Resurrection of Anopheles darlingi FREP1 Ancestor Reveals Adaptive Evolution Characterized by Changes in Protein Stability and Plasmodium falciparum Interaction

Fibrinogen-related protein 1 (FREP1), a midgut-localized fibrinogen-like lectin in Anopheles mosquitoes, mediates Plasmodium ookinete attachment by binding α-tubulin-1. To elucidate the evolutionary forces shaping this interaction, the study analysed FREP1 sequences from 29 Anopheles species using codon-based tests, ancestral sequence reconstruction, stability modelling, and docking. Both aBSREL and branch-site codeml identified the Anopheles darlingi lineage as the sole branch experiencing episodic diversifying selection, with MEME detecting a single positively selected site (codon 173/residue 218) within the FBG domain. Ancestral reconstruction revealed a Ser to Asn substitution at this position in the extant protein. Although Rosetta ΔΔG analyses indicated only modest local effects of this substitution, docking showed that extant A. darlingi FREP1 exhibits markedly reduced predicted binding affinity to Plasmodium falciparum α-tubulin-1 relative to its reconstructed ancestor. Complementary cophylogenetic analyses (PACo and ParaFit) identified significant global phylogenetic congruence between Anopheles and Plasmodium lineages, consistent with broad lineage-level structuring of vector-parasite compatibility rather than strict co-speciation. Within this evolutionary backdrop, the lineage-specific adaptive change at residue 218 suggests fine-scale molecular tuning of the FREP1 ligand-binding interface, potentially reflecting an arms-race-like dynamic in which A. darlingi FREP1 functionally diverged in response to historical parasite pressures despite the absence of tight tree-wide coevolution.