A. gambiae Keele strain mosquitoes were maintained on a 10% sugar solution in laboratory culture at 27°C and 70% humidity with a 12 hrs light/dark cycle according to standard rearing procedures [49] . A single cohort of adult female mosquitoes were collected immediately after eclosion, and either maintained under normal, non-sterile insectary conditions or placed into a sterile environment. Following, adult female mosquitoes were daily given fresh filtered sterilized 10% sucrose solution containing 15 µg gentamicin sulphate (Sigma) and 10 units/10 µg of penicillin-streptomycin (Invitrogen) per ml, respectively. Each cohort of mosquitoes was simultaneously membrane-fed freshly washed human erythrocytes resuspended to 40% haematocrit using human serum. As far as possible, every care was taken to maintain the sterility of the blood and membrane-feeding apparatus used to feed the mosquitoes, in order to prevent the antibiotic-treated mosquitoes acquiring bacterial infection during the process of membrane-feeding. The mosquitoes were starved for 8 hrs before feeding to encourage engorgement, and sugar solution was replaced once blood feeding had finished. At 24 hrs after blood feeding, 20 mosquitoes from each replicate of each cohort was collected and dissected on ice. RNA was extracted from dissected tissues at the assayed time points using the RNeasy kit (Qiagen). The quantification of RNA concentrations was performed using a Spectrophotometer (Eppendorf).
Free full text: Click here
