In this sense, the study of Wolbachia dynamics in recently infected populations represents an excellent opportunity to estimate infection prevalence and to assess effects on mtDNA evolution in host species populations. Here, Wolbachia infection prevalence this website is evaluated in natural D. willistoni populations of the Atlantic Forest biome, southern Brazil. The effect of the infection
on mitochondrial haplotypic diversity is also assessed. Specimens were collected in April 2010 at seven Atlantic Forest sites corresponding to the municipalities São João do Polêsine (29°39′08.94″S, 53°31′43.74″W), Osório (29°53′08.20″S, 50°16′39.81″W), and Torres (29°22′33.3″S, 49°45′69.2″W), (in the state of Rio Grande do Sul), Maracajá (28°50′16.5″ S, 49°24′45.6″W) and Laguna (28° 24′56.0″S, 48°47′47.0″W), (state of Santa Catarina) and Guaratuba (25°51′12.4″S, 48° 33′73.8″W) and Pontal do Paraná (25°33′33.2″S, 48°33′27.0″W), (state of Paraná). Genomic DNA was extracted from one single fly according to the non-phenolic protocol by Gloor et al. (1993). PCR reactions were run in a 25-μL volume using 1 μL of the DNA to amplify Cytochrome Oxidase I (COI) and 2 μL for Wolbachia Surface Protein (wsp), with 12.5 μL of the PCR Master Mix 2X (Fermentas, Lithuania) (0.05 U/μL Taq DNA polymerase, 10X buffer, 4 mM MgCl2,
0.4 mM Forskolin solubility dmso of each dNTP), 1 μL of each primer (20 μM each) and ultrapure water to the final volume. The primers used were TY-J-1460 5′-TACAATCTATCGCCTAAACTTCAGCC-3′ and C1-N-2329 5′-ACTGTAAATATATGATGAGCTCA-3′ ( Simon et al., 1994) to amplify an approximately 950-bp fragment of the mitochondrial gene COI. Temperature cycles were: 5 min 95 °C, 35 cycles of 94 °C for 40 s, 55 °C for 40 s and 72 °C for 1 min, then 72 °C for 3 min. PCR screening for Wolbachia infection was conducted using the primers Wsp-F 5′-TGGTCCAATAAGTGATGAAGAAACTAGCTA-3′ and Wsp-R 5′-AAAAATTAAACGCTACTCCAGCTTCTGCAC-3′ ( Jeyaprakash and Hoy, 2000), which amplify an approximately 600 bp
Dimethyl sulfoxide fragment of the gene wsp. Cycling conditions were 95 °C for 2 min, followed by 35 cycles of 94 °C for 1 min, 55 °C for 1 min and 72 °C for 1 min, then 72 °C for 5 min. A negative (ultrapure water) and a positive control (Drosophila melanogaster Oregon line that is infected by Wolbachia) were included in both reactions. PCR products were electrophoresed on agarose gels 1% stained with ethidium bromide and visualized under UV transillumination. Amplicons were submitted to purification and direct sequencing in Macrogen (Macrogen Inc., Seoul, Korea). Each sample was sequenced from both directions. Quality of the chromatogram was evaluated using the Chromas Pro 1.5 software (http://www.technelysium.com.au). Sequence identity was obtained by comparison of similarity values to the sequences deposited in GenBank using the BLASTn program (NCBI, available online). Sequence alignment was carried out using the ClustalW tool, Mega 5 software (Tamura et al.