Collection sites and laboratory conditions
Field collections of Ae. aegypti were carried out in 2018 from three states in Mexico: Nuevo Leon in the northeast with two locations, Monterrey and Guadalupe; Yucatan in the southeast with two locations, Merida and San Antonio Kaua; and Chiapas in the south with one location.
Mosquitoes collected in the field were at immature stages, and they were reared to adults under laboratory conditions at 25±4°C and a 12:12 h photoperiod. They were morphologically identified and stored at -20°C until DNA extraction.
DNA was isolated from ~30 individual mosquitoes per location using the salt extraction technique  and resuspended in 50 mL of water (Cellgro® sterile purified water, molecular biology grade and free of proteases, DNases and RNases). The concentration and quality of each DNA sample was determined on a ThermoScientific NanoDrop 2000 spectrophotometer.
Development of the multiplex PCR method
The amplification primers used for the variants of loci 410, 1,016 and 1,534 are given in Table 1.
The specific oligonucleotides V410fw and L410fw amplify a region of 113 bp, corresponding to the L410 allele (resistant). The specific oligonucleotides V410fw and 410rev amplify a region of 133 bp, corresponding to the V410 allele (susceptible). The specific oligonucleotides I1,016f and I1,016r amplify a region of 82 bp, corresponding to the I1,016 allele (resistant). Oligonucleotides V1,016f and I1,016r amplify a region of 102 bp, corresponding to the V1,016 allele (susceptible). The oligonucleotides c1,534-f and c1,534-r amplify a region of 368 bp with which the specific oligonucleotides Ae1,534F-r and Ae1,534C-f hybridize, resulting in products of 180 bp for the C1,534 allele (resistant) and of 232 bp for the F1,534 allele (susceptible) (Figure 1).
Tests for optimization of PCR conditions resulted in the following multiplex PCR protocol. The DNA samples used in the amplification process were in a concentration range of 20-250 ng/mL. The final reaction mixture was 19.12 mL and contained: 1.02X buffer, 1.53 mM MgCl2, 0.2 mM dNTPs, oligonucleotides for genotyping 410 at a final reaction concentration of 1.27 pmol/mL, for 1,016 at 1.02 pmol/mL and for 1,534 at 0.82 pmol/mL (Table 1), and also 5 U Taq DNA polymerase.
The reaction was carried out in a Multigene Optimax thermal cycler (Labnet International, Edison, NJ, USA). The reaction conditions were as follows: 95°C for 2 min for the initial separation of the DNA strands, followed by 45 cycles of 95°C (30 s), 58.6°C (1 min) and 72°C ( 30 s) and a final extension for 2 min.
A PCR tube containing all the components except genomic DNA was run with the primers as a contamination control. Controls were included in each PCR performed, the New Orleans strains was used as susceptible control.
After amplification, 4 µl of the products of the PCR reaction mixture were analyzed by horizontal electrophoresis on a 2.5% agarose gel. The electrophoresis conditions were 110 V for 1 h using 1X SB buffer (200 mM sodium borate buffer, pH 8) along with a 25-bp molecular weight marker to determine the size of the fragments and staining with GelRed® (Biotium, Hayward CA, USA). The PCR products were visualized with a transilluminator (UVITEC, Cambridge, UK). At the end of amplification, it was possible to obtain up to 7 PCR products per sample, whose size indicated the genotypic combination for loci 410, 1,016 and 1,534.
Validation of the study
For the validation of the results obtained in multiplex PCR, AS-PCR was performed according to Saavedra et al. , Yanola et al.  and Villanueva-Segura et al. .