PROJECT SUMMARY Most, if not all, animals form beneficial symbioses with bacteria and live in contact with these microbes for the majority of their lives. Understanding the mechanisms by which animals associate with their symbionts is important to human health as we maintain consortia of benefical bacteria at multiple body sites such as the gut and skin. While both partners ultimately benefit from mutualistic interactions, hosts and symbionts both need to maintain mechanisms by which they can ensure cooperation by their partner, also called “sanctioning”. While sanctioning of symbionts is a widespread phenomenon, it can be difficult to study due to the large number and diversity of microbial species present in many interactions and the accessibility of symbiotic interfaces within the host. The proposed work will determine the molecular determinants of sanctioning in two different model systems: The Hawaiian Bobtail Squid Euprymna scolopes and its symbiont Vibrio fischeri, and the skin microbiome of the newt Taricha granulosa. Both of these associations are defensive mutualisms where the symbiont provides a product to the host that enables the host to escape predation. We will leverage the tractability and accessibility of these systems, as well as techniques that we have already developed, to answer crucial questions pertaining to sanctioning. First, we will determine how the host detects symbiotic currency, the first step in some sanctioning efforts, through candidate gene and unbiased approaches. Second, we will define the mechanisms by which hosts sanction symbionts by determining which cells and molecules lead to sanctioning and how they influence bacterial symbionts. Overall, this work will determine the molecular mechanisms that allow animal hosts to ensure the production of valuable symbiotic currency by their symbionts, filling a major gap in our understanding of how mutualistic associations are maintained over the life of an animal.