Wolbachia

Tóth, Judit Bereczki Szilárd Póliska Alex Váradi János P., 2020, Incipient sympatric speciation via host race formation in Phengaris arion (Lepidoptera: Lycaenidae), Organisms Diversity & Evolution (New York, N. Y.) 20 (1), pp. 63-76 : 66

Wolbachia studies

The same DNA extracts used for microsatellite studies were screened for Wolbachia . Each specimen was screened by the amplification of the highly conservative 16S ribosomal RNA gene with Wolbachia specific W-Spec primers developed by Werren and Windsor (2000). The amplification procedure described in Rácz et al. (2015) was followed. We used positive (confirmed infected samples) and negative controls (master mix without any DNA sample) in each reaction. The success of PCRs, i.e. Wolbachia presence, was checked by running 2 μl of product on 1% agarose gels stained with GelRed Nucleic Acid Stain (Biotium Inc., Fremont, CA, U.S.A.).

Wolbachia strain identification was carried out by the amplification of Wolbachia surface protein (WSP) ( Baldo et al. 2006). WSP typing was completed in 41 individuals (Suppl. Table S1). After sequencing, we identified the strains in the Wolbachia MLST database (http://pubmlst.org/wolbachia/).

The relative abundance of Wolbachia was quantified using real-time quantitative PCR (RT-QPCR) technology. Wolbachia copy number was determined by measuring 16S rRNA gene level using the W-Spec primers of Werren and Windsor (2000). To calculate the relative abundance of Wolbachia , the copy number of the host-specific histone 3 (H3) gene was measured from the same DNA extract (using H3 primers from Talavera et al. (2013)). Measurements were made with a Quantstudio 12K Flex instrument (Thermo Fisher Scientific, Waltham, MA, USA). The 10-μl reaction mixture contained 5 μl of SYBR Green I Master mix (Roche Life Science, Penzberg, Germany), 0.2 μl of 10 μM primer mixture of forward and reverse primers and 4.8 μl of DNA extract (10 ng /μl). Run profile was as follows: 95 °C for 10 min, followed by 40 cycles of amplification (95 °C for 15 s, 60 °C for 30 s). Specificity of the used primer pairs was checked by the melting curve analysis of the amplified products.

The differences in Wolbachia quantity were tested between the two forms and we also completed pairwise comparisons by sampling sites. As the dataset does not follow normal distribution, we performed a Kolmogorov-Smirnov test using PAST 2.17 ( Hammer et al. 2001).