Adult Mosquitoes Infected with Bacteria Early in Life Have Stronger Antimicrobial Responses and More Hemocytes after Reinfection Later in Life

Abstract

The immunological strategies employed by insects to overcome infection vary with the type of infection and may change with experience. We investigated how a bacterial infection in the hemocoel of the African malaria mosquito, Anopheles gambiae, prepares the immune system to face a subsequent bacterial infection. For this, adult female mosquitoes were separated into three groups-unmanipulated, injured, or infected with Escherichia coli-and five days later all the mosquitoes were infected with a different strain of E. coli. We found that an injury or a bacterial infection early in life enhances the ability of mosquitoes to kill bacteria later in life. This protection results in higher mosquito survival and is associated with an increased hemocyte density, altered phagocytic activity by individual hemocytes, and the increased expression of nitric oxide synthase and perhaps prophenoloxidase 6. Protection from a second infection likely occurs because of heightened immune awareness due to an already existing infection instead of memory arising from an earlier, cured infection. This study highlights the dynamic nature of the mosquito immune response and how one infection prepares mosquitoes to survive a subsequent infection.

Keywords: Anopheles gambiae; immunity; insect; nitric oxide synthase; phagocytosis; prophenoloxidase; survival.

Figure 1

Diagrammatic representations of the experimental approach. At 3 days after eclosion, mosquitoes were subjected to one of three primary treatments: naïve (unmanipulated), injured, or infected with E. coli-K12. At day 8, all the mosquitoes were infected with E. coli-GFP and several phenotypes were measured. Infection intensity was measured at 1, 3, and 7 days after the E. coli-GFP infection. The number of hemocytes and immune gene expression were assayed 1 day after the E. coli-GFP infection. Phagocytosis was measured 1 hr after the E. coli-GFP infection, and survival was tracked every day after the E. coli-GFP infection until all the mosquitoes had died.

Figure 2

Infection intensity after secondary treatment. Five days after the primary treatment of naïve, injury, or infection with E. coli-K12, all the mosquitoes were infected with E. coli-GFP and the number of E. coli-GFP in their hemocoel was quantified 1 (A), 3 (B), or 7 (C) days later. Column heights mark the mean and whiskers denote the standard error of the mean (SEM). Circles mark the infection intensity of individual mosquitoes.

Figure 3

Number of circulating hemocytes after secondary treatment. Five days after the primary treatment of naïve, injury, or infection with E. coli-K12, all the mosquitoes were infected with E. coli-GFP and the number of circulating hemocytes was counted 1 day later. Column heights mark the mean and whiskers denote the SEM. Circles mark the number of hemocytes in individual mosquitoes.

Figure 4

Phagocytic index and phagocytic capacity after secondary treatment. Five days after the primary treatment of naïve, injury, or infection with E. coli-K12, all the mosquitoes were infected with E. coli-GFP and 1 hr later the following phenotypes were measured: the percentage of hemocytes that phagocytosed E. coli-GFP (A), the average number of E. coli-GFP phagocytosed by each hemocyte (B), and the average number of E. coli-GFP phagocytosed by each phagocytic hemocyte (C). Column heights mark the mean and whiskers denote the SEM. Circles mark the phagocytic activity in individual mosquitoes.

Figure 5

Immune gene expression after secondary treatment. Five days after the primary treatment of naïve, injury, or infection with E. coli-K12, all the mosquitoes were infected with E. coli-GFP and 1 day later the relative expressions of PPO6 (A), NOS (B), LYSC1 (C), CEC1 (D), TEP1 (E), and RPS17 (F) were measured by RT-PCR using RPS7 as the reference. Column heights mark the mean and whiskers denote the SEM.

Figure 6

Mosquito survival after the secondary treatment. Five days after the primary treatment of naïve, injury, or infection with E. coli-K12, all the mosquitoes were infected with E. coli-GFP and their survival was tracked every day until all the mosquitoes had died.