Determination of validamycin concentration
We next wondered if the ToCV transmission by B. tabaci would be influenced by know inhibitors of trehalase. Validamycin was chosen for this experiment because its ability to inhibit trehalase has been established with other insects. To determine the optimal inhibitive dose of validamycin in B. tabaci, a range of validamycin concentrations were tested. We found that trehalase activity in B. tabaci was inhibited gradually with increase of validamycin from 0–200 mg/L. It therefore had a linear negative correlation with the validamycin concentration. The inhibitory effect of trehalase activity was similar in 1–3 days. There was no significant difference in trehalase activity between 200 and 500 mg/L. It also fluctuated around 40 U/mg protein (Fig. 2). On the fourth day, the inhibitory effect of each concentration on trehalase activity was weakened and stabilized at 55 U/mg protein. Therefore, 200 mg/L validamycin were chosen as treatment levels to be applied to plants in the follow-up experiment.
Trehalase activity and trehalose content of B. tabaci after validamycin treatment
Upon treatment with 200 mg/L validamycin, differences in trehalose content in B. tabaci were observed after 12 h feeding on ToCV-infected and uninfected tomato plants. Compared to the control group, the trehalose content of B. tabaci increased with the extension of feeding time (Fig. 3a, c). Our study showed that validamycin treatment significantly increased the treholase content in whiteflies fed on ToCV-infected tomato, but also in those fed on uninfected tomato (H: F1, 28 = 152.3, P < 0.001; V: F1, 28 = 96.6, P < 0.001).
By contrast, trehalase activity in B. tabaci that fed on both uninfected and infected tomato plants decreased significantly, after 12 hours of feeding. The trehalase activity in B. tabaci that fed on the infected tomato plants was 36.07 U/mg prot, and for those that fed on the uninfected tomato plants was 35.02 U/mg prot. (Fig. 3b, d) With the extension of feeding time, the trehalase in whiteflies had lower activity compared to the control group. Our results suggested that when whiteflies fed on uninfected and ToCV-infected tomato plants treated with 200 mg/L validamycin, the trehalase activity was lower than that of the control group (H: F1, 28 = 55.47, P < 0.001; V: F1, 28 = 81.04, P < 0.001).
ToCV transmission efficiency by B. tabaci after validamycin treatment
We then tested whether validamycin treatment affected the ability of B. tabaci to transmit ToCV. As shown in Fig. 4, the transmission rate of whiteflies was 91.7% after water treatment, but was reduced to 66.7% after treatment with validamycin (Table 1). The amount of virus transmitted by whiteflies was reduced to 54.4% of the untreated control (Fig. 4). In conclusion, treating B. tabaci with validamycin significantly reduced the transmission rate and transmission efficiency of ToCV.
Table 1
Transmission rate of whiteflies after different treatments
Treat
|
Concentration(mg/L)
|
Total number of tested plants
|
Number of tested plants infected with ToCV
|
Virus transmission rate
|
Water
|
0
|
24
|
22
|
91.7 %
|
Validamycin
|
200
|
24
|
16
|
66.7 %
|
Preference of B. tabaci after validamycin treatment
To determine whether validamycin treatment altered the preference of B. tabaci for certain type of tomato (ToCV-infected or uninfected), we next carried out a selectivity experiment. As shown in Fig. 5, upon control treatment with water, an average of 16.4 viruliferous whiteflies landed on healthy tomato plants 3 days later. This compared to just 6 for whiteflies treated with validamycin. At 4 days post treatment, these numbers were 21.7 and 9.5, respectively (Fig. 5). Therefore, after validamycin treatment, the number of viruliferous whiteflies that preferred healthy tomato plants was significantly reduced (F1, 46 = 35.1, P < 0.001).
Flight behavior of B. tabaci after validamycin treatment
We next determined whether the flight behavior of B. tabaci was affected by validamycin treatment. As shown in Fig. 6, validamycin treatment reduced the flight ability of non-viruliferous whiteflies. Compared to the control group, the flight distance of whiteflies was significantly reduced (F1, 28 = 11.6, P < 0.05), and the flight time was significantly shortened (F1, 28 = 5.4, P < 0.05). The flight velocity also decreased significantly (Fig. 6a-c) (F1, 28 = 27.6, P < 0.001).
Validamycin treatment also markedly inhibited the flight distance, time and velocity of the viruliferous whiteflies. Compared to the control group, flight distance (F1, 28 = 17.5, P < 0.001), time (F1, 28=18.9, P < 0.001) and velocity (F1, 28=10.145, P < 0.001) of whiteflies all significant decreased (Fig. 6d-f). Our results indicated that validamycin effectively inhibited the flight behavior of B. tabaci.
Stylet activities of B. tabaci after validamycin treatment
The total duration of probes (F1, 28 = 42.922, P < 0.001), total duration of waveform C (F1, 28 = 17.615, P < 0.001), time of waveform E2 (F1, 28 = 53.943, P < 0.001) and number of waveform E1 and E2 (E1: F1, 28 = 39.628, P < 0.001; E2: F1, 28 = 39.628, P < 0.001) of non-viruliferous whiteflies which fed on tomato plants treated with validamycin, were significantly shorter compared to the control group (Fig. 7a, c, g-h). The time from first probe to first E (pd) (F1, 28 = 61.691, P < 0.001) was significantly longer (Fig. 7d). However, there were no significant differences in the number of probes (F1, 28 = 1.784, P = 0.187) and the time of waveform E1 (F1, 28 = 2.314, P = 0.134) (Fig. 7b, e). In general, after validamycin treatment, the duration of waveform np was longer, while the duration of waveform C and E were shorter than that of non-viruliferous whiteflies (Table S1, Fig. 9a-b).
The total duration of waveform C (F1, 28 = 8.474, P = 0.007), duration of waveform E1 and E2 (E1: F1, 28 = 13.250, P < 0.001; E2: F1, 28 = 20.972, P < 0.001) and number of waveform E1 and E2 (E1: F1, 28 = 46.873, P < 0.001; E2: F1, 28 = 46.873, P < 0.001) of viruliferous whiteflies which fed on tomato plants treated with validamycin were significantly shorter, compared to the control group (Fig. 8a, c, e-h). The total duration of probes (F1, 28 = 43.169, P < 0.001) and time from first probe to first E(pd) (F1, 28 = 47.092, P < 0.001) were significantly longer (Fig. 8d, e). There were no significant differences in the number of probes (F1, 28 = 0.149, P = 0.702) (Fig. 8b). In summary, after validamycin treatment, the duration of waveform np was longer, while the duration of waveform C and E were shorter in viruliferous whiteflies (Table S1, Fig. 9c-d). Our findings suggested that validamycin significantly disrupted the feeding of the phloem of whiteflies.