Reliable protocols for whole-mount fluorescent in situ hybridization （FISH） in the pea aphid Acyrthosiphon pisum： A comprehensive survey and analysis

RNA in situ hybridization （ISH）, including chromogenic ISH （CISH） and fluorescent ISH （FISH）, has become a powerful tool for revealing the spatial distribution ofgene transcripts in model organisms. Previously, we developed a robust protocol for wholemount RNA CISH in the pea aphid Acyrthosiphon pisum, an emerging insect genomicmodel. In order to improve the resolving capacity of gene detection, we comprehensively surveyed current protocols of whole-mount RNA-FISH and developed protocols that allow,using confocal microscopy, clearer visualization of target messenger RNAs （mRNAs） - including those subcellularly localized and those with spatially overlapping expression. Wefind that Fast dye-based substrate fluorescence （SF）, tyramide signal amplification （TSA）, and TSA Plus all enable identifying gene expression thanks to multiplex amplificationof fluorescent signals. By contrast, methods of direct fluorescence （DF） do not allow visualizing signals. Detection of a single gene target was achieved with SF and TSA Plusfor most mRNAs, whereas TSA only allowed visualization of abundant transcripts such as Apvasl andAppiwi2 in the germ cells. For detection of multiple gene targets using doubleFISH, we recommend： （i） TSA/TSA, rather than TSA Plus/TSA Plus for colocalized mRNAs abundantly expressed in germ ceils, as proteinase K treatment can be omitted;and （ii） SF/TSA Plus for other gene targets such as Apenl and Apen2 as inactivation of enzyme conjugates is not required. SF/SF is not ideal for double FISH experiments due tosignal blurring. Based on these new conditions for RNA-FISH, we have obtained a better understanding of germline specification and embryonic segmentation in the pea aphid.We anticipate that the RNA-FISH protocols for the pea aphid may also be used for other aphids and possibly other insect species, thus expanding the range of species from whichuseful insights into development and evolution may be obtained.