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.
Baravuga, M. E., Bodde, M. M., Makunin, A., Michael, P., Mlalwe, A. T., Mwalimu, C. D., Korlevic, P., Ferguson, H. M., Salje, H., Lawniczak, M. K. N., Govella, N. J.
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