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Mosquito physiology and immunity are integral determinants of malaria vector competence. This includes the principal role of hormonal signaling in  Anopheles gambiae  initiated shortly after blood-feeding, which stimulates immune induction and promotes vitellogenesis through the function of 20-hydroxyecdysone (20E). Previous studies demonstrated that manipulating 20E signaling through the direct injection of 20E or the application of a 20E agonist can significantly impact  Plasmodium  infection outcomes, reducing oocyst numbers and the potential for malaria transmission. In support of these findings, we demonstrate that a 20E agonist, halofenozide, is able to induce anti- Plasmodium  immune responses that limit  Plasmodium  ookinetes. We demonstrate that halofenozide requires the function of ultraspiracle (USP), a component of the canonical heterodimeric ecdysone receptor, to induce malaria parasite killing responses. Additional experiments suggest that the effects of halofenozide treatment are temporal, such that its application only limits malaria parasites when applied prior to infection. Unlike 20E, halofenozide does not influence cellular immune function or AMP production. Together, our results further demonstrate the potential of targeting 20E signaling pathways to reduce malaria parasite infection in the mosquito vector and provide new insight into the mechanisms of halofenozide-mediated immune activation that differ from 20E.

The 20-hydroxyecdysone agonist, halofenozide, promotes anti-Plasmodium immunity in Anopheles gambiae via the ecdysone receptor