Paired metagenomics and metatranscriptomics reveal metabolic capabilities of uncultivated Verrucomicrobia and other bacteria in the Hawaiian bobtail squid reproductive symbiosis

Host-associated microbiomes often consist of complex bacterial consortia, many of whose members remain uncultivated and/or have poorly characterized functions. In this study, we used metagenomics and metatranscriptomics to better understand the reproductive defensive symbiosis of the accessory nidamental gland (ANG) of the Hawaiian bobtail squid, Euprymna scolopes. We recovered 23 high- and medium-quality metagenome-assembled genomes (MAGs) from the four major ANG symbiont taxa (Alphaproteobacteria, Verrucomicrobia, Gammaproteobacteria, and Flavobacteriia) that dominate the E. scolopes ANG community. Three Puniceicoccaceae MAGs represent the first Verrucomicrobia genomes from a cephalopod ANG and are potentially novel species in Verrucomicrobia subdivision four. These Verrucomicrobia encode the highest diversity of carbohydrate-active enzymes (CAZymes) among the analyzed strains. Metatranscriptomes revealed no differential expression between the ANG and eggs, indicating metabolic stability during symbiont transfer from the ANG tubules to egg jelly coats. Community-wide expression of glycoside hydrolases may enable shared degradation of host O- and N-glycosylated mucins. Genes often important in host-association, such as motility, chemotaxis, and quorum sensing, were broadly expressed amongst the major taxa. Expression of diverse secondary metabolite clusters (e.g., NRPS, PKS, bacteriocins) and competitive mechanisms, like the T6SS, were also expressed and may contribute to mediating competition within the ANG or in egg defense. Overall, this work reveals the genomic and transcriptomic repertoire of the E. scolopes ANG microbiome and provides insight on members of the marine Verrucomicrobia, a group with growing recognition as important in symbiotic associations. SignificanceThe Verrucomicrobia are a ubiquitous group of growing interest, particularly due to the role of Akkermansia in the human gut. However, little is known about aquatic Verrucomicrobia, especially those that switch between a free-living and host associated lifestyle. This study furthers our understanding of potential mucin degradation among novel Puniceicoccaceae of the E. scolopes ANG reproductive symbiosis. Shared expression of carbohydrate utilization pathways among ANG symbionts may reveal networks of competition and cooperation that parallel the complex networks found in the human gut. As well, this study lays the groundwork for further understanding the mechanisms of bacteria-mediated host egg defense, serving as a model for study of other marine defensive symbioses.