The tick Ixodes scapularis transmits several pathogens of relevance to public health, including Borrelia burgdorferi (Lyme disease), Anaplasma phagocytophilum (human granulocytic anaplasmosis), B. miyamotoi (tick-borne relapsing fever), Babesia microti (babesiosis) and Powassan virus (tick-borne encephalitis). To date, while diverse areas of immunity have been studied in insects, the underpinnings of the tick immune system are in its infancy, and the molecules involved in microbial sensing remain chiefly undefined. Similarly, the molecular characterization of tick immune cells or hemocytes remains mostly elusive. Over the previous funding period, we discovered a non-canonical immune deficiency (IMD) network in I. scapularis and showed that the IMD pathway is important against B. burgdorferi and A. phagocytophilum infection. Specifically, we uncovered the role of molecules that act on the I. scapularis IMD pathway and provided mechanistic support for an observation that the immune system of ticks is fundamentally different than insects. We made substantial progress in addressing the three specific aims, which resulted in several peer-reviewed publications. For this R01 project (competing renewal), we will build on our earlier findings and investigate the nexus between metabolism and immunology. Our central hypothesis states that changes in metabolic status impact the immune system and microbial infection of ticks. Accordingly, in Aim #1 of this proposal, we will characterize the signaling flexibility of the IMD pathway in ticks. The classical textbook definition of an immune pathway consists of a fixed stack of proteins that amplifies the immune signal from a receptor to an effector. We will reveal that the tick IMD pathway is malleable and reprograms itself upon metabolic changes and/or microbial infection. In Aim #2 of this grant application, we will categorize tick hemocyte populations under distinct metabolic and microbial conditions through a platform that involves coupling of single cell RNA sequencing (scRNA-seq) with a monoclonal antibody screening strategy. This major resource to the vector biology community will show that metabolism and microbial infection affect tick hemocyte biology. In Aim #3 of this submission, we will assess whether metabolism affects fitness parameters associated with the life cycle of I. scapularis. We will also evaluate transstadial and transgenerational passage of microbes in ticks. Altogether, this R01 project will demonstrate that bioenergetic processes likely define cost-effective and/or energetically expensive tick-microbe interactions.