Mosquito cellular immunity at single-cell resolution

Insect hemocytes are the functional equivalents of leukocytes and limit the capacity of mosquitoes to transmit human pathogens through phagocytosis, encapsulation, secretion of immune factors and immune priming (1, 2). Here we profile the transcriptomes of 8506 hemocytes of Anopheles gambiae and Aedes aegypti, two important mosquito vectors. Blood feeding, infection with malaria parasites and other immune challenges reveal a previously unknown functional diversity of hemocytes, with different types of granulocytes expressing distinct and evolutionarily conserved subsets of effector genes. A new cell type, which we term megacyte, is defined in Anopheles by a unique transmembrane protein marker (TM7318) and high expression of LPS-Induced TNF-alpha transcription factor 3 (LL3). Knock-down experiments indicate that LL3 mediates hemocyte differentiation during immune priming. We identify two main hemocyte lineages and find evidence of proliferating granulocyte populations. We validate our analysis with RNA in-situ hybridization and highlight the mobilization of sessile hemocytes into circulation upon infection. Our data (https://hemocytes.cellgeni.sanger.ac.uk/) provide the first atlas of medically relevant invertebrate immune cells at single cell resolution. It provides an important resource for invertebrate immunology by identifying cellular events that underpin mosquito immunity to malaria infection.