Abstract: 【Aim】 This study aims to investigate whether clear lipid-exchanged acrylamide-hybridized rigid imaging/immunostaining/in situ hybridizationcompatible tissue-hYdrogel (CLARITY) technique is feasible in insect tissue treatments, using guts of Apis mellifera workers as the test samples, and to test the feasibility of CLARITY in combination with fluorescence in situ hybridization (FISH) in the clarification of insect gut tissues. 【Methods】 A. mellifera worker guts were fixed and solidified according to the regular CLARITY protocol and clarified using the passive method. The in situ hybridization of the clarified gut tissues was conducted using species-specific oligonucleotide probes including Nosema ceranae-specific 16S rRNA-targeted oligonucleotide probe labeled with fluorescein isothiocyanate (FITC) and a universal eukaryotic 18S rRNAtargeted oligonucleotide probe labeled with Texas RED. The nuclei were counter-stained with DAPI (in blue), and the stained tissues were observed under a confocal laser scanning microscope. 【Results】 For the first time, A. mellifera guts were successfully processed and clarified.  The original morphology of Malpighian tubules in the abdomen was observed, and the spatial distribution of Nosema with higher density at the distal part of the midgut was clearly labeled in the 3D scanning using confocal laser scanning microscope. 【Conclusion】 CLARITY can be used in the clarity of honey bee gut tissues, and the clarified gut tissues can be stained by FISH and observed with confocal laser scanning microscopy. CLARITY in combination with FISH provides a reliable and specific labeling method for insect physiological and pathological studies without the troubles of antibody preparation and paraffin sectioning usually required for a similar study.

Key words: Apis mellifera, Nosema ceranae, CLARITY, gut, confocal laser scanning microscopy, in situ hybridization