Genome-wide transcriptomic profiling of Anopheles gambiae hemocytes reveals pathogen-specific signatures upon bacterial challenge and Plasmodium berghei infection.
ABSTRACT: 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:Background: 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. Results: 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. Conclusion: 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. In order to identify hemocyte-specific and immune-responsive transcripts, we first compared transcripts expressed in hemocytes from one day old sugar-fed mosquitoes to transcripts detected in whole mosquitoes of the same age and feeding status. This resulted in identification of the hemocyte-enriched transcriptome. We then compared hemocytes from 1 day old mosquitoes, 1 hour after immune challenge with heat-killed Escherichia coli or Micrococcus luteus, to control female mosquitoes injected with sterile PBS to determine the bacteria challenge responsive transcriptomes. We used heat-killed bacteria in these assays, because our primary interest was in identifying the bacterial responsive transcriptome and to avoid the potentially confounding effects of altered gene expression due to the lethal effects of a systemic infection associated with injection of living bacteria. Lastly, we compared hemocytes from mosquitoes at 24 hours and 19 days after ingestion of a blood meal infected with Plasmodium berghei to mosquitoes of the same age fed a non-infected blood meal to determine the ookinete and sporozoite infection responsive transcriptomes, respectively. This design resulted in a total of five experimental treatments. The following samples are not included in this submission: Hemo E coli vs. hemo unchallenged A Hemo E coli vs. hemo unchallenged B Hemo m luteus vs. hemo unchallenged A Hemo m luteus vs. hemo unchallenged B
Project description:Insect hemocytes mediate important cellular immune responses including phagocytosis and encapsulation and also secrete immune factors such as opsonins, melanization factors, and antimicrobial peptides. However, the molecular composition of these important immune cells has not been elucidated in depth, because of their scarcity in the circulating hemolymph, their adhesion to multiple tissues and the lack of primary culture methods to produce sufficient material for a genome-wide analysis. In this study, we report a genome-wide molecular characterization of circulating hemocytes collected from the hemolymph of adult female Anopheles gambiae mosquitoes--the major mosquito vector of human malaria in subSaharan Africa. Their molecular profile identified 1,485 transcripts with enriched expression in these cells, and many of these genes belong to innate immune gene families. This hemocyte-specific transcriptome is compared to those of Drosophila melanogaster and two other mosquitoes, Aedes aegypti and Armigeres subalbatus. We report the identification of two genes as ubiquitous hemocyte markers and several others as hemocyte subpopulation markers. We assess, via an RNAi screen, the roles in development of Plasmodium berghei of 63 genes expressed in hemocytes and provide a molecular comparison of the transcriptome of these cells during malaria infection.
Project description: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 infection in mosquitoes, the specific contribution of hemocytes to immune responses and the hemocyte-specific 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 Drosophila 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.
Project description:Hemocytes are crucial players of the mosquito immune system and critically affect transmission of pathogens including malaria parasites. We and others discovered previously that a blood meal is a major immune stimulus for mosquito hemocytes. To determine whether these blood meal-induced hemocyte changes in Anopheles gambiae constitute steps in cell differentiation or demonstrate transient cell activation, we analyzed the temporal pattern of these changes over the first three days post blood meal (dpbm). Flow cytometry and immunofluorescence analyses revealed a global shift of the entire hemocyte population, peaking at 1 dpbm. All hemocyte activation markers returned to pre-blood meal baseline levels within the following 24-48 h. Our observations are consistant with An. gambiae hemocytes undergoing transient activation rather than terminal differentiation upon blood feeding. Interestingly, the temporal pattern followed the gonotrophic cycle of the mosquito, strongly suggesting hormonal control of mosquito hemocyte activation and deactivation.
Project description:Reverse genetics in the mosquito Anopheles gambiae by RNAi mediated gene silencing has led in recent years to an advanced understanding of the mosquito immune response against infections with bacteria and malaria parasites. We developed RNAi screens in An. gambiae hemocyte-like cells using a library of double-stranded RNAs targeting 109 genes expressed highly or specifically in mosquito hemocytes to identify novel regulators of the hemocyte immune response. Assays included phagocytosis of bacterial bioparticles, expression of the antimicrobial peptide CEC1, and basal and induced expression of the mosquito complement factor LRIM1. A cell viability screen was also carried out to assess dsRNA cytotoxicity and to identify genes involved in cell growth and survival. Our results identify 22 novel immune regulators, including proteins putatively involved in phagosome assembly and maturation (Ca²? channel, v-ATPase and cyclin-dependent protein kinase), pattern recognition (fibrinogen-domain lectins and Nimrod), immune modulation (peptidase and serine protease homolog), immune signaling (Eiger and LPS-induced factor), cell adhesion and communication (Laminin B1 and Ninjurin) and immune homeostasis (Lipophorin receptor). The development of robust functional cell-based assays paves the way for genome-wide functional screens to study the mosquito immune response to infections with human pathogens.
