Induction of RNAi Core Machinery’s Gene Expression by Exogenous dsRNA and the Effects of Pre-exposure to dsRNA on the Gene Silencing Efficiency in the Pea Aphid (Acyrthosiphon pisum)

Abstract

The pea aphid, Acyrthosiphon pisum, is an important agricultural pest and biological model organism, and RNA interference (RNAi) is an important tool for functional genomics and for insect pest management. However, the efficiency of RNAi in pea aphids is variable, limiting its application in aphids. In this study, we present optimized conditions for inducing and increasing the gene silencing efficiency of RNAi in pea aphids. The optimal gene silencing of the target Aphunchback gene was achieved by injecting 600 ng double-stranded (ds) RNA, and the highest mRNA depletion rate (74%) was detected at 36 h after injection. Moreover, the same gene silencing conditions were used to achieve transcript silencing for nine different genes in the pea aphid, although the silencing efficiencies for the different genes varied. Furthermore, the pre-exposure of aphids to dsRNA (600 ng dsGFP) led to significant hunchback silencing following a secondary exposure to 60 ng of dshunchback, a dose which did not lead to gene silencing when independently injected. The information presented here can be exploited to develop more efficient RNAi bioassays for pea aphids, both as gene functional study tools and an insect pest control strategy.

Topical dsRNA delivery induces gene silencing and mortality in the pea aphid

“…(a)). To evaluate whether the topically applied dsRNAs could induce gene silencing in aphids, we determined the RNAi efficiency based on two factors: dsRNA dose (termed as dose‐dependent RNAi efficiency) and time (h) after application (termed as time‐dependent RNAi efficiency), using our previous target gene, Aphunchback , as the indicator . For the dose‐dependent RNAi efficiency, serial doses of dsRNA in 1 μL per aphid, was applied (Fig.…”

“…The most important achievement of this study is that a novel dsRNA delivery method for aphids, topical RNAi, was established. Currently, dsRNA is mainly delivered to aphids by microinjections or ingestion . The new method will serve as an important tool in gene function studies and could allow further exploration of RNAi as an aphid control strategy .…”

“…Moreover, the injury caused by the injection results in a difficult recovery period and sometimes a significant mortality. These factors can confound the observed physiological changes and deter molecular studies required to elucidate the functions of the silenced genes in aphids . Alternatively, plant‐mediated feeding of dsRNA through ingestion in aphids is widely used .…”

“…The tests of the dsRNA's dose‐ and time‐dependent effects on RNAi efficiency in aphids showed that approximately 60 ng dsRNA per adult aphid could induce sufficient gene silencing. Using microinjection, the significant inhibition of the expression of this same targeted gene by RNAi required approximately 600 ng dsRNA . In addition, with the same dsRNA dose, the topical application resulted in the earliest gene silencing being detected at approximately 2 h after the dsRNA treatment .…”

“…Two reference genes in each aphid species, ApRps20 and ApEF1α for Ac. pisum , Acβact and AcEF1α for Ap. citricidus , and Mpactin and Mppara for M. persicae (Table ), were included to normalize the qPCR data, and the relative expression levels of the tested genes were calculated following the framework of qBase +…”