Do bed bugs transmit human viruses, or do humans spread bed bugs and their viruses? A worldwide survey of the bed bug RNA virosphere.

BED BUGS: (Hemiptera: Cimicidae) are a globally distributed hematophagous pest that routinely feed on humans. Unlike many blood-sucking arthropods, they have never been linked to pathogen transmission in a natural setting, and despite increasing interest in their role as disease vectors, little is known about the viruses that bed bugs naturally harbor. Here, we present a global-scale survey of the bed bug RNA virosphere. We sequenced the metatranscriptomes of 22 individual bed bugs (Cimex lectularius and Cimex hemipterus) from 8 locations around the world. We detected sequences from two known bed bug viruses (Shuangao bedbug virus 1 and Shuangao bedbug virus 2) which extends their geographical range. We identified three novel bed bug virus sequences from a tenui-like virus (Bunyavirales), a toti-like virus (Ghabrivirales), and a luteo-like virus (Tolivirales). Interestingly, some of the bed bug viruses branch near to insect-transmitted plant-infecting viruses, opening questions regarding the evolution of plant virus infection. When we analyzed the viral sequences by their host's collection location, we found unexpected patterns of geographical diversity that may reflect humans' role in bed bug dispersal. Additionally, we investigated the effect that Wolbachia, the primary bed bug endosymbiont, may have on viral abundance and found that Wolbachia infection neither promotes nor inhibits viral infection. Finally, our results provide no evidence that bed bugs transmit any known human pathogenic viruses.

Ex vivo characterization of the circulating hemocytes of bed bugs and their responses to bacterial exposure.

Bed bugs (Cimex spp.) are urban pests of global importance. Knowledge of the immune system of bed bugs has implications for understanding their susceptibility to biological control agents, their potential to transmit human pathogens, and the basic comparative immunology of insects. Nonetheless, the immunological repertoire of the family Cimicidae remains poorly characterized. Here, we use microscopy, flow cytometry, and RNA sequencing to provide a basal characterization of the circulating hemocytes of the common bed bug, Cimex lectularius. We also examine the responses of these specialized cells to E. coli exposure using the same techniques. Our results show that circulating hemocytes are comprised of at least four morphologically distinct cell types that are capable of phagocytosis, undergo degranulation, and exhibit additional markers of activation following stimulation, including size shift and DNA replication. Furthermore, transcriptomic profiling reveals expression of predicted Toll/IMD signaling pathway components, antimicrobial effectors and other potentially immunoresponsive genes in these cells. Together, our data demonstrate the conservation of several canonical cellular immune responses in the common bed bug and provide a foundation for additional mechanistic immunological studies with specific pathogens of interest.

Developmental and comparative perspectives on mosquito immunity.

Diseases spread by mosquitoes have killed more people than those spread by any other group of arthropod vectors and remain an important factor in determining global health and economic stability. The mosquito innate immune system can act to either modulate infection with human pathogens or fight off entomopathogens and increase the fitness and longevity of infected mosquitoes. While work remains towards understanding the larval immune system and the development of the mosquito immune system, it has recently become clearer that environmental factors heavily shape the developing mosquito immune system and continue to influence the adult immune system as well. The adult immune system has been well-studied and is known to involve multiple tissues and diverse molecular mechanisms. This review summarizes and synthesizes what is currently understood about the development of the mosquito immune system and includes comparisons of immune components unique to mosquitoes among the blood-feeding arthropods as well as important distinguishing factors between the anopheline and culicine mosquitoes. An explanation is included for how mosquito immunity factors into vector competence and vectorial capacity is presented along with a model for the interrelationships between nutrition, microbiome, pathogen interactions and behavior as they relate to mosquito development, immune status, adult female fitness and ultimately, vectorial capacity. Novel discoveries in the fields of mosquito ecoimmunology, neuroimmunology, and intracellular antiviral responses are highlighted.

The Selection of a Hepatocyte Cell Line Susceptible to Plasmodium falciparum Sporozoite Invasion That Is Associated With Expression of Glypican-3.

