Late-phase immune responses limiting oocyst survival are independent of TEP1 function yet display strain specific differences in Anopheles gambiae

BACKGROUND: There is emerging evidence that mosquito anti-Plasmodium immunity is multimodal with distinct mechanisms for killing malaria parasites at either the ookinete or oocyst stages. Early-phase responses targeting the ookinete require complement-like components circulating in the mosquito hemolymph that result in TEP1-mediated lysis or melanization. Additional responses mediated by the LL3 and STAT pathways limit oocyst survival through unknown mechanisms that require mosquito hemocyte function. While previous experiments argue that these mechanisms of parasite killing are independent, the transient nature of gene-silencing has rendered these experiments inconclusive. To address this issue, we outline experiments using a TALEN-derived TEP1 mutant line to examine the role of TEP1 in the Anopheles gambiae late-phase immune response. RESULTS: Despite higher early oocyst numbers in the TEP1 mutant line, no differences in oocyst survival were observed when compared to control mosquitoes, suggesting that TEP1 function is independent of the late-phase immune response. To further validate this phenotype in the TEP1 mutant, oocyst survival was evaluated in the TEP1 mutant background by silencing either LL3 or STAT-A. Surprisingly, only STAT-A silenced mosquitoes were able to reconstitute the late-phase immune phenotype increasing oocyst survival in the TEP1 mutant line. Additional experiments highlight significant differences in LL3 expression in the M/S hybrid genetic background of the TEP1 mutant line compared to that of the Keele strain (M form) of An. gambiae, and demonstrate that LL3 is not required for granulocyte differentiation in the M/S hybrid G3 genetic background in response to malaria parasite infection. CONCLUSIONS: Through the combination of genetic experiments utilizing genetic mutants and reverse genetic approaches, new information has emerged regarding the mechanisms of mosquito late-phase immunity. When combined with previously published experiments, the body of evidence argues that Plasmodium oocyst survival is TEP1 independent, thus establishing that the mechanisms of early- and late-phase immunity are distinct. Moreover, we identify that the known components that mediate oocyst survival are susceptible to strain-specific differences depending on their genetic background and provide further evidence that the signals that promote hemocyte differentiation are required to limit oocyst survival. Together, this study provides new insights into the mechanisms of oocyst killing and the importance of genetics in shaping mosquito vector competence. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13071-017-2308-0) contains supplementary material, which is available to authorized users.