In this paper, we used some of these markers in order to estimate the feasibility of a MLVA system for Wolbachia. We isolated markers with tandem repeats from the wMel
genome  and applied them to a number of Wolbachia strains from supergroups A, B and C to assess their applicability and resolution for Wolbachia strain typing. We chose two types of loci containing tandem repeats, two intergenic VNTR loci and two genes encoding proteins containing ankyrin repeats. The two VNTR loci, VNTR-105 and VNTR-141 were originally isolated from supergroup A strain wMel and were polymorphic between wMel, wMelCS and wMelPop isolates from different D. melanogaster lines . VNTRs are also polymorphic between the closely selleck screening library related wAu from D. PD-1/PD-L1 Inhibitor 3 research buy simulans and wWil from Drosophila willistoni , and serve as highly diagnostic marker sets for fingerprinting conspecific Wolbachia strains in the Drosophila
paulistorum species cluster . Recently, a polymorphic VNTR locus was isolated from supergroup B strain wPip . Ankyrin repeat genes are abundant in the genomes of Wolbachia and a number of other intracellular bacteria [42, 43]. The number and distribution of these repeats varies substantially between strains that induce different host phenotypes, suggesting that they may be involved in host manipulation . We extended our click here analysis to include a wider range of Wolbachia strains from supergroup A, B and C in order to evaluate the usefulness of the four markers VNTR-105, VNTR-141, WD0550 and WD0766,
originally isolated from wMel, in discriminating between Wolbachia strains. Methods Wolbachia strains and hosts We used 14 supergroup A Wolbachia isolates from 8 different Drosophila species and 2 tephritid species, Rhagoletis cerasi, a host that is naturally infected, and Ceratitis capitata, microinjected with Wolbachia originating from R. cerasi (Table 1). Based on previous strain typing using 16S rRNA, ftsZ, wsp and some MLST loci, these 14 strains are moderately or closely related, yet they reveal different phenotypic characteristics, such as varying levels Decitabine supplier of CI induction (strong, weak, or non-CI inducers), and different CI rescue phenotypes (reviewed in ). Wolbachia DNA was isolated from Drosophila fly stocks reared on standard corn-flour-sugar-yeast medium at 25°C. Wolbachia-free controls D. melanogaster yw 67c23T and D. simulans Riverside-DSRT were established by tetracycline treatment using standard techniques . Wolbachia of R. cerasi was isolated from field collected samples from Austria and Hungary . Wolbachia from C. capitata was isolated from the WolMed 88.6 lab line that was artificially infected with wCer2 from R. cerasi . We also included strains from B (wNo, wBol1, wMau) and C (wDim) supergroups. wNo and wMau were isolated from D. simulans, wBol1 from Hypolimnas bolina  and wDim from dog heart worm Dirofilaria immitis .