Diversity Of Sponge-associated Microbes And N-cycle Related Genes

Many novel bioactive natural products have been isolated from South China Sea sponge Phakellia fusca, however, its microbial symbionts is still unknown. The quantitative analysis on the sponge microbial community is rarely carried out compared with qualitative investigation. Compared with sponge-associated microbial diversity, little is known about the roles microbial symbionts play in sponge biology and ecology. In this study, the community structure and relative abundance of bacteria, actinobacteria, archaea and ammonia-oxidizing archaea in Phakellia fusca were wholly revealed by 16S rRNA gene library and quantitative real time PCR (qRT-PCR). Meanwhile, the ammonia-oxidizing populations were investigated by PCR based on amoA gene and anammox-specific 16S rRNA gene. Bacterial symbionts of sponge Phakellia fusca consist of proteobacteria including Gamma- (78%), Alpha- (7%) and Delta-proteobacteria (3%) and cyanobacteria (12%). The diversity of actinobacterial symbionts in Phakellia fusca is higher, which is composed of Corynebacterineae (48%), Acidimicrobidae (22%), Frankineae (15%), Micrococcineae (13%) and Streptosporangineae (2%). All the observed archaea in sponge Phakellia fusca belong to Crenarchaeota. The detected ammonia-oxidizing populations are ammonia-oxidizing archaea. According to qRT-PCR analysis, bacterial symbionts dominated the prokaryotic microbial symbionts including bacteria, actinobacteria, archaea and ammonia-oxidizing archaea in Phakellia fusca, while archaea represent the second predominant symbints followed by actinobacteria. This study expends our knowledge of the diversity, abundance of microbial symbionts in sponge as well as the ecological roles microbial symbionts play in sponge ecology. And this is useful for cultivation of sponge-associated microbes and natural products. Sponges harbor phylogenetically complex and abundant microbial communities, however the role sponge-associated microorganisms play in nitrogen cycling remains largely unknown, particularly, we hardly know ammonia-oxidizing bacteria (AOB) and denitrification in sponges. In this study, the phylogenetic diversity of nitrogen cycling genes amoA, nifH, nxrA, nirS and nirK and anaerobic ammonia-oxidizing bacteria and nitrite-oxidizing bacteria associated with seven different sponges were investigated. As a result, AOB were found based on amoA genes in five sponges Haliclona sp., Lamellomorpha sp., Placospongia sp., Acanthella sp. and Pericharax heteroraphis. Besides AOB, ammonia-oxidizing archaea (AOA) were detected in sponges Haliclona sp., Lamellomorpha sp., Spirastrellidae diplastrella and Mycale fibrexilis. Meanwhile, we investigated diversity of archaea in these four sponges. Particularly, only AOB were found in sponges Placospongia sp., Acanthella sp. and Pericharax heteroraphis suggesting the main role of AOB in ammonia-oxidization of sponges. Nitrite reductase nirS was detected only in sponge Spirastrellidae diplastrella, while nirK was only observed in sponge Lamellomorpha sp.. Phylogenetic analysis of ammonia monooxygenase and nitrite reductase genes indicates diverse microbial symbionts of sponges involved in the process of nitrification and denitrification. Most of the detected functional gene sequences matched to uncultured clones with low similarity, suggesting the novelty of nitrogen-cycling genes in these sponges. And it was the first time to find nirK genes in sponges. According to this study, sponge species-dependent different nitrification and denitrification mediated by microbial symbionts was suggested. This study extends our knowledge of complex nitrogen transformation mediated by sponge-associated microorganisms.