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.
Highlights
Across Metazoa, immune cells are vital to promoting wound healing, maintaining homeostasis, and providing anti-pathogen defenses (Chaplin, 2010)
Cells were stained with a live-dead viability stain to select for live cells, with mosquito immune cells distinguished by labeling with FITC-conjugated wheat germ agglutinin (WGA) as a general hemocyte marker and the far-red stain DRAQ5 to label DNA content as previously (Kwon and Smith, 2019)
Monocle3 analysis supports that precursor cells (Cluster 5) first differentiate into an intermediate, immature oenocytoid stage (Cluster 7) before maturation into the mature oenocytoid cells of Cluster 8 (Figure 4). Based on these cell trajectories (Figure 4A–C), as well as the transcriptional differences that likely define immature cell types (Figure 3—figure supplement 4), our results support a model for immune cell differentiation and the progression of cells within each lineage (Figure 4D). These data corroborate the differentiation of cells from a prohemocyte precursor as previously proposed (Ramirez et al, 2014; Rodrigues et al, 2010; Smith et al, 2015), while providing insight into the potential role of cell intermediates undergoing maturation before terminal differentiation of mosquito immune cell subtypes (Figure 4D) similar to those recently described in Drosophila (Tattikota et al, 2020)
Summary
Across Metazoa, immune cells are vital to promoting wound healing, maintaining homeostasis, and providing anti-pathogen defenses (Chaplin, 2010). The advent of single-cell sequencing (scRNAseq) has continued to delineate and provide further resolution into new cell types and immune cell functions in mammals (Szabo et al, 2019; Villani et al, 2017). In lesser studied invertebrates lacking adaptive immunity, single-cell technologies have enhanced descriptions of previously described cell types and have redefined cell complexity (Cattenoz et al, 2020; Cho et al, 2020; Raddi et al, 2020; Severo et al, 2018; Tattikota et al, 2020). Mosquito immune cells (hemocytes) have proven integral to the cellular and humoral responses that limit invading pathogens in the mosquito host
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