ScRNA-seq of Anopheles gambiae hemocytes after blood-feeding, Plasmodium berghei infection, or sugar feeding
ABSTRACT: Anopheline mosquitoes transmit Plasmodium parasites to humans, and are responsible for an estimated 219 million cases of malaria, leading to over 400,000 deaths annually. The mosquito’s immune system limits Plasmodium infection in several ways, and hemocytes, the insect white blood cells, are key players in these defense responses. However, the full functional diversity of mosquito hemocytes and their developmental trajectories have not been established. We use single cell RNA sequencing (scRNA-seq) to analyze the transcriptional profiles of individual mosquito hemocytes in response to blood feeding or infection with Plasmodium. Circulating hemocytes were collected from adult A. gambiae M form (A. coluzzii) females that were either kept on a sugar meal or fed on a healthy or a Plasmodium berghei-infected mouse. Transcriptomes from 5,383 cells (collected 1, 3, and 7 days after feeding) revealed nine major cell clusters.
Project description:We aimed at characterizing the diverse hemocyte populations present in the hemolymph of the Drosophila larvae. The hemocytes were collected from wandering larvae infested by wasp (WI) or not infested (NI). The hemocytes were then sequenced using 10x genomics technology.
Project description:Plasmodium-specific CD4+ T cells from mice infected with Plasmodium chabaudi chabaudi AS parasites were recovered at Days 0, 7, and 28 to undergo processing and generate scRNA-seq dataset. At Day 28, mice were administered with either saline or artesunate (intermittent artesunate therapy - IAT). scRNA-seq dataset was analysed to investigate transcriptome dynamics of CD4+ T cells from effector to memory states.
Project description:Anopheline mosquitoes transmit Plasmodium parasites to humans, and are responsible for an estimated 219 million cases of malaria, leading to over 400,000 deaths annually. The mosquito’s immune system limits Plasmodium infection in several ways, and hemocytes, the insect white blood cells, are key players in these defense responses. However, the full functional diversity of mosquito hemocytes and their developmental trajectories have not been established. We use bulk RNA sequencing (scRNA-seq) to analyze the transcriptional profiles of hemocytes, of guts, and of carcasses of mosquito hemocytes in response to blood feeding or infection with Plasmodium. Data from three independent biological replicates for each condition and time-point (day 0, 1, 2, 3, and 7 after sugar-feeding, blood-feeding or P. berghei infection).
Project description:Single-cell RNA-seq was carried out for control embryos and embryos produced from gd7, Tollrm9/rm10, and Toll10B mutant mothers. The embryos from mutant mothers produce only a single type along the dorsal-ventral axis. We used these embryos to compare single-cell gene expression patterns across tissues.
Project description:Anopheles gambiae mosquitoes that have been infected with Plasmodium mount a more effective immune response to a subsequent infection. Priming is established when Plasmodium invasion of the mosquito midgut allows contact of the gut microbiota with epithelial cells. This event is followed by a systemic release of a hemocyte differentiation factor (HDF) consisting of Lipoxin A4 bound to Evokin, a lipocalin carrier, which increases the proportion of circulating hemocytes. We show that mosquito midgut cells produce and release prostaglandin E2 (PGE2), which attracts hemocytes to the midgut surface and enhances their patrolling activity. Systemic injection of prostaglandins (PGs) recapitulates the priming response and enhances antiplasmodial immunity by triggering HDF production. Although insects lack cyclooxygenases, two heme peroxidases, HPX7 and HPX8, catalyze essential steps in PG biosynthesis in mosquitoes. Mosquito midgut PGE2 release attracts hemocytes and establishes a long-lasting enhanced systemic cellular immune response to Plasmodium infection.
