d. dilatatus (wDil, Sainte-Marguerite) selleck products (Grève, unpublished results). Wolbachia strains inducing feminization
have been described in A. vulgare (wVulC, Celles sur Belle and wVulM, Mery sur Cher) [44, 45], A. nasatum (wNas, Poitiers) [46], Oniscus asellus (wAse, Quinçay) [38], Porcellionides pruinosus (wPruIII, Nevers) [47]. An uninfected lineage of A. vulgare (originating from Nice, France) was used as negative control for PCR and Southern blotting experiments. Total DNA was extracted from male and female gonads of all isopod species as described previously [48]. Infection status of each individual was confirmed by a PCR-assay based on the bacterial 16S rDNA gene using Wolbachia-specific primers ( Additional file 1: Table S1) [49]. Distribution of pk1 and pk2 genes The genome of the feminizing wVulC Wolbachia strain is at the final assembly step (whole-genome shotgun-sequencing project: European Wolbachia EuWol (contract QLK3-CT2000-01079, coordinated by K. Bourtzis, University of Ioannina, Greece). This includes phage contigs of which sequences are homologous to the BLZ945 concentration Wolbachia WO prophage. Annotation of the pk1 and pk2 genes was performed by protein and DNA homology searches with BLASTP and BLASTN programs [50] using the wPip-Pel pk1 and pk2 alleles as queries (see Table 1). Ankyrin and other functional
motif predictions were performed by the SMART web server [51] RANTES on protein sequences. Specific primers were designed to amplify full-length or 200–500 bp fragments of the wVulC pk1 and pk2 alleles using a standard PCR protocol as previously described ( Additional file 1: Table S1) [52]. The purified PCR products were directly sequenced on both strands on an ABI PRISM 3100 Genetic Analyzer using Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer’s instructions. pk1 or pk2 copy number variation among Wolbachia strains was assessed by Southern blotting. About 15 μg of
total DNA were digested at 37° overnight with EcoRI or BamHI enzymes that did not cut any of the wVulC pk1 and pk2 alleles. Digested DNA as well as undigested DNA from non-infected ovaries used as controls (data not shown) was electrophoresed on 0.8% agarose gels and blotted to nylon membranes. Probes were obtained by PCR amplification of the wVulC full-length pk1 (pk1a and pk1b types) and pk2 (pk2b type) ank genes ( Additional file 1: Table S1), labelled using [α-32P]-dCTP by the random selleckchem primer method and hybridized overnight to membranes. The final wash was performed at 52° in 0.1X SSC. Hybridized blots were imaged and analyzed using a PhosphoImager (Molecular Dynamics, Sunnyval, CA, USA). Sequence analyses of pk1 and pk2 genes Homologous sequences of both genes were first aligned in the server-based program MAFFT (http://align.bmr.kyushu-u.ac.jp/mafft/online/server/) using automatic settings.