Hemocyte differentiation mediates innate immune memory in Anopheles gambiae mosquitoes

Abstract

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.

Fig. 1

Effect of pre-exposure to Plasmodium infection on the immune response to subsequent infections. (A) 7 days post priming (dpp) or (B) 14 dpp with P. berghei in naïve (Nv) or challenged (Ch) mosquitoes. (C) Effect of pre-challenge with P. falciparum on a second infection at 7 dpp. Plasmodium infections were evaluated 7 days after the second infection. Effect of eliminating the gut microbiota with oral antibiotics before the (D) first or (E) second challenge with P. berghei on a second infection 7 dpp. Each circle represents the number of parasites in an individual midgut, and the line indicates the median.

Fig. 2

Effect of immune priming with Plasmodium/bacteria on commensal bacteria, hemocyte populations, and gut-associated hemocyte-specific antiplasmodial mRNAs. (A) Effect of priming on whole-body bacterial levels in naïve (Nv) and challenged (Ch) mosquitoes 7 days post priming (dpp). (B) Total midgut bacteria before (Be) or 24 hours after (Af) a second infectious meal was given 7 dpp. Bacterial 16S rRNA levels were determined using real-time PCR. Effect of immune priming on the relative abundance of (C) granulocytes and (D) prohemocytes 7dpp in the presence or absence of gut microbiota. Effect of immune priming on the relative abundance of TEP1 and LRIM1 mRNAs associated with the midgut or circulating hemocytes in (E) the presence or (F) the absence of gut microbiota determined before (grey bars) or 24 hours after (red bars) a second infectious meal was given 7 dpp. Bars represent the Mean ± SEM.

Fig. 3

Effect of immune priming on granulocyte morphology and binding properties, and of water injection on or hemolymph transfer on hemocyte populations and Plasmodium infection. (A) Morphology of naïve (Nv) and challenged (Ch) granulocytes in a plastic Neubauer chamber (bar = 7.5 µm). (B) Effect of priming on lectin-binding properties of granulocytes. WGA, wheat germ agglutinin; LCA, Lens culinaris agglutinin (bar = 5 µm). Effect of water injection (69 nl) on circulating (C) granulocytes and prohemocytes. (D) Effect of water injection 7 days post priming on Plasmodium infection. Effect of cell-free hemolymph transfer from Nv or Ch mosquito on (E) circulating granulocytes, oenocytoids, and prohemocytes and (F) on Plasmodium infection 4 days post transfer. Each circle represents the number of parasites in an individual midgut, and the line indicates the median. Bars represent the Mean ± SEM.