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

Kwon, Hyeogsun; Franzén, Oscar; Mohammed, Mubasher; Ankarklev, Johan; Smith, Ryan C.

doi:10.1101/2020.07.11.197939

Cited by 9 publications

(21 citation statements)

References 97 publications

(333 reference statements)

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“…These cell identifications are supported herein by the use of clodronate liposomes (16) to demonstrate that AgPGE2R is unaffected by phagocyte depletion experiments, and recent single-cell RNA-seq studies of An. gambiae immune cell populations (19). Our findings are also consistent with recent evidence that the M. sexta PGE2R is immunolocalized to oenocytoid immune cell populations (8).…”

Section: Discussionsupporting

confidence: 92%

“…qRT-PCR analysis demonstrates that AgPGE2R transcript remains unchanged in perfused hemolymph samples from mosquitoes treated with CLD, while the expression of eater (a known phagocytic maker (17,18)) was significantly reduced ( Figure S4B). Taken together, these results support that AgPGE2R is expressed primarily in oenocytoid immune cell populations and confirmed by recent scRNA-seq studies of mosquito immune cell populations (19).…”

Section: Characterization Of the Agpge2 Receptorsupporting

confidence: 87%

“…As a result, these data support that prostaglandin signaling is central to the regulation of PPO1, -3, -7 and -8. This same subset of PPOs is highly enriched in mosquito oenocytoid popoulations and display regulation by the transcription factor lozenge (19), an important regulator of oenocytoid (or comparable Drosophila crystal cell) differentiation (19,38,39). Recent data suggest that the remaining PPO genes, PPO2, -4, -5, -6, -9 are highly expressed in granulocyte populations (16,19), providing additional support for distinct mechanisms of PPO regulation in mosquito immune cell populations.…”

Section: Discussionmentioning

confidence: 91%

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Identification of a prostaglandin E2 receptor that regulates mosquito oenocytoid immune cell function in limiting bacteria and parasite infection

Kwon

Smith

2020

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Lipid-derived signaling molecules known as eicosanoids have integral roles in mediating immune and inflammatory processes across metazoans. This includes the function of prostaglandins and their cognate G protein-coupled receptors (GPCRs) to employ their immunological actions. In insects, prostaglandins have been implicated in the regulation of both cellular and humoral immune responses, yet studies have been limited by the absence of a described prostaglandin receptor. Here, we characterize a prostaglandin E2 receptor (AgPGE2R) in the mosquito Anopheles gambiae and examine its contributions to innate immunity. AgPGE2R expression is most abundant in circulating hemocytes where it is primarily localized to oenocytoid immune cell populations. Through the administration of prostaglandin E2 (PGE2) and AgPGE2R-silencing by RNAi, we demonstrate that PGE2 signaling regulates the expression of a subset of prophenoloxidases (PPOs) and antimicrobial peptides (AMPs). PGE2 priming via the AgPGE2R significantly limited bacterial replication and suppressed Plasmodium oocyst survival. Additional experiments establish that PGE2 priming increases phenoloxidase (PO) activity through the increased expression of PPO1 and PPO3, which significantly influence Plasmodium oocyst survival. We also provide evidence that PGE2 priming is concentration-dependent, where high concentrations of PGE2 promote oenocytoid lysis, negating the protective effects of PGE2 priming on anti-Plasmodium immunity. Taken together, our results characterize the AgPGE2R and the role of prostaglandin signaling on immune cell function, providing new insights into the role of PGE2 on anti-bacterial and anti-Plasmodium immune responses in the mosquito host.