Project description:Hemocytes synthesize key components of the mosquito complement-like system, but their role in the activation of antiplasmodial responses has not been established. The effect of activating Toll signaling in hemocytes on Plasmodium survival was investigated by transferring hemocytes or cell-free hemolymph from donor mosquitoes in which the suppressor cactus was silenced. These transfers greatly enhanced antiplasmodial immunity, indicating that hemocytes are active players in the activation of the complement-like system, through an effector/effectors regulated by the Toll pathway. A comparative analysis of hemocyte populations between susceptible G3 and the refractory L3-5 Anopheles gambiae mosquito strains did not reveal significant differences under basal conditions or in response to Plasmodium berghei infection. The response of susceptible mosquitoes to different Plasmodium species revealed similar kinetics following infection with P. berghei,P. yoelii or P. falciparum, but the strength of the priming response was stronger in less compatible mosquito-parasite pairs. The Toll, Imd,STAT or JNK signaling cascades were not essential for the production of the hemocyte differentiation factor (HDF) in response to P. berghei infection, but disruption of Toll, STAT or JNK abolished hemocyte differentiation in response to HDF. We conclude that hemocytes are key mediators of A. gambiae antiplasmodial responses.
Project description:The mosquito Anopheles gambiae is a major vector of human malaria. Increasing evidence indicates that blood cells (hemocytes) comprise an essential arm of the mosquito innate immune response against both bacteria and malaria parasites. To further characterize the role of hemocytes in mosquito immunity, we undertook the first genome-wide transcriptomic analyses of adult female An. gambiae hemocytes following infection by two species of bacteria and a malaria parasite.We identified 4047 genes expressed in hemocytes, using An. gambiae genome-wide microarrays. While 279 transcripts were significantly enriched in hemocytes relative to whole adult female mosquitoes, 959 transcripts exhibited immune challenge-related regulation. The global transcriptomic responses of hemocytes to challenge with different species of bacteria and/or different stages of malaria parasite infection revealed discrete, minimally overlapping, pathogen-specific signatures of infection-responsive gene expression; 105 of these represented putative immunity-related genes including anti-Plasmodium factors. Of particular interest was the specific co-regulation of various members of the Imd and JNK immune signaling pathways during malaria parasite invasion of the mosquito midgut epithelium.Our genome-wide transcriptomic analysis of adult mosquito hemocytes reveals pathogen-specific signatures of gene regulation and identifies several novel candidate genes for future functional studies.
Project description:The mosquito complement-like system is a major defense mechanism that limits Plasmodium infection. Ookinete midgut invasion results in irreversible damage to invaded cells and triggers epithelial nitration and complement activation. Several lines of evidence suggest that hemocytes participate in early antiplasmodial responses that target ookinetes, but their role remains unclear. The fate of hemocytes in response to Plasmodium infection was investigated by labeling this cell population in vivo. We found that midgut nitration triggers the local release of hemocyte-derived microvesicles (HdMv) into the basal labyrinth of the midgut. Several different strategies, such as gene silencing, immune priming, or systemic injection of polystyrene beads, were used to either enhance or reduce HdMv release. We provide direct experimental evidence that contact of hemocytes with the nitrated midgut basal surface triggers HdMv release and that this response is necessary for effective activation of mosquito complement. Our studies suggest that hemocyte-derived microvesicles may deliver some critical factor(s) that promote activation of thioester-containing protein 1, a key effector of the mosquito antiplasmodial immunity.
Project description:Mosquito midgut invasion by ookinetes of the malaria parasite Plasmodium disrupts the barriers that normally prevent the gut microbiota from coming in direct contact with epithelial cells. This triggers a long-lived response characterized by increased abundance of granulocytes, a subpopulation of hemocytes that circulates in the insect's hemocoel, and enhanced immunity to bacteria that indirectly reduces survival of Plasmodium parasites upon reinfection. In mosquitoes, differentiation of hemocytes was necessary and sufficient to confer innate immune memory.
Project description:Spermatogenesis in mammals is a complex and highly orchestrated process, which involves the differentiation of diploid (2n-DNA content) spermatogonia into haploid (n) sperm. This process begins with spermatogonia, a niche of stem cells, which drive this process. Spermatogonial stem cells (SSCs) undergo mitotic self-renewal to maintain this niche, while sub-populations of spermatogonia progressively undergo differentiation and later gain competence to initiate meiosis and form sperm. SSCs undergo extensive chromatin remodelling and major morphological changes, while maintaining genome integrity and parental imprints. To study this in greater detail we performed single-cell RNA sequencing of spermatogenic cells derived from the testis of the non-human primate Macaca fascicularis.