Coupling clearing and Hybridization Chain Reaction approaches to
investigate gene expression in organs inside whole-mount intact insect.
Abstract
Detecting RNA molecules within their natural environment inside intact
arthropods has long been challenging, particularly in small organisms
covered by a tanned and pigmented cuticle. Here, we have developed a
methodology that enables high-resolution analysis of the spatial
distribution of transcripts of interest without having to dissect tiny
organs or tissues, thereby preserving their integrity. We have combined
an in situ amplification approach based on Hybridization Chain
Reaction, which enhances the signal-to-noise ratio, and a clearing
approach that allows the visualization of inner organs beneath the
cuticle. We have implemented this methodology for the first time in
Hemiptera, mapping two salivary aphid ( Acyrthosiphon pisum)
transcripts, the effector c002 and the salivary sheath protein SHP. With
a multiplex approach, we could simultaneously detect different mRNAs in
whole-mount pea aphid head-thorax samples and show that they were
distributed in distinct secretory cells of salivary glands.