Badillo-Vargas Ismael E, Rotenberg Dorith, Schneweis Brandi A, Whitfield Anna E
Department of Plant Pathology, Kansas State University, Manhattan, KS, USA.
Department of Plant Pathology, Kansas State University, Manhattan, KS, USA.
J Insect Physiol. 2015 May;76:36-46. doi: 10.1016/j.jinsphys.2015.03.009. Epub 2015 Mar 18.
The insect order Thysanoptera is exclusively comprised of small insects commonly known as thrips. The western flower thrips, Frankliniella occidentalis, is an economically important pest amongst thysanopterans due to extensive feeding damage and tospovirus transmission to hundreds of plant species worldwide. Geographically-distinct populations of F. occidentalis have developed resistance against many types of traditional chemical insecticides, and as such, management of thrips and tospoviruses are a persistent challenge in agriculture. Molecular methods for defining the role(s) of specific genes in thrips-tospovirus interactions and for assessing their potential as gene targets in thrips management strategies is currently lacking. The goal of this work was to develop an RNA interference (RNAi) tool that enables functional genomic assays and to evaluate RNAi for its potential as a biologically-based approach for controlling F. occidentalis. Using a microinjection system, we delivered double-stranded RNA (dsRNA) directly to the hemocoel of female thrips to target the vacuolar ATP synthase subunit B (V-ATPase-B) gene of F. occidentalis. Gene expression analysis using real-time quantitative reverse transcriptase-PCR (qRT-PCR) revealed significant reductions of V-ATPase-B transcripts at 2 and 3 days post-injection (dpi) with dsRNA of V-ATPase-B compared to injection with dsRNA of GFP. Furthermore, the effect of knockdown of the V-ATPase-B gene in females at these two time points was mirrored by the decreased abundance of V-ATPase-B protein as determined by quantitative analysis of Western blots. Reduction in V-ATPase-B expression in thrips resulted in increased female mortality and reduced fertility, i.e., number of viable offspring produced. Survivorship decreased significantly by six dpi compared to the dsRNA-GFP control group, which continued decreasing significantly until the end of the bioassay. Surviving female thrips injected with dsRNA-V-ATPase-B produced significantly fewer offspring compared to those in the dsRNA-GFP control group. Our findings indicate that an RNAi-based strategy to study gene function in thrips is feasible, can result in quantifiable phenotypes, and provides a much-needed tool for investigating the molecular mechanisms of thrips-tospovirus interactions. To our knowledge, this represents the first report of RNAi for any member of the insect order Thysanoptera and demonstrates the potential for translational research in the area of thrips pest control.
缨翅目昆虫完全由通常被称为蓟马的小型昆虫组成。西花蓟马（Frankliniella occidentalis）是缨翅目中一种具有重要经济意义的害虫，因其广泛的取食危害以及能将番茄斑萎病毒传播给全球数百种植物。西花蓟马在地理上不同的种群已对多种传统化学杀虫剂产生了抗性，因此，蓟马和番茄斑萎病毒的防治在农业中一直是一项挑战。目前缺乏用于确定特定基因在蓟马 - 番茄斑萎病毒相互作用中的作用以及评估其作为蓟马防治策略中基因靶点潜力的分子方法。这项工作的目标是开发一种能够进行功能基因组分析的RNA干扰（RNAi）工具，并评估RNAi作为一种基于生物学的方法控制西花蓟马的潜力。我们使用显微注射系统，将双链RNA（dsRNA）直接注射到雌性蓟马的血腔中，以靶向西花蓟马的液泡型ATP合酶亚基B（V - ATPase - B）基因。使用实时定量逆转录 - PCR（qRT - PCR）进行基因表达分析显示，与注射绿色荧光蛋白（GFP）的dsRNA相比，注射V - ATPase - B的dsRNA后2天和3天，V - ATPase - B转录本显著减少。此外，通过蛋白质印迹定量分析确定，在这两个时间点雌性蓟马中V - ATPase - B基因敲低的效果反映在V - ATPase - B蛋白丰度的降低上。蓟马中V - ATPase - B表达的降低导致雌性死亡率增加和繁殖力下降，即产生的存活后代数量减少。与dsRNA - GFP对照组相比，到注射后6天，存活率显著下降，并且在生物测定结束前一直持续显著下降。与dsRNA - GFP对照组相比，注射dsRNA - V - ATPase - B的存活雌性蓟马产生的后代明显更少。我们的研究结果表明，基于RNAi的策略用于研究蓟马中的基因功能是可行的，能够产生可量化的表型，并为研究蓟马 - 番茄斑萎病毒相互作用的分子机制提供了急需的工具。据我们所知，这是关于缨翅目昆虫任何成员的RNAi的首次报道，并证明了在蓟马害虫防治领域进行转化研究的潜力。
