Jinzhi Niu

Plant defense has driven the evolution of species-dependent adaptive strategies in many herbivores, but the molecular mechanisms of adaptation in most arthropods remain largely unknown. The two-spotted spider mite ( Tetranychus urticae ) is a generalist herbivore that feeds by sucking the contents of mesophyll cells, a feeding strategy distinct from that of phloem-feeding insects. Here, we show the unexpected differential expression of apolipoprotein D ( ApoD ) genes during feeding when mites are transferred to different host plant species, as well as extreme gene family expansion (64 ApoD paralogs, the largest reported in any organism). We find that ApoD proteins protect mites against reactive oxygen species (ROS) during feeding, and we identify a dual-layer mechanism by which ApoD proteins counteract plant ROS defenses during ingestion and digestion. First, the salivary protein TuApoD2 is secreted during the ingestion of mesophyll cell contents, where it interacts with glycolate oxidase 2 (GOX2) to inhibit H ₂ O ₂ generation. After ingestion, TuApoD33 in the gut cells also interacts with GOX2 to maintain the inhibition of H ₂ O ₂ generation. These findings suggest that the evolutionary expansion of the ApoD gene family is a key component of the molecular arms race between herbivorous mites and host plants.

RNA interference (RNAi) is a form of gene silencing triggered by double-stranded RNA (dsRNA) that operates in all eukaryotic organisms, including insects, mites, nematodes and fungi. In the last two decades, many dsRNAs have been synthesized to silence the target genes for exploration of the underlying function in these pests. Some of them are lethal to pests or inhibit the growth of the pest population, leading to a new concept of pesticides. The generation of these environment-friendly pesticides requires precise in silico design of dsRNA molecules that are specific to target pests but do not target non-pest organisms. Current efforts for dsRNA design are mostly focused on gene sequence level, lacking comprehensive analysis of RNAi-based mode-of-action at the whole transcriptome level of given species, causing low efficiency and imprecise dsRNA target exploration. To address these limitations, we have created dsRNAEngineer, a publicly available online tool that allows a comprehensive and rational dsRNA design, incorporating hundreds of pests and non-pests transcriptomes. Developed functionalities include screen-target (screen conserved genes for co-targets of various pest species), on-target, off-target, and multi-target to generate optimal dsRNA for precise pest control. dsRNAEngineer is available at https://dsrna-engineer.cn/ . Graphical

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.

RNA interference (RNAi) is a form of gene silencing triggered by double‐stranded RNA (dsRNA) that operates in all eukaryotic cells. RNAi has been widely investigated in insects to determine the underlying molecular mechanism, to investigate its role in systemic antiviral defense, and to develop strategies for pest control. When insect cells are infected by viruses, viral dsRNA signatures trigger a local RNAi response to block viral replication and generate virus‐derived DNA that confers systemic immunity. RNAi‐based insect pest control involves the application of exogenous dsRNA targeting genes essential for insect development or survival, but the efficacy of this approach has limited potency in many pests through a combination of rapid dsRNA degradation, inefficient dsRNA uptake/processing, and ineffective RNAi machinery. This could be addressed by dsRNA screening and evaluation, focusing on dsRNA design and off‐target management, as well as dsRNA production and delivery. This review summarizes recent progress to determine the role of RNAi in antiviral defense and as a pest control strategy in insects, addressing gaps between our fundamental understanding of the RNAi mechanism and the exploitation of RNAi‐based pest control strategies.

Systemic RNA interference deficient‐1‐like (SIL1) is considered a core component in dsRNA uptake in some insect species. Investigation related to the potential function of SIL1 in dsRNA uptake can contribute to a further understanding of RNA interference (RNAi) mechanisms in insects and agricultural pest control. However, the role of SIL1 in dsRNA uptake in insects such as aphids remains controversial. We have thoroughly analyzed the role of SIL1 from the model aphid Acyrthosiphon pisum (ApSIL1) in cellular dsRNA to clarify its function. First, the induced expression of ApSIL1 upon dsRNA oral exposure provided a vital clue for the possible involvement of ApSIL1 in cellular dsRNA uptake. Subsequent in vivo experiments using the RNAi‐of‐RNAi approach for ApSIL1 supported our hypothesis that the silencing efficiencies of reporter genes were reduced after inhibition of ApSIL1 expression. The impaired biological phenotypes of aphids, including cumulative average offspring, deformities of the nymph, and mortality upon pathogen infection, were then observed in the treatment group. Thereafter, in vitro dual‐luciferase reporter assay showed compelling evidence that the luciferin signal was significantly attenuated when dsluciferase or dsGFP was transferred into ApSIL1‐transfected Drosophila S2 cells. These observations further confirmed that the signal of Cy3‐labeled dsRNA was rapidly attenuated with time in ApSIL1‐transfected Drosophila S2 cells. Overall, these findings conclusively establish that ApSIL1 is involved in dsRNA uptake in A. pisum.

Spider mites (Tetranychidae) are destructive agricultural pests which have evolved strategies to overcome plant defenses, such as the ability to puncture the leaves of their hosts to feed. The expression of many genes with unknown functions is altered during feeding, but little is known about the role of these genes in plant–mite interactions. Here, we identified 3 novel gene families through analysis of genomic and transcriptomic data from 3 spider mite species. These GARP family genes encode glycine and alanine‐rich proteins; they are present in mites (Acariformes) but absent in ticks (Parasitiformes) in the subclass Acari, indicating that these genes have undergone a significant expansion in spider mites and thus play important adaptive roles. Transcriptomic analysis revealed that the expression of GARP genes is strongly correlated with feeding and the transfer to new hosts. We used RNA interference to silence GARP1d in the two‐spotted spider mite Tetranychus urticae, which inhibited feeding and egg laying and significantly increased mortality when the mites were transferred to soybean shoots; a similar effect was observed after TuVATPase was silenced. However, no changes in mite mortality were observed after TuGARP1d‐silenced mites were placed on an artificial diet, which was different from the effect of TuVATPase silencing. Our results indicate that GARP family members play important roles in mite–plant interactions. Additional studies are needed to clarify the mechanisms underlying these interactions.