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Abstract

To survive in the nutrient-poor waters of the tropics, reef-building corals evolved the capacity to engage in a mutualistic symbiosis with unicellular dinoflagellates. This symbiosis creates both the structural and trophic foundation of the entire ecosystem – a highly productive ecosystem that is astonishingly rich in biodiversity. Most coral species reestablish this symbiosis with each new generation, meaning that coral progeny must select compatible symbionts from the environment. The symbionts are phagocytosed by theendodermal cells of the coral, where they reside in a specialized vacuole, i.e., the symbiosome, and a bidirectional nutrient exchange ensues. While immune suppression has been implicated in mediating symbiosis in corals, the cellular mechanisms connected to immune suppression that influence symbiont selection and maintenance are unknown. Furthermore, it is unclear how the modulation of immune pathways can simultaneously promote symbiont maintenance while thwarting invasion by non-beneficial microorganisms. To better understand how symbionts are stably integrated into host cells, we established a comparative framework using the endosymbiosis model Aiptasia, a small sea anemone closely related to corals. This comparative analysis was complemented with confocal microscopy and immunofluorescence, live imaging, transcriptomic analyses, and exogenous immune modulation to analyze various aspects of symbiosis establishment, from uptake to maintenance. We found that initial uptake of microalgae is largely indiscriminate as Aiptasia larvae phagocytose a vast array of particles, including symbionts, non-symbiotic microalgae, heat-killed microalgae, and beads. After phagocytosis, we found that non-symbiotic or heat-killed particles are expelled via vomocytosis, a process that is stochastic and dependent on extracellular-regulated kinase 5. Not only are these particles expelled, but they are frequently reacquired and expelled again, in what resembles a search for symbionts based on trial-and-error. The symbionts evade this expulsion and establish an intracellular lysosomal associated membrane protein 1 (LAMP1)-positive niche. Additionally, we found that LAMP1 accumulates around heat-killed microalgae, possibly in an attempt to digest the particles. However, even though the heat-killed non-symbiotic microalgae are intracellular longer than their healthy counterparts, they too are expelled, eventually. Thus, we revealed dual functionality for LAMP1 in intracellular niche establishment and its canonical association with degradative lysosomes. Analyzing transcriptomic data from symbiotic cells compared with non-symbiotic microalgae-containing cells revealed that symbiont-uptake induces broad immune suppression, sufficient to halt their expulsion. Exogenously activating the innate immune system in Aiptasia larvae during infection with symbionts impairs symbiosis establishment. Using live imaging, we demonstrated that this compromised infection is a direct consequence of enhanced expulsion. Conversely, we showed that symbiosis establishment is bolstered when the toll-like receptor pathway is inhibited, specifically by interfering with MyD88 homodimer formation. In summary, we found that although some pre-phagocytic selection mechanisms exist, as heat-killing of microalgae influences uptake, the truly decisive symbiont selection mechanisms occur post-phagocytosis. Furthermore, our findings demonstrated that local immune suppression during symbiosis establishment is essential to bypass vomocytosis and initiate LAMP1-niche formation. This work revealed the role of an evolutionarily ancient innate immune response involved in symbiont selection and symbiosis establishment.