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.
