ABSTRACT
Optical imaging of gene expression by fluorescence in situ hybridisation (FISH) in insects is often impeded by their pigmented cuticle. As most chemical bleaching agents are incompatible with FISH, we developed an RNA interference (RNAi)-based method for clearing cuticular pigmentation which enables the use of whole-mount body appendages for RNA FISH (termed RNA-i-FISH). Silencing laccase2 or tyrosine hydroxylase in two leaf beetles species (Chrysomela populi and Phaedon cochleariae) cleared their pigmented cuticle and decreased light absorbance. Subsequently, intact appendages (palps, antennae, legs) from RNAi-cleared individuals were used to image the expression and spatial distribution of antisense mRNA of two chemosensory genes encoding gustatory receptor and odorant-binding protein. Imaging did not work for RNAi controls because the pigmentation was retained, or for FISH controls (sense mRNA). Several bleaching agents were incompatible with FISH, because of 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 insect appendages.
Footnotes
Author contributions
Conceptualization: S.P., W.B., A.B.; Methodology: S.P., V.G., A.O., L.S., A.B.; Validation: S.P., V.G., A.O., L.S.; Formal analysis: S.P., V.G., A.O.; Investigation: S.P., V.G., A.O., L.S.; Data curation: S.P.; Writing - original draft: S.P.; Writing - review & editing: S.P., V.G., W.B., A.B.; Visualization: S.P., V.G.; Supervision: S.P., W.B., A.B.; Project administration: S.P., A.B.; Funding acquisition: S.P., W.B., A.B.
Funding
S.P. and A.B. received project funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 705151. Further financial support to all authors from the Max Planck Society is acknowledged.
Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request. Sequences generated in this study have been deposited in GenBank (www.ncbi.nlm.nih.gov/genbank) under the following accession numbers: CpopLac2: MH253687; PcoLac2: MH253688; CpopTH: MH253689; PcoTH: MH253690; CpopGR1: MH253691; and CpopOBP13: MH253692.
