Background Vector populations are a key target for malaria control and elimination. In Honduras there are at least 12 reported anopheline species, however, the definitive number of species remains uncertain. Due to the inherent limitations of morphological identification of Anophelesspecies, molecular approaches have been developed to provide accurate identification and robust surveillance of local malaria vectors. The aim of this study was to design and assess three PCR-RFLP assays to identify anopheline species in Honduras. Methods Mosquitoes captured between 2018 and 2022 in seven malaria-endemic and non-endemic departments in Honduras were analysed. The ITS2 ribosomal region and three restriction enzyme-based assays were evaluated in silico and experimentally. Results A total of 132 sequences from 12 anopheline species were analysed. The ITS2 marker showed length polymorphisms that generated products between 388 bp and 592 bp and no relevant intraspecies polymorphisms were found. Furthermore, the three PCR-RFLP assays were able to differentiate eleven species with sufficient precision and resolution. Conclusion The ITS2 region showed to be a useful molecular marker for identifying local Anophelesspecies. In addition, the PCR-RFLP assays evaluated here proved to be capable of discriminating most of the anopheline species present in Honduras. These methods provide alternatives to improve entomological surveillance of Anophelesin Honduras and other Mesoamerican countries.

Background Vector populations are a key target for malaria control and elimination. In Honduras, there are at least 12 reported anopheline species, however, the definitive number of species remains uncertain. Due to the inherent limitations of morphological identification of Anopheles species, molecular approaches have been developed to provide accurate identification and robust surveillance of local malaria vectors. The aim of this study was to design and assess three PCR–RFLP assays to identify anopheline species known to presently occur in Honduras. Methods Mosquitoes captured between 2018 and 2022 in seven malaria-endemic and non-endemic departments in Honduras were analysed. The ITS2 ribosomal region and three restriction enzyme-based assays were evaluated in silico and experimentally. Results A total of 132 sequences from 12 anopheline species were analysed. The ITS2 marker showed length polymorphisms that generated products between 388 and 592 bp and no relevant intraspecies polymorphisms were found. Furthermore, the three PCR–RFLP assays were able to differentiate 11 species with sufficient precision and resolution. Conclusion The ITS2 region was shown to be a useful molecular marker for identifying local Anopheles species. In addition, the PCR–RFLP assays evaluated here proved to be capable of discriminating most of the anopheline species present in Honduras. These methods provide alternatives to improve entomological surveillance of Anopheles in Honduras and other Mesoamerican countries.