In vitro studies of liver stage (LS) development of the human malaria parasite Plasmodium falciparum are technically challenging; therefore, fundamental questions about hepatocyte receptors for invasion that can be targeted to prevent infection remain unanswered. To identify novel receptors and to further understand human hepatocyte susceptibility to P. falciparum sporozoite invasion, we created an optimized in vitro system by mimicking in vivo liver conditions and using the subcloned HC-04.J7 cell line that supports mean infection rates of 3-5% and early development of P. falciparum exoerythrocytic forms-a 3- to 5-fold improvement on current in vitro hepatocarcinoma models for P. falciparum invasion. We juxtaposed this invasion-susceptible cell line with an invasion-resistant cell line (HepG2) and performed comparative proteomics and RNA-seq analyses to identify host cell surface molecules and pathways important for sporozoite invasion of host cells. We identified and investigated a hepatocyte cell surface heparan sulfate proteoglycan, glypican-3, as a putative mediator of sporozoite invasion. We also noted the involvement of pathways that implicate the importance of the metabolic state of the hepatocyte in supporting LS development. Our study highlights important features of hepatocyte biology, and specifically the potential role of glypican-3, in mediating P. falciparum sporozoite invasion. Additionally, it establishes a simple in vitro system to study the LS with improved invasion efficiency. This work paves the way for the greater malaria and liver biology communities to explore fundamental questions of hepatocyte-pathogen interactions and extend the system to other human malaria parasite species, like P. vivax.

Whole genome sequence of two Rathayibacter toxicus strains reveals a tunicamycin biosynthetic cluster similar to Streptomyces chartreusis.

Rathayibacter toxicus is a forage grass associated Gram-positive bacterium of major concern to food safety and agriculture. This species is listed by USDA-APHIS as a plant pathogen select agent because it produces a tunicamycin-like toxin that is lethal to livestock and may be vectored by nematode species native to the U.S. The complete genomes of two strains of R. toxicus, including the type strain FH-79, were sequenced and analyzed in comparison with all available, complete R. toxicus genomes. Genome sizes ranged from 2,343,780 to 2,394,755 nucleotides, with 2079 to 2137 predicted open reading frames; all four strains showed remarkable synteny over nearly the entire genome, with only a small transposed region. A cluster of genes with similarity to the tunicamycin biosynthetic cluster from Streptomyces chartreusis was identified. The tunicamycin gene cluster (TGC) in R. toxicus contained 14 genes in two transcriptional units, with all of the functional elements for tunicamycin biosynthesis present. The TGC had a significantly lower GC content (52%) than the rest of the genome (61.5%), suggesting that the TGC may have originated from a horizontal transfer event. Further analysis indicated numerous remnants of other potential horizontal transfer events are present in the genome. In addition to the TGC, genes potentially associated with carotenoid and exopolysaccharide production, bacteriocins and secondary metabolites were identified. A CRISPR array is evident. There were relatively few plant-associated cell-wall hydrolyzing enzymes, but there were numerous secreted serine proteases that share sequence homology to the pathogenicity-associated protein Pat-1 of Clavibacter michiganensis. Overall, the genome provides clear insight into the possible mechanisms for toxin production in R. toxicus, providing a basis for future genetic approaches.

Plasmodium falciparum CRK4 directs continuous rounds of DNA replication during schizogony.

Plasmodium parasites, the causative agents of malaria, have evolved a unique cell division cycle in the clinically relevant asexual blood stage of infection1. DNA replication commences approximately halfway through the intracellular development following invasion and parasite growth. The schizont stage is associated with multiple rounds of DNA replication and nuclear division without cytokinesis, resulting in a multinucleated cell. Nuclei divide asynchronously through schizogony, with only the final round of DNA replication and segregation being synchronous and coordinated with daughter cell assembly2,3. However, the control mechanisms for this divergent mode of replication are unknown. Here, we show that the Plasmodium-specific kinase PfCRK4 is a key cell-cycle regulator that orchestrates multiple rounds of DNA replication throughout schizogony in Plasmodium falciparum. PfCRK4 depletion led to a complete block in nuclear division and profoundly inhibited DNA replication. Quantitative phosphoproteomic profiling identified a set of PfCRK4-regulated phosphoproteins with greatest functional similarity to CDK2 substrates, particularly proteins involved in the origin of replication firing. PfCRK4 was required for initial and subsequent rounds of DNA replication during schizogony and, in addition, was essential for development in the mosquito vector. Our results identified an essential S-phase promoting factor of the unconventional P. falciparum cell cycle. PfCRK4 is required for both a prolonged period of the intraerythrocytic stage of Plasmodium infection, as well as for transmission, revealing a broad window for PfCRK4-targeted chemotherapeutics.

A Malaria Transmission-Blocking (+)-Usnic Acid Derivative Prevents Plasmodium Zygote-to-Ookinete Maturation in the Mosquito Midgut.

