Periodic Reporting for period 1 - SquidVibrio (Characterization of multi-species bacterial interactions underlying the beneficial symbiosis of a Mediterranean squid.)
Reporting period: 2022-09-01 to 2024-08-31
Beneficial host-microbe interactions play a crucial role in maintaining animal health, yet their complexity makes them challenging to study. Invertebrate models for mutualism, however, offer valuable insights into the function of these associations. One such model is the squid-vibrio symbiosis that examines the partnership that occurs within a symbiotic light organ. The symbiosis has been studied extensively in the USA focusing on the Hawaiian bobtail squid Euprymna scolopes and it’s bioluminescent bacterial symbiont Vibrio fischeri. The SquidVibrio Project aims to expand studies of the squid-vibrio symbiosis into Europe by studying Sepiola affinis, a species of Mediterranean squid. Intriguingly, S. affinis is colonized by multiple Vibrio spp., allowing for examination of how interspecies bacterial interactions persist within the symbiosis. The overarching aim of the project is to establish the first experimental system to study the squid-vibrio symbiosis within Europe. Furthermore, the project will serve to (1) establish a reliable husbandry system for S. affinis, (2) examine multiple Vibrio species within the squid’s symbiotic light organ and (3) characterize small molecule patterns within the light organ. The experimental framework outlined here demonstrates how the SquidVibrio Project is positioned to introduce an exciting new model organism to Europe while making significant contributions to the fields of symbiosis and marine biology.
The SquidVibrio Project is structured into three objectives that have all yielded important achievements. The first main objective was establishing a squid husbandry facility for S. affinis. The facility consisted of two parallel systems: one for maintaining adult squid and one for rearing juvenile squid. The adult system was first established and can house up to 20 adult squid, with both S. affinis and Sepietta neglecta successfully being maintained and producing viable eggs. To date, the adult squid system has produced over 200 eggs that were transferred to the rearing system. The rearing system successfully incubated squid eggs and then efficiently raised them upon hatching, with survival rates > 75% achieved for both S. affinis and S. neglecta. In addition, the squid rearing system allowed for experimental control of the colonization state, with squid able to be raised nonsymbiotic or colonized by genetic mutants. The squid husbandry facility ultimately allowed for the closing of the life cycle for both S. affinis and S. neglecta. The second main objective was a characterization of the S. affinis light organ microbiome. From field-caught squid, a library of bacterial symbionts was built that allowed for examination of strain composition. Using whole genome sequencing, two uncharacterized species of Vibrio bacteria were discovered as symbionts of the S. affinis light organ. Furthermore, some of the strains were able to be labeled with fluorescent proteins, allowing for experimental tracking and imaging. Using confocal microscopy, the first visualization within the S. affinis light organ was performed, providing intriguing images of the symbiotic crypts. The third main objective consisted of examining the biochemical environment within the S. affinis light organ. Using mass spectrometry imaging, metabolomic profiles of the novel Vibrio strains and S. affinis light organs were able to be generated. Furthermore, distinct metabolites linked to both the host and bacterial symbiont were able to be identified and localized within tissue sections. The achievements described here represent an intriguing foundation for future experiments with S. affinis.
The SquidVibrio Project has produced intriguing results that align with its overall goal of establishing a Mediterranean squid model for symbiosis within Europe. The first result was the establishment of an efficient squid husbandry system. The system was able to successfully close the life cycles of both S. affinis and S. neglecta with survival rates > 75 %, and is capable of efficiently producing hundreds of squid hatchlings per year. The husbandry system will allow for future studies examining the squid-vibrio symbiosis within a Mediterranean host. Specifically, experiments will be able to examine how two distinct Vibrio species are maintained within the host through different seasons. The next major result was the discovery of two uncharacterized Vibrio species within the S. affinis light organ. In addition, the S. affinis light organ was also discovered to contain 12 symbiotic crypts, which is the most ever reported in a squid light organ. These surprising results exemplify how symbioses of European squid are nearly completely unexplored and provide a valuable comparison to other mutualistic associations. Finally, small molecule analysis through mass spectrometry imaging was able to identify metabolites within tissue sections that could be specifically linked to either the host or bacterial symbiont. Future experiments will allow for examination of molecules linked to interspecies bacterial communication under symbiotic conditions and molecules used for host-symbiont communication. Overall, these results show how the SquidVibrio Project has expanded the state of the art for studying the squid-vibrio symbiosis and beneficial host-microbe interactions in general.