Bongrand, C. | bioRxiv Science

bioRxiv2025

Symbiont diversity and light-organ morphology in Sepiola affinis

The squid-vibrio symbiosis has illuminated fundamental mechanisms of beneficial animal-microbe associations, yet the interactions within sepiolid squid in the Mediterranean Sea remain underexplored. Here we characterize the Sepiola affinis squid-vibrio symbiosis by combining whole-genome sequencing of light-organ isolates, confocal microscopy, and temperature-dependent growth assays. Comparative genomic analyses (ANI, phylogenomics, and functional analyses) revealed two previously undescribed Vibrio species associated with the S. affinis light organ. One species clusters more distantly from other Vibrio while the other species is closer to established Vibrio clades, with the second species exhibiting an expanded repertoire of mobile elements and Type VI secretion components, suggesting heightened capacity for genetic exchange and interbacterial interaction. Confocal microscopy of juvenile squid established that the S. affinis light organ comprises twelve crypts connected by pores and ducts, expanding the number of symbiotic niches relative to other sepiolid squid. In addition, fluorescently labeled isolates from the two Vibrio species colonized juveniles in both mono- and co-colonization patterns within crypts. Finally, growth assays across 16-24{degrees}C identified species-specific temperature differences, indicating temperature preference that may align with seasonal variability in the Mediterranean Sea. Together, these findings position S. affinis as a tractable model for studying how symbiont diversity, organ architecture, and interbacterial interactions contribute to the stability of a mutualistic symbiosis.

microbiology↗

bioRxiv2024

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.

microbiology↗