Resistance to pyridaben in Canadian greenhouse populations of two-spotted spider mites, Tetranychus urticae (Koch).

Two-spotted spider mite (TSSM) Tetranychus urticae (Koch) is an important agricultural pest that causes considerable yield losses to over 150 field and greenhouse crops. Mitochondrial electron transport inhibitors (METI) acaricides are commonly used to control mite species in commercial Canadian greenhouses. Development of resistance to METIs in TSSM populations have been reported worldwide, but not until recently in Canada. The objectives of this study were to: 1) monitor the acaricide-susceptibility in greenhouse TSSM populations, and 2) investigate the resistance to pyridaben, a METI acaricide, in greenhouse resistant and pyridaben-selected (SRS) mite strains. The increased mortality to the pyridaben sub-lethal concentration (LC30) when SRS mites were exposed to piperonyl butoxide (PBO), a general cytochrome P450 monooxygenase inhibitor, and higher P450 activity compared to the greenhouse strain (RS) mites, indicated that P450s may be at least partially responsible for the resistance. The molecular mechanisms of target site insensitivity-mediated resistance in the pyridaben resistant strain of TSSM were investigated by comparing the DNA sequence of NADH dehydrogenase subunits TYKY and PSST, NADH-ubiquinone oxidoreductase chain 1 and 5 (ND1, ND5) and the NADH-ubiquinone oxidoreductase subunit 49 kDa from SRS to the reference strain (SS) and RS. Despite a number of nucleotide substitutions, none correlated with the pyridaben resistance. Understanding the underlying mechanisms of TSSM adaptation to acaricides is an essential part of resistance management strategy in any IPM program. The findings of this study will encourage growers to apply acaricides with different modes of action to reduce the rate at which acaricide resistance will occur in greenhouse TSSM populations.

RNAi-based reverse genetics in the chelicerate model Tetranychus urticae: A comparative analysis of five methods for gene silencing.

RNA interference (RNAi) can be used for the protection against agricultural pests through the silencing of genes required for pest fitness. To assess the potential of RNAi approaches in the two-spotted spider mite, Tetranychus urticae, we compared 5 methods for the delivery of double-stranded RNA (dsRNA). These methods include mite feeding on either (i) leaves floating on a dsRNA solution, (ii) dsRNA-expressing plants, (iii) artificial diet supplemented with dsRNA, or (iv) dsRNA-coated leaves, and (v) mite soaking in a dsRNA solution. In all cases, the gene targeted for method validation was the Vacuolar-type H+-ATPase (TuVATPase), encoding a constitutively expressed ATP-driven proton pump located in the membrane. Down-regulation of TuVATPase increased mortality and/or reduced fecundity in all methods, but with variable efficiency. The most efficient methods for dsRNA delivery were direct soaking of mites in the dsRNA solution and mite feeding on dsRNA-coated leaves that mimics dsRNA application as a sprayable pesticide. Both resulted in a dark-body phenotype not observed in mites treated with a control dsRNA. Although with lower efficiency, dsRNA designed for TuVATPase silencing and expressed in transgenic Arabidopsis plants impacted the fitness of mites feeding on these plants. RNAi may thus be a valuable strategy to control spider mite populations, either as a sprayable pesticide or through transgenic crops. This comparative methodological study focusing on the induction of RNAi-based gene silencing in T. urticae paves the way for reverse genetics approaches in this model chelicerate system and prepares large-scale systematic RNAi screens as a first step towards the development of specific RNA-based pesticides. Such alternative molecules may help control spider mites that cause significant damages to crops and ornamental plant species, as well as other chelicerates detrimental to agriculture and health.