Insect-specific RNA virus affects the stylet penetration activity of brown citrus aphid (Aphis citricidus) to facilitate its transmission.

Sap-sucking insects often transmit plant viruses but also carry insect viruses, which infect insects but not plants. The impact of such insect viruses on insect host biology and ecology is largely unknown. Here, we identified a novel insect-specific virus carried by brown citrus aphid (Aphis citricidus), which we tentatively named Aphis citricidus picornavirus (AcPV). Phylogenetic analysis discovered a monophyletic cluster with AcPV and other unassigned viruses, suggesting that these viruses represent a new family in order Picornavirales. Systemic infection with AcPV triggered aphid antiviral immunity mediated by RNA interference, resulting in asymptomatic tolerance. Importantly, we found that AcPV was transmitted horizontally by secretion of the salivary gland into the feeding sites of plants. AcPV influenced aphid stylet behavior during feeding and increased the time required for intercellular penetration, thus promoting its transmission among aphids with plants as an intermediate site. The gene expression results suggested that this mechanism was linked with transcription of salivary protein genes and plant defense hormone signaling. Together, our results show that the horizontal transmission of AcPV in brown citrus aphids evolved in a manner similar to that of the circulative transmission of plant viruses by insect vectors, thus providing a new ecological perspective on the activity of insect-specific viruses found in aphids and improving the understanding of insect virus ecology.

First Report of a Mesonivirus and Its Derived Small RNAs in an Aphid Species Aphis citricidus (Hemiptera: Aphididae), Implying Viral Infection Activity.

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.

Evaluation of a cuticle protein gene as a potential RNAi target in aphids.

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.

Characterization of the Geranylgeranyl Diphosphate Synthase Gene in Acyrthosiphon pisum (Hemiptera: Aphididae) and Its Association With Carotenoid Biosynthesis.

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, ß-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.

Characterization of a new bunyavirus and its derived small RNAs in the brown citrus aphid, Aphis citricidus.

High-throughput sequencing is widely used for virus discovery, and many RNA viruses have been discovered and identified. A new negative-sense single-stranded RNA virus was identified in the brown citrus aphid and named Aphis citricidus bunyavirus. The genome consists of large (7037 nt), medium (3462 nt), and small (1163 nt) segments. Phylogenetic analysis and amino acid sequences identities of this virus with other bunyaviruses suggest that it is a new species belonging to the family Phenuiviridae. The small interfering RNA pathway could be involved against the infection of this virus in brown citrus aphid as supported by the viral derived small RNAs. The discovery of this virus illustrates the diversity of RNA viruses and contributes to the classification of bunyaviruses.

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

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. © 2019 Society of Chemical Industry.

RNA-sequencing of a citrus bud-feeder, Podagricomela weisei (Coleoptera: Chrysomelidae), reveals xenobiotic metabolism/core RNAi machinery-associated genes and conserved miRNAs.

The citrus leaf-mining beetle, Podagricomela weisei Heikertinger, is an important citrus pest that ingests the mesophyll and new shoots. The mechanism underlying the xenobiotic metabolism of P. weisei is not well understood, in part because of a lack of available genomic and transcriptomic data, which has hampered the development of novel pest management approaches [e.g., RNA interference (RNAi)]. In this study, we completed the deep sequencing of the P. weisei transcriptome to identify factors potentially involved in xenobiotic metabolism and the core RNAi machinery. The sequencing of the P. weisei transcriptome generated >27 million clean reads, ultimately yielding 90,410 unigenes with an N50 of 1065 bp. The unigenes were used as queries to search the Nr database, which revealed that 21,847 unigenes were homologous to known genes in various species. Transcripts encoding genes involved in xenobiotic metabolism were identified, including genes encoding cytochrome P450 monooxygenase (P450, 47 unigenes), glutathione S-transferase (GST, 12 unigenes), esterase (EST, 25 unigenes), and the ATP-binding cassette transporter (ABC transporter, 32 unigenes). A parallel sequencing of small RNAs detected 30 conserved miRNAs, with the most abundant being Pwe-miR-1-3p, with an expression level reaching 517,996 reads in the prepared library, followed by Pwe-miR-8-3p (149,402 reads). Genes encoding components of the miRNA, siRNA, and piRNA pathways were also identified, and the results indicated that P. weisei possesses only one of each gene in all three pathways. In summary, this is the first detailed analysis of the transcriptome and small RNAs of P. weisei. The datasets presented herein may form the basis for future molecular characterizations of P. weisei as well as the development of enhanced pest control strategies.