Quenching autofluorescence of insect tissues for in situ detection of endosymbionts

Oligonucleotide-probed fluorescent in situ hybridization (FISH) targeting 16S rRNA is a powerful technique for detecting and characterizing bacterial cells in environmental samples without cultivation; however, general application of the technique to insect endosymbionts has been hindered by the strong autofluorescence frequently observed in insect tissues. Here we describe a protocol that markedly reduces autofluorescence of insect tissues by hydrogen peroxide (H₂O₂) treatment, whereby 16S rRNA of bacterial endosymbionts is kept in a FISH-detectable condition. Among various histological fixatives, Carnoy's solution was superior in that whole insects were successfully fixed and autofluorescence of insect tissues was suppressed in comparison with the widely used formaldehyde-based fixatives. Treatment with both alcoholic 6% H₂O₂ solution and aqueous 6% H₂O₂ solution markedly reduced autofluorescence of the fixed insect tissues, wherein the former kept 16S rRNA of bacterial endosymbiont in a FISH-detectable condition while the latter failed to do so. The protocol was applicable to endosymbionts of diverse insects such as aphids, lice and bat flies. The protocol was applicable not only to fresh insect samples but also to archival insect samples preserved in acetone for several years. We propose a general and robust protocol for quenching autofluorescence of insect tissues for FISH detection of bacterial endosymbionts, which is potentially applicable to endosymbionts of a wider range of organisms with considerable autofluorescence.