Visualizing chemical transport from extracellular symbionts into squid host tissues using NanoSIMS

Chemical communication is the basis of host-microbe interaction. Insights into this communication will provide a deeper understanding of the mechanisms that govern these complex associations, in beneficial as well as pathogenic contexts. In this dissertation, we used the beneficial relationship between the Hawaiian bobtail squid Euprymna scolopes and its symbiont, bioluminescent bacterium Vibrio fischeri as a model system to investigate the molecular transfer from symbiont to host. Using correlative transmission electron microscopy and quantitative ion microprobe isotopic imaging (NanoSIMS; nano-scale secondary ion mass spectrometry), the transfer and localization of 15N-labeled V. fischeri-derived molecules into tissues of newly-hatched squids were studied. Together, these data suggest that the squid is able to take up and incorporate bacterial products released from the symbiont and from other Gram-negative bacteria. Inoculating squids with strongly 15N-labeled V. fischeri cells, or with outer membrane vesicles (OMVs) released by these cells, resulted in widespread 15N-enrichment inside the host light organ. 15N-enrichment was observed in the light-organ epithelia and in other squid tissues, such as the gills and gut. 15N-enrichment in light-organ tissues was most notable inside cell nuclei: preferentially in the euchromatin regions and the nucleoli. Squids exposed to a mixture of 15N-enriched amino acids showed comparable cellular 15N-distributions, suggesting that the majority of bacterial molecules were transferred to the host as part of a more general chemical transfer from the environment. Inoculating squids with non-symbiotic Vibrionaceae cells (Vibrio parahaemolyticus and Photobacterium leiognathi) produced 15N-enrichment patterns similar and comparable to V. fischeri-induced patterns. It is likely that the similar host 15N-enrichment patterns between all bacterial treatments is induced by molecules that have shared structural and functional features and/or are processed by similar pathways within the host cells. On the other hand, substantial differences in the host 15N-enrichment were observed when squids where exposed to OMVs derived from different bacteria; specifically, V. fischeri, V. parahaemolyticus, and Escherichia coli. These ‘OMV 15N-enrichment patterns’ were distinct, especially between the Vibrio strains and E. coli, demonstrating a clear host capacity for specific uptake of molecules derived from different bacteria. This thesis provides new insights into the chemical transfer that might occur in the squid’s natural habitat. The results indicate that, in addition to the highly specific molecular interactions V. fischeri is known to have with its host, a general molecular exchange takes place. Although the nature of this molecular uptake cannot be inferred from the observations presented here, the molecules involved might have an essential role in interactions between the bacteria and their animal host. In broader terms, this work demonstrates the unique link between the spatial and functional information provided by the NanoSIMS imaging, which has the potential to open a new frontier for the study of communication between host and symbiont.