Chemical depletion of phagocytic immune cells in Anopheles gambiae reveals dual roles of mosquito hemocytes in anti- Plasmodium immunity

Abstract

Mosquito immunity is composed of both cellular and humoral factors that provide protection from invading pathogens. Immune cells known as hemocytes, have been intricately associated with phagocytosis and innate immune signaling. However, the lack of genetic tools has limited hemocyte study despite their importance in mosquito anti-Plasmodiumimmunity. To address these limitations, we employ the use of a chemical-based treatment to deplete phagocytic immune cells inAnopheles gambiae,demonstrating the role of phagocytes in complement recognition and prophenoloxidase production that limit the ookinete and oocyst stages of malaria parasite development, respectively. Through these experiments, we also define specific subtypes of phagocytic immune cells inAn. gambiae, providing insights beyond the morphological characteristics that traditionally define mosquito hemocyte populations. Together, this study represents a significant advancement in our understanding of the roles of mosquito phagocytes in mosquito vector competence and demonstrates the utility of clodronate liposomes as an important tool in the study of invertebrate immunity.

20-Hydroxyecdysone Primes Innate Immune Responses That Limit Bacterial and Malarial Parasite Survival in Anopheles gambiae

“…Immune cells, known as hemocytes, serve as primary immune sentinels that recognize and destroy invading pathogens by phagocytosis or through the production of humoral immune factors (34,35). To examine how blood feeding and 20E influence these phagocytic properties, we injected fluorescent beads to evaluate phagocytosis under different physiological conditions, as previously described (29,36). Similar to the findings of Kwon and Smith (29), we demonstrate that blood feeding, independent of pathogen challenge, significantly increased the percentage of phagocytic cells ( Fig.…”

“…More direct evidence demonstrates the role of 20E in prophenoloxidase (PPO) expression in vitro (27) and that of LRIM9, a leucine-rich immunomodulatory protein, through experiments in vivo (28). Previous studies show that both LRIM9 (28) and multiple PPOs (29) influence Plasmodium killing, supporting a model in which 20E promotes an anticipatory immune response to immune challenge immediately following a blood meal (28). When combined with previously described antibacterial and anti-Plasmodium responses of other immune genes identified in our RNA-seq analysis (43-47), our experiments demonstrate the activation of mosquito innate immunity by 20E.…”

“…For mosquito infections, an mCherry strain of Plasmodium berghei (62) was passaged into female Swiss Webster mice and monitored for exflagellation as previously described (29,36,63). Mosquitoes were fed on an infected, anesthetized mouse, and then maintained at 19°C.…”

“…4B; Table S1). PPO3 is a known Plasmodium antagonist (29), while three other genes, LYSC1, CLIPB15, and MDL1, are antagonists against both bacteria and malaria parasites (43)(44)(45)(46)(47). The antimicrobial peptides CEC1 and CEC3 ( Fig.…”

“…In Drosophila, evidence suggests that 20E mediates cellular immunity (26) and regulates a subset of antimicrobial peptides (AMPs) involved in antibacterial defense through peptidoglycan recognition protein LC (PGRP-LC)-dependent and PGRP-LCindependent mechanisms of the immunodeficiency (IMD) pathway activation (23,24). However, few studies have examined the influence of 20E on mosquito innate immunity, thus far implicating 20E in the regulation of prophenoloxidase (PPO) expression (27) and leucine-rich repeat immune protein 9 (LRIM9) (28), both of which are known effectors of anti-Plasmodium immunity (28,29). Additional studies have suggested that 20E may also mediate mosquito immune cell activation following blood feeding (30)(31)(32), implying that 20E is an important determinant of An.…”