Department of Entomology, University of Nebraska, Lincoln, NE 68583-0816, USA.
Pest Manag Sci. 2012 Apr;68(4):587-91. doi: 10.1002/ps.2301.
RNA interference (RNAi) is commonly used in insect functional genomics studies and usually involves direct injection of double-stranded RNA (dsRNA). Only a few studies have involved exposure to dsRNAs through feeding. For western corn rootworm (Diabrotica virgifera virgifera) larvae, ingestion of dsRNA designed from the housekeeping gene, vacuolar ATPase (vATPase) triggers RNAi causing growth inhibition and mortality; however, the effect of dsRNA feeding on adults has not been examined. In this research, WCR adults were fed with vATPase-dsRNA-treated artificial diet containing a cucurbitacin bait, which is a proven feeding stimulant for chrysomelid beetles of the subtribe Diabroticina to which rootworms belong.
Real-time PCR confirmed suppression of vATPase expression and western blot analysis indicated reduced signal of a protein that cross-reacted with a vATPase polyclonal antiserum in WCR adults exposed to artificial diet treated with dsRNA and cucurbitacin bait. Continuous feeding on cucurbitacin and dsRNA-treated artificial diet resulted in more than 95% adult mortality within 2 weeks while mortality in control treatments never exceeded 20%.
This research clearly demonstrates the effect of RNAi on WCR adults that have been exposed to dsRNA by feeding and establishes a tool to screen dsRNAs of potential target genes in adults. This technique may serve as an alternative to target screening of larvae which are difficult to maintain on artificial diets.
RNA 干扰（RNAi）常用于昆虫功能基因组学研究，通常涉及双链 RNA（dsRNA）的直接注射。只有少数研究涉及通过喂食暴露于 dsRNA。对于西部玉米根虫（Diabrotica virgifera virgifera）幼虫，摄入源自管家基因液泡型三磷酸腺苷酶（vATPase）的 dsRNA 会引发 RNAi，导致生长抑制和死亡；然而，dsRNA 喂食对成虫的影响尚未得到检验。在这项研究中，WCR 成虫以含有葫芦素诱饵的 vATPase-dsRNA 处理的人工饲料喂食，该诱饵已被证明是属于玉米根虫亚科的金刚烷类甲虫的进食刺激物。
实时 PCR 证实了 vATPase 表达的抑制，Western blot 分析表明，在暴露于 dsRNA 和葫芦素诱饵处理的人工饲料中的 WCR 成虫中，与 vATPase 多克隆抗血清发生交叉反应的蛋白质信号减少。连续以葫芦素和 dsRNA 处理的人工饲料喂食，在两周内导致超过 95%的成虫死亡，而对照处理中的死亡率从未超过 20%。
这项研究清楚地表明，喂食 dsRNA 对 WCR 成虫的 RNAi 效应，并建立了一种筛选成虫中潜在靶基因 dsRNA 的工具。该技术可能替代幼虫的靶标筛选，因为幼虫难以在人工饲料上维持。
