Species Diversity Of Benthic Ciliates And Symbiotic Bacteria On Astomatous Ciliate In The Sublittoral Zone Of Sanshan Island,Laizhou

Marine benthic ciliates are an important part of the microbial food web,which inhabit the bottom of the ocean and sediments.Due to the difficulties in isolation and identification,research on the species diversity of benthic ciliates lags behind that of planktonic groups,and there is still a lot of blank in the research on the species diversity of benthic ciliates including the subtidal area of Sanshan Island,Laizhou.From August 2020 to December 2021,the species diversity of benthic ciliates in the littoral zone of Sanshan Island,Laizhou,was carried out.In total,42 benthic ciliates species were found,and 22 of them were identified based on live observations,protargol staining method,and 18 S SSU r RNA gene sequences,which belong to 20 genera(Pseudokeronopsis,Condylostoma,Dysteria,Cardiostomatella,Loxophyllum,Parauronema,Uronychia,Euplotes,Diophrys,Geleia,Frontonia,Hartmannula,Strombidium,Pleuronema,Chlamydodon,Pseudourostyl,Lacrymaria,Holosticha,Tracheloraphos,Pinacocoleps).The interactions between symbiotic bacterial consortia and their protist hosts in benthic environments have gained increasing concerns in recent years.In the present study,we investigated the diversity of potentially associated bacteria for an astomatous ciliate Kentrophoros flavus,collected in the intertidal zone of Yantai,China.For the first time,the diversity of associated bacteria in the species K.flavus was examined using 16 S r RNA-based techniques of clone libraries and Pac Bio sequencing,and the fluorescence in situ hybridization(FISH)technique.The 16 S r RNA-based sequencing revealed a higher diversity of associated bacteria in K.flavus than previously expected.In addition to the genus-typical thiotrophic symbionts "Candidatus Kentron" YE,we provided evidence to show the consistent existence of at least one other Muribaculaceae-like bacterium,which was secondary abundant among the bacterial OTUs.Fluorescence in situ hybridization(FISH)with three specific probes and crossing validations with "Candidatus Kentron" probes showed that the Muribaculaceae-like bacterium was abundant and merged with "Candidatus Kentron" YE on the cell surface of the host.Phylogenetic analysis of the bacterial 16 S r RNA gene showed that the bacterium was a distinct branch in Muribaculaceae,which were mainly members of the gut microbiome.The name“Muribaculaceae-like bacterium associated with Kentrophoros flavus(MLAKF)” was proposed for the new bacterium.The higher 16 S r RNA diversity in K.flavus,and the discovery of MLAKF on the cell surface,both suggest potentially bacterial consortium interacting with the host K.flavus.