Molecular identification of methane monooxygenase and quantitative analysis of methanotrophic endosymbionts under laboratory maintenance in Bathymodiolus platifrons from the South China Sea

Deep-sea mussels of the genus Bathymodiolus are numerically dominant macrofauna in many cold seep and hydrothermal vent ecosystems worldwide, and they depend on organic carbon produced by symbionts present in the epithelial cells of the gills. Although Bathymodiolus platifrons represents typical methanotrophic endosymbiosis, our understanding of molecular mechanisms of methane oxidization and carbon fixation is still in its infancy. Moreover, the laboratory maintenance of B. platifrons and the symbiont abundance dynamics during maintenance has not been reported. In the present study, we report the first systematic identification and phylogenetic analysis of three subunits of methane monooxygenase (pmoA, pmoB, and pmoC) obtained from the endosymbiotic bacteria found in B. platifrons. The coding sequences (CDS) of the three genes in the B. platifrons endosymbiont were 750, 1,245, and 753 bp, encoding 249, 414, and 250 amino acids, respectively. Sequence alignment and phylogenetic analysis revealed that the symbiont of B. platifrons belongs to the type I methanotrophs. In order to clarify the impact of environmental methane on symbiont abundance, a 34-day laboratory maintenance experiment was conducted in which B. platifrons individuals were acclimatized to methane-present and methane-absent environments. Symbiont abundance was evaluated by calculating the relative DNA content of the methane monooxygenase gene using quantitative real-time PCR. We found that symbiont quantity immediately decreased from its initial level, then continued to gradually decline during maintenance. At 24 and 34 days of maintenance, symbiont abundance in the methane-absent environment had significantly decreased compared to that in the methane-present environment, indicating that the maintenance of symbionts relies on a continuous supply of methane. Our electron microscopy results validated the qPCR analysis. This study enriches our knowledge of the molecular basis and the dynamic changes of the methanotrophic endosymbiosis in B. platifrons, and provides a feasible model biosystem for further investigation of methane oxidization, the carbon fixation process, and environmental adaptations of deep-sea mussels.