Molecular Identification of Anopheles Mosquitoes

Mosquitoes were morphologically identified as Anopheles gambiae sensu lato (s.l.) in the field prior to CDC bottle bioassay testing. A sub-set of 480 samples were further identified using an end point PCR assay developed by Santolamazza et al. [12 (link)]. This assay amplifies the SINE200 insertion, a highly repetitive ~ 200 bp element which is widespread in the An. gambiae sensu stricto (s.s.) genome [12 (link)]. Samples were prepared with forward (5′-TCGCCTTAGACCTTGCGTTA-3′) and reverse (5′-CGCTTCAAGAATTCGAGATAC-3′) primers, and amplifications performed in 20 µL reactions containing 2 µL cDNA, 2 µL of each primer (10 µM), 4 µL H₂O, and 10 µL 2× Hot Start Taq PCR Master Mix (New England Biolabs). The cycling conditions in the Bio-Rad T100™ Thermal Cycler were: 10 min at 94 °C; 35 cycles of 30 s at 94 °C, 30 s at 54 °C, and 60 s at 72 °C; then 10 min at 72 °C. Products were run on 2% agarose gels in an Invitrogen E-gel iBase™ Real-Time Transilluminator. Amplification products of 479 bp or 249 bp were considered indicative of Anopheles coluzzii or An. gambiae s.s., respectively; both bands indicate a hybrid individual. The An. gambiae s.s. form has an identical banding pattern to that of Anopheles melas and Anopheles quadriannulatus (249 bp), however, due to the geographical location of sampling, it is highly unlikely that these species were present.