Single-cell analysis of mosquito hemocytes identifies signatures of immune cell subtypes and cell differentiation

Abstract

Mosquito immune cells, known as hemocytes, are integral to cellular and humoral responses that limit pathogen survival and mediate immune priming. However, without reliable cell markers and genetic tools, studies of mosquito immune cells have been limited to morphological observations, leaving several aspects of their biology uncharacterized. Here, we use single-cell RNA sequencing (scRNA-seq) to characterize mosquito immune cells, demonstrating an increased complexity to previously defined prohemocyte, oenocytoid, and granulocyte subtypes. Through functional assays relying on phagocytosis, phagocyte depletion, and RNA-FISH experiments, we define markers to accurately distinguish immune cell subtypes and provide evidence for immune cell maturation and differentiation. In addition, gene-silencing experiments demonstrate the importance of lozenge in defining the mosquito oenocytoid cell fate. Together, our scRNA-seq analysis provides an important foundation for future studies of mosquito immune cell biology and a valuable resource for comparative invertebrate immunology.

Prostaglandin E2 Signaling Mediates Oenocytoid Immune Cell Function and Lysis, Limiting Bacteria and Plasmodium Oocyst Survival in Anopheles gambiae

“…this subset of PPO genes was significantly upregulated, while AgPGE2R-silencing significantly reduced their expression, supporting the novel contributions of PGE2 signaling on a subset of PPOs. Recent studies have also demonstrated that the expression of PPO1, -3, and -8 is mediated by the transcription factor lozenge ( 22), an integral regulator of oenocytoid (or comparable Drosophila crystal cell) differentiation (22,43,44), however our initial experiments suggest that lozenge expression is not directly influenced by PGE2 signaling. In addition, the regulation of a subset of PPOs via PGE2 signaling further highlight differences in gene regulation between PPO genes expressed in oenocytoids (PPO1, -3, -7 and -8) and granulocytes (PPO2, -4, -5, -6, -9) (14,22), providing support for distinct mechanisms of PPO regulation in mosquito immune cell populations that require further study.…”

“…This is in agreement with recent single-cell RNA-seq analysis of An. gambiae immune cell populations (22). However, our experiments demonstrate that AgPGE2R is unaffected by phagocyte depletion, suggesting that AgPGE2R expression in oenocytoids is responsible for the majority of its expression.…”

“…Focusing on the influence of AgPGE2R in mosquito immune function, we demonstrate that PGE2 treatment influences PPO gene expression and subsequent downstream PO activity. Of interest, we found that PGE2 signaling influences the expression of a subset of PPO genes (PPO1, -3, -7 and -8), which coincides with the same subset of PPO genes that are enriched in oenocytoid cell populations (22). Following PGE2 treatment, The prevalence of infection (% infected/total) is depicted for mosquitoes under each experimental condition and examined by X 2 analysis to determine significance.…”

“…We also further validate AgPGE2R localization to oenocytoids through the use of clodronate liposomes (CLD) to ablate phagocytic granulocyte populations as previously described (14,17). qRT-PCR analysis demonstrates that AgPGE2R transcript remains unchanged in perfused hemocyte samples from mosquitoes treated with CLD, while the expression of eater [a known phagocytic marker (22)(23)(24)] was significantly reduced (Figure S4B). Together, these results provide strong support that AgPGE2R is strongly expressed in oenocytoid immune cell populations.…”

“…Previous studies have also implicated PGE2 signaling on the synthesis of antimicrobial peptides (AMPs) in Anopheles albimanus (4). Therefore, we examined the expression of major AMPs that display strong expression in Anopheles oenocytoid cell populations (22). Following PGE2 treatment or AgPGE2Rsilencing as in Figures 2A, B examining PPO expression, PGE2 treatment significantly increased the expression of cecropin 1 (CEC1) and defensin 1 (DEF1) (Figure 2E), while the silencing of AgPGE2R reduced CEC1, CEC4 and DEF1 (Figure 2F).…”