Tsetse flies, trypanosomes, hosts and microbiome diversity at agricultural–conservation interfaces in Mozambique

African animal trypanosomoses (AAT), caused by protozoans of the genus Trypanosoma and transmitted by tsetse flies ( Glossina spp.), significantly impede socio-economic growth in Africa. Mozambique harbours four main tsetse species, and there is widespread presence of pathogenic trypanosomes in both wildlife and domestic animals. Moreover, human–livestock–wildlife interfaces increase the risk of disease spillover. The objective of this study was to identify the diversity of tsetse flies, trypanosomes, vertebrate blood meal hosts and microbiome composition at the human–wildlife–livestock interface in Mozambique, and to explore the associations among these components relevant to AAT transmission cycle. We collected tsetse flies in two Mozambican National Parks (Maputo National Park—MNP, Gorongosa National Park—GNP) and their respective surrounding buffer zones (MBZ, GBZ) where agricultural activities occur. Each collected specimen was identified using taxonomic keys. A subsample (200) was then selected for DNA extraction from the whole flies, and several barcodes were amplified and sequenced to identify the diversity of trypanosomes, blood meal sources, and microbiome diversity. We identified 2291 tsetse flies representing four species ( G. austeni, G. brevipalpis, G. morsitans, G. pallidipes ) with varying trypanosome prevalences (65–90%). Seven Trypanosoma species/subspecies were detected: T. brucei (35%) and T. godfreyi (20%) were the most abundant, alongside T. congolense Kilifi, T. congolense Savannah, T. simiae , T. simiae Tsavo, and T. vivax . Blood meal source analysis revealed 17 host species, with wildlife predominating. Warthogs (26%) and humans (25%) were the most common hosts. Microbiome profiling identified dominant symbionts Wigglesworthia and Wolbachia. Our analyses demonstrate that tsetse species strongly shape trypanosome diversity, host selection, and microbiome composition, with location (difference between parks and their respective buffer zones) playing a secondary role. This study provides a comprehensive molecular snapshot of vector–host–parasite diversity in Mozambican tsetse populations. Our findings highlight the complex diversity and association of the AAT transmission cycle at the interface of conservation and agricultural areas, underscoring the role of wildlife, domestic animals, and humans in sustaining trypanosome cycles and their influence on the distribution of circulating tsetse species. These insights are crucial for developing targeted, effective One Health control strategies against African trypanosomosis.