m. KU55933 mouse morsitans female and male adult flies from the Yale University laboratory colony. Dissections were performed in 1X PBST ((3.2 mM Na2HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mM NaCl, 0.05% Tween 20, pH 7.4), and dissected tissues were placed in 200

μl of lysis buffer (Qiagen, Valencia, CA). The DNA was isolated using a Qiagen DNeasy kit (Qiagen, Valencia, CA) following the manufacturer’s instructions. PCR amplication of 16S rRNA, fbpA, and wsp were performed using the primers wspecF/wspecR, fbpA_F1 / fbpA_R1 and 81F / 691R, respectively [2, 41, 57] (see Additional file 1- Supplementary Table 1). PCR mixes of 25 μl contained 5 μl of 5x reaction buffer (Promega, Madison, WI), 3 μl MgCl2 (25mM), 0.5 μl deoxynucleotide triphosphate mixture (25 mM each), 0.5 μl of each primer (10 μM), 0.125 μl of Taq (Promega, ��-Nicotinamide purchase Valencia, CA) (1U/μl), 14.375 μl water and 1 μl of template DNA. The PCR protocol was: 35 cycles of 30 sec at 95°C, 30 sec at 54°C and 1 min at 72 °C. Phylogenetic analysis All Wolbachia gene sequences generated in this study were

manually edited with SeqManII by DNAStar and aligned using MUSCLE [58] and ClustalW [59], as implemented in Geneious 5.3.4 [60], and adjusted by eye. Phylogenetic analyses were performed using Bayesian Inference (BI) and Maximum-Likelihood (ML) estimation for a concatenated data set of the protein-coding genes (gatB, fbpA, hcpA, ftsZ and coxA) and for wsp separately. For the Bayesian inference of phylogeny, PAUP version 4.0b10 [61] was used to select the optimal evolution model by critically evaluating the selected parameters using the Akaike Information Criterion [62]. For the concatenated data and the wsp set, the submodel GTR+I+G was

selected. Bayesian analyses were performed as implemented in MrBayes 3.1 [63]. Analyses were initiated from random starting trees. Four separate runs, each composed of four chains, were run for 6,000,000 generations. The cold chain was sampled every 100 generations, and the first 20,000 generations were discarded. Posterior probabilities were computed for the remaining trees. ML trees were constructed using MEGA 5.0 [64], with gamma distributed rates with 1000 bootstrap replications, and the method of Jukes and Cantor [65] as genetic distance model. Nucleotide sequence accession numbers. All MLST, wsp and 16S rRNA gene sequences generated in this Vorinostat mouse study have been deposited into GenBank under accession numbers JF494842 to JF494922 and JF906102 to JF906107. Results Wolbachia infection prevalence in different populations The presence of Wolbachia was investigated in nine species within the three subgenera of Glossina. A total of 551 laboratory and 3199 field-collected adult flies, originating from 10 African countries, were NCT-501 supplier tested using a Wolbachia specific 16S rRNA-based PCR assay (Table 1). The prevalence of Wolbachia infections differed significantly between the various populations of Glossina (Table 1).