Abstract

AbstractRNA interference (RNAi) is a technique used in many insects to study gene function. However, prior research suggests possible off-target effects when usingGreen Fluorescent Protein(GFP) sequence as a non-target control. We used a transcriptomic approach to study the effect ofGFPRNAi (GFP-i) inNasonia vitripennis, a widely used parasitoid wasp model system. Our study identified 3.4% of total genes being differentially expressed in response toGFP-i. A subset of these genes appears involved in microtubule and sperm functions.In silicoanalysis identified 17 potential off-targets, of which only one was differentially expressed afterGFP-i. We suggest the primary cause for differential expression afterGFP-i is the non-specific activation of the RNAi machinery at the injection site, and a potentially disturbed spermatogenesis. Still, we advise that any RNAi study involving the genes deregulated in this study, exercises caution in drawing conclusions and uses a different non-target control.

Highlights

  • Nasonia vitripennis is a widely used parasitoid wasp model system to study major topics such as genetics, development, ecology, and behavior (Beukeboom & Desplan, 2003; Lynch, 2015)

  • We investigate the potential for off-target effects of using Green Fluorescent Protein (GFP) doublestrand RNA as non-target control in N. vitripennis RNA interference (RNAi) experiments

  • In one case we were able to relate this effect to endogenous GFP sequence similarity in N. vitripennis, and this leads us to assume that GFP RNAi (GFP-i) does not cause specific off-target effects, but instead results in a general response to doublestrand RNA (dsRNA)

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Summary

Introduction

Nasonia vitripennis is a widely used parasitoid wasp model system to study major topics such as genetics, development, ecology, and behavior (Beukeboom & Desplan, 2003; Lynch, 2015). Knockdown of genes by RNA interference (RNAi) or by parental RNAi (pRNAi) is still the most popular method to study gene function and maternal transcript provision in N. vitripennis (Dalla Benetta et al, 2020; Lynch & Desplan, 2006; Wang et al, 2020; Werren et al, 2009). In other Hymenoptera such as the honeybee, RNAi has been reported to have undesirable off-target effects, for example when using GFP dsRNA as a control (Jarosch & Moritz, 2011; Nunes et al, 2013). We investigate the potential for off-target effects of using GFP doublestrand RNA (dsRNA) as non-target control in N. vitripennis RNAi experiments

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