PROJECT SUMMARY/ABSTRACT More than 5% of US children and 45% of US adolescents have anxiety or depression; these rates have increased for over a decade. Anxiety and depression are highly comorbid internalizing mental disorders that often start during childhood and are associated with abnormal hypothalamic-pituitary-adrenal (HPA) axis function. Early life stress (ELS) increases vulnerability to anxiety and depression throughout life. Accumulating evidence suggests that ELS may produce long-lasting changes in gut microbiota contributing to abnormal HPA axis function and behavior, which could play a pivotal role in internalizing behaviors. Many avenues exist whereby microbiome composition may influence behavior and physiology, one of which is through altering host epigenetics. ELS reduces folate-producing strains in the gut microbiome and folate is a necessary component for DNA methylation. Recent work highlights potential interactions between microbiome and host epigenome, with microbiota involved in regulating the host response to environment signals such as ELS. It is unknown whether ELS influences gut microbiome composition in early development in humans or how the microbiome influences HPA activity and internalizing behavior. We hypothesize that early psychosocial stress decreases folate-producing genre in the gut microbiome which influences HPA DNA methylation and internalizing behavior. Elucidating how environment and gut microbiome influences behavior will provide insights for a new generation of effective interventions and treatments for mental health. This proposal capitalizes on two existing cohorts: the Environmental influences on Child Health Outcomes (ECHO; K99 Phase) and the Arizona Twin Project (ATP; R00 Phase). Both cohorts have extensive longitudinal, affective behavioral phenotyping during infancy, toddlerhood, and early childhood. Aim 1 will use existing ECHO longitudinal microbiome and ELS metadata to determine if maternal depression during early childhood contributes to folate-producing relative abundance. Aim 2 will determine if host DNA methylation is a mechanism by which the gut microbiota composition influences internalizing behavior. The R00 Aims will extend our K99 results and determine if ELS influences microbiome composition in adolescence and assess DNA methylation in relation to internalizing behaviors. Using twin ACE models we can estimate the genetic and environmental influences of the relationships between microbiome, DNA methylation, and internalizing behaviors. ACE model results will pinpoint aspects of the environment, unconfounded by genetic influences that are crucial players in the mechanistic pathway for mental health. Results will provide valuable information for the wider scientific community in understanding early environmental influences on mental health through development.