Development of molecular methods to study hemocyte biology and functions in Aedes aegypti

The mosquito innate immune system is a critical determinant of vector competence. Mosquito immune cells known as hemocytes serve primary roles in immune recognition and pathogen killing, with important functions in Anopheles gambiae that limit malaria parasite survival in the mosquito host. However, the role of mosquito hemocytes in antiviral defense has yet to be established. Previous studies suggest potential roles of hemocytes in arbovirus infection and dissemination in the mosquito host, yet these studies have been limited by the lack of genetic tools to assess the functional contributions of mosquito hemocytes. To approach these questions, we have identified potential genetic markers for mosquito hemocyte populations to study their biology and developed methods to chemically deplete phagocytic cell populations in Aedes aegypti to determine the functional contribution of these immune cells on arbovirus infection. Our results demonstrate that nimrod, peroxidasin and lozenge are potential candidate marker genes for granulocytes and oenocytoids respectively, that can be utilized to create transgenic constructs to label hemocytes. To enable the study of hemocyte functions, phagocytic cell populations were effectively depleted through chemical treatment as validated through light microscopy, reduced expression of hemocyte-specific genes, and impaired immune function following bacterial challenge. Analysis of subsets using flow cytometry argue for presence of additional subsets of hemocytes that vary in phagocytic ability and morphology. Current studies look to further develop these molecular tools to examine viral-host interactions and better understand the role of mosquito cellular immunity in shaping arbovirus infection and transmission

Development of molecular methods to study hemocyte biology and functions in Aedes aegypti

The mosquito innate immune system is a critical determinant of vector competence. Mosquito immune cells known as hemocytes serve primary roles in immune recognition and pathogen killing, with important functions in Anopheles gambiae that limit malaria parasite survival in the mosquito host. However, the role of mosquito hemocytes in antiviral defense has yet to be established. Previous studies suggest potential roles of hemocytes in arbovirus infection and dissemination in the mosquito host, yet these studies have been limited by the lack of genetic tools to assess the functional contributions of mosquito hemocytes. To approach these questions, we have identified potential genetic markers for mosquito hemocyte populations to study their biology and developed methods to chemically deplete phagocytic cell populations in Aedes aegypti to determine the functional contribution of these immune cells on arbovirus infection. Our results demonstrate that nimrod, peroxidasin and lozenge are potential candidate marker genes for granulocytes and oenocytoids respectively, that can be utilized to create transgenic constructs to label hemocytes. To enable the study of hemocyte functions, phagocytic cell populations were effectively depleted through chemical treatment as validated through light microscopy, reduced expression of hemocyte-specific genes, and impaired immune function following bacterial challenge. Analysis of subsets using flow cytometry argue for presence of additional subsets of hemocytes that vary in phagocytic ability and morphology. Current studies look to further develop these molecular tools to examine viral-host interactions and better understand the role of mosquito cellular immunity in shaping arbovirus infection and transmission.</p