Deciphering the role of Amblyomma maculatum hemocytes during Rickettsia parkeri infection

The increase in tick-borne diseases is a significant threat to public health without preventive measures. The innate immune system of hematophagous ticks is divided into two primary mechanisms of immune defense: soluble effector molecules that mediate humoral responses and cellular immune responses such as phagocytosis that promote pathogen killing. Immune cells, referred to as hemocytes, are a critical component of the immune system and produce immune factors that either facilitate or suppress pathogen development in a tick. Despite their importance in pathogen development, understanding of their basic biology and molecular mechanisms is limited. In this study, a combination of pharmacological and molecular tools was utilized to investigate the functional role of tick hemocytes in rickettsiae infection. In Amblyomma maculatum, four groups of distinct morphological types of hemocytes were classified according to their defined cytoplasmic projections, intracytoplasmic granules, and the nuclear-cytoplasmic ratio. Phagocytosis assay led to identifying phagocytic and non-phagocytic populations of hemocytes. Clodronate liposome was used to deplete phagocytic hemocytes and elucidate their role in immune response and survival against bacterial infection. Finally, we conducted a comprehensive mRNA profiling of Am. maculatum hemocytes with and without Rickettsia parkeri infection. Bulk RNA sequencing of hemocytes revealed a total of 39,249 mRNA transcripts, with 11,301 identified as immune-related. Using a reverse genetic approach (RNAi), we functionally characterized two differentially expressed marker genes in hemocyte phagocytosis. Together, these results represent a significant advancement in our understanding of the basic biology of tick hemocytes and their role in the tick immune response.