Environmental RNA interference in two-spotted spider mite,Tetranychus urticae, reveals dsRNA processing requirements for efficient RNAi response

Abstract

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 Leaf-Mimicking Method for Oral Delivery of Bioactive Substances Into Sucking Arthropod Herbivores

“…The TuVATPase transcript level reached >75% reduction in mites collected 4 days after treatment ( Figure 3E). Although the reduction of the transcript abundance was 2 to 3 times higher than that reported in previous studies (Kwon et al, 2013;Suzuki et al, 2017b;Bensoussan et al, 2020), this might be due to the high concentration of orally administered dsRNA used in the present study. Optimizing the observation period (~10 days) and dsRNA concentration (>160 ng µl −1 ) in the mesh method should allow for high-throughput screening of candidate genes for RNAi-based T. urticae control.…”

“…To evaluate the usefulness of the mesh method to deliver genetic materials, we tested environmental RNAi targeting the TuVATPase gene with exogenously supplied dsRNA in mites. The dark-body phenotype ( Figure 3C) associated with RNAi targeting the TuVATPase gene was observed in approximately 90% of mites ( Figure 3D), which was 2 to 3 times higher than that reported by Suzuki et al (2017b) and comparable to that noted by Bensoussan et al (2020), who tracked dsRNA ingestion using a tracer dye. In addition, we observed reductions in mite survivorship and fecundity by RNAi targeting the TuVATPase gene ( Figures 3A, B).…”

“…In addition, we observed reductions in mite survivorship and fecundity by RNAi targeting the TuVATPase gene ( Figures 3A, B). According to Suzuki et al (2017b) and Bensoussan et al (2020), a marked reduction in mite survivorship was observed at 6 to 10 days after treatment. In…”

“…Thus, an observation period of about 10 days should be used to accurately assess the lethal effect of environmental RNAi in mites. Although the dsRNA concentration used (1 µg µl −1 ) was higher than that used by Suzuki et al (2017b) and Bensoussan et al (2020) (20 to 320 ng µl −1 ), the reductions in mite survivorship and fecundity were comparable. Bensoussan et al (2020) have reported no significant difference in the effects of RNAi targeting the TuVATPase and a subunit of coatomer protein complex (TuCOPB2) genes at dsRNA concentrations higher than 160 ng µl −1 , which suggest that the lethal RNAi effect may have already been saturated at 1 µg µl −1 dsRNA.…”

“…The fecundity was significantly lower in mites fed on dsRNA-TuVATPase than in the control group at 2 to 6 days after treatment ( Figure 3B). The dark-body phenotype that is associated with VATPase gene silencing in T. urticae ( Figure 3C; Suzuki et al, 2017b;Bensoussan et al, 2020) was observed in around 80 and 90% of mites fed on dsRNA-TuVATPase at 2 days and at 3-6 days after treatment, respectively, whereas no mites showed the dark coloration in the control group even at 6 days after treatment ( Figure 3D). The expression level of endogenous TuVATPase transcripts was significantly lower in the treatment group than in the control group at 2-4 days after treatment ( Figure 3E).…”