The evolution of drug resistance is a recurrent problem that has plagued efforts to treat and control malaria. Recent emergence of artemisinin resistance in Southeast Asia underscores the need to develop novel antimalarials and identify new targetable pathways in Plasmodium parasites. Transmission-blocking approaches, which typically target gametocytes in the host bloodstream or parasite stages in the mosquito gut, are recognized collectively as a strategy that when used in combination with antimalarials that target erythrocytic stages will not only cure malaria but will also prevent subsequent transmission. We tested four derivatives of (+)-usnic acid, a metabolite isolated from lichens, for transmission-blocking activity against Plasmodium falciparum using the standard membrane feeding assay. For two of the derivatives, BT37 and BT122, we observed a consistent dose-response relationship between concentration in the blood meal and oocyst intensity in the midgut. To explore their mechanism of action, we used the murine model Plasmodium berghei and found that both derivatives prevent ookinete maturation. Using fluorescence microscopy, we demonstrated that in the presence of each compound zygote vitality was severely affected, and those that did survive failed to elongate and mature into ookinetes. The observed phenotypes were similar to those described for mutants of specific kinases (NEK2/NEK4) and of inner membrane complex 1 (IMC1) proteins, which are all vital to the zygote-to-ookinete transition. We discuss the implications of our findings and our high-throughput screening approach to identifying next generation, transmission-blocking antimalarials based on the scaffolds of these (+)-usnic acid derivatives.

Molecular Profiling of Phagocytic Immune Cells in Anopheles gambiae Reveals Integral Roles for Hemocytes in Mosquito Innate Immunity.

The innate immune response is highly conserved across all eukaryotes and has been studied in great detail in several model organisms. Hemocytes, the primary immune cell population in mosquitoes, are important components of the mosquito innate immune response, yet critical aspects of their biology have remained uncharacterized. Using a novel method of enrichment, we isolated phagocytic granulocytes and quantified their proteomes by mass spectrometry. The data demonstrate that phagocytosis, blood-feeding, and Plasmodium falciparum infection promote dramatic shifts in the proteomic profiles of An. gambiae granulocyte populations. Of interest, large numbers of immune proteins were induced in response to blood feeding alone, suggesting that granulocytes have an integral role in priming the mosquito immune system for pathogen challenge. In addition, we identify several granulocyte proteins with putative roles as membrane receptors, cell signaling, or immune components that when silenced, have either positive or negative effects on malaria parasite survival. Integrating existing hemocyte transcriptional profiles, we also compare differences in hemocyte transcript and protein expression to provide new insight into hemocyte gene regulation and discuss the potential that post-transcriptional regulation may be an important component of hemocyte gene expression. These data represent a significant advancement in mosquito hemocyte biology, providing the first comprehensive proteomic profiling of mosquito phagocytic granulocytes during homeostasis blood-feeding, and pathogen challenge. Together, these findings extend current knowledge to further illustrate the importance of hemocytes in shaping mosquito innate immunity and their principal role in defining malaria parasite survival in the mosquito host.

The fucomic potential of mosquitoes: Fucosylated N-glycan epitopes and their cognate fucosyltransferases.

Fucoconjugates are key mediators of protein-glycan interactions in prokaryotes and eukaryotes. As examples, N-glycans modified with the non-mammalian core α1,3-linked fucose have been detected in various organisms ranging from plants to insects and are immunogenic in mammals. The rabbit polyclonal antibody raised against plant horseradish peroxidase (anti-HRP) is able to recognize the α1,3-linked fucose epitope and is also known to specifically stain neural tissues in the fruit fly Drosophila melanogaster. In this study, we have detected and localized the anti-HRP cross-reactivity in another insect species, the malaria mosquito vector Anopheles gambiae. We were able to identify and structurally elucidate fucosylated N-glycans including core mono- and difucosylated structures (responsible for anti-HRP cross reactivity) as well as a Lewis-type antennal modification on mosquito anionic N-glycans by applying enzymatic and chemical treatments. The three mosquito fucosyltransferase open reading frames (FucT6, FucTA and FucTC) required for the in vivo biosynthesis of the fucosylated N-glycan epitopes were identified in the Anopheles gambiae genome, cloned and recombinantly expressed in Pichia pastoris. Using a robust MALDI-TOF MS approach, we characterised the activity of the three recombinant fucosyltransferases in vitro and demonstrate that they share similar enzymatic properties as compared to their homologues from D. melanogaster and Apis mellifera. Thus, not only do we confirm the neural reactivity of anti-HRP in a mosquito species, but also demonstrate enzymatic activity for all its α1,3- and α1,6-fucosyltransferase homologues, whose specificity matches the results of glycomic analyses.

