When a mosquito acquires an infection in the hemocoel, dedicated immune cells called hemocytes aggregate around the valves of the heart. These sessile hemocytes are called periostial hemocytes. In the present study we scrutinized the immune response mounted by the periostial hemocytes of the malaria mosquito, Anopheles gambiae, against bacterial pathogens, and tested the relationship between periostial hemocyte aggregation, immune activity, and hemolymph flow. Initially, we quantified the process of periostial hemocyte aggregation and found that hemocytes migrate to the periostial regions in response to infection with Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Micrococcus luteus (all infections tested). Then, we investigated whether the periostial hemocytes are evenly distributed along the six periostial regions of the heart, and found that they preferentially aggregate in the periostial regions of the mid-abdominal segments (4, 5 and 6). This distribution perfectly correlates with the spatial distribution of phagocytic activity along the surface of the heart, and to a lesser extent, with the distribution of melanin deposits. Finally, experiments measuring circulatory physiology found that the majority of hemolymph enters the heart through the ostia located in the periostial regions of abdominal segments 4, 5, and 6. These data show that periostial hemocytes aggregate on the surface of the heart in response to diverse foreign stimuli, and that both hemocytes and immune activity preferentially occur in the regions that experience the swiftest hemolymph flow. Thus, these data show that two major organ systems - the immune and circulatory systems - interact to control infections.

The dengue, Zika and chikungunya viruses are transmitted by the mosquito Aedes aegypti and pose a substantial threat to global public health. Current vaccines and mosquito control strategies have limited efficacy, so novel interventions are needed 1,2. Wolbachia are bacteria that inhabit insect cells and have been found to reduce viral infection-a phenotype that is referred to as viral 'blocking' 3. Although not naturally found in A. aegypti 4 , Wolbachia were stably introduced into this mosquito in 2011 4,5 and were shown to reduce the transmission potential of dengue, Zika and chikungunya 6,7. Subsequent field trials showed Wolbachia's ability to spread through A. aegypti populations and reduce the local incidence of dengue fever 8. Despite these successes, the evolutionary stability of viral blocking is unknown. Here, we utilized artificial selection to reveal genetic variation in the mosquito that affects Wolbachia-mediated dengue blocking. We found that mosquitoes exhibiting weaker blocking also have reduced fitness, suggesting the potential for natural selection to maintain blocking. We also identified A. aegypti genes that affect blocking strength, shedding light on a possible mechanism for the trait. These results will inform the use of Reprints and permissions information is available at www.nature.com/reprints.

Haemocytes respond to infection by phagocytosing pathogens, producing the enzymes that drive the phenoloxidase-based melanization cascade, secreting lytic factors, and producing other humoral proteins. A subset of haemocytes, called periostial haemocytes, aggregate on the surface of the heart of mosquitoes and kill pathogens in areas of high haemolymph flow. Periostial haemocytes are always present, but an infection induces the recruitment of additional haemocytes to these regions. Here, we tested whether members of the Nimrod gene family are involved in the periostial immune response of the African malaria mosquito, Anopheles gambiae. Using organismal manipulations, RNA interference (RNAi) and microscopy, we show that, following an infection with Escherichia coli, nimrod - the orthologue of Drosophila NimB2 - is not involved in periostial responses. At 4 h postinfection, however, RNAi-based knockdown of draper results in a marginal increase in the number of periostial haemocytes and a doubling of E. coli accumulation at the periostial regions. Finally, at 24 h postinfection, knockdown of eater decreases the number of periostial haemocytes and decreases the phagocytosis of E. coli on the surface of the heart. Phagocytosis of bacteria is more prevalent in the periostial regions of the mid abdominal segments, and knockdown of draper, nimrod or eater does not alter this distribution. Finally, knockdown of Nimrod family genes did not have a meaningful effect on the accumulation of melanin at the periostial regions. This study identifies roles for eater and draper in the functional integration of the mosquito immune and circulatory systems.

A powerful immune system protects mosquitoes from pathogens and influences their ability to transmit disease. The mosquito's immune and circulatory systems are functionally integrated, whereby intense immune processes occur in areas of high hemolymph flow. The primary circulatory organ of mosquitoes is the dorsal vessel, which consists of a thoracic aorta and an abdominal heart. In adults of the African malaria mosquito, Anopheles gambiae, the heart periodically alternates contraction direction, resulting in intracardiac hemolymph flowing toward the head (anterograde) and toward the posterior of the abdomen (retrograde). During anterograde contractions, hemolymph enters the dorsal vessel through ostia located in abdominal segments 2–7, and exits through an excurrent opening located in the head. During retrograde contractions, hemolymph enters the dorsal vessel through ostia located at the thoraco-abdominal junction, and exits through posterior excurrent openings located in the eighth abdominal segment. The ostia in abdominal segments 2 to 7—which function in anterograde intracardiac flow—are sites of intense immune activity, as a subset of hemocytes, called periostial hemocytes, respond to infection by aggregating, phagocytosing, and killing pathogens. Here, we assessed whether hemocytes are present and active at two sites important for retrograde intracardiac hemolymph flow: the thoraco-abdominal ostia and the posterior excurrent openings of the heart. We detected sessile hemocytes around both of these structures, and these hemocytes readily engage in phagocytosis. However, they are few in number and a bacterial infection does not induce the aggregation of additional hemocytes at these locations. Finally, we describe the process of hemocyte attachment and detachment to regions of the dorsal vessel involved in intracardiac retrograde flow.