Exploring the diversity and spatial distribution of Anopheles mosquitoes and their associated Plasmodium species in Tanzania using the ANOSPP amplicon panel

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Abstract Species-level identification of malaria vectors and parasites is essential for targeted vector control, surveillance, and clinical case management. However, routine surveillance often relies on morphology, species group-restricted PCR assays, and parasite-detection workflows optimised for Plasmodium falciparum , potentially masking wider vector and parasite diversity. To address this gap, we applied the previously published ANOSPP amplicon sequencing panel to Anopheles mosquitoes collected across 25 districts in mainland Tanzania between 2020 and 2023, using it to characterise vector and parasite diversity, evaluate routine species assignments, and examine how primary-vector occurrence related to national malaria endemicity patterns. Of 71,146 mosquitoes collected, 10,020 were morphologically identified as Anopheles . ANOSPP-based identification resolved 15 Anopheles taxa, including the first Tanzanian record of Anopheles funestus -like mosquitoes (n = 10) and the first resolution of Anopheles longipalpis to Type C in the country (n = 13). Using ANOSPP as the reference, morphology misclassified 6.2% of ANOSPP-analysed specimens, with Anopheles rufipes and Anopheles maculipalpis frequently misclassified, and Anopheles pretoriensis and Anopheles marshallii sensu lato completely missed. Five Plasmodium species were detected across 37 parasite-positive mosquitoes: P. falciparum , P. vivax , P. ovale , P. malariae , and P. caprae . Plasmodium caprae was detected only in Anopheles arabiensis (n = 6), representing the first record of this parasite in Tanzania and its first detection in An. arabiensis globally. Co-occurrence of An. arabiensis , An. funestus s.s., and An. gambiae s.s. was more common in higher malaria transmission strata, whereas occurrence of An. arabiensis alone predominated in very-low and low strata. These findings show that integrating ANOSPP into surveillance can substantially improve vector and parasite resolution, uncover overlooked diversity, and strengthen species-informed malaria surveillance. Author summary Effective malaria control requires accurate data on mosquito species distributions and the parasites they carry. However, in many settings surveillance still relies mainly on morphological identification or molecular tests targeting limited vector groups, while parasite detection often focuses on Plasmodium falciparum . This limits understanding of mosquito and parasite diversity, obscuring transmission dynamics. We used the previously published ANOSPP amplicon panel to analyse Anopheles mosquitoes across Tanzania. This revealed hidden biological complexity with direct implications for malaria transmission, resolving 15 mosquito and five Plasmodium species. Notably, for the first time in Tanzania, we detected the goat-associated parasite Plasmodium caprae in Anopheles arabiensis and identified An. funestus -like, whose vectorial capacity remains unclear, and An. longipalpis Type C, implicated in malaria transmission in dryland Kenya. Interestingly, co-occurrence of all three major Tanzanian vectors aligned with higher transmission settings, whereas areas where An. arabiensis was the sole vector were mainly in lower transmission settings. These findings show that species-resolved surveillance can reveal hidden biological complexity and provide a stronger basis for locally targeted malaria control.

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