Sex-partitioning of the Plasmodium falciparum stage V gametocyte proteome provides insight into falciparum-specific cell biology.

One of the critical gaps in malaria transmission biology and surveillance is our lack of knowledge about Plasmodium falciparum gametocyte biology, especially sexual dimorphic development and how sex ratios that may influence transmission from the human to the mosquito. Dissecting this process has been hampered by the lack of sex-specific protein markers for the circulating, mature stage V gametocytes. The current evidence suggests a high degree of conservation in gametocyte gene complement across Plasmodium, and therefore presumably for sex-specific genes as well. To better our understanding of gametocyte development and subsequent infectiousness to mosquitoes, we undertook a Systematic Subtractive Bioinformatic analysis (filtering) approach to identify sex-specific P. falciparum NF54 protein markers based on a comparison with the Dd2 strain, which is defective in producing males, and with syntenic male and female proteins from the reanalyzed and updated P. berghei (related rodent malaria parasite) gametocyte proteomes. This produced a short list of 174 male- and 258 female-enriched P. falciparum stage V proteins, some of which appear to be under strong diversifying selection, suggesting ongoing adaptation to mosquito vector species. We generated antibodies against three putative female-specific gametocyte stage V proteins in P. falciparum and confirmed either conserved sex-specificity or the lack of cross-species sex-partitioning. Finally, our study provides not only an additional resource for mass spectrometry-derived evidence for gametocyte proteins but also lays down the foundation for rational screening and development of novel sex-partitioned protein biomarkers and transmission-blocking vaccine candidates.

The acute transcriptomic and proteomic response of HC-04 hepatoma cells to hepatocyte growth factor and its implications for Plasmodium falciparum sporozoite invasion.

The routine study of human malaria liver-stage biology in vitro is hampered by low infection efficiency of human hepatocellular carcinoma (HCC) lines (<0.1%), poor understanding of steady-state HCC biology, and lack of appropriate tools for trace sample analysis. HC-04 is the only HCC that supports complete development of human malaria parasites. We hypothesized that HCCs are in various intermediate stages of the epithelial-mesenchymal transition (EMT) and HC-04s retain epithelial characteristics that permit infection. We developed a facile analytical approach to test this hypothesis viz. the HC-04 response to hepatocyte growth factor (HGF). We used online two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) to quantify protein expression profiles in HC-04 pre-/post-HGF treatment and validated these results by RT-qPCR and microscopy. We successfully increased protein identification efficiency over offline-2D methods by 12-fold, using less sample material, allowing robust protein quantification. We observed expected up-regulation and down-regulation of EMT protein markers in response to HGF, but also unexpected cellular responses. We also observed that HC-04 is generally more susceptible to HGF-mediated signaling than what was observed for HepG2, a widely used, but poor malaria liver stage-HCC model. Our analytical approach to understanding the basic biology of HC-04 helps us understand the factors that may influence its utility as a model for malaria liver-stage development. We observed that HC-04 treatment with HGF prior to the addition of Plasmodium falciparum sporozoites did not facilitate cell invasion, which suggests unlinking the effect of HGF on malaria liver stage development from hepatocyte invasion. Finally, our 2D-LC-MS/MS approach and broadly applicable experimental strategy should prove useful in the analysis of various hepatocyte-pathogen interactions, tumor progression, and early disease events.

Spatial and temporal in vivo analysis of circulating and sessile immune cells in mosquitoes: hemocyte mitosis following infection.

BACKGROUND: Mosquitoes respond to infection by mounting immune responses. The primary regulators of these immune responses are cells called hemocytes, which kill pathogens via phagocytosis and via the production of soluble antimicrobial factors. Mosquito hemocytes are circulated throughout the hemocoel (body cavity) by the swift flow of hemolymph (blood), and data show that some hemocytes also exist as sessile cells that are attached to tissues. The purpose of this study was to create a quantitative physical map of hemocyte distribution in the mosquito, Anopheles gambiae, and to describe the cellular immune response in an organismal context. RESULTS: Using correlative imaging methods we found that the number of hemocytes in a mosquito decreases with age, but that regardless of age, approximately 75% of the hemocytes occur in circulation and 25% occur as sessile cells. Infection induces an increase in the number of hemocytes, and tubulin and nuclear staining showed that this increase is primarily due to mitosis and, more specifically, autonomous cell division, by circulating granulocytes. The majority of sessile hemocytes are present on the abdominal wall, although significant numbers of hemocytes are also present in the thorax, head, and several of the appendages. Within the abdominal wall, the areas of highest hemocyte density are the periostial regions (regions surrounding the valves of the heart, or ostia), which are ideal locations for pathogen capture as these are areas of high hemolymph flow. CONCLUSIONS: These data describe the spatial and temporal distribution of mosquito hemocytes, and map the cellular response to infection throughout the hemocoel.

