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Abstract:
Optical imaging of gene expression by fluorescence in situ hybridisation (FISH) in insects is often impeded by their pigmented cuticle. Since most chemical bleaching agents are incompatible with FISH, we developed a RNA interference-based method for clearing cuticular pigmentation which enables using whole-mount body appendages for RNA FISH. Silencing laccase2 or tyrosine hydroxylase in two leaf beetles species (Chrysomela populi, Phaedon cochleariae) cleared their pigmented cuticle and decreased light absorbance. Subsequently, intact appendages (palps, antennae, legs) from RNAi-cleared individuals were used to image expression and spatial distribution of antisense mRNA of two chemosensory genes (gustatory receptor, odorant-binding protein). Imaging did neither work for RNAi-controls due to retained pigmentation, nor for FISH-controls (sense mRNA). Several bleaching agents were incompatible with FISH, either due to degradation of RNA, lack of clearing efficacy or long incubation times. Overall, silencing pigmentation genes is a significant improvement over bleaching agents enabling FISH in intact appendages.
