Enhancement of Bacillus thuringiensis toxicity by feeding Spodoptera littoralis larvae with bacteria expressing immune suppressive dsRNA

Figure 1C

Calibration curves used for qRT-PCR absolute quantification of Sl 102 and GFP dsRNA present in E. coli suspensions used in the bioassays

Figure 2

Transcript levels of Sl 102 gene in S. littoralis 4th instar larvae orally treated for 3 days with dsRNA. The Sl 102 gene was down-regulated upon ingestion of Sl 102 dsRNA administered by oral gavage, both in the case of dsRNA synthesized in vitro (Sl 102 dsRNA-synt) and suspensions of sonicated bacteria expressing Sl 102 dsRNA (Sl 102 dsRNA-bac). Delivery with artificial diet showed a silencing response that was dose-dependent and more pronounced when bacteria were used as delivery vectors. GFP dsRNA synthesized in vitro and bacteria expressing GFP dsRNA were used in control experiments. The values reported are the mean ± standard errors (*P < 0.0001, Student’s t test)

Figure 3

Encapsulation assay in S. littoralis 4th larvae treated for 3 days with Sl 102 dsRNA synthesized in vitro (Sl 102 dsRNA-synt) or transformed HT115 E. coli expressing Sl 102 dsRNA (Sl 102 dsRNA-bac). Chromatography beads injected into the body cavity of control larvae were encapsulated and melanized (a). On the contrary, the efficiency of encapsulation was lower in silenced larvae, independently from the dsRNA administration method (gavage or with artificial diet) (b). The encapsulation index was affected by oral delivery method and, in the case of oral administration on artificial diet, by dsRNA quantity. GFP dsRNA synthesized in vitro and bacteria expressing GFP dsRNA were used in control experiments. The values reported are the mean ± standard errors (*P < 0.0001, Student’s t test)

Figure 4

Bioassay with S. littoralis 4th instar larvae exposed to dsRNA before Bt treatment. Newly molted larvae were treated for 3 days with artificial diet layered with transformed HT115 E. coli expressing Sl 102 dsRNA (Sl 102 dsRNA-Bac, corresponding to 200 ng of dsRNA) and then with 12 µg/cm2 of Xentari™ for 3 more days (see “Materials and methods” section for experimental details). Survival was monitored until day 8 (a), when the weight was assessed on the surviving experimental larvae (b). Bacteria expressing GFP dsRNA were used in control experiments. The timing of the treatments is indicated with arrows. The values reported are the mean ± standard errors (in a *P < 0.0001 based on log-rank test; in b different letters denote statistical difference based on Kruskal–Wallis test, followed by Dunn’s multiple-comparison post hoc test)

Fig. 5

Bioassay with S. littoralis 4th instar larvae simultaneously exposed to dsRNA and Bt. Newly molted larvae were treated for 3 days with artificial diet layered with transformed HT115 E. coli expressing Sl 102 dsRNA (Sl 102 dsRNA-Bac, corresponding to 200 ng of dsRNA) and with 9 µg/cm2 of Xentari (see “Materials and methods” section for experimental details). Survival was monitored until day 8 (a) when the weight was assessed on the surviving experimental larvae (b). Bacteria expressing GFP dsRNA were used in control experiments. The timing of the treatments is indicated by arrows The values reported are the mean ± standard errors (in a **P < 0.0001 and *P < 0.0046 based on log-rank test; in b different letters denote statistical difference based on Kruskal–Wallis, followed by Dunn’s multiple comparisons post hoc test)

Fig. 6

Bioassays with S. littoralis 5th instar larvae simultaneously exposed to dsRNA and Bt. Newly molted larvae were treated for 3 days with artificial diet layered with transformed HT115 E. coli expressing Sl 102 dsRNA (Sl 102 dsRNA-Bac, corresponding to 200 ng of dsRNA) and with 12 µg/cm2 of Xentari (see “Materials and methods” section for experimental details). Survival was monitored until day 8 (a), when the weight was assessed on the surviving experimental larvae (b). Bacteria expressing GFP dsRNA were used in control experiments. The timing of the treatments is indicated by arrows. The values reported are the mean ± standard errors (in a *P < 0.0001 based on log-rank test; in b different letters denote statistical difference based on Kruskal–Wallis test followed by Dunn’s multiple-comparison post hoc test)