BACKGROUND: With the growing number of available aphid genomes and transcriptomes, an efficient and easy-to-adapt tool for gene function study is urgently required. RNA interference (RNAi), as a post-transcriptional gene silencing mechanism, is important as a research tool for determining gene functions and has potential as a novel insect control strategy. However, these applications have been hampered by the lack of effective dsRNA delivery approaches in aphids. RESULTS:Here, we developed a convenient and efficient dsRNA delivery method, topical RNAi, in aphids. An investigation of its dose and time-dependent RNAi efficiencies revealed that with as little as 60 ng dsRNA per adult pea aphid (Acyrthosiphon pisum), the indicator gene, Aphunchback, could be significantly silenced within 2 h of exposure. The method was further validated by successfully silencing other different genes, and it was also efficient toward two other aphid species, Aphis citricidus and Myzus persicae. Furthermore, a noticeable mortality was also observed in pea aphids using topical RNAi-mediated gene silencing, within 4 days post-dsRNA application for four out of seven tested genes. CONCLUSION: Compared with the currently used dsRNA delivery methods in aphids, microinjection and ingestion, topical RNAi is time-and cost-effective, which could greatly influence RNAi-based gene functional studies and potential candidate gene selection for developing RNAi-based aphid control strategies in the future.

BACKGROUND RNA interference (RNAi) has potential as a pest insect control technique. One possible RNAi target is the cuticle protein, which is important in insect molting and development. As an example, here we evaluate the possibility of designing double‐stranded RNA (RNA) that is effective for silencing the cuticle protein 19 gene (CP19) in aphids but is harmless to non‐target predator insects. RESULTS The sequences of CP19s were similar (86.6–94.4%) among the tested aphid species (Aphis citricidus, Acyrthosiphon pisum, and Myzus persicae) but different in the predator Propylaea japonica. Ingestion of species‐specific dsRNAs of CP19 by the three aphids produced 39.3–64.2% gene silencing and 45.8–55.8% mortality. Ingestion of non‐species‐specific dsRNA (dsAcCP19) by Ac. pisum and M. persicae gave gene silencing levels ranging from 40.4% to 50.3% and 43.3–50.8% mortality. The dsApCP19 did not affect PjCP19 expression or developmental duration in P. japonica. CONCLUSION The results demonstrate that CP19 is a promising RNAi target for aphid control via one dsRNA design. The targeting of genes that are conserved in insect pests but not present in beneficial insects is a useful RNAi‐based pest control strategy. © 2019 Society of Chemical Industry

Carotenoids play many crucial roles in organisms. Recently, the de novo synthesis of carotenoids has been reported in pea aphid (Acyrthosiphon pisum) through horizontally transferred genes. However, their upstream pathway in the pea aphid is poorly understood. Geranylgeranyl diphosphate synthase (GGPPS) is the functional enzyme in the synthesis of geranylgeranyl diphosphate (GGPP) which is a precursor for the biosynthesis of many biological metabolites, including carotenoid synthesis. In this study, we performed a series of experiments to characterize GGPPS gene and its association with carotenoid biosynthesis. (1) determining the transcript abundance and carotenoid content in two geographical strain with red and green morphs, and (2) examining the abundance of carotenoid related genes and carotenoid levels after silencing of GGPPS in both red and green morphs. We observed that GGPPS was more highly expressed in the green morph than in the red morph of two strains of the pea aphid. The total level of carotenoids was also higher in green morphs than in red morphs in both strains. In addition to the total carotenoid difference, the carotenoids found in the two morphs also differed. There were α-carotene, β-carotene, and γ-carotene in the green morphs, but three additional carotenoids, including cis-torulene∗, trans-torulene∗, and 3,4-didehydrolycopene∗, were present in the red morphs. Silencing the GGPPS by RNAi in both the red and green morphs decreased the expression of some carotenoid biosynthesis-related genes, including carotenoid synthase/cyclase genes and carotenoid desaturase genes in green morphs. Carotenoid levels were decreased in both green and red morphs. However, the specific carotenoids present were not changed after silencing GGPPS. These results demonstrated that GGPPS may act as the upstream enzyme to influence the synthesis of the total amount of carotenoids. The present study provided important molecular evidence for the conserved roles of GGPPS associated with carotenoids biosynthesis and will enhance further investigation on the mechanisms of carotenoid biosynthesis in pea aphid.

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We report a new positive-sense single-stranded RNA (ss RNA+) virus from the brown citrus aphid Aphis citricidus. The 20,300 nucleotide (nt)-long viral genome contains five open-reading frames and encodes six conserved domains (TM2, 3CLpro, TM3, RdRp, Zm, and HEL1). Phylogenetic analysis and amino acid sequence analysis revealed this virus might belong to an unassigned genus in the family Mesoniviridae. The presence of the virus was also confirmed in the field population. Importantly, analysis of the virus-derived small RNAs showed a 22-nt peak, implying that viral infection triggers the small interfering RNA pathway as antiviral immunity in aphids. This is the first report of a mesonivirus in invertebrates other than mosquitoes.