Infection-induced interaction between the mosquito circulatory and immune systems.

Insects counter infection with innate immune responses that rely on cells called hemocytes. Hemocytes exist in association with the insect's open circulatory system and this mode of existence has likely influenced the organization and control of anti-pathogen immune responses. Previous studies reported that pathogens in the mosquito body cavity (hemocoel) accumulate on the surface of the heart. Using novel cell staining, microdissection and intravital imaging techniques, we investigated the mechanism of pathogen accumulation in the pericardium of the malaria mosquito, Anopheles gambiae, and discovered a novel insect immune tissue, herein named periostial hemocytes, that sequesters pathogens as they flow with the hemolymph. Specifically, we show that there are two types of endocytic cells that flank the heart: periostial hemocytes and pericardial cells. Resident periostial hemocytes engage in the rapid phagocytosis of pathogens, and during the course of a bacterial or Plasmodium infection, circulating hemocytes migrate to the periostial regions where they bind the cardiac musculature and each other, and continue the phagocytosis of invaders. Periostial hemocyte aggregation occurs in a time- and infection dose-dependent manner, and once this immune process is triggered, the number of periostial hemocytes remains elevated for the lifetime of the mosquito. Finally, the soluble immune elicitors peptidoglycan and ß-1,3-glucan also induce periostial hemocyte aggregation, indicating that this is a generalized and basal immune response that is induced by diverse immune stimuli. These data describe a novel insect cellular immune response that fundamentally relies on the physiological interaction between the insect circulatory and immune systems.

The evolutionary imprint of domestication on genome variation and function of the filamentous fungus Aspergillus oryzae.

The domestication of animals, plants, and microbes fundamentally transformed the lifestyle and demography of the human species [1]. Although the genetic and functional underpinnings of animal and plant domestication are well understood, little is known about microbe domestication [2-6]. Here, we systematically examined genome-wide sequence and functional variation between the domesticated fungus Aspergillus oryzae, whose saccharification abilities humans have harnessed for thousands of years to produce sake, soy sauce, and miso from starch-rich grains, and its wild relative A. flavus, a potentially toxigenic plant and animal pathogen [7]. We discovered dramatic changes in the sequence variation and abundance profiles of genes and wholesale primary and secondary metabolic pathways between domesticated and wild relative isolates during growth on rice. Our data suggest that, through selection by humans, an atoxigenic lineage of A. flavus gradually evolved into a "cell factory" for enzymes and metabolites involved in the saccharification process. These results suggest that whereas animal and plant domestication was largely driven by Neolithic "genetic tinkering" of developmental pathways, microbe domestication was driven by extensive remodeling of metabolism.

Complex effects of temperature on mosquito immune function.

Over the last 20 years, ecological immunology has provided much insight into how environmental factors shape host immunity and host-parasite interactions. Currently, the application of this thinking to the study of mosquito immunology has been limited. Mechanistic investigations are nearly always conducted under one set of conditions, yet vectors and parasites associate in a variable world. We highlight how environmental temperature shapes cellular and humoral immune responses (melanization, phagocytosis and transcription of immune genes) in the malaria vector, Anopheles stephensi. Nitric oxide synthase expression peaked at 30°C, cecropin expression showed no main effect of temperature and humoral melanization, and phagocytosis and defensin expression peaked around 18°C. Further, immune responses did not simply scale with temperature, but showed complex interactions between temperature, time and nature of immune challenge. Thus, immune patterns observed under one set of conditions provide little basis for predicting patterns under even marginally different conditions. These quantitative and qualitative effects of temperature have largely been overlooked in vector biology but have significant implications for extrapolating natural/transgenic resistance mechanisms from laboratory to field and for the efficacy of various vector control tools.

Members of the salivary gland surface protein (SGS) family are major immunogenic components of mosquito saliva.

