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, Karenia caelatata and Tanna 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 Candidatus Sulcia muelleri (Sulcia) and a yeast-like fungal symbiont (YLS) were harbored in the bacteriomes and fat bodies, respectively. Both of Sulcia and YLS can be transmitted to the offspring via ovaries, forming a "symbiont ball" in each egg. Neither Sulcia nor YLS were harbored in the salivary glands, gut tissues and testes. Phylogenetic trees of both Sulcia and cicadas confirm that K. caelatata is a member of the tribe Dundubiini, and the tribe Leptopsaltriini that comprises Ta. sp. is not monophyletic. YLS of K. caelatata is embedded inside the lineage of YLS of Dundubiini, whereas YLS of Ta. sp. is closely related to the clade comprising both cicada-parasitizing fungi Ophiocordyceps and YLS of Mogannia conica and Meimuna mongolica, suggesting an evolutionary replacement of YLS in Ta. sp. from an Ophiocordyceps fungus to another Ophiocordyceps 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.