Abstract
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.
Highlights
Thrips (Thysanoptera: Thripidae) are major insect pests of ornamental crops and soft fruit and inflict mechanical damage arising from feeding and oviposition (Mouden, Sarmiento, Klinkhamer, & Leiss, 2017)
We previously demonstrated that BFo2, a bacterial symbiont of western flower thrips (WFT) (De Vries, Van der Wurff, Jacobs, & Breeuwer, 2008; Facey et al, 2015), can be used to successfully deliver RNA interference (RNAi) to these insects via an apramycin‐ resistant double‐strand RNA (dsRNA)‐expressing BFo2 clone (Whitten et al, 2016)
Luria broth (LB) diet was compared with Tryptone soy broth and yeast extract (TSBY)+ inoculated with 1.25 × 107/ml GFP‐expressing BFo2 to determine whether the reintroduction of such concentrations of symbiotic bacteria would have an impact host insect survival
Summary
Thrips (Thysanoptera: Thripidae) are major insect pests of ornamental crops and soft fruit and inflict mechanical damage arising from feeding and oviposition (Mouden, Sarmiento, Klinkhamer, & Leiss, 2017). Several criteria must be considered and optimized when using an RNAi approach, such as the method of dsRNA synthesis (transcription efficiency, yield and quality, time, and overall cost), adequate nutritional support, the integrity of dsRNA before and after internalization by the insect (which is especially important for dietary delivery), and the relative ease with which phenotype assays can be set up and the insects manipulated. All these factors impact on the ultimate efficiency of knockdown and success of the RNAi strategy, and each may be more or less important depending on the intended application. The article highlights possible challenges that could be faced using dietary RNAi delivery techniques
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