Mosquitoes transmit Plasmodium and certain arboviruses during blood feeding, when they are injected along with saliva. Mosquito saliva interferes with the host's hemostasis and inflammation response and influences the transmission success of some pathogens. One family of mosquito salivary gland proteins, named SGS, is composed of large bacterial-type proteins that in Aedes aegypti were implicated as receptors for Plasmodium on the basal salivary gland surface. Here, we characterize the biology of two SGSs in the malaria mosquito, Anopheles gambiae, and demonstrate their involvement in blood feeding. Western blots and RT-PCR showed that Sgs4 and Sgs5 are produced exclusively in female salivary glands, that expression increases with age and after blood feeding, and that protein levels fluctuate in a circadian manner. Immunohistochemistry showed that SGSs are present in the acinar cells of the distal lateral lobes and in the salivary ducts of the proximal lobes. SDS-PAGE, Western blots, bite blots, and immunization via mosquito bites showed that SGSs are highly immunogenic and form major components of mosquito saliva. Last, Western and bioinformatic analyses suggest that SGSs are secreted via a non-classical pathway that involves cleavage into a 300-kDa soluble fragment and a smaller membrane-bound fragment. Combined, these data strongly suggest that SGSs play an important role in blood feeding. Together with their role in malaria transmission, we propose that SGSs could be used as markers of human exposure to mosquito bites and in the development of disease control strategies.

Contraction of the ventral abdomen potentiates extracardiac retrograde hemolymph propulsion in the mosquito hemocoel.

BACKGROUND: Hemolymph circulation in mosquitoes is primarily controlled by the contractile action of a dorsal vessel that runs underneath the dorsal midline and is subdivided into a thoracic aorta and an abdominal heart. Wave-like peristaltic contractions of the heart alternate in propelling hemolymph in anterograde and retrograde directions, where it empties into the hemocoel at the terminal ends of the insect. During our analyses of hemolymph propulsion in Anopheles gambiae, we observed periodic ventral abdominal contractions and hypothesized that they promote extracardiac hemolymph circulation in the abdominal hemocoel. METHODOLOGY/PRINCIPAL FINDINGS: We devised methods to simultaneously analyze both heart and abdominal contractions, as well as to measure hemolymph flow in the abdominal hemocoel. Qualitative and quantitative analyses revealed that ventral abdominal contractions occur as series of bursts that propagate in the retrograde direction. Periods of ventral abdominal contraction begin only during periods of anterograde heart contraction and end immediately following a heartbeat directional reversal, suggesting that ventral abdominal contractions function to propel extracardiac hemolymph in the retrograde direction. To test this functional role, fluorescent microspheres were intrathoracically injected and their trajectory tracked throughout the hemocoel. Quantitative measurements of microsphere movement in extracardiac regions of the abdominal cavity showed that during periods of abdominal contractions hemolymph flows in dorsal and retrograde directions at a higher velocity and with greater acceleration than during periods of abdominal rest. Histochemical staining of the abdominal musculature then revealed that ventral abdominal contractions result from the contraction of intrasegmental lateral muscle fibers, intersegmental ventral muscle bands, and the ventral transverse muscles that form the ventral diaphragm. CONCLUSIONS/SIGNIFICANCE: These data show that abdominal contractions potentiate extracardiac retrograde hemolymph propulsion in the abdominal hemocoel during periods of anterograde heart flow.

Structural mechanics of the mosquito heart and its function in bidirectional hemolymph transport.

The insect circulatory system transports nutrients, signaling molecules, wastes and immune factors to all areas of the body. The primary organ driving circulation is the dorsal vessel, which consists of an abdominal heart and a thoracic aorta. Here, we present qualitative and quantitative data characterizing the heart of the mosquito, Anopheles gambiae. Visual observation showed that the heart of resting mosquitoes contracts at a rate of 1.37 Hz (82 beats per minute) and switches contraction direction, with 72% of contractions occurring in the anterograde direction (toward the head) and 28% of contractions occurring in the retrograde direction (toward the tip of the abdomen). The heart is tethered to the midline of the abdominal tergum by six complete and three incomplete pairs of alary muscles, and propels hemolymph at an average velocity of 8 mm s(-1) by sequentially contracting muscle fibers oriented in a helical twist with respect to the lumen of the vessel. Hemolymph enters the heart through six pairs of incurrent abdominal ostia and one pair of ostia located at the thoraco-abdominal junction that receive hemolymph from the abdominal hemocoel and thoracic venous channels, respectively. The vessel expels hemolymph through distal excurrent openings located at the anterior end of the aorta and the posterior end of the heart. In conclusion, this study presents a comprehensive revision and expansion of our knowledge of the mosquito heart and for the first time quantifies hemolymph flow in an insect while observing dorsal vessel contractions.