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

Abstract

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.

Keywords: Aliivibrio fischeri; amplicon sequencing; microbiome; symbiosis.

Figure 1.. hsp60 amplicon sequencing differentiates Vibrio fischeri from other closely related vibrios at the species level.

(A) Known Input corresponds to bacterial isolates added to each known community, and Observed Taxa reports the relative abundance (%) of taxa identified from each known community using hsp60 amplicon sequencing. All known input strains were detected in their corresponding samples, including in mixed communities comprised of multiple Vibrio fischeri strains and closely related vibrios. The number of unique ASVs that were assigned to each taxonomic identifier is reported here. (B) Relative abundance (%) of V. fischeri ASVs comprising > 0.1% of total abundance in any given sample. Of ASVs that meet this criteria, two ASVs were identified as V. fischeri ES114, two ASVs were identified as V. fischeri MB13B2, and one ASV was identified as MB13B1. Abundance of any additional unique ASVs identified in panel A fell below the abundance cutoff 0.1%. (C) Neighbor-joining phylogenetic tree analysis based on distance matrix calculated by multiple sequence alignment (MSA) of 73 Vibrio fischeri hsp60 sequences obtained from NCBI and trimmed to the region amplified by the primers used in this study (see table S1). Orange boxes represent strains with 100% identity between hsp60 sequences compared to the average nucleotide identity (ANI) between the corresponding strains at the whole-genome level as calculated by FastANI (98.26 – 99.99% ANI).

Figure 2.. Relative abundance of V. fischeri and other conserved taxa associated with juvenile and adult squid.

(A) Relative abundance of V. fischeri, other vibrios, and non-vibrio taxa in juvenile squid, juvenile squid ventate, and adult squid. The percentage of reads identified as V. fischeri in each sample is indicated on the y-axis (% Vf). In wild-caught adult panel, MB* indicates animal identifier, with A or B indicating paired samples from each lobe of the same light organ. (B) Corresponding relative abundance of non-V. fischeri taxa in the samples described in panel A. Taxa are plotted at the family level, and Vibrionaceae excludes ASVs identified as V. fischeri. Unidentified Bacteria correspond to ASVs that were not assigned taxonomy past the kingdom level, likely due to a lack of similar sequences in available hsp60 reference sequences. Other Taxa correspond to ASVs that were assigned taxonomy to at least the family level but make up < 0.1 % of the total read abundance. Sample order is consistent between panel A and panel B.