Comparisons Of Sponge Holobionts Across Different Niches And Genomic Study Of Uncultured Sponge Symbiotic Bacteria

As the most ancient multicellular animal,sponges have formed the close symbiotic relationship with microbes during the long history of evolution.Based on systematic biology,sponges and their symbionts are defined as holobionts.For both historical and technical reasons,our understanding towards sponge holobionts is mostly focused on the 16 S rRNA gene-based phylogenetic diversity of sponge-associated bacteria.Our knowledge of the relationships between sponge holobionts and niches,the functional genomics and ecological roles of sponge symbionts,the eukaryotic symbionts,and the sponge-microbe interaction remain rudimentary.In the first part of this thesis(Chapter 2-4),samples of Hawaiian encrusting sponge Mycale grandis that grew under three niches,i.e.the algal mats,on the surface of corals,and on the rocky matrix,were collected.Combined strategy including 454 pyrosequencing,GeoChip,and transcriptome sequencing was used to investigate and compare the in situ transcriptionally active microbial communities and functional genes,host gene expression,and the host-microbe interaction pattern in the context of niche heterogeneity.First,16 S rRNA(454 pyrosequencing)and gyrB(GeoChip)was used to analyse the in situ transcriptionally active bacterial symbionts of M.grandis.The two phylogenetic markers indicated the similar phylogenetic composition of M.grandis symbiotic bacterial communities from three niches,which were dominated by Proteobacteria,particularly ?-proteobacteria.Yet gyrB analysis uncovered the microdiversity that 16 S rRNA failed to detect,indicating a clear separation across the three niches.Moreover,the expression pattern of gyrB suggested the distinct highly active bacterial subgroups existed in each niche.At the level of functional categories,GeoChip indicated the metabolism repertoire of M.grandis-associated bacterial communities from different niches appeared to be similar.Upon inspection of individual genes,90% of the genes were shared by samples from three niches(core genes).Nonetheless,the diversity and expression patterns of functional genes were distinct from one niche to another.Analysis at sequence-level indicated a high functional redundancy of core functional genes.Additionally,little overlapping was detected among the highly expressed variants of different niches.Overall,it appears that the differences of M.grandis symbiotic bacterial communities are the reflections of the bacterial microdiversity and the functional redundancy of microbial communities.Next,we used 28 S rRNA-454 pyrosequencing and GeoChip to compare the in situ transcriptionally active symbiotic fungal communities of M.grandis in three niches.The results of 28 S rRNA-454 pyrosequencing indicated the fungal diversity within M.grandis across different niches was largely cohesive and distinct from that in the ambient seawater.Capnodiales dominated the fungal communities with transcriptionally activity in M.grandis.GeoChip analysis illustrated that fungal symbionts of M.grandis mainly participated in the carbon degradation,aromatics degradation,and denitrification.Similar to the results of bacterial communities,the functional gene diversity and gene expression patterns of fungal symbiotic communities in three niches were distinct from each other.The sequence-level inspection also indicated the high functional redundancy of core fungal functional genes and the existence of distinct highly active subgroups in each niche.At last,the transcriptome sequencing of M.grandis from three niches indicated a clear separation between niches.The functional annotations of differentially expressed genes suggested the stress and physiological status of M.grandis varied from niche to niche.When it grew under algal mats,it might be under the stress of unfavourable conditions.When it grew on coral reefs,it might be under the stress of competing against corals for space or in the early reproductive stage.When it grew on the rocky matrix,it might need to control the overflow.Based on the Pearson Correlation Coefficients between all differentially expressed CDS and the microbial features(i.e.functional gene diversity and expression rate),we found eukaryotic-like proteins and proteins related to the innate immune system were the major groups that correlated to the microbial features.Based on the above results,we proposed a hypothesis that M.grandis might be in different physiological status in differetn niches,which affects the expression of eukaryotic-like proteins and proteins related to the innate immune system.Subsequently,the differential expression of these proteins presents varied selective pressure against sponge symbionts.The differences of M.grandis symbiotic bacterial and fungal communities across different niches are the results of the interaction of niche-specific selective pressure,microbial microdiversity,and functional redundancy.In the second part of the thesis,we focused on the genomes of uncultured sponge symbionts.“Entotheonella”(phylum “Tectomicrobia”)is a filamentous symbiont that produces almost all known bioactive compounds derived from the Lithistida sponge Theonella swinhoei.In contrast to the comprehensive knowledge of its secondary metabolism,knowledge of its lifestyle,resilience,and interaction with the sponge host and other symbionts remains rudimentary.In this study,we obtained two “Entotheonella” genomes from T.swinhoei from the South China Sea through metagenome binning.The high average nucleotide index(ANI)values suggested they were the same phylotypes as the two “Entotheonella” phylotypes from T.swinhoei from the Japan Sea.Genomic analysis indicated the amino acid auxotrophic mixotrophic nature of “Entotheonella”.The features related to dissimilatory sulphate reduction,multiple anaerobic respiration,and denitrification indicated that “Entotheonella” could survive in anaerobic conditions.The endospore-forming potential along with metal-and antibiotic resistance indicated “Entotheonella” was highly resilient to harsh conditions.The discovery of Type II and Type VI secretion systems in “Entotheonella” suggested it could secrete extracellular hydrolases,form tight adhesion,act against phagocytes,and kill other prokaryotes.The newly discovered genomic features suggest “Entotheonella” is a highly competitive member of the symbiotic community of T.swinhoei and provide background knowledge for cultivating this uncultured genus.