Whitefly Vg Silencing Effects

To investigate whether silencing Vg influences Vg localization, symbiont localization and abundance, and the numbers of ovarioles and eggs, approximately 2,000 female adult whiteflies infected with Portiera at day 1 after emergence were injected with 1.0 μg/μL dsVg in injection buffer by using an Eppendorf microinjection system (Hamburg, Germany) and incubated on cotton leaf disks as described above. Control whiteflies were injected with 1.0 μg/μL dsGFP. Whiteflies were collected at 3 days after dsRNA injection. The survival rates of injected whiteflies were 75% for dsGFP and 40% for dsVg at day 3 after injection. RNA was extracted from eight female adult whiteflies for each of three biological replicates to examine the expression of Vg 3 days after dsRNA injection. To examine whether silencing whitefly Vg affects Vg localization in ovarioles and bacteriocytes and Portiera localization in bacteriocytes, whiteflies were collected at days 1, 3, and 5 after injection. Whitefly ovarioles and bacteriocytes were dissected, fixed, permeabilized, and incubated with antibodies against Vg for ovarioles as well as antibodies against Vg and a fluorescent probe for Portiera in bacteriocytes as described above. To examine whether silencing whitefly Vg affects Hamiltonella localization in bacteriocytes, whiteflies were collected at day 3 after injection. Whitefly bacteriocytes were dissected, fixed, permeabilized, and hybridized with the fluorescent probe for Portiera and Hamiltonella in bacteriocytes. Three biological replicates were conducted. Images were analyzed using a FV3000 confocal microscope (Olympus, Japan). The fluorescence intensity of Vg and Portiera was analyzed by Image J software. In each of three biological replicates, three ovarioles and bacteriocytes of dsGFP-injected whiteflies and dsVg-injected whiteflies were used for fluorescence intensity analysis of Vg and three bacteriocytes of dsGFP-injected whiteflies and dsVg-injected whiteflies were used for fluorescence intensity analysis of Portiera. To test whether silencing whitefly Vg influences the abundance of symbionts Portiera and Hamiltonella, DNA was extracted from the whole body of individual female adult whiteflies for each of 10 biological replicates and from bacteriocytes and ovaries of eight female adult whiteflies for each of five biological replicates at day 3 after the whiteflies were microinjected with dsVg. Then, qPCR was performed as described above. In parallel, ovarioles were dissected in PBS at pH 7.4, and the number of ovarioles was scored in 10 individuals for dsVg-injected and dsGFP-injected female adult whiteflies at day 3 postinjection. To determine if silencing Vg influences whitefly fecundity, individual Vg-injected and dsGFP-injected whiteflies were transferred onto cotton leaf disks and kept on 1.5% agar plates at 26 ± 2°C, with a 14-h-light:10-h-dark photoperiod and 60% to 80% relative humidity (RH). Egg numbers were recorded for the surviving whiteflies. Nineteen biological replicates of individuals were conducted at day 3 postinjection.