Cell Culture and Mosquito Maintenance Protocols

Vero cells (African green monkey kidney) were cultured in serum free Opti-Pro medium (Invitrogen) as previously described [42 (link)]. Mosquito C6/36 (derived from A. albopictus; ATCC), C₇10 and Aag2 (derived from A. albopictus and A. aegypti, respectively; generous gift from Dr. A. Fallon, University of Minnesota) cells were maintained in Dulbecco minimal essential medium (DMEM) (Invitrogen) supplemented with 5% FBS (Lonza), 1x penicillin-streptomycin-glutamine (PSG) solution (Invitrogen), 1x MEM nonessential amino acids (Cellgro, Swedesboro, NJ), 1 × MEM vitamin solution (Invitrogen) and 5 μg/L of gentamicin (Invitrogen) at 32°C in an atmosphere of 5% CO₂. The C6/36 cells were used in all but the initial experiments, because they exhibit superior viability in our experimental conditions compared to C₇10 cells.
Galveston colony of A. albopictus (generous gift of Dr. S. C. Weaver, UTMB) and NIH strain of A. aegypti mosquitoes have been described earlier [53 (link),68 (link)]. Both mosquito species were maintained in carton containers supplemented with 10% sucrose on cotton balls at 28°C, 80% humidity and with a 16-hr daylight cycle.