Bacterial Diversity Of Bacteriomes Associated With Two Cicada Species (Hemiptera: Cicadidae) Based On 16S RRNA High-throughput Sequencing

Insects of the family Cicadidae have the piercing-sucking mouthparts,with nymphs and adults exclusively feeding on xylem sap of host-plants.Xylem sap is an extremely dilute food source of any plant material,which is limited in carbohydrate,amino acid and vitamin.During the long evolution process,cicadas have developed intimate symbioses with bacteria to obtain nutrients and compounds that are lacking in their diet.Symbionts often play potential roles on host’s niche expansion and species differentiation,and are co-evolutionary with host insects.The endosymbionts of cicadas are typically restricted to a specialized organ,bacteriome,which consists of a set of distinctive host cells,called bacteriocytes.To date,the comparison of bacterial diversity in the bacteriomes and reproductive organs of different populations/different development stages has been little investigated,and the bacterial community in these organs remains also a puzzle.Here,using 16S rRNA high-throughput sequencing,we investigated the bacterial diversity in bacteriomes and reproductive organs of two populations of Subpsaltria yangi Chen and also the individuals of Meimuna mongolica（Distant）across the life history.In addition,the gross morphology of bacteriomes and reproductive organs in this two cicada species were observed using the optical microscopy.（1）Subpsaltria yangi was collected from the Helan Mountains（HM）of Ningxia Hui Autonomous Region and Hancheng City（HC）in Shaanxi Province,respectively,whose geography and habitat are greatly divergent.Morphological differences of bacteriomes and reproductive organs between these two populations are not significant.Each tissue of the HC population had a richer bacterial diversity than the corresponding tissue of the HM population,and meanwhile,the testes had the most bacterial diversity than other tissues of both populations.The obligated endosymbiont Sulcia muelleri was observed in all samples,mostly with high levels of relative abundance in bacteriomes and ovaries（relative abundance>85%）.In addition,this symbiont was also the most abundant bacterium in eggs,suggesting that S.muelleri are stably maintained through host generations by vertical transmission from mothers to their offspring via the ovaries.The usually co-resident endosymbiont Candidatus Hodgkinia cicadicola was not detected.Instead,a novel Rhizobiales bacterium which shows⁸1%similarity to H.cicadicola was detected.It is probable that this novel Rhizobiales bacterium act as a symbiont similar to Hodgkinia cicadicola with beneficial trophic functions.These results provide new clues to help establish the definition of the bacterial genus Candidatus Hodgkinia,and are informative for further studies of evolutionary divergence of related endosymbionts hosted in cicadas.（2）The number of spherical units of bacteriomes increased with the nymphs of Meimuna mongolica（Distant）growing.The reproductive organs（ovaries and testes）emerged in the5^th-instar and the number of ovarioles and testis follicles increased with age.The bacterial diversity in testis was significantly higher than that in other tissues.The obligate symbiont Candidatus Sulcia muelleri was dominated（relative abundance>85%）and stable in eggs and bacteriomes among development stages.In addition,this endosymbiont got established in the reproductive organs of nymphs till the 5^th-instar once ovaries and testes emerged,and its abundance increased significantly in ovaries with age.The co-primary obligate symbiont Candidatus Hodgkinia cicadicola,previous known in some other cicada species,was not detected also.Instead,the species Spiroplasma sp.of Entomoplasmatales was detected in all samples and especially dominated in reproductive organs.Whether Spiroplasma as a novel symbiont has replaced Hodgkinia to supply host nutritions needs further investigation.（3）Our results suggest that the bacterial communities of reproductive organs between different populations within a species are different,which may possibly be effected by the different habitats and host-plants,and particularly involved in potential evolutionary of the cicada host.The bacterial community composition in bacteriomes across the life history of the same cicada species is not significantly different.The obligate endosymbiont S.muelleri is transmitted vertically,and got established in the reproductive organs of nymphs till the5^th-instar once ovaries and testes emerged.The co-resident symbiont Hodgkinia cicadicola was not detected in both cicada species,which is possibly that Hodgkinia cicadicola was lost or had been replaced.