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

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 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 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 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 V. fischeri inoculum, as well as ventate samples and light organ cores from wild-caught adults. V. fischeri 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 V. fischeri is the dominant, if not sole, symbiont typically 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 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.