Bioassay to validate Sf-SR-C as the receptor for Vip3Aa.

Abstract

(A) Scheme for the S. exigua bioassays. The 1st instar S. exigua larvae were first fed with the HT-pET-Se-SRi, HT-pET-Hypi and HT-pET28a strains for 7 d, respectively (For the first three days, the number of bacteria in each hole was 2 × 107; for the next four days, the number of bacteria in each hole was 4 × 107 per well.). And then 3st instar larvae were selected from each group to detect the transcription level of the Se-SR-C gene in the midgut of the larvae. The larvae were then exposed to Vip3Aa (0.8 μg/cm2) and the strains (4 × 107 per well) respectively for another 5 d to detect the mortality rate. (B) qRT-PCR analysis of the relative transcript levels of the SR-C gene in the midgut of S. exigua larvae after feeding with the HT-pET-Se-SRi, HT-pET-Hypi and HT-pET28a strains for 7 d, respectively. Data are expressed as the mean ± SD from three independent experiments; ns, non-significant; *** P S.exigua larvae feed with the bacterially-expressed dsRNA (feeding with HT-pET-Se-SRi, HT-pET-Hypi and HT-pET28a, respectively) after exposure to Vip3Aa (0.8 μg/cm2) (n = 20). The survival rates of each group were analyzed every day. Data are expressed as the mean ± SEM from three independent experiments; ns, non-significant; * P Drosophila bioassays. The adult flies of different strains were transferred to fresh medium and reared at 25 °C for 4 days. Then the flies strains (adult flies and their larvae) were shifted to 29 °C (Gal4 ‘‘on”) or 18 °C (Gal4 ‘‘off”) for 3 days. The 2st instar larvae of each strains were picked separately and treated with Vip3Aa or dialysis buffer for 48 h. The survival rates of each group were analyzed every 12 h. (E) Survival rate of different fly strains treated with Vip3Aa (100 μg/mL) or dialysis buffer (n = 20). The survival rates of each group were analyzed every 12 h. Data are expressed as the mean ± SD from three independent experiments; ** P < 0.01 by two-tailed Student’s t tests compared with the corresponding control value.</p