Aphid Colonization with Fluorescent Bacteria

As described in the injection methods for EcR dsRNA, A. pisum LSR1 were first reared at a low density for at least two generations. CWBI-2.3^T-GFP with the dsEcR-800 or dsE2C-800 plasmid were grown and resuspended in PBS as described for the C002 experiments. Forty aphids per condition were fed overnight on a diet mix containing 98 μL of Febvay diet, 1 μL of blue food dye, and 1 μL of resuspended bacterial cells. After feeding, the aphids were transferred to plants and reared to adulthood (about 5 days post-feeding). At this point the aphids were screened for colonization with fluorescent CWBI-2.3^T bacteria on a blue light transilluminator. Successfully colonized aphids were moved to fresh plants (four aphids per plant) and these groups were transferred to new plants every 24 h for 3 consecutive days. After 24 h on the final plants, these colonized adult aphids were removed to prevent the accumulation of additional offspring. Offspring produced by the colonized adults were reared to the 4th instar and scored for the development of wing buds.