Aedes albopictus larvae microbiota from breeding site
The bacterial community of larvae was studied amplifying fragments of the 16S rRNA genes V4 region, which was performed using the primers 515F (5CGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGCCAGCMGCCGCGGA 3) and 806R (5GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACHVGGGTWTCA 3), both containing the adapter for the Illumina platform (Caporaso et al., 2011, 2010). PCR was conducted with the Platinum Taq DNA Polymerase High Fidelity kit (Invitrogen, Carlsbad, CA, USA) in final volume of 25 L containing: 1 X High Fidelity PCR Buffer, 2U of Taq Polymerase, 2 mM of MgSO4, 0.2 mM of dNTP, 25 ng of ultra-pure BSA (Invitrogen, Carlsbad, CA, USA), 0.1 M of each primer, 50 ng of DNA extracted and ultra-pure water to complete the final volume. Initial denaturation at 94 C for 5 min was followed by 30 cycles of: 94 C for 45 s, 56 C for 45 s and 68 C for 1 min and the final extension occurred at 68 C for 10 min. Amplifications were done using an Mastercycler Personal 5332 Thermocycler (Eppendorf) device. Amplifications were purified using the Agencount AMPure beads (Beckman Coulter) and quantified with a Qubit apparatus (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Indexes were added to the DNA libraries (Illumina Inc, San Diego, CA, USA) and sequencing was conducted with the Illumina MiSeq v2 500 kit platform to generate readings of ~290 bp.