dsRNA-treated insects showed lethal phenotype at pupal-adult molt. Exon 8 and exon 8a dsRNA-treatment led to a weaker phenotype with split elytra. (B) Specific down-regulation of <i>TcKnk3-3′</i> transcripts by RNAi. dsRNA (200 ng per insect n = 60) for <i>TcKnk3-3′</i> (corresponding to exon 8a) was injected into pharate pupae. Five days after injection, total RNA was extracted from pharate adult insects (n = 3) and used for cDNA synthesis. Depletion of different transcripts was checked by using pairs of transcript-specific primers (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004537#pgen.1004537.s005" target="_blank">Table S1</a>). For detection of transcripts with or without exon 8a, a pair of forward and reverse primers flanking exon 8a was used (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004537#pgen.1004537.s005" target="_blank">Table S1</a>). <i>TcRPS6</i> was used as internal loading control. <i>TcVer</i> dsRNA was injected as a control. RT-PCR for the <i>TcRpS6</i> transcripts was carried our prior to these analyses to ensure that equal amounts of cDNA templates from different developmental stages were being used in these comparisons. Results are from 28, 28, 28 and 24 cycles of RT-PCR for <i>TcKnk3-FL TcKnk3</i>-<i>5′ and TcKnk3-3′</i> and <i>TcRpS6</i>, respectively. The RT-PCR products were run on separate gels for each RT-PCR product (with different sizes for each transcript), but the figure as shown is composite showing the relevant regions only to avoid “white space”. The grainy quality of the amplification products for <i>TcKnk3-3′</i> relative to the other RT-PCR products is due to different camera settings used to reveal the minor band from the alternatively spliced product with exon 8a sequences. (C) <i>TcKnk2</i> or <i>TcKnk3</i> (exon-9)-specific dsRNA was injected into last instar larvae and pharate pupae. Four to five days after injections, pharate pupal and pharate adult (n = 5) insects were collected for chitin content analysis by a modified Morgan–Elson method as described earlier <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004537#pgen.1004537-Arakane3" target="_blank">[21]</a>. dsRNA for <i>TcVer</i> and <i>TcChs-A</i> were injected as negative and positive controls, respectively. The mean chitin content for TcKnk dsRNA treated insects is adapted from previously published data <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004537#pgen.1004537-Chaudhari1" target="_blank">[11]</a>.