Wing dimorphism is a phenomenon of phenotypic plasticity in aphid dispersal. However, the signal transduction for perceiving environmental cues (e.g., crowding) and the regulation mechanism remain elusive. Here, we found that aci-miR-9b was the only down-regulated microRNA (miRNA) in both crowding-induced wing dimorphism and during wing development in the brown citrus aphid Aphis citricidus. We determined a targeted regulatory relationship between aci-miR-9b and an ABC transporter (AcABCG4). Inhibition of aci-miR-9b increased the proportion of winged offspring under normal conditions. Overexpression of aci-miR-9b resulted in decline of the proportion of winged offspring under crowding conditions. In addition, overexpression of aci-miR-9b also resulted in malformed wings during wing development. This role of aci-miR-9b mediating wing dimorphism and development was also confirmed in the pea aphid Acyrthosiphon pisum. The downstream action of aci-miR-9b-AcABCG4 was based on the interaction with the insulin and insulin-like signaling pathway. A model for aphid wing dimorphism and development was demonstrated as the following: maternal aphids experience crowding, which results in the decrease of aci-miR-9b. This is followed by the increase of ABCG4, which then activates the insulin and insulin-like signaling pathway, thereby causing a high proportion of winged offspring. Later, the same cascade, “miR-9b-ABCG4-insulin signaling,” is again involved in wing development. Taken together, our results reveal that a signal transduction cascade mediates both wing dimorphism and development in aphids via miRNA. These findings would be useful in developing potential strategies for blocking the aphid dispersal and reducing viral transmission.

Vitellogenin (Vg) and its receptor (VgR) play a key role in the reproductive process and development of insects. Aphids are a group of high-fecundity insect species with pseudoplacental viviparity, but the roles of their Vg and VgR genes have not been investigated yet. The brown citrus aphid, Aphis (Toxoptera) citricidus, is a major insect pest of citrus and the main vector of Citrus tristeza closterovirus. In this study, we identified and characterized these two genes, designated as AcVg and AcVgR, from the brown citrus aphid. We found that AcVg has lost the DUF1943 domain that is present in other insect Vgs. Silencing of AcVg and AcVgR led to a delay in the nymph-adult transition, a prolonged prereproductive period, and a shortened reproductive period, which in turn resulted in slower embryonic development and fewer new-born nymphs. Interestingly, silencing of AcVg decreased the transcript level of AcVgR, but silencing of AcVgR resulted in increased transcript levels of AcVg. In addition, silencing of Vg/VgR had similar phenotypes between alate and apterous morphs, suggesting that the functions of these two genes are the same in the two wing morphs of the aphid. Our results demonstrate that Vg and VgR are involved in various aspects of aphid development and reproduction. Further studies on the synthesis of Vg could help to elucidate the reproductive mechanism and provide information that will be useful for developing new pest control strategies.

Insulin receptors play key roles in growth, development, and polymorphism in insects. Here, we report two insulin receptor genes (AcInR1 and AcInR2) from the brown citrus aphid, Aphis (Toxoptera) citricidus. Transcriptional analyses showed that AcInR1 increased during the nymph–adult transition in alate aphids, while AcInR2 had the highest expression level in second instar nymphs. AcInR1 is important in aphid development from fourth instar nymphs to adults as verified by dsRNA feeding mediated RNAi. The silencing of AcInR1 or/and AcInR2 produced a variety of phenotypes including adults with normal wings, malformed wings, under-developed wings, and aphids failing to develop beyond the nymphal stages. Silencing of AcInR1 or AcInR2 alone, and co-silencing of both genes, resulted in 73% or 60%, and 87% of aphids with problems in the transition from nymph to normal adult. The co-silencing of AcInR1 and AcInR2 resulted in 62% dead nymphs, but no mortality occurred by silencing of AcInR1 or AcInR2 alone. Phenotypes of adults in the dsInR1 and dsInR2 were similar. The results demonstrate that AcInR1 and AcInR2 are essential for successful nymph–adult transition in alate aphids and show that RNAi methods may be useful for the management of this pest.

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.

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.

Winged and wingless morphs in insects represent a trade-off between dispersal ability and reproduction. We studied key genes associated with apterous and alate morphs in Toxoptera citricida (Kirkaldy) using RNAseq, digital gene expression (DGE) profiling, and RNA interference. The de novo assembly of the transcriptome was obtained through Illumina short-read sequencing technology. A total of 44,199 unigenes were generated and 27,640 were annotated. The transcriptomic differences between alate and apterous adults indicated that 279 unigenes were highly expressed in alate adults, whereas 5,470 were expressed at low levels. Expression patterns of the top 10 highly expressed genes in alate adults agreed with wing bud development trends. Silencing of the lipid synthesis and degradation gene (3-ketoacyl-CoA thiolase, mitochondrial-like) and glycogen genes (Phosphoenolpyruvate carboxykinase [GTP]-like and Glycogen phosphorylase-like isoform 2) resulted in underdeveloped wings. This suggests that both lipid and glycogen metabolism provide energy for aphid wing development. The large number of sequences and expression data produced from the transcriptome and DGE sequencing, respectively, increases our understanding of wing development mechanisms.

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.