First molecular confirmations of Anopheles dirus and Anopheles scanloni in Indonesia, with DNA of zoonotic, enzootic and human malarias detected in An. dirus

Despite Indonesia reporting high numbers of Plasmodium knowlesi malaria cases in North Sumatra Province, the Anopheles mosquito vectors remain unknown. This study identified the Leucosphyrus Group species present in Langkat Regency, North Sumatra, and the Plasmodium species DNA in their heads and thoraces. Mosquitoes collected by human landing catch were morphologically identified and their species identification subsequently confirmed using ITS2 sequencing as well as Dirus Complex (DiCSIP) and Anopheles scanloni-specific PCR. Reverse-transcription real-time and nested PCR assays targeting the 18 S rRNA gene were applied for Plasmodium species detection and identification. Of 597 morphologically identified Leucosphyrus Group mosquitoes, two species of the Dirus Complex were confirmed for the first time in Indonesia: 97.8% of specimens were Anopheles dirus with 2.2% being Anopheles scanloni. Seven An. dirus specimens were Plasmodium-positive, including mixed infections with P. inui, P. knowlesi, and/or P. vivax and one equivocal sample positive for P. coatneyi and P. knowlesi. BLAST analysis indicated possible cross-reactivity of P. fieldi primers with P. inui. This study provides the first molecular confirmation of An. dirus and confirms the presence of An. scanloni, two species of the Dirus Complex in North Sumatra. In addition, it demonstrates the presence of both macaque and human Plasmodium species DNA in An. dirus, suggesting the potential role of this species in zoonotic and human malaria transmission in this Indonesian region.