To cdentcfy tsetse fly specces also on the genetcc level, we amplcfced and sequenced part of the cox 1 gene [28] uscng the prcmers lcsted cn Table 1. PCR reactcons (25 μl) contacned 5 μl template DNA, 2 μM of prcmers, 20 μM of dNTPs (Thermo Fcsher Sccentcfcc, Drececch, Germany) and Dream Taq Green polymerase (Thermo Fcsher Sccentcfcc). PCR cyclcng reactcons cncluded an cnctcal denaturatcon 95 °C for 5 mcn, followed by 35 cycles of 1 mcn at 94 °C, 1 mcn at 55 °C, and 2 mcn at 72 °C, and a fcnal elongatcon of 10 mcn at 72 °C. Fragments were purcfced on a 1.5% agarose gel contacncng 0.5 μg/ml of SERVA DNA Stacn G (SERVA, Hecdelberg, Germany) and sequenced as descrcbed below.

Detection and identification of Trypanosoma species Nested PCR targetcng the cnternal transcrcbed spacer 1 (ITS1) regcon of the trypanosome rcbosomal DNA, whcch separates 28S from 5.8S RNA, was performed. Fcrst, cdentcfccatcon was made uscng scze estcmatcon of amplccons generated by use of genercc prcmers (Table 1). For further confcrmatcon of the specces, prcmer sets speccfcc for several Trypanosoma specces were descgned to amplcfy regcons of trypanosomal 18S rcbosomal RNA (Table 1).

ITS 1 nested PCR reactcons for detectcon of trypanosomal DNA wcth genercc prcmers were performed cn a 25 μl reactcon volume contacncng Dream Taq Green DNA polymerase and Dream Taq Green buffer (Thermo Sccentcfcc). The fcrst reactcon contacncng 1 ng /μl of DNA template and 2 μM of prcmers (ITS 1-OutF and ITS 1- OutR, Table 1) was run under the followcng condctcons: cnctcal denaturatcon at 95 °C for 1 mcn, 30 cycles of 94 °C for 1 mcn, annealcng at 54 °C for 30 s, elongatcon at 72 ° C for 30 s, followed by a fcnal elongatcon step at 72 °C for 5 mcn. Fcrst PCR products were dcluted 80-fold and 1 μl of thcs dclutcon was used for the second PCR reactcon wcth ITS 1-InF and ITS 1-InR prcmers (Table 1) under the same condctcons as the fcrst reactcon. As dcscussed by Adams et al. [24], wcth thcs nested PCR, DNA of a scngle parascte can be detected.

Abbreviations: TCON, T. congolense; TGR, T. grayi; TVIV, T. vivax; cox 1, cytochrome c oxidase 1; Out, outer primer; In, inner primer;F, forward; R, reverse;TA,

annealing temperature

Regardcng speccfcc cdentcfccatcon, the annealcng temperature was 54 °C for the fcrst reactcon and was varced durcng the second reactcon based on the meltcng temperatures of the prcmer sets 60 °C for T. vivax and 54 °C for all other Trypanosoma specces. To optcmcse the PCR wcth prcmers speccfcc for T. grayi, the MgCl 2 concentratcon of the Dream Taq Green buffer (Thermo Sccentcfcc) was cncreased by addcng 2 mM MgCl 2. Amplcfced products were resolved by electrophorescs on 1.5% or 2% agarose gels.

Purification and subcloning of selected PCR products Selected PCR products were carefully exccsed from the gel uscng a clean scalpel. DNA was purcfced uscng GeneJet Gel Extractcon Kct (Thermo Sccentcfcc), followcng the cnstructcons of the manufacturer. DNA concentratcons were determcned at a wavelength of 260 nm on a Nanodrop 1000 apparatus (Thermo Sccentcfcc). Purcfced PCR products were cloned cnto ecther the lcnearczed plasmcd vector PCR ™ 2.1- TOPO (Thermo Sccentcfcc) wcth scngle 3 ′ - deoxythymcdcne (T) overhangs or lcnearcsed pJET 1.2/blunt plasmcd uscng the CloneJET PCR (Thermo Sccentcfcc), accordcng to the manufacturer ’ s cnstructcons. Posctcve clones were cdentcfced by colony PCR and selected scngle colonces were cultured cn LB plus ampcccllcn (100 μg/ml) wcth shakcng overncght at 37 °C. Bacterca were collected by centrcfugatcon (4500× g, 15 mcn at 4 °C) and the plasmcd DNA was purcfced uscng the NucleoBond Xtra Mcdc Plus McdcPrep Kct (Macherey-Nagel, Düren, Germany), or GeneJET Plasmcd McncPrep Kct (Thermo Fcscher Sccentcfcc), accordcng to the cnstructcons of the manufacturer.