In insect reverse genetics, dietary delivery of interfering RNAs is a practical approach in nonmodel species, such as thrips, whose small size, and feeding behavior restricts the use of other delivery methods. In a laboratory context, an unsuitable diet could confound the interpretation of an RNA interference (RNAi) phenotype, however well‐formulated artificial diets can minimize experimental variability, reduce the need for insect handling, and can further be used for roles, such as delivering double‐strand RNA (dsRNA)‐expressing recombinant bacteria. In this study, artificial diets for oral delivery of dsRNA were developed for two important pest thrips species, western flower thrips (Frankliniella occidentalis) and onion thrips (Thrips tabaci), with the goal of (a) stimulating feeding behavior, (b) supporting optimal growth rates of dsRNA‐expressing symbiotic bacteria, and (c) nutritionally supporting the thrips for sufficient periods to observe RNAi phenotypes. The efficacy of artificial diets for ingesting “naked” dsRNA or dsRNA‐expressing symbionts and dsRNA delivery via host plant uptake was evaluated. Compared with previously published diet formulations, new combinations based on tryptone, yeast, and soy were superior for enhancing feeding and longevity. However, simply adding “naked” dsRNA to an artificial diet was an unreliable form of RNAi delivery in our hands due to dsRNA degradation. Delivery via host plants was more successful, and the new diet formulation was suitable for symbiont‐mediated dsRNA delivery, which we believe is the most convenient approach for large‐scale knockdown experiments. This study, therefore, provides alternative methodologies for thrips rearing, dietary RNAi delivery, and insights into the challenges of performing dietary RNAi in nonmodel insects.

中文翻译：

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优化西花蓟马（Frankliniella occidentalis）和洋葱蓟马（Thrips tabaci）的膳食 RNA 干扰传递。

在昆虫反向遗传学中，通过膳食递送干扰RNA对于非模式物种来说是一种实用方法，例如蓟马，其体型较小，进食行为限制了其他递送方法的使用。在实验室环境中，不合适的饮食可能会混淆对 RNA 干扰 (RNAi) 表型的解释，但是精心配制的人工饮食可以最大限度地减少实验变异性，减少对昆虫处理的需要，并且可以进一步用于各种角色，例如传递表达双链RNA（dsRNA）的重组细菌。在这项研究中，为两种重要的害虫蓟马物种——西花蓟马（ Frankliniella occidentalis）和洋葱蓟马（Thrips tabaci）开发了用于口服 dsRNA 的人工饲料。），目标是（a）刺激摄食行为，（b）支持表达 dsRNA 的共生细菌的最佳生长速率，以及（c）在足够长的时间内为蓟马提供营养支持以观察 RNAi 表型。评估了人工饮食摄入“裸露”dsRNA 或表达 dsRNA 的共生体以及通过宿主植物吸收传递 dsRNA 的功效。与之前发表的饮食配方相比，基于胰蛋白胨、酵母和大豆的新组合在增强喂养和延长寿命方面具有优越性。然而，由于 dsRNA 降解，简单地将“裸”dsRNA 添加到人工饮食中是一种不可靠的 RNAi 传递形式。通过宿主植物的传递更加成功，并且新的饮食配方适合共生体介导的 dsRNA 传递，我们认为这是大规模敲除实验最方便的方法。因此，这项研究为蓟马饲养、膳食 RNAi 传递提供了替代方法，并深入了解了在非模式昆虫中进行膳食 RNAi 的挑战。