Individual mosquitoes were identified to species level according to Santolamazza et al. (77 (link)). PCRs contained 2 μl of 10 μM forward primer (5′-TCGCCTTAGACCTTGCGTTA -3′), 2 μl of 10 μM reverse primer (5′-CGCTTCAAGAATTCGAGATAC -3′), 1 μl of extracted DNA, and 10 μl of Hot Start Taq 2X Master Mix (New England Biolabs, UK) for a final reaction volume of 20 μl. Prepared reactions were run on a Bio-Rad T100 thermal cycler with the following conditions: 10 min denaturation time at 94°C, followed by 35 amplification cycles of 94°C for 30 s, 54°C for 30 s, and 72°C for 60 s, followed by a final extension at 72°C for 10 min. PCR products were visualized on 2% E-gel agarose gels in an Invitrogen E-gel iBase real-time transilluminator. A Quick-Load 100-bp DNA ladder (New England Biolabs) was used to determine band size. Amplified PCR products of 479 or 249 bp were indicative of An. coluzzii or An. gambiae s.s., respectively. As the dominant species, only An. coluzzii individuals of the same age and resistance phenotype were selected and pooled for 16S rRNA sequencing.
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