ABSTRACT This proposal will characterize the influence of tick gut microbiota on the ability of Borrelia burgdorferi, the Lyme disease agent, to be transmitted from ticks to the vertebrate host. Our recent work (Narasimhan et al, Cell Host Microbe 15:58-71, 2014) demonstrated that tick gut microbiota modulate B. burgdorferi colonization of Ixodes scapularis. Our preliminary data suggest that normal gut microbiota facilitate efficient B. burgdorferi transmission to the mammalian host. This is a critical event in the context of human Lyme disease and understanding how tick gut microbiota influence Borrelia transmission to the mammalian host will reveal new ways to prevent Lyme disease transmission. In Aim 1 we will characterize and determine tick gut bacterial profiles that are associated with decreased B. burgdorferi transmission to the vertebrate host. Further, we will also begin to assess the microbial compositions of nymphal ticks obtained from endemic regions in Connecticut to co-relate our observations on lab-reared ticks. Application of this knowledge to field studies in future efforts might provide new predictors/biomarkers of transmission risk in specific geographic areas. In Aim 2 we will perform a transcriptome analysis of the tick gut to describe the molecular pathways of the tick that are influenced by gut microbiota during B. burgdorferi transmission. With a focus on up to 10 genes that are significantly altered by gut microbiota we will determine their functional consequence on B. burgdorferi transmission. Potential vaccine targets to impair Borrelia transmission may be identified from this functional analysis. Finally, we will determine if dysbiosis might modulate the expression profiles of Borrelial virulence genes, and additionally influence spirochete transmission to the host. We will focus on the lipoprotein encoding virulence genes of B. burgdorferi and unravel a new understanding of how B. burgdorferi senses the tick gut environment to regulate the expression of virulence genes. This research effort will move the field beyond a descriptive understanding of tick gut microbiota, and unfold a mechanistic understanding of the influence of tick gut bacteria on tick-pathogen interactions in particular, and on vector-pathogen interactions in general. Tick gut microbiota provides a new vantage point to understand the biology of B. burgdorferi transmission to mice and to develop new paradigms to explore the factors that contribute to the spread of Lyme disease.