Euprymna scolopes microbiome - hsp60 amplicon sequencing

The symbiotic relationship between Vibrio (Aliivibrio) fischeri and the Hawaiian bobtail squid, Euprymna scolopes, serves as a key model for understanding host-microbe interactions. Traditional culture-based methods have primarily isolated V. fischeri from wild-caught squid, yet culture-independent analyses of the symbiotic microbiome remain limited. This study aims to enhance species-level resolution of bacterial communities associated with E. scolopes using hsp60 amplicon sequencing. We validated our hsp60 sequencing approach using pure cultures and mixed bacterial populations, demonstrating its ability to distinguish V. fischeri from other closely-related vibrios. This approach was applied to whole-animal juvenile squid exposed to seawater and clonal V. fischeri inoculate, as well as ventate samples and light organ cores from wild-caught adults. V. fischeri accounted for 31%-62% of amplicon sequence variants (ASVs) from whole juvenile samples, and 94%-99% of ASVs for adult light organ core samples, confirming that V. fischeri is the dominant symbiont associated with E. scolopes light organs. In one ventate sample, V. fischeri comprised 82% of reads, indicating the potential for non-invasive community assessments using this approach. Analysis of non-V. fischeri ASVs revealed that Bradyrhizobium spp. and other members of the Rhodobacterales order are conserved across juvenile and adult samples. These findings suggest additional microbial associations with the squid host that have not been previously detected through traditional culture methods. Further, our results affirm the utility of hsp60 amplicon sequencing in investigating microbial diversity within the squid-vibrio symbiosis and identified the presence of potentially significant, yet uncultured, bacterial taxa.