Knock down of Whitefly Gut Gene Expression and Mortality by Orally Delivered Gut Gene-Specific dsRNAs

<div><p>Control of the whitefly <i>Bemisia tabaci</i> (Genn.) agricultural pest and plant virus vector relies on the use of chemical insecticides. RNA-interference (RNAi) is a homology-dependent innate immune response in eukaryotes, including insects, which results in degradation of the corresponding transcript following its recognition by a double-stranded RNA (dsRNA) that shares 100% sequence homology. In this study, six whitefly ‘gut’ genes were selected from an <i>in silico</i>-annotated transcriptome library constructed from the whitefly alimentary canal or ‘gut’ of the B biotype of <i>B</i>. <i>tabaci</i>, and tested for knock down efficacy, post-ingestion of dsRNAs that share 100% sequence homology to each respective gene target. Candidate genes were: <i>Acetylcholine receptor subunit α</i>, <i>Alpha glucosidase 1</i>, <i>Aquaporin 1</i>, <i>Heat shock protein 70</i>, <i>Trehalase1</i>, and <i>Trehalose transporter1</i>. The efficacy of RNAi knock down was further tested in a gene-specific functional bioassay, and mortality was recorded in 24 hr intervals, six days, post-treatment. Based on qPCR analysis, all six genes tested showed significantly reduced gene expression. Moderate-to-high whitefly mortality was associated with the down-regulation of osmoregulation, sugar metabolism and sugar transport-associated genes, demonstrating that whitefly survivability was linked with RNAi results. Silenced <i>Acetylcholine receptor subunit α</i> and <i>Heat shock protein 70</i> genes showed an initial low whitefly mortality, however, following insecticide or high temperature treatments, respectively, significantly increased knockdown efficacy and death was observed, indicating enhanced post-knockdown sensitivity perhaps related to systemic silencing. The oral delivery of gut-specific dsRNAs, when combined with qPCR analysis of gene expression and a corresponding gene-specific bioassay that relates knockdown and mortality, offers a viable approach for functional genomics analysis and the discovery of prospective dsRNA biopesticide targets. The approach can be applied to functional genomics analyses to facilitate, species-specific dsRNA-mediated control of other non-model hemipterans.</p></div

Percent mortality post- <i>AQP1</i> and <i>AGLU1</i> dsRNA knockdown.

<p>Percent mortality post- <i>AQP1</i> and <i>AGLU1</i> dsRNA knockdown.</p

Results of qPCR analysis for gene targets involved in sugar metabolism and transport.

<p>The qPCR amplification of expressed whitefly genes involved in sugar metabolism and transport, post-RNAi knock down. (A) Comparison of fold-change expression of <i>Trehalose transporter 1</i> (<i>Tret1</i>) for whiteflies treated with 30μg/ml of <i>Tret1</i> dsRNA (tan), and the buffer control (blue bars) on day one, four, and six, post-treatment (x-axis). (B) Comparison of fold-change in expression of <i>Trehalase1</i> (<i>Tre1</i>) for whiteflies treated with 30μg/ml of <i>Tre1</i> dsRNA (purple) on day four and six (x-axis). Sugar metabolism genes were ~ 30–40% down regulated. The “asterisk” indicates a significant <i>p-</i>value (< 0.05) (<i>t</i>-test).</p

Primers used for PCR and RT-PCR amplification and <i>in vitro</i> transcription.

<p>The T7 promoter sequence is underlined.</p

Percent whitefly mortality for <i>Tre1</i> and <i>Tret1</i> knockdowns.

<p>Percent whitefly mortality for <i>Tre1</i> and <i>Tret1</i> knockdowns.</p

Percent mortality post-<i>AChRα</i> dsRNA knock down, and exposure to <i>I MaxxPro</i>^®^&.

<p>Percent mortality post-<i>AChRα</i> dsRNA knock down, and exposure to <i>I MaxxPro</i>^®<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168921#t004fn002" target="_blank">^&</a>.</p

Percentage mortality, post- <i>AChRα</i> dsRNA knockdown.

<p>Percentage mortality, post- <i>AChRα</i> dsRNA knockdown.</p

Results of real-time qPCR analysis of a neurotransmission gene target.

<p>The qPCR expression of target whitefly genes involved in neurotransmission, post-RNAi knock down. (A) The bar graphs show the comparisons in fold-change of expression of the <i>Acetylcholine receptor subunit alpha</i> (<i>AChRα</i>) when whiteflies were treated with 30μg/ml of <i>AChRα</i> dsRNA (gray bars), and the buffer treated control (blue bars) at day one, four, and six (x-axis). The “asterisk” indicates a significant <i>p-</i>value (< 0.05) (<i>t-</i>test).</p

Results of qPCR analysis of gene targets involved in osmoregulation and thermo tolerance.

<p>Real-time qPCR amplification of expressed whitefly genes involved in osmoregulation, post-RNAi knock down. (A) Comparison of fold-changes in expression of <i>Alpha glucosidase1</i> (<i>AGLU1</i>) for whitefly treated with 30 μg/ml of <i>AGLU1</i> dsRNA (red) and buffer control (blue) at day one, four, and six, post-treatment (x-axis). (B) Comparison of fold-changes in expression of <i>Aquaporin1</i> (<i>AQP1</i>) for whiteflies treated with 30μg/ml of <i>AQP1</i> dsRNA (green), and the buffer treated control (blue) at day one, four and six, post-treatment (x-axis). (C) Comparison of fold-changes in expression of <i>Heat shock protein 70</i> (<i>Hsp70</i>) for whiteflies treated with 30μg/ml of <i>Hsp70</i> dsRNA (yellow), and the buffer control (blue bars) at day one, four, and six (x-axis). Of the three gene targets, <i>AGLU1</i> showed the most significant decrease in gene expression, which occurred on day six. The “asterisk” indicates a significant <i>p-</i>value (< 0.05) (<i>t</i>-test).</p

Gene-specific quantitative PCR primer-probe combinations.

<p>Gene-specific quantitative PCR primer-probe combinations.</p