Abstract
RNA interference (RNAi) in insects is routinely used to ascertain gene function, but also has potential as a technology to control pest species. For some insects, such as beetles, ingestion of small quantities of double-stranded RNA (dsRNA) is able to knock down a targeted gene's expression. However, in other species, ingestion of dsRNA can be ineffective owing to the presence of nucleases within the gut, which degrade dsRNA before it reaches target cells. In this study, we observed that nucleases within the gut of the Queensland fruit fly (Bactrocera tryoni) rapidly degrade dsRNA and reduce RNAi efficacy. By complexing dsRNA with liposomes within the adult insect's diet, RNAi-mediated knockdown of a melanin synthesis gene, yellow, was improved significantly, resulting in strong RNAi phenotypes. RNAi efficiency was also enhanced by feeding both larvae and adults for several days on dsRNAs that targeted two different dsRNase gene transcripts. Co-delivery of both dsRNase-specific dsRNAs and yellow dsRNA resulted in almost complete knockdown of the yellow transcripts. These findings show that the use of liposomes or co-feeding of nuclease-specific dsRNAs significantly improves RNAi inhibition of gene expression in B. tryoni and could be a useful strategy to improve RNAi-based control in other insect species.
Highlights
RNA interference (RNAi) has become a widely used reverse genetics tool in insects, owing largely to the relative ease of knocking down a targeted gene’s transcripts by injecting double-stranded RNA into the insect’s haemocoel
Evidence supporting that nucleases can have significant impacts on RNAi efficacy has been confirmed by reducing nuclease activity by delivering nuclease-specific double-stranded RNA (dsRNA) to the insects
RNAi efficiencies can vary considerably in different insect species, and, in many instances, the lack of effective RNAi has been attributed to endogenous nucleases that can destroy the dsRNA before it can reach its intracellular targets yellow transcript levels relative to actin royalsocietypublishing.org/journal/rsob Open Biol. 9: 190198
Summary
RNA interference (RNAi) has become a widely used reverse genetics tool in insects, owing largely to the relative ease of knocking down a targeted gene’s transcripts by injecting double-stranded RNA (dsRNA) into the insect’s haemocoel (reviewed in [1]). Because of its sequence specificity, RNAi has the potential to provide a new generation of species-specific pesticides that target transcripts within a pest species, but do not adversely affect beneficial or non-target species [2,3,4] To this end, transgenic plants expressing insecticidal dsRNAs have been produced that provide effective control against specific pests [5,6] and foliar dsRNA sprays have proven effective in controlling herbivorous insect pests in laboratory trials [7]. Chung et al [21] induced knockdown of bacterial symbiosis genes in the pea aphid Acyrthosiphon pisum by co-feeding a mixture of dsRNAs targeting both symbiosis-related genes and a gut nuclease gene In these cases, enough nuclease-specific dsRNAs are thought to have entered the gut cells of the insects in the early feedings, reducing nuclease activity in the gut and thereby improving the efficacy of RNAi within a few days of further dsRNA consumption
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