Whiteflies

All plants used in the study were grown under greenhouse conditions at the International Institute of Tropical Agriculture (IITA), Dar es Salaam, Tanzania. The varieties of cassava and tomato used were Albert and Moneymaker, respectively, while sweet potato and cotton were local landraces. All greenhouse plants were grown in pots with a soil mix of forest soil and manure mixed in a 4:1 ratio. Plants were typically 20–30 cm tall or with a minimum of five leaves. “Albert” is known to be preferred by whiteflies, while the tomato cultivar—Moneymaker—is extensively used in the scientific literature.
Considering the numerous reports of vector feeding behavior manipulation by viruses, it was important to ensure the use of virus-free cassava plants (Liu et al., 2013 (link); Moreno-Delafuente et al., 2013 (link); Lu et al., 2017 (link)). All cassava planting material was obtained from CMD and CBSD asymptomatic fields in Mtwara Region, Tanzania. Furthermore, leaf samples were taken from each stem and tested for the presence of Cassava Brown Streak Ipomoviruses using real-time RT-PCR to exclude the possibility of asymptomatic infections. The CBSI virus testing was done using the protocol described for cassava by Shirima et al. (2017 (link)). Leaf samples in the form of the middle leaflet were taken from the fifth youngest leaf from each cassava stem cutting used for planting. Samples were dried between two sheets of paper at room temperature for 4 days. Total RNA was extracted using the acetyltrimetyl ammonium bromide (CTAB) protocol, cassava complementary DNA (cDNA) was synthetized and real-time polymerase chain reaction was performed using primers, probes, and cycling conditions described in Shirima et al. (2017 (link)). “Albert” is resistant to CMD, and since only symptomless plants were selected for planting material, the risk of infection was considered low and the material was not tested for the presence of CMBs. The assumption of the absence CMD was further supported as none of the grown plants exhibited the symptoms. Sweet potato plants were asymptomatic. Vegetative material used to plant them has been maintained under insect-proof screenhouse conditions, without symptoms of virus infection, for 3 years. Tomato and cotton were grown from certified seed.

To study the effect of the JH pathway on Vg expression and Vg localization in whiteflies, approximately 800 female adult whiteflies at day 1 after emergence were injected with 1.5 μg/μL dsJHAMT and incubated on cotton leaf disks by using a previously described method (13 (link), 45 (link), 47 (link), 57 (link), 61 (link)). Control whiteflies were injected with 1.5 μg/μL dsGFP. Whiteflies were collected at 5 and 6 days after dsRNA injection. The survival rates of injected whiteflies were 60% for dsGFP and 40% for dsJHAMT at day 5 after injection. RNA was extracted from the whole body of 10 female adult whiteflies for each of three biological replicates. The expression of JHAMT at days 5 and 6 after dsJHAMT injection and the expression of Vg at day 5 after dsJHAMT injection were examined using qRT-PCR. To detect whether silencing whitefly JHAMT affects Vg localization in ovarioles and bacteriocytes, whiteflies were collected at day 5 after injection. For each biological replicate, ovarioles and bacteriocytes were dissected from 30 female adult whiteflies, fixed, permeabilized, and incubated with antibodies against Vg for ovarioles and bacteriocytes. The samples were incubated with no antibodies against Vg as the negative control. Three biological replicates were conducted. Images were analyzed using an FV3000 confocal microscope (Olympus, Japan). The fluorescence intensity of Vg was analyzed by Image J software. In each of three biological replicates, four ovarioles and three bacteriocytes of dsGFP-injected whiteflies and dsJHAMT-injected whiteflies were used for fluorescence intensity analysis. To further test the effect of JH on Vg expression, approximately 300 female adult wild-type whiteflies at 3 to 4 days after emergence were injected with 0.5 μg/μL pyriproxyfen (JH analog) dissolved in distilled water by using an Eppendorf microinjection system (Hamburg, Germany). Distilled water-injected whiteflies were used as the control. Expression of Vg in female adult whiteflies at days 1 and 2 after the whiteflies were microinjected with pyriproxyfen was examined using qRT-PCR with three biological replicates.

The interaction between Vg and Portiera was examined using an IC-PCR assay by previously described protocols (29 (link)). PCR tubes were coated with 25 μL antibody against Vg (1:1,000 diluted in coating buffer), for 1.5 h at 37°C, and then washed five times for 5 min each time with 50 μL washing buffer. Homogenates of bacteriocytes and heads that were collected from 8 to 10 whiteflies in 5 μL PBS were incubated for 18 h at 4°C in the coated PCR tubes. The tubes were washed five times, 5 min each time, with 25 μL washing buffer and dried. PCR amplification of Portiera bound to the Vg protein, which was itself bound to the antibody-coated tubes, was performed with Portiera-specific 16S rRNA gene fragment primers (Table S1). The control no-antibody-coated tubes were incubated with a homogenate of the bacteriocytes and heads of whiteflies, and antibody-coated tubes incubated with a homogenate of whitefly heads not containing bacteriocytes served as controls.

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.