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Section: Discussionsupporting

confidence: 92%

Section: Characterization Of the Agpge2 Receptorsupporting

confidence: 87%

Section: Discussionmentioning

confidence: 91%

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Identification of a prostaglandin E2 receptor that regulates mosquito oenocytoid immune cell function in limiting bacteria and parasite infection

Kwon

Smith

2020

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“…In case of the Sua5.1 cells, the functional analysis involved the top 118 genes (expression ranging from 1681.8 to 1.0 TPM), which were enriched for GO terms related to cellular carbohydrate metabolism and chemosensory perception (Supplementary Table S4). Overrepresentation of chemosensory genes is consistent with their enrichment in oenocytoid hemocytes isolated from perfused haemolymph of adult A. gambiae females 44 .…”

Section: Resultssupporting

confidence: 58%

A male-specific cell line from the African malaria mosquito Anopheles gambiae: Establishment and a comparative transcriptomic analysis

Krzywińska

Ferretti

Krzywinski

2022

Preprint

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Cell lines allow studying various biological processes that may not be easily tractable in whole organisms. Here, we have established the first male-specific cell line from the African malaria mosquito, Anopheles gambiae. The cells, named AgMM and derived from the sex-sorted neonate larvae, were able to undergo spontaneous contractions for a number of passages following establishment, indicating their myoblast origin. Comparison of their transcriptome to the transcriptome of an A. gambiae-derived Sua5.1 hemocyte cells revealed distinguishing molecular signatures of each cell line, including numerous muscle-related genes that were highly and uniquely expressed in the AgMM cells. Moreover, the AgMM cells express the primary sex determiner gene Yob and support male sex determination and dosage compensation pathways. Therefore, the AgMM cell line represents a valuable tool for molecular and biochemical in vitro studies of these male-specific processes. In a broader context, a rich transcriptomic data set generated in this study contributes to a better understanding of transcribed regions of the A. gambiae genome and sheds light on the biology of both cell types, facilitating their anticipated use for various cell-based assays.

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“…gambiae : Castillo et al ., 2006), and Kwon et al . (2021) utilized WGA labeling for hemocyte separation in An. gambiae .…”

Section: Discussionmentioning

confidence: 99%

Characterization and mode of action analysis of black soldier fly (Hermetia illucens) larva‐derived hemocytes

et al. 2021

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With the growing importance of the black soldier fly (Hermetia illucens) for both sustainable food production and waste management as well as for science, a great demand of understanding its immune system arises. Here, we present the first description of the circulating larval hemocytes with special emphasis on uptake of microorganisms and distinguishing hemocyte types. With histological, zymographic, and cytometric methods and with a set of hemocyte binding lectins and antibodies, the hemocytes of H. illucens are identified as plasmatocytes, crystal cells, and putative prohemocytes. Total hemocyte counts (THC) are determined, and methods for THC determination are compared. Approximately 1100 hemocytes per microliter hemolymph are present in naive animals, while hemocyte density decreases dramatically shortly after wounding, indicating a role of hemocytes in response to wounding (and immune response in general). The determination of the relative abundance of each hemocyte type (differential hemocyte count, DHC) revealed that plasmatocytes are highly abundant, whereas prohemocytes and crystal cells make up only a small percentage of the circulating cells. Plasmatocytes are not only the most abundant but also the professional phagocytes in H. illucens. They rapidly engulf and take up bacteria both in vivo and in vitro, indicating a very potent cellular defense against invading pathogens. Larger bioparticles such as yeasts are also removed from circulation by phagocytosis, but slower than bacteria. This is the first analysis of the potent cellular immune response in the black soldier fly, and a first toolbox that helps to identify hemocyte (types) is presented.

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

Cited by 9 publications

References 97 publications

Identification of a prostaglandin E2 receptor that regulates mosquito oenocytoid immune cell function in limiting bacteria and parasite infection

Identification of a prostaglandin E2 receptor that regulates mosquito oenocytoid immune cell function in limiting bacteria and parasite infection

A male-specific cell line from the African malaria mosquito Anopheles gambiae: Establishment and a comparative transcriptomic analysis

Characterization and mode of action analysis of black soldier fly (Hermetia illucens) larva‐derived hemocytes

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