Hemocyte-targeted gene expression in the female malaria mosquito using thehemolectinpromoter fromDrosophila

Abstract

Hemocytes, the immune cells in mosquitoes, participate in immune defenses against pathogens including malaria parasites. Mosquito hemocytes can also be infected by arthropod-borne viruses but the pro-or anti-viral nature of this interaction is unknown.Although there has been progress on hemocyte characterization during pathogen infections in mosquitoes, the specific contribution of hemocytes to immune responses and the hemocytespecific functions of immune genes and pathways remain unresolved due to the lack of genetic tools to manipulate gene expression in these cells specifically. Here, we used the Gal4-UAS system to characterize the activity of the Drosophila hemocyte-specific hemolectin promoter in the adults of Anopheles gambiae, the malaria mosquito. We established an hml-Gal4 driver line that we further crossed to a fluorescent UAS responder line, and examined the expression pattern in the adult progeny driven by the hml promoter. We show that the hml regulatory region drives hemocyte-specific transgene expression in a subset of hemocytes, and that transgene expression is triggered after a blood meal. The hml promoter drives transgene expression in differentiating prohemocytes as well as in differentiated granulocytes.Analysis of different immune markers in hemocytes in which the hml promoter drives transgene expression revealed that this regulatory region could be used to study phagocytosis as well as melanization. Finally, the hml promoter drives transgene expression in hemocytes in which o'nyong'nyong virus replicates. Altogether, the hml promoter constitutes a good tool to drive transgene expression in hemocyte only and to analyze the function of these cells and the genes they express during pathogen infection in Anopheles gambiae.

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

“…Similar to Drosophila, our data in mosquitoes support the developmental progression and specialization of immune cells from precursor populations, the presence of multiple phagocytic cell populations, and multiple shared markers that delineate the oenocytoid/crystal cell lineage (Cattenoz et al, 2020;Tattikota et al, 2020). However, several differences are also noted in mosquitoes, including the absence of well-characterized Drosophila immune cell markers such as hemolectin (Goto et al, 2003;Pondeville et al, 2020) and hemese (Kurucz et al, 2003), and the lack of lamellocyte cell populations. We also identify several mosquito immune cell markers, such as LRIM15 and the expansion of PPO genes, which are unique to mosquito immune cell populations.…”

“…gambiae based on gene expression, previous proteomics studies (Smith et al, 2016), and phagocytic properties. This expansion of the general "granulocyte" classification is supported by previous morphological observations of granulocytes in mosquitoes (Kwon and Smith, 2019;Pondeville et al, 2020), and more recently by parallel scRNA-seq experiments in Drosophila (Cattenoz et al, 2020;Tattikota et al, 2020) and An. gambiae (Raddi et al, 2020).…”

“…prohemocytes, oenocytoids, and granulocytes (Castillo et al, 2006). However, with the advent of additional molecular tools to study mosquito immune cell function, several studies have supported an increased complexity of hemocyte populations beyond these generalized cell subtype classifications (Bryant and Michel, 2016;Kwon and Smith, 2019;Pondeville et al, 2020;Raddi et al, 2020;Severo et al, 2018;Smith et al, 2016). Herein, we demonstrate through scRNA-seq experiments and additional molecular characterization that there are at least seven conservatively defined immune cell populations in An.…”