Many animals enter into life-long beneficial associations with bacteria. These symbioses are often species-specific, so that effective mechanisms must ensure the establishment of stable partnerships. The symbiosis between the squid, Euprymna scolopes, and the bioluminescent bacterium Vibrio fischeri is a useful model for understanding the influence of beneficial bacteria on animal colonization and development. Juvenile squid hatch without V. fischeri and must obtain them from seawater that contains few potential symbionts and many non-symbiotic bacteria. The host must maintain these symbionts throughout its life while excluding other bacteria. Recent studies suggest that the immune system plays a significant role in mediating these interactions. Host blood cells (hemocytes) have different responses to symbiotic and non-symbiotic bacteria; colonization alters the ability of these cells to recognize symbiotic bacteria, suggesting that the immune system of the host becomes "educated" to the presence of the symbionts. Because invertebrates lack adaptive immunity (the ability to produce antibodies that lead to immunological memory), this specificity must be mediated through the host's innate immune system. This project will explore how host hemocytes mediate specificity in the squid/vibrio symbiosis. The genes and proteins expressed by these hemocytes will be characterized in response to colonization and to symbiotic and non-symbiotic bacteria. Hemocyte responses to V. fischeri and environmental bacteria will be characterized during the early onset of the association to understand the potential mechanisms by which the host's immune system recognizes and becomes adapted ("educated") to the symbiont. This project will integrate research with education by contributing to the training of undergraduate, graduate and post-doctoral students including those from underrepresented groups. Results will be disseminated through scientific publications as well as a public symbiosis website.