Blood feeding induces hemocyte proliferation and activation in the African malaria mosquito, Anopheles gambiae Giles

Abstract

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.

Keywords: Blood meal; Immunology; Infectious disease; Innate immunity; Phenoloxidase.

Fig. 1.

Blood feeding induces hemocyte proliferation. (A) Hemocyte numbers are increased significantly in blood-fed (BF) compared with sugar-fed (SF) mosquitoes (Mann–Whitney U-test). N=11 for each group from two independent biological replicates, shown as median with interquartile range. (B) EdU incorporation is increased significantly in hemocytes from blood-fed females (Mann–Whitney U-test). N=14 for each group, shown as median with interquartile range. For each data point, 300–400 cells were assessed from a hemocyte pool collected from two mosquitoes. (C) Confocal images of representative EdU-positive hemocytes. Blue, DAPI; green, EdU; scale bar, 10 μm. (D) Flow cytometry analysis of PI-stained hemocytes from sugar-fed and blood-fed mosquitoes. Dot plots of propidium iodide (PI) fluorescence signal area over width (expressed in arbitrary units) illustrate DNA content per cell. Gates were drawn to eliminate putative aggregates based on high signal width from further analysis. Histograms show measurements for PI staining and thus DNA content for the gated cells. Markers designate three hemocyte populations based on DNA content, M1 representing euploid, M2 and M3 representing aneuploid cells. Percentages of cells within the three markers are indicated above the brackets. The figure shows a representative result from three independent biological replicates.

Fig. 2.

Blood feeding induces changes in hemocyte population and morphology. (A) The percentage of euploid and aneuploid cells obtained from the flow cytometry analyses of PI-stained hemocytes (means ± s.e.m.). (B) Euploid cells were gated and analyzed for their size (FSC) and granularity (SSC). Axes are shown in arbitrary units. (C) Density dot plots illustrating size and granularity (expressed as arbitrary units) and their intensity on a blue to white color scale. (D) Overlaying histograms of sugar-fed and blood-fed euploid hemocytes revealing an increase in average cell size and granularity after a blood meal. Data shown are representative of three independent experiments.

Fig. 3.

Blood feeding increases expression of several cell activation markers. Hemocytes from sugar-fed and blood-fed females were analyzed for blood meal-induced activation markers. N=91 for WGA (A), N=126 for pERK (B), N=103 for TEP-1 (C) and N=136 for PPO6 (D). Confocal maximum intensity projections are shown for all stains: blue, DAPI; green, WGA; red, pERK (B), TEP-1 (C) and PPO6 (D). Scale bar, 10 μm. Quantification of activation markers is shown as median with interquartile range. Blood feeding led to a significant increase in fluorescence for all immunofluorescence analyses (Mann–Whitney U-test, P<0.0001). Experiments were performed in triplicate with one representative experiment image and graph shown for each hemocyte activation marker. Results from all replicates are shown in supplementary material Fig. S2.