A Study on Symbiotic Systems of Cicadas Provides New Insights into Distribution of Microbial Symbionts and Improves Fluorescence In Situ Hybridization Technique.

Nutritional symbionts of sap-sucking auchenorrhynchan insects of Hemiptera are usually confined to the bacteriomes and/or fat bodies. Knowledge is limited about the distribution of microbial symbionts in other organs. We investigated the distribution of obligate symbionts in the salivary glands, gut tissues, reproductive organs, bacteriomes, and fat bodies of two cicada species, and sp., using integrated methods, including a modified fluorescence in situ hybridization (FISH) technique, which can greatly enhance the FISH signal intensity of related symbionts. We revealed that Sulcia muelleri () and a yeast-like fungal symbiont (YLS) were harbored in the bacteriomes and fat bodies, respectively. Both of and YLS can be transmitted to the offspring via ovaries, forming a "symbiont ball" in each egg. Neither nor YLS were harbored in the salivary glands, gut tissues and testes. Phylogenetic trees of both and cicadas confirm that is a member of the tribe Dundubiini, and the tribe Leptopsaltriini that comprises sp. is not monophyletic. YLS of is embedded inside the lineage of YLS of Dundubiini, whereas YLS of sp. is closely related to the clade comprising both cicada-parasitizing fungi and YLS of and , suggesting an evolutionary replacement of YLS in sp. from an fungus to another fungus. Our results provide new insights into the symbiosis between Cicadidae and related symbionts. Modification through the addition of helpers and heat shock greatly enhanced the FISH signal intensity of YLS, which may provide guidelines for enhancement of the hybridization signal intensity of other symbiont(s) in the FISH experiments.