Fig 1.
Map of Zambia and Zimbabwe showing areas of tsetse fly collection.
The map was sourced from Simarro P, et al, 2012 [40] and modified in Adobe Illustrator CC 2019 v23.0.1. Areas where tsetse flies were caught are marked in red stars: (A) Kafue in Zambia, (B) Rufunsa in Zambia and (C) Hurungwe in Zimbabwe.
Table 1.
Primers used in this study.
Fig 2.
Workflow for read analysis using AMPtk pipeline.
Fig 3.
Representative replicates analysis results.
(A) Gel analysis of Rufunsa samples done in replicate showing matching bands per sample. (B) Amplicon sequence analysis of the same samples in showing number of reads detected per species in each sample.
Table 2.
Read data of all samples analyzed.
Fig 4.
Representative gel and sequence analysis results.
(A) Arrows showing bands are not visible after the 1st PCR become visible after 2nd PCR. (B) By gel analysis, amplicon bands of samples 5, 7 and 10 are indistinguishable by size and are deemed to be all T. godfreyi while sequencing reveals that the amplicon of sample 10 is, in fact, T. vivax. Positive controls comprise; Tbg (T. brucei gambiense), Tbr (T. brucei rhodesiense), Tb/Tc (an artificial mixture of equal amounts of T. brucei gambiense and T. congolense DNA).
Fig 5.
Phylogenetic tree of unique ASVs generated from amplicon sequence data.
A Bodo caudatus ITS1 sequence was included as outgroup. Individual Trypanosome species and subspecies cluster into distinct clades. ASV are named after their respective blast best hit matches.
Fig 6.
The distribution of Trypanosome species amongst infected tsetse flies.
(A1) Pie chart showing the prevalence of Trypanosome species in wild-caught tsetse flies collected from Kafue, Zambia. (A2) Bar graph showing the Trypanosome species infecting male and female flies from flies collected in Kafue, Zambia. (A3) Graphical representation showing the infection rates of male and female flies collected in Kafue, Zambia. (B) Pie chart showing the prevalence of Trypanosome species in wild-caught tsetse flies collected from Hurungwe, Zimbabwe. (C) Pie chart showing the prevalence of Trypanosome species in wild-caught tsetse flies collected from Rufunsa, Zambia. TBE = T. brucei/T. evansi, TV = T. vivax, TS = T. simiae, TG = T. godfreyi, and TC = T. congolense.
Table 3.
Prevalence of Trypanosome species infection in wild-caught tsetse flies.