Tick hemocytes have pleiotropic roles in microbial infection and arthropod fitness.

Uncovering the complexity of systems in non-model organisms is critical for understanding arthropod immunology. Prior efforts have mostly focused on Dipteran insects, which only account for a subset of existing arthropod species in nature. Here, we describe immune cells or hemocytes from the clinically relevant tick using bulk and single cell RNA sequencing combined with depletion via clodronate liposomes, RNA interference, Clustered Regularly Interspaced Short Palindromic Repeats activation (CRISPRa) and RNA-fluorescence hybridization (FISH). We observe molecular alterations in hemocytes upon tick infestation of mammals and infection with either the Lyme disease spirochete or the rickettsial agent . We predict distinct hemocyte lineages and reveal clusters exhibiting defined signatures for immunity, metabolism, and proliferation during hematophagy. Furthermore, we perform a mechanistic characterization of two hemocyte markers: and . Depletion of phagocytic hemocytes affects and levels, which impacts blood feeding and molting behavior of ticks. Hemocytin specifically affects the c-Jun N-terminal kinase (JNK) signaling pathway, whereas astakine alters hemocyte proliferation in . Altogether, we uncover the heterogeneity and pleiotropic roles of hemocytes in ticks and provide a valuable resource for comparative biology in arthropods.