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

2025 · doi:10.32942/x2965c
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Abstract

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.
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This is a Preprint and has not been peer reviewed. This is version 2 of this Preprint. You must log in to post a comment. There are no comments or no comments have been made public for this article. This is a Preprint and has not been peer reviewed. This is version 2 of this Preprint. Add a Comment You must log in to post a comment. Comments There are no comments or no comments have been made public for this article. Fibrinogen-related proteins (FREPs) contribute to mosquito-parasite interactions, yet the evolutionary processes shaping their functional diversification remain poorly resolved. The mosquito protein FBN30 has been implicated in restricting Plasmodium development, but its molecular basis of action is unknown. Here, the study examines the evolutionary history of FBN30 across Anopheles mosquitoes to test whether lineage-specific adaptive evolution has modified its functional properties. Codon-based analyses of FBN30 orthologs from 29 Anopheles species reveal a single episode of strong episodic diversifying selection confined to the Anopheles darlingi lineage. Site-level tests identify a positively selected residue within the conserved fibrinogen-like (FBG) domain. Ancestral sequence reconstruction shows that this site underwent a serine-to-asparagine substitution along the A. darlingi lineage, with structural modeling indicating only modest local effects on protein stability. Using protein-protein docking and binding affinity prediction as a proxy for functional engagement, the study finds that the reconstructed ancestral FBN30 exhibits significantly stronger predicted affinity for Plasmodium falciparum α-tubulin-1 than the extant A. darlingi protein, whereas the derived substitution alone does not account for this difference. These results indicate that evolutionary divergence in FBN30 is associated with reduced predicted engagement at a parasite-facing interface and support a model in which inhibitory mosquito proteins undergo fine-scale adaptive refinement under parasite-mediated selective pressures. https://doi.org/10.32942/X2965C Life Sciences Anopneles, Plasmodium, codeml, episodic selection, ASR Published: 2025-11-21 18:33 Last Updated: 2026-01-27 21:59 CC-BY Attribution-NonCommercial 4.0 International Language: English

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