There are an estimated 219 million cases of malaria per year, leading to more than 400,000 deaths annually according to the World Health Organization. Hemocytes (insect white blood cells) comprise the mosquito immune system and are the basis for immunity to malaria. There are three known hemocyte types: granulocytes (highly phagocytic cells of about 10 to 20 μm in diameter); oenocytoids (8- to 12-μm round cells that produce melanin involved in pathogen encapsulation); and prohemocytes (round cells (4 to 6 μm) with a high nuclear-to-cytoplasmic ratio and are thought to be precursors of the other two cell types). These can be circulating in the hemolymph or resident in tissues like the gut epithelium (where the ookinete stage of the Plasmodium migrates). Upon infection, hemocytes activate and kill Plasmodium parasite by complement activation. Repeated infection is prevented by immunological memory termed "priming" in mosquitos; subsequent infections stimulate release of hemocyte differentiation factor into the hemolymph, which causes granulocyte induction. This priming is effectuated by release of hemocyte differentiation factor (HDF), a combination of lipoxin 4 and evokin, which is a lipocalin carrier.
Against this backdrop of basic mosquito biology, an international team* of researchers reported their results of an in-depth study of mosquito immune system in a paper published in Science entitled "Mosquito cellular immunity at single cell resolution." Using single cell RNA sequencing techniques on individual hemocytes, these researchers analyzed transcriptomes from 5,383 Anopheles gambiae specimens, comparing transcriptomes of circulating hemocytes from mature adult female mosquitos fed either a sugar meal or with a blood meal from Plasmodium-infected or healthy mice. Their results showed the following major cell clusters: two from adipose tissue, one from muscle tissue, and six hemocyte clusters. One adipose-tissue derived cluster expressed several immune-modulatory genes such as CAP-Gly domain containing linker proteins (CLIPs, including CLIPA1, -7, -8, -9, and -14), homeobox transcription factors LRIM1, -4A, -8A, -8B, -9, and -17, lectins (CTL4 and MA2), and Serpin 2 (SRPN2). The other adipose-tissue derived cluster expressed high levels of vitellogenin, a canonical fat-body marker. The six hemocyte clusters had diverse characteristics:
• HC1 showed high mRNAs levels of prophenoloxidases, including PPO4 and PPO9, characteristic of oenocytoids, and contained low levels of leucine-repeat protein 8 (LRR8) mRNA.
• HC2 showed low or absent PPO4 and high LLR8 levels; had a morphology typical of prohemocytes and granulocytes; expressed SPARC, cathepsin-L, and LRR8; had 73% fewer unique molecular identifiers (UMIs) (mean UMI of 413) than cells of the HC3 cluster; and were less differentiated and were thought to constitute prohemocytes.
• HC3 showed low or absent PPO4 and high LLR8 levels; had a morphology typical of prohemocytes and granulocytes; expressed SPARC, cathepsin-L, and LRR8; had a greater number of UMI than cells of the HC2 (mean of 1516); and typical granulocyte morphology, with prominent pseudopodia and abundant granules.
• HC4 showed low or absent PPO4 and high LLR8 levels; had a morphology typical of prohemocytes and granulocytes; shared markers with cells of the HC3 cluster; expressed cyclin B, aurora kinase, and other mitotic markers, which suggests that they are proliferating hemocytes -- consistent with this hypothesis, cells in this cluster expressed mitotic markers consistent with proliferation in response to a blood meal which was also consistent with blood-feeding induced DNA synthesis; and a correlation analysis shows these cells to be granulocytes.
• HC5 showed low levels of LLR8 and expressed no PPO4; expressed high levels of an uncharacterized transmembrane protein AGAP007318 (TM7318) and lipopolysaccharide-induced tumor necrosis factor–α transcription factor 3 (LL3); and showed two different morphologies, wherein TM7318-positive cells were present in low abundance (0.5% of granulocytes) that represented a novel, separate giant cell type (25 to 40 μm) they termed "megacytes".
• HC6 showed low levels of LLR8 and no PPO4, were negative to HC4 and HC5 markers but expressed antimicrobial peptides such as defensin 1, cecropins 1, and C-type lysozyme; and also exhibited differential morphologies, whereas cells negative for TM7318 represented small granulocytes that expressed antimicrobial genes (AM Gran) (16.4% of granulocytes).
The transcriptomes were further analyzed and these researchers found that granulocytes formed three major subclusters, one representing the basal state (Gran1) and the others represented by cells activated by blood meal (Gran2) and Plasmodium infection (Gran3). Prohemocytes, on the other hand, clustered into two population subclusters (PHem1 and PHem2), wherein PHem2 seems to be an intermediate between PHem1 and Gran1. The Gran1 cluster was linked to Gran2 and Gran3, with Gran3 being linked to dividing granulocytes. Gran2 also linked to megacytes, and Gran1 linked to antimicrobial granulocytes, as shown in the accompanying figure:
The status of immunological cells in An. gambiae was compared with another insect vector species, Aedes aegypti, known to transmit dengue fever, yellow fever, chikungunya, and Zika virus. When compared with transcriptomes of An. gambiae, cross-species correlation analysis revealed four different cell states, including a proliferating S-phase granulocyte cluster (AaHC6) without a clear An. gambiae equivalent. These researchers appreciated two clusters (AaHC1 and AaHC2) with conserved transcriptome signatures for oenocytoids (99 and 77% correlation, respectively, with AgHC1) and different granulocyte types, including antimicrobial peptide–expressing cells (94% with AgHC6) and proliferating granulocytes (87% with AgHC4). Granulocytes expressed laminins, leucine-rich repeat proteins, scavenger receptors, Toll-like receptor 5, and the transcription factor Rel2. However, megacytes (AgHC5) detected in An. gambiae lacked an obvious counterpart in Aedes, and their characteristic marker (TM7318) was present only in anophelines of the Cellia subgenus (unlike Aedes, these mosquitos are malaria vectors in Africa and Asia).
The researchers summarized their results by saying:
Together, these analyses suggest the existence of a proliferative, oligopotent cell population that can replenish the pool of granulocytes and differentiate into more specialized hemocytes, such as megacytes and antimicrobial granulocytes.
And further:
The conservation of diverse and molecularly well-defined hemocyte types between distantly related mosquito genera and the apparent absence of megacytes in our Ae. aegypti mosquito dataset raise questions as to how the immune systems of these mosquito species have evolved to limit their capacity to transmit parasites and arboviruses to humans. This knowledge will ultimately underpin immunological strategies aimed at interrupting disease transmission by rendering mosquitoes resistant to such pathogens.
* Wellcome Sanger Institute, Cambridge; Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health USA; Zoology Department, Stockholm University; Departamento de Biología del Neurodesarrollo, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo; Institute and Department of Physics, University of Cambridge; Molecular Infection Medicine Sweden, Molecular Biology Department, Umeå University
