morsitans, G. fuscipes, G. pallidipes, and G. brevipalpis) was performed using the Holmes–Bonner protocol (Holmes & Bonner, 1973). Nucleic acid extraction for C. columbae was performed using the QIAamp tissue mini kit (Qiagen, Valencia, CA). All samples were resuspended in 1 × Tris-EDTA following DNA isolation. DNA samples were subjected to PCR amplification of genes encoding putative outer membrane components; specifically ompA, the outer membrane protein A, ompC, the osmoporin protein C, and rcsF, ycfM, slyB, and spr, producing various outer membrane lipoproteins. PCR annealing temperatures, primers, and respective amplicon sizes are included in Supporting Information, Table S1. Notably, amplification reactions

of ycfM from C. columbae and Selleck GDC0068 C. melbae PF 01367338 and rcsF and slyB from C. columbae were not successful. Negative controls were included in each set of amplification reactions. The amplification products were analyzed by agarose gel electrophoresis and visualized with Kodak 1d image analysis software. The amplicons were purified using QIAquick PCR purification kit (Qiagen) and subject to DNA sequencing at the West Virginia University’s Department of Biology Genomics Center on an ABI 3130xl analyzer (Applied Biosystems, Foster City, CA) using a 3.1 BigDye protocol (Applied Biosystems). For each

sample, three to five amplicons were sequenced in both directions and contigs were assembled using Ridom Trace Edit (Ridom GmbH, Wurzburg Germany). The Sodalis ompA gene was amplified from two G. morsitans, G. fuscipes, G. brevipalpis, and G. pallidipes individuals. Amplicons were ligated into pGEM-T vector (Promega) and Escherichia coli JM109 cells were transformed. Four colonies per individual tsetse were verified for an ompA insertion Ixazomib clinical trial and sequenced as described above. All analyses included sequence data collected in this study or publicly available at NCBI GenBank. DNA sequences were aligned using the clustal x algorithm with default settings, and refined manually when necessary. Maximum parsimony (MP) and neighbor joining (NJ) analyses were performed with 1000 replicates in paup 4.0 (Swofford, 2002). MP heuristic searches utilized the tree-bisection-reconnection

(TBR) branch-swapping algorithm with 200 Max trees and starting trees were created using stepwise additions. All MP analyses were performed twice, where gaps were treated either as ‘missing data’ or as a ‘fifth character state,’ with no differences noted between the results. NJ analyses implemented Kimura’s two-parameter model (Kimura, 1980). Lineage support was measured by calculating nonparametric bootstrap values (n=1000) (Felsenstein, 1985). The evolutionary models used for Bayesian analyses were determined using the Akaike Information Criterion in mrmodeltest 2.3 (Nylander, 2004). Bayesian analyses were performed in mrbayes 3.1.2 (Ronquist & Huelsenbeck, 2003), and the number of categories used to approximate the gamma distribution was set at four.