Molecular characterization of midgut Peritrophin-44 and Peritrophin-48 and their role in Plasmodium development

The peritrophic matrix (PM) is a semi-permeable, chitin-peritrophin layer lining the insect midgut. It facilitates digestive compartmentalization, maintains midgut homeostasis, mitigates the oxidative cytotoxicity of free heme, and acts as a mechanical barrier impeding the midgut invasion of pathogens like Plasmodium. The specific roles of individual peritrophins during parasite transmission are not well characterized. This study provides the first functional characterization of two peritrophins, Peritrophin-44 (Per-44) and Peritrophin-48 (Per-48), in Anopheles stephensi. Sequence analysis confirmed that both proteins contain peritrophin A-domain (PAD) with conserved six-cysteine motifs, identifying them as Class III chitin-binding proteins. Both Per-44 and Per-48 genes were predominantly expressed in the midgut. These transcripts are expressed throughout the lifecycle (i.e. larvae, pupae, and adults), with peak expression in fourth-instar larvae and adults. Following an infectious blood meal, both are significantly upregulated during ookinete invasion. RNA interference (RNAi)-mediated silencing of Per-44 and Per-48 impaired mosquito fitness, increasing mortality and reducing fecundity in infected vectors. Notably, Per-48 knockdown induced the most severe phenotype, causing a significant reduction in oocyst burden and demonstrating its essential role in Plasmodium transmission. These results demonstrate that Per-44 and Per-48 are essential PM components that may modulate midgut integrity and vector competence, highlighting them as viable targets for transmission-blocking strategies against malaria.