2.1. Mosquito Colonies

Colony Maintenance: Four mosquito colonies: A. aegypti, New Orleans (PERM-S), A. aegypti, Vergel (PERM-R), A. albopictus, ATM-NJ95 (PERM-S), and A. albopictus, Coatzacoalcos (unknown PERM sensitivity) were reared at the Arthropod-Borne Infectious Disease Laboratory at Colorado State University (CSU), Fort Collins, Colorado, USA. All mosquito colonies were maintained at a constant temperature of 28–29 °C and 70–80% relative humidity on an approximately 12 h:12 h light:dark cycle. Eggs were hatched in 150 mL of deionized room temperature water and larvae were fed fishfood daily. Pupae were placed in plastic cups within netted cages prior to their emergence as adults. Adult mosquitoes were given raisins ad libitum.

Aedes aegypti: New Orleans (PERM-S) mosquitoes were maintained in our insectary without exposure to insecticides. Vergel (PERM-R) mosquitoes of the 20th generation were used as a resistant colony [15]. Permethrin resistance was maintained in Vergel by exposing adults to permethrin every third generation. Permethrin concentrations ranged from 10 to 25 ug/bottle to select between 40% to 60% of the mosquitoes. The New Orleans (PERM-S) strain had no kdr alleles associated with knockdown resistance [6] and the Vergel (PERM-R) strain has been extensively characterized genetically and had an 88% frequency of the I1016 allele in the F₂ and F₃ generations [15,16,17].

Aedes albopictus: The ATM-NJ95 (PERM-S) population was obtained from BEI Resources (generation F₁₂) as a confirmed insecticide susceptible PERM-S population of A. albopictus [18]. The permethrin susceptibility of A. albopictus collected from the city of Coatzacoalcos in Veracruz, Mexico was unknown but assumed to be more resistant than ATM-N95 due to the heavy use of pyrethroids in the collection region. Prior to performing the repellency or insecticide assays with nootkatone, all four colonies were tested in bottle bioassays to determine their current level of resistance (if any) to permethrin (see results). Coatzacoalcos proved not to have a significant resistant phenotype and the strain was similar to ATM-NJ95 in its sensitivity to permethrin (Supplementary Figures S1 and S2). A. aegypti and A. albopictus strains and their PERM-resistance status are summarized in Table 1.

Mosquito strains used to analyze permethrin resistance and reaction to Nootkatone.

* PERM-status at the beginning of trials. ** PERM-status following bottle bioassays to analyze whether (or not) the mosquito colony was still resistant to permethrin.