Project description:The immune system of adult mosquitoes has received significant attention because of the ability of females to vector disease-causing pathogens while ingesting blood meals. However, few studies have focused on the immune system of larvae, which, we hypothesize, is highly robust due to the high density and diversity of microorganisms that larvae encounter in their aquatic environments and the strong selection pressures at work in the larval stage to ensure survival to reproductive maturity. Here, we surveyed a broad range of cellular and humoral immune parameters in larvae of the malaria mosquito, Anopheles gambiae, and compared their potency to that of newly-emerged adults and older adults.We found that larvae kill bacteria in their hemocoel with equal or greater efficiency compared to newly-emerged adults, and that antibacterial ability declines further with adult age, indicative of senescence. This phenotype correlates with more circulating hemocytes and a differing spatial arrangement of sessile hemocytes in larvae relative to adults, as well as with the individual hemocytes of adults carrying a greater phagocytic burden. The hemolymph of larvae also possesses markedly stronger antibacterial lytic and melanization activity than the hemolymph of adults. Finally, infection induces a stronger transcriptional upregulation of immunity genes in larvae than in adults, including differences in the immunity genes that are regulated.These results demonstrate that immunity is strongest in larvae and declines after metamorphosis and with adult age, and suggest that adaptive decoupling, or the independent evolution of larval and adult traits made possible by metamorphosis, has occurred in the mosquito lineage.
Project description:Malaria is a global public health problem, especially in sub-Saharan Africa, where the mosquito Anopheles gambiae Giles serves as the major vector for the protozoan Plasmodium falciparum Welch. One determinant of malaria vector competence is the mosquito's immune system. Hemocytes are a critical component as they produce soluble immune factors that either support or prevent malaria parasite development. However, despite their importance in vector competence, understanding of their basic biology is just developing. Applying novel technologies to the study of mosquito hemocytes, we investigated the effect of blood meal on hemocyte population dynamics, DNA replication and cell cycle progression. In contrast to prevailing published work, the data presented here demonstrate that hemocytes in adult mosquitoes continue to undergo low basal levels of replication. In addition, blood ingestion caused significant changes in hemocytes within 24 h. Hemocytes displayed an increase in cell number, size, granularity and Ras-MAPK signaling as well as altered cell surface moieties. As these changes are well-known markers of immune cell activation in mammals and Drosophila melanogaster Meigen, we further investigated whether a blood meal changes the expression of hemocyte-derived immune factors. Indeed, hemocytes 24 h post-blood meal displayed higher levels of critical components of the complement and melanization immune reactions in mosquitoes. Taken together, this study demonstrates that the normal physiological process of a blood meal activates the innate immune response in mosquitoes. This process is likely in part regulated by Ras-MAPK signaling, highlighting a novel mechanistic link between blood feeding and immunity.
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 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:The mosquito Culex quinquefasciatus poses a substantial threat to human and veterinary health as a primary vector of West Nile virus (WNV), the filarial worm Wuchereria bancrofti, and an avian malaria parasite. Comparative phylogenomics revealed an expanded canonical C. quinquefasciatus immune gene repertoire compared with those of Aedes aegypti and Anopheles gambiae. Transcriptomic analysis of C. quinquefasciatus genes responsive to WNV, W. bancrofti, and non-native bacteria facilitated an unprecedented meta-analysis of 25 vector-pathogen interactions involving arboviruses, filarial worms, bacteria, and malaria parasites, revealing common and distinct responses to these pathogen types in three mosquito genera. Our findings provide support for the hypothesis that mosquito-borne pathogens have evolved to evade innate immune responses in three vector mosquito species of major medical importance.
Project description:The resident gut flora is known to have significant impacts on the life history of the host organism. Endosymbiotic bacterial species in the Anopheles mosquito gut are potent modulators of sexual development of the malaria parasite, Plasmodium, and thus proposed as potential control agents of malaria transmission.Here we report a melanotic pathology in the major African malaria vector Anopheles gambiae, caused by the dominant mosquito endosymbiont Elizabethkingiameningoseptica. Transfer of melanised tissues into the haemolymph of healthy adult mosquitoes or direct haemolymph inoculation with isolated E. meningoseptica bacteria were the only means for transmission and de novo formation of melanotic lesions, specifically in the fat body tissues of recipient individuals. We show that E. meningoseptica can be vertically transmitted from eggs to larvae and that E. meningoseptica-mono-associated mosquitoes display significant mortality, which is further enhanced upon Plasmodium infection, suggesting a synergistic impact of E. meningoseptica and Plasmodium on mosquito survival.The high pathogenicity and permanent association of E. meningoseptica with An. Gambiae through vertical transmission constitute attractive characteristics towards the potential design of novel mosquito/malaria biocontrol strategies.