Immune Regulation of Plasmodium Is Anopheles Species Specific and Infection Intensity Dependent

Abstract

Malaria parasite ookinetes must traverse the vector mosquito midgut epithelium to transform into sporozoite-producing oocysts. The Anopheles innate immune system is a key regulator of this process, thereby determining vector competence and disease transmission. The role of Anopheles innate immunity factors as agonists or antagonists of malaria parasite infection has been previously determined using specific single Anopheles-Plasmodium species combinations. Here we show that the two C-type lectins CTL4 and CTLMA2 exert differential agonistic and antagonistic regulation of parasite killing in African and South American Anopheles species. The C-type lectins regulate both parasite melanization and lysis through independent mechanisms, and their implication in parasite melanization is dependent on infection intensity rather than mosquito-parasite species combination. We show that the leucine-rich repeat protein LRIM1 acts as an antagonist on the development of Plasmodium ookinetes and as a regulator of oocyst size and sporozoite production in the South American mosquito Anopheles albimanus Our findings explain the rare observation of human Plasmodium falciparum melanization and define a key factor mediating the poor vector competence of Anopheles albimanus for Plasmodium berghei and Plasmodium falciparumIMPORTANCE Malaria, one of the world's deadliest diseases, is caused by Plasmodium parasites that are vectored to humans by the bite of Anopheles mosquitoes. The mosquito's innate immune system is actively engaged in suppressing Plasmodium infection. Studies on mosquito immunity revealed multiple factors that act as either facilitators or inhibitors of Plasmodium infection, but these findings were mostly based on single Anopheles-Plasmodium species combinations, not taking into account the diversity of mosquito and parasite species. We show that the functions of CTL4 and CTLMA2 have diverged in different vector species and can be both agonistic and antagonistic for Plasmodium infection. Their protection against parasite melanization in Anopheles gambiae is dependent on infection intensity, rather than the mosquito-parasite combination. Importantly, we describe for the first time how LRIM1 plays an essential role in Plasmodium infection of Anopheles albimanus, suggesting it is a key regulator of the poor vector competence of this species.

Keywords: Anopheles; Plasmodium; innate immunity; malaria; melanization.

FIG 1

Plasmodium infection in A. gambiae. (A) Dots indicate the number of parasites in the individual midguts of female A. gambiae (Keele strain) mosquitoes infected with P. berghei (standard infection) or P. falciparum (standard and high infection). L, live parasites (orange dots); M, melanized parasites (black dots); A, all parasites (sum of L and M parasites). Horizontal red bars indicate the median. Pie charts show the percentage of A. gambiae midguts not infected (clear), containing live parasites only (orange), melanized parasites only (black), or containing both live and melanized (dark brown) parasites. Median (M) numbers of oocysts are shown above the pie charts. Two-tailed P values by Mann-Whitney test are shown: **, P < 0.01; ***, P < 0.001. Detailed statistical information concerning the infection assays is summarized in Table S2. (B) Images illustrate P. falciparum-infected A. gambiae midguts showing live parasites, melanized parasites (indicated by white arrows), and both live and melanized parasites. Scale bars, 100 µm. (C) A model of the parasite phenotypes observed in this study during the Plasmodium invasion of the A. gambiae midgut. Blue squares indicate the phenotypes observed. From left to right are shown a regular-size A. gambiae oocyst, a lysed ookinete, and a melanized ookinete. MGE, midgut epithelium; BL, basal lamina; HC, hemocytes; SPZ, sporozoites.

FIG 2

Plasmodium infection in A. albimanus. (A and B) Dots indicate the number of parasites in individual midguts of female A. albimanus infected with (A) P. berghei or (B) P. falciparum. L, live parasites; M, melanized parasites; M/SD, melanized and smaller and darker parasites; A, all parasites. Horizontal red bars indicate the median. Bars in the infection prevalence graphs show the percentage of mosquitoes harboring at least one oocyst. Pie charts show the percentage of A. albimanus midguts not infected (clear); containing live parasites only (orange), melanized parasites only (black), live and melanized parasites (dark brown), live, melanized, and smaller and darker parasites, live and smaller and darker parasites (light brown), or melanized and smaller and darker parasites (white). The median (M) number of oocysts is shown above each pie chart; red arrows indicate significant increase in prevalence (Fisher’s exact test). Two-tailed P values by Mann-Whitney test (infection intensity) or Fisher exact test (infection prevalence) are shown: *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) Images illustrate P. berghei-infected A. albimanus midguts showing melanized (M) and both live and melanized (A [all]) parasites. Scale bars are 20 µm in the far left image and 100 µm in the other images. (D) P. falciparum-infected A. albimanus midguts showing particular A. albimanus infection phenotypes: differently sized live parasites (left image), uniform and large live parasites (central image), or smaller and darker (indicated by black arrows) parasites (right image). Scale bars, 100 µm. (E) Model of the parasite phenotypes during the Plasmodium invasion of the A. albimanus midgut. Blue squares indicate the phenotypes observed. From left to right are shown a large AaLRIM1-silenced oocyst, a regular-size A. albimanus oocyst, a smaller and darker early oocyst, a lysed ookinete, and a melanized ookinete.

FIG 3

Evolutionary relationships in various mosquito species. (A and B) Phylogenetic trees (neighbor-joining) of (A) A. gambiae CTL family members and their putative A. albimanus orthologues and A. gambiae CTLMA2 and (B) A. gambiae CTL4, CTLMA2, and LRIM1 and their putative orthologs in A. albimanus, A. dirus, and A. stephensi. (Note that AgCTLMA2 has no ortholog in A. stephensi.) (C) Pairwise amino acid alignment of full-length A. gambiae CTL4 and its putative orthologs in A. albimanus, A. dirus, and A. stephensi.