Antibiotic Treatment

Approximately 100–200 C. nubeculosus and C. sonorensis pupae were collected during three consecutive days and placed in a Petri dish containing moistened cotton wool and filter paper in separate buckets (diameter 12.2 cm, height 12.2 cm; Jokey). For a period of 6 days, they were allowed to emerge and had direct access to 6% glucose solution (untreated group), or 6% glucose solution containing a combination of 10 μg/ml penicillin and 10 μg/ml streptomycin (Duchefa Biochemie B.V., Haarlem, The Netherlands) (antibiotic-treated group) [36]. Penicillin was chosen because it is a broad-spectrum antibiotic against gram-positive bacteria, and streptomycin was chosen because it is a broad-spectrum antibiotic against gram-negative bacteria. Biting midges in the antibiotic-treated group were allowed to feed on a glucose solution with antibiotics for 3 to 6 days before being transferred to the Biological Safety Level 3 (BSL3) facility at Wageningen University & Research, where arbovirus infections were performed. Antibiotic treatment was continued throughout the duration of the experiments.

Aedes aegypti adults were collected from larval trays and divided into two groups of approximately 100–200 mosquitoes in Bugdorm-1 cages. One cage was maintained on 6% glucose solution (untreated group), whereas the other cage was maintained on 6% glucose solution with 20 U/ml penicillin (Sigma-Aldrich, Saint Louis, MO, USA) and 20 μg/ml streptomycin (PenStrep) (antibiotic-treated group; Sigma-Aldrich) for 4 days [32]. Females were then transferred to plastic buckets (diameter 12.2 cm, height 12.2 cm; Jokey) and transported to the BSL3 facility for arbovirus infection studies.