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

Abstract Background Species-level identification of malaria vectors and parasites is essential for tailored vector control and clinical case management. However, routine surveillance often relies on morphology, PCR assays restricted to major vector complexes, and parasite-detection workflows optimised for Plasmodium falciparum , potentially masking wider vector and parasite diversity. We applied the previously published ANOSPP amplicon sequencing panel to characterise Anopheles and Plasmodium diversity across Tanzania, evaluate its utility for routine surveillance, and examine whether spatial primary-vector occurrence was associated with malaria endemicity patterns. Methods Between 2020 and 2023, we surveyed Anopheles mosquitoes across 25 districts in mainland Tanzania. Specimens were identified by a combination of morphology, PCR and/or ANOSPP amplicon sequencing, with ANOSPP assignments used as the molecular benchmark for evaluating morphological identification. Associations between vector species occurrence and malaria endemicity were assessed by linking district-level primary-vector co-occurrence patterns to National transmission strata and the underlying composite malaria risk score. Results Of 71,146 mosquitoes collected, 10,020 (14.0%) were Anopheles ; molecular identification resolved 15 species, including a first Tanzanian record of Anopheles funestus -like ( n = 10) and the first resolution of Anopheles longipalpis to Type C in the country ( n = 13). Using ANOSPP as the molecular benchmark, morphology misclassified 6.2% of ANOSPP-analysed specimens, with An. rufipes and An. maculipalpis frequently misclassified, and An. pretoriensis and An. marshallii sensu lato completely missed. Five Plasmodium species were detected across 37 Plasmodium -positive mosquitoes: P. falciparum , P. vivax , P. ovale , P. malariae , and P. caprae . Plasmodium caprae was found only in An. 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. Conclusions Routine surveillance tools remain valuable for monitoring major malaria vectors, but integrating ANOSPP substantially improved both vector and parasite resolution, uncovering new species records, and detecting atypical and non-falciparum Plasmodium species that routine workflows miss.