Kentaro Imaizumi, Reiko Nozaki, Hidehiro Kondo, Ikuo Hirono
Symbiotic bacteria belonging to the class Mollicutes are widely associated with arthropods, yet their ecological roles in crustaceans remain poorly understood. Our previous 16S rRNA gene amplicon surveys revealed that Mollicutes consistently dominated the gastric microbiota of the whiteleg shrimp Penaeus vannamei. To characterize this dominant lineage, genome-resolved metagenomic analysis was conducted on Mollicutes-dominated samples from aquaculture ponds in Thailand, where a single lineage accounted for >95% of the community. A high-quality metagenome-assembled genome (MAG) was reconstructed (98.55% completeness, 1.33% contamination), with a small genome (~1.28 Mb) and low GC content (29.7%). Phylogenomic analyses placed this lineage within the order Enteroplasmatales but clearly separated it from Enteroplasma. Genomic relatedness metrics, including average nucleotide identity (~68%), average amino acid identity (~57%), and POCP values (43-46%), support its classification as a novel genus and species. Functional annotation revealed a highly reduced metabolic repertoire consistent with host association, including loss of amino acid biosynthesis and respiratory chain components, while retaining glycolysis and fermentative metabolism and expanded ABC transport systems for nutrient uptake. Notably, genes for nearly complete peptidoglycan biosynthesis were retained, an unusual feature in Mollicutes that may reflect incomplete cell wall loss or functional repurposing of these genes. Overall, this study identifies a previously uncharacterized Enteroplasmatales lineage dominating the shrimp gastric microbiota and exhibiting strong signatures of reductive evolution and host adaptation. This lineage is proposed as Candidatus Penaeiplasma gastricola gen. Nov., sp. nov., representing a distinct crustacean-associated clade within Enteroplasmatales and likely encompassing lineages previously assigned to Candidatus Bacilloplasma based on 16S rRNA gene similarity. These findings provide a genomic framework for understanding the ecological roles and evolutionary history of dominant gastric symbionts in penaeid shrimp, and a foundation for future studies of shrimp microbiome function and health.