Microsatellite Analysis of Aedes albopictus

DNA was extracted from individual mosquitoes using a cetyltrimethylammonium bromide (CTAB) protocol [39 ]. A total of 564 individuals were genotyped (24–30 individuals per site). In each site, half of the analyzed individuals were males and the other half females. A set of 16 microsatellite loci previously developed for Ae. albopictus was used [32 (link), 34 (link)] (Additional file 2: Table S2). Microsatellite locus primers were pooled in three PCR-mixes based on non-overlapping size ranges and amplified by multiplex PCRs using fluorescent primers (Additional file 2: Table S2). All PCR reactions (10 µl) contained 0.5 U of Taq DNA polymerase, 6 µM of dNTPs, 37.5 µM of MgCl₂, 1.2–7 µM of each primer (Additional file 2: Table S2) and 10 ng of mosquito DNA. The following program was used to amplify all 16 loci: 10 min at 95 °C followed by 35 cycles at 95 °C for 30 s, 57 °C for 30 s and 72 °C for 1 min and a final elongation step of 72 °C for 10 min. PCR products were run on a 3730XL DNA Genetic Analyzer (Applied Biosystems, California, USA) with a GeneScan 500LIZ size standard. Microsatellite alleles were read using the software GeneMapper v. 4.0 (Applied Biosystems).