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

Mosquito innate immunity is comprised of both cellular and humoral factors that provide protection from invading pathogens. Immune cells, known as hemocytes, have been intricately associated with these immune responses through direct roles in phagocytosis and immune signaling. Recent studies have implicated hemocytes as integral determinants of anti-Plasmodium immunity, yet little is known regarding the specific mechanisms by which hemocytes limit malaria parasite survival. With limited genetic tools to enable their study, we employed a chemical-based treatment widely used for macrophage depletion in mammalian systems for the first time in an invertebrate organism. Upon its application in Anopheles gambiae, we observe distinct populations of phagocytic immune cells that are significantly depleted, causing high mortality following bacterial challenge and an increased intensity of malaria parasite infection. Through these studies, we demonstrate that phagocytes are required for mosquito complement recognition of invading ookinetes, as well as the production of prophenoloxidases that limit oocyst survival. Through these experiments, we also define specific sub-types of phagocytic immune cells in An. gambiae, providing new insights beyond the morphological characteristics that traditionally define mosquito hemocyte populations. Together, this study provides the first definitive insights into the dual roles of mosquito phagocytes in limiting malaria parasite survival, and illustrates the use of clodronate liposomes as an important advancement in the study of invertebrate immunity.

A single vertebrate DNA virus protein disarms invertebrate immunity to RNA virus infection

Virus-host interactions drive a remarkable diversity of immune responses and countermeasures. We found that two RNA viruses with broad host ranges, vesicular stomatitis virus (VSV) and Sindbis virus (SINV), are completely restricted in their replication after entry into Lepidopteran cells. This restriction is overcome when cells are co-infected with vaccinia virus (VACV), a vertebrate DNA virus. Using RNAi screening, we show that Lepidopteran RNAi, Nuclear Factor-κB, and ubiquitin-proteasome pathways restrict RNA virus infection. Surprisingly, a highly conserved, uncharacterized VACV protein, A51R, can partially overcome this virus restriction. We show that A51R is also critical for VACV replication in vertebrate cells and for pathogenesis in mice. Interestingly, A51R colocalizes with, and stabilizes, host microtubules and also associates with ubiquitin. We show that A51R promotes viral protein stability, possibly by preventing ubiquitin-dependent targeting of viral proteins for destruction. Importantly, our studies reveal exciting new opportunities to study virus-host interactions in experimentally-tractable Lepidopteran systems.