Comprehensive understanding of pleiotropic roles of RNAi machinery highlighted the conserved chromosomal functions of RNA interference. The consequences of the evolutionary variation in the core RNAi pathway genes are mostly unknown, but may lead to the species-specific functions associated with gene silencing. The two-spotted spider mite, Tetranychus urticae, is a major polyphagous chelicerate pest capable of feeding on over 1100 plant species and developing resistance to pesticides used for its control. A well annotated genome, susceptibility to RNAi and economic importance, make T. urticae an excellent candidate for development of an RNAi protocol that enables high-throughput genetic screens and RNAi-based pest control. Here, we show that the length of the exogenous dsRNA critically determines its processivity and ability to induce RNAi in vivo. A combination of the long dsRNAs and the use of dye to trace the ingestion of dsRNA enabled the identification of genes involved in membrane transport and 26S proteasome degradation as sensitive RNAi targets. Our data demonstrate that environmental RNAi can be an efficient reverse genetics and pest control tool in T. urticae. In addition, the species-specific properties together with the variation in the components of the RNAi machinery make T. urticae a potent experimental system to study the evolution of RNAi pathways.

Comprehensive understanding of pleiotropic roles of RNAi machinery highlighted the conserved chromosomal functions of RNA interference. The consequences of the evolutionary variation in the core RNAi pathway genes are mostly unknown, but may lead to the species-specific functions associated with gene silencing. The two-spotted spider mite, Tetranychus urticae, is a major polyphagous chelicerate pest capable of feeding on over 1,100 plant species and developing resistance to pesticides used for its control. A well annotated genome, susceptibility to RNAi and economic importance, make T. urticae an excellent candidate for development of an RNAi protocol that enables high-throughput genetic screens and RNAibased pest control. Here, we show that the length of the exogenous dsRNA critically determines its processivity and ability to induce RNAi in vivo. A combination of the long dsRNAs and the use of dye to trace the ingestion of dsRNA enabled the identification of genes involved in membrane transport and 26S proteasome degradation as sensitive RNAi targets. Our data demonstrate that environmental RNAi can be an efficient reverse genetics and pest control tool in T. urticae. In addition, the species-specific properties together with the variation in the components of the RNAi machinery make T. urticae a potent experimental system to study the evolution of RNAi pathways.

A recently developed biopesticide made of safflower and cottonseed oils has excellent ovicidal activity against the hard‐to‐control spider mite Tetranychus urticae Koch (Acari: Tetranychidae). It has attracted attention as a sustainable treatment for controlling T. urticae because it has low potential for promoting resistance and little effect on the predatory mite Neoseiulus californicus (McGregor) (Acari: Phytoseiidae), which is an important natural enemy of spider mites. Here, we investigated the mechanism of its ovicidal activity against T. urticae. The oil droplets in the oil‐in‐water emulsion of the biopesticide strongly adhered to T. urticae eggs, seeped through the chorion being cut during hatching, and inhibited the embryonic rotational movement necessary for cutting and hatching. No adverse effect was observed on N. californicus eggs even in undiluted biopesticide. We conclude that this biopesticide and N. californicus can be used simultaneously in the integrated management of T. urticae in oily biopesticide‐tolerant plant species.

Double-stranded RNA (dsRNA) as a trigger of RNAi-mediated sequence-specific gene silencing is a promising next-generation pesticide that could enable selective pest control ultimately at the species level. The discovery that orally administrated dsRNA induces RNAi in the two-spotted mite, Tetranychus urticae Koch (Trombidiformes: Tetranychidae), has led to a dramatic increase in research toward the development of RNAi-based biopesticides for spider mites.

RNA interference (RNAi) or post-transcriptional gene silencing is a biological process conserved in a broad range of eukaryotes and triggered by endogenous or exogenous double-stranded RNA (dsRNA). RNAi suppresses the endogenous expression of the target gene of which the mRNA sequence is complementary to the dsRNA sequence. RNAi has been widely used as a gene functional analysis technology, and recently, exogenous dsRNA has begun to be applied to a sprayable biopesticide with a different mode-of-action from conventional synthetic pesticides. The two-spotted spider mite, Tetranychus urticae Koch (Trombidiformes: Tetranychidae), is distributed worldwide and has been known as one of the most difficult agricultural pests to control due to its rapid development of resistance to synthetic pesticides. Therefore, the development of RNAi-based biopesticide targeting this species is highly anticipated. However, the function of RNAi machinery in this species remains unclear. Processing dsRNA into small RNA is a fundamental process contributing to RNAi efficacy. Here we performed functional analysis of TuDcr1 and TuDcr2 genes, homologues of Drosophila melanogaster Dicer-1 and Dicer-2, which belong to the RNase III family. The nucleotide sequences of TuDcr1 (tetur19g00520), TuDcr2 (tetur07g00990), and an intergenic region as a negative control (NC) were obtained from the ORCAE database and used for PCR amplification of each DNA fragment. RNA synthesized by in vitro transcription from each DNA fragment was used to prepare dsRNA specific to TuDcr1, TuDcr2, and NC (dsRNA-TuDcr1, dsRNA-TuDcr2, and dsRNA-NC, respectively). A nylon-mesh-based feeding device was used to orally deliver dsRNA to age-synchronized adult female mites for 24 h. Both TuDcr1 and TuDcr2 were upregulated after oral administration of dsRNA-NC, which indicates the activation of RNAi machinery by orally-delivered dsRNA. Downregulations of TuDcr1 and TuDcr2 were observed in mites fed on dsRNA-TuDcr1 and dsRNA-TuDcr2 when compared to dsRNA-NC. However, no significant effects were observed in the susceptibility to RNAi targeting an essential gene Vacuolar-type H+-ATPase after oral administration of dsRNA-TuDcr1, dsRNA-TuDcr2, or dsRNA-NC. The dsRNA-cleaving assay revealed that the dicing activity was reduced in mites fed on dsRNA-TuDcr1 and dsRNA-TuDcr2 when compared with dsRNA-NC and that the activity seemed to be higher in TuDcr2 than TuDcr1. Our results suggest that TuDcr1 and TuDcr2 possess the activity of dsRNA cleavage and that TuDcr2 is responsible for dicing exogenous dsRNA.