Unbiased classification of mosquito blood cells by single-cell genomics and high-content imaging

Severo, Maiara S.; Landry, Jonathan J. M.; Lindquist, Randall L.; Goosmann, Christian; Brinkmann, Volker; Collier, Paul; Hauser, Anja E.; Beneš, Vladimír; Henriksson, Johan; Teichmann, Sarah A.; Levashina, Elena A.

doi:10.1101/234492

Cited by 3 publications

(9 citation statements)

References 68 publications

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“…In addition, similar to Severo et al (57), we identify variable (high and low) expression of PPO6 expression in PPO6 + immune cells with and without phagocytic ability (Fig. S13).…”

Section: Resultssupporting

confidence: 87%

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

Kwon

Smith

2018

Preprint

109

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24Mosquito innate immunity is comprised of both cellular and humoral factors that provide 25 protection from invading pathogens. Immune cells, known as hemocytes, have been 26 intricately associated with these immune responses through direct roles in phagocytosis 27 and immune signaling. Recent studies have implicated hemocytes as integral 28 determinants of anti-Plasmodium immunity, yet little is known regarding the specific 29 mechanisms by which hemocytes limit malaria parasite survival. With limited genetic tools 30 to enable their study, we employed a chemical-based treatment widely used for 31 macrophage depletion in mammalian systems for the first time in an invertebrate 32 organism. Upon its application in Anopheles gambiae, we observe distinct populations of 33 phagocytic immune cells that are significantly depleted, causing high mortality following 34 bacterial challenge and an increased intensity of malaria parasite infection. Through these 35 studies, we demonstrate that phagocytes are required for mosquito complement 36 recognition of invading ookinetes, as well as the production of prophenoloxidases that 37 limit oocyst survival. Through these experiments, we also define specific sub-types of 38 phagocytic immune cells in An. gambiae, providing new insights beyond the 39 morphological characteristics that traditionally define mosquito hemocyte populations. 40

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“…In addition, similar to Severo et al (57), we identify variable (high and low) expression of PPO6 expression in PPO6 + immune cells with and without phagocytic ability (Fig. S13).…”

Section: Resultssupporting

confidence: 87%

“…While PPO6-silencing does not produce a discernable oocyst phenotype, several reports have described PPO6 as a reliable marker of mosquito hemocytes (8, 9, 56, 57) and phagocytes (10, 57). Following CLD-treatment, the number of circulating PPO6 + phagocytes are significantly depleted (Fig.…”

Section: Resultsmentioning

confidence: 97%

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

Kwon

Smith

2018

Preprint

109

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“…Similarly, PPO2 was not expressed in all granulocytes, sometimes co-expressed with the transgene and sometimes not, highlighting at least four populations of granulocytes. Similar results were obtained with the PPO6-tdTomato line (Volohonsky, Terenzi et al 2015, Severo, Landry et al 2018) in which we detected transgene expression in some granulocytes only. Our data are in agreement with previous studies in Anopheles mosquitoes.…”

Section: Discussionsupporting

confidence: 89%

“…Our data are in agreement with previous studies in Anopheles mosquitoes. Indeed, different immune markers such as Sp22D, PPO4 and PPO6 were expressed only in a subset of granulocytes (Castillo, Robertson et al 2006, Bryant and Michel 2014, Severo, Landry et al 2018, Kwon and Smith 2019. Therefore, different populations of granulocytes are likely to be present in Anopheles mosquitoes, even though they display the same morphology.…”

Section: Discussionmentioning

confidence: 99%

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

Pondeville

Puchot

Parvy

et al. 2019

Preprint

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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.

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“…1b-c) and PPO9, characteristic of oenocytoids. This expression profile agrees with previously reported scRNA-seq data for a small number of hemocytes (25 cells (15)). To select markers for major hemocyte lineages, we used bulk RNAseq data from different tissues to guide us towards hemocyte specific genes ( Fig.…”

Section: Scrnaseq Reveals New Granulocyte Types and Statessupporting

confidence: 92%

Mosquito cellular immunity at single-cell resolution

Raddi

Ferreira

Efremova

et al. 2020

Preprint

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Insect hemocytes are the functional equivalents of leukocytes and limit the capacity of mosquitoes to transmit human pathogens through phagocytosis, encapsulation, secretion of immune factors and immune priming (1,2). Here we profile the transcriptomes of 8506 hemocytes of Anopheles gambiae and Aedes aegypti, two important mosquito vectors. Blood feeding, infection with malaria parasites and other immune challenges reveal a previously unknown functional diversity of hemocytes, with different types of granulocytes expressing distinct and evolutionarily conserved subsets of effector genes. A new cell type, which we term megacyte, is defined in Anopheles by a unique transmembrane protein marker (TM7318) and high expression of LPS-Induced TNF-alpha transcription factor 3 (LL3). Knock-down experiments indicate that LL3 mediates hemocyte differentiation during immune priming.We identify two main hemocyte lineages and find evidence of proliferating granulocyte populations.We validate our analysis with RNA in-situ hybridization and highlight the mobilization of sessile hemocytes into circulation upon infection. Our data (https://hemocytes.cellgeni.sanger.ac.uk/) provide the first atlas of medically relevant invertebrate immune cells at single cell resolution. It provides an important resource for invertebrate immunology by identifying cellular events that underpin mosquito immunity to malaria infection.

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Unbiased classification of mosquito blood cells by single-cell genomics and high-content imaging

Cited by 3 publications

References 68 publications

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

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

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

Mosquito cellular immunity at single-cell resolution

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