Gene silencing in the spider mite Tetranychus urticae: dsRNA and siRNA parental silencing of the Distal-less gene.

A major prerequisite to understanding the evolution of developmental programs includes an appreciation of gene function in a comparative context. RNA interference (RNAi) represents a powerful method for reverse genetics analysis of gene function. However, RNAi protocols exist for only a handful of arthropod species. To extend functional analysis in basal arthropods, we developed a RNAi protocol for the two-spotted spider mite Tetranychus urticae focusing on Distal-less (Dll), a conserved gene involved in appendage specification in metazoans. First, we describe limb morphogenesis in T. urticae using confocal and scanning electron microscopy. Second, we examine T. urticae Dll (Tu-Dll) mRNA expression patterns and correlate its expression with appendage development. We then show that fluorescently labeled double-stranded RNA (dsRNA) and short interfering RNA (siRNA) molecules injected into the abdomen of adult females are incorporated into the oviposited eggs, suggesting that dsRNA reagents can be systemically distributed in spider mites. Injection of longer dsRNA as well as siRNA induced canonical limb truncation phenotypes as well as the fusion of leg segments. Our data suggest that Dll plays a conserved role in appendage formation in arthropods and that such conserved genes can serve as reliable starting points for the development of functional protocols in nonmodel organisms.