Wolbachia

Bereczki, Judit, Rácz, Rita, Varga, Zoltán & Tóth, János P., 2015, Controversial patterns of Wolbachia infestation in the social parasitic Maculinea butterflies (Lepidoptera: Lycaenidae), Organisms Diversity & Evolution (New York, N. Y.) 15 (3), pp. 591-607 : 593-594

Wolbachia screening and strain identification

DNA was extracted by homogenizing the heads or thoraxes of butterflies following the protocol in Bereczki et al. (2014). Altogether 506 individuals were screened (1–11 individuals/ population) from 18 geographical regions (Online Resource 1) by the amplification of the highly conservative 16S ribosomal RNA gene with Wolbachia specific primers W-Spec of Werren and Windsor (2000). Amplification from 1 μl of DNA extracts was carried out in 25 μl final reaction volumes containing 5X PCR buffer, 0.2 mM dNTPs, 2 mM MgCl 2, 0.5 mg /ml bovine serum albumin, 0.02 units/μl of Taq DNA polymerase (Phusion Hot Start II High-Fidelity, Thermo Scientific) and 0.3 μM of each primer. Amplification was carried out in an ABI Veriti thermal cycler programmed for the following: initial denaturation for 2 min at 98 °C; 40 cycles of 15 s at 98 °C, 45 s at 60 °C, 1 min 30 s at 72 °C; final elongation of 10 min at 72 °C. Amplification procedure was repeated twice in M. teleius and M. nausithous since we could obtain faint bands as a result of the first 40 cycles. We used positive (surely infected samples) and negative controls (master mix without any DNA sample) in each reaction. The success of PCRs was checked by running 2 μl of product on 1 % agarose gels stained with GelRed Nucleic Acid Stain (Biotium Inc.). The infection rates of the populations were plotted on geographic maps using QGIS v. 2.6 (2014). PCR products originating from 70 individuals (Online Resource 2) were sequenced by commercial service provider Macrogen Inc. (Seoul, South Korea). Sequences were edited and revised manually by Chromas Lite v. 2.01 and aligned by MEGA v. 6 ( Tamura et al. 2013). Bayesian phylogenetic relationships were assessed in MrBayes v. 3.2.2 ( Ronquist et al. 2012) as described below.

Wolbachia strain identification was carried out in M. alcon and M. arion according to Wolbachia multilocus sequence typing (MLST) system ( Baldo et al. 2006) by the amplification of Wolbachia surface protein (WSP) and five conserved gene regions (gatB, coxA, hcpA, ftsZ, fbpA) following the PCR protocol mentioned above. The latter five genes determine together the sequence type (ST). WSP typing was achieved in a single individual each in M. alcon and M. arion . Wolbachia MLST was completed altogether in 32 specimens in the two species (Online Resource 2). After sequencing, we defined the strains using the reference sequences of Wolbachia MLST database (http:// pubmlst.org/wolbachia/) and searched the identified sequence types in other organisms in the mentioned database.