Aedes mosquito, DNA

Fontaine, Albin, Mignotte, Antoine, Lacour, Guillaume, Nguyen, Agnès, Gomez, Nicolas, Chanaud, Lionel, L’Ambert, Grégory & Briolant, Sébastien, 2024, Multiplexed amplicon sequencing reveals the heterogeneous spatial distribution of pyrethroid resistance mutations in AEdES AlbopICTUS mosquito populations in southern France, Parasites & Vectors (539) 17 (1), pp. 1-10 : 2

Mosquito DNA extraction

A two-step approach was implemented to screen for kdr alleles in Ae. albopictus mosquitoes: (i) an initial screening by sequencing pooled mosquito DNA from each site, followed by (ii) sequencing individual mosquito DNA to determine kdr allele prevalence and genotype. A total of 3–80 (mean = 24.5, SD = 15) mosquitoes were selected by site and grouped into 100 different pools. Heads from larvae or adult mosquitoes were dissected under magnifying glasses. Each pool was made up of mosquito heads sampled at the beginning and the end of the sampling period for each site when possible. All mosquitoes from sites in which kdr alleles were detected in step i were selected for single-mosquito DNA sequencing, excluding damaged mosquitoes. Tis second selection also included sites without detection of kdr alleles in step i, with a total of 56 sites throughout 50 municipalities. Mosquito heads or bodies were ground in a 96 wells plate using a Tissue- Lyser (Qiagen) for 2 min at 30 oscillation/s. Genomic DNA was then extracted from homogenates using the NucleoSpin 96 Tissue Core Kit (Macherey–Nagel) and stored at −20 °C until use.