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.

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.