Application of <i>hsp60</i> amplicon sequencing to characterize microbial communities associated with juvenile and adult <i>Euprymna scolopes</i> squid

The symbiotic relationship between <i>Vibrio (Aliivibrio) fischeri</i> and the Hawaiian bobtail squid, <i>Euprymna scolopes</i>, serves as a key model for understanding host-microbe interactions. Traditional culture-based methods have primarily isolated <i>V. fischeri</i> from the light organs of wild-caught squid, yet culture-independent analyses of this symbiotic microbiome remain limited. This study aims to enhance species-level resolution of bacterial communities associated with <i>E. scolopes</i> using <i>hsp60</i> amplicon sequencing. We validated our <i>hsp60</i> sequencing approach using pure cultures and mixed bacterial populations, demonstrating its ability to distinguish <i>V. fischeri</i> from other closely related vibrios and the possibility of using this approach for strain-level diversity with further optimization. This approach was applied to whole-animal juvenile squid exposed to either seawater or a clonal <i>V. fischeri</i> inoculum, as well as ventate samples and light organ cores from wild-caught adults. <i>V. fischeri</i> accounted for the majority of the identifiable taxa for whole-animal juvenile samples and comprised 94%-99% of amplicon sequence variants (ASVs) for adult light organ core samples, confirming that <i>V. fischeri</i> is the dominant, if not sole, symbiont typically associated with <i>E. scolopes</i> light organs. In one ventate sample, <i>V. fischeri</i> comprised 82% of reads, indicating the potential for non-invasive community assessments using this approach. Analysis of non-<i>V. fischeri</i> ASVs revealed that <i>Bradyrhizobium spp.</i> and other members of the Rhodobacterales order are conserved across juvenile and adult samples. These findings provide insight into the presence of additional microbial associations with the squid host tissue outside of the light organ that have not been previously detected through traditional culture methods.