Some strains of Plasmodium falciparum, a human malaria parasite, evade the complement-like system of Anopheles gambiae mosquitoes

Abstract

Plasmodium falciparum lines differ in their ability to infect mosquitoes. The Anopheles gambiae L3-5 refractory (R) line melanizes most Plasmodium species, including the Brazilian P. falciparum 7G8 line, but it is highly susceptible to some African P. falciparum strains such as 3D7, NF54, and GB4. We investigated whether these lines differ in their ability to evade the mosquito immune system. Silencing key components of the mosquito complement-like system [thioester-containing protein 1 (TEP1), leucine-rich repeat protein 1, and Anopheles Plasmodium-responsive leucine-rich repeat protein 1] prevented melanization of 7G8 parasites, reverting the refractory phenotype. In contrast, it had no effect on the intensity of infection with NF54, suggesting that this line is able to evade TEP1-mediated lysis. When R females were coinfected with a line that is melanized (7G8) and a line that survives (3D7), the coinfection resulted in mixed infections with both live and encapsulated parasites on individual midguts. This finding shows that survival of individual parasites is parasite-specific and not systemic in nature, because parasites can evade TEP1-mediated lysis even when other parasites are melanized in the same midgut. When females from an extensive genetic cross between R and susceptible A. gambiae (G3) mosquitoes were infected with P. berghei, encapsulation was strongly correlated with the TEP1-R1 allele. However, P. falciparum 7G8 parasites were no longer encapsulated by females from this cross, indicating that the TEP1-R1 allele is not sufficient to melanize this line. Evasion of the A. gambiae immune system by P. falciparum may be the result of parasite adaptation to sympatric mosquito vectors and may be an important factor driving malaria transmission.

Fig. 1.

Survival of different P. falciparum lines (GB4, 3D7, NF54, and 7G8) in the R A. gambiae (L3-5 strain) 7–9 d postfeeding. (A) Mercurochrome staining of infected midguts. (B) Live and melanized parasites on individual mosquito midguts. The medians are indicated with red lines. (C) Proportion of live (orange) and melanized (black) parasites. (D) Prevalence of mosquito infection. All parasite phenotypes were confirmed in two or three independent experiments.

Fig. 2.

Effect of TEP1 silencing in R A. gambiae (L3-5 strain) females on P. falciparum (7G8) infection. R females were injected with dsLacZ control or dsTEP1 3 d before feeding on a P. falciparum (7G8) gametocyte culture, and midgut infection was assessed 8 d postfeeding. (A) Mercurochrome staining of midguts. (B) Live and melanized parasites on individual mosquito midguts. The medians are indicated with red lines. (C) Proportion of live (orange) and melanized (black) parasites. (D) Prevalence of mosquito infection. All gene silencing phenotypes were confirmed in two or three independent experiments.

Fig. 3.

Effect of TEP1 and LRIM1 silencing in R A. gambiae (L3-5 strain) females on P. falciparum (NF54) infection. R females were injected with dsLacZ control, dsTEP1, or dsLRIM1 3 d before feeding on a P. falciparum (NF54) gametocyte culture, and midgut infection was assessed 8 d postfeeding. (A) Mercurochrome staining of midguts. (B) Live and melanized parasites on individual mosquito midguts. The medians are indicated with red lines. (C) Proportion of live (orange) and melanized (black) parasites. (D) Prevalence of mosquito infection. All gene silencing phenotypes were confirmed in two or three independent experiments.

Fig. 4.

Coinfection of P. falciparum 3D7 and 7G8 strains in R A. gambiae (L3-5) females. Gametocyte cultures of P. falciparum 3D7 and 7G8 strains were fed to R females either separately or as a 1:1 mixture of the two strains. Midgut infection was assessed 8 d postfeeding. (A) Mercurochrome staining of midguts. (B) Live and melanized parasites on individual mosquito midguts. The medians are indicated with red lines. (C) Proportion of live (orange) and melanized (black) parasites. (D) Prevalence of mosquito infection. All parasite phenotypes were confirmed in two or three independent experiments.

Fig. 5.

Survival of P. berghei or P. falciparum 7G8 (black dots) infections in a laboratory cross line of A. gambiae with different genotypes for the TEP1 locus. Infections were analyzed 6 d postinfection for P. berghei or 8 d postinfection for P. falciparum. (A) Number of live and melanized P. berghei parasites (Left; blue dots) and P. falciparum 7G8 (Right; black dots) on individual mosquito midguts. (B) Number of live and melanized P. berghei parasites on individual mosquitoes with the R1/R1, R1/S3, or S3/S3 TEP1 genotypes. (C) Proportion of live (green) and melanized (black) P. berghei parasites in mosquitoes with the R1/R1, R1/S3, or S3/S3 TEP1 genotypes. (D) Prevalence of infection in mosquitoes with the R1/R1, R1/S3, or S3/S3 TEP1 genotypes.

Fig. P1.

A. gambiae L3-5 mosquitoes are refractory to infection with most Plasmodium species, including the P. falciparum 7G8 strain isolated in Brazil. The parasites are killed, and a black insoluble pigment, melanine, is deposited on their surface as they emerge from the mosquito midgut (Left; the black arrowheads indicate melanized parasites). However, this mosquito strain is highly susceptible to infection by some African P. falciparum strains, such as NF54 (Right). Disruption of the mosquito complement-like allows P. falciparum 7G8 to survive, showing that this parasite strain is actively eliminated by the mosquito immune system. In contrast, this treatment has no effect in mosquitoes infected with P. falciparum NF54 from Africa, indicating that this parasite strain evades the mosquito complement-like system. This ability of some parasites to evade the mosquito immune response may be promoting transmission of human malaria.