DOI: 10.5281/zenodo.3754633
title: Enhancement of Bacillus thuringiensis toxicity by feeding Spodoptera littoralis larvae with bacteria expressing immune suppressive dsRNA

description: <strong>Figure 1C</strong> Calibration curves used for qRT-PCR absolute quantification of <em>Sl 102</em> and <em>GFP</em> dsRNA present in <em>E. coli</em> suspensions used in the bioassays <strong>Figure 2</strong> Transcript levels of <em>Sl 102</em> gene in <em>S. littoralis</em> 4th instar larvae orally treated for 3 days with dsRNA. The <em>Sl</em> <em>102</em> gene was down-regulated upon ingestion of <em>Sl</em> <em>102</em> dsRNA administered by oral gavage, both in the case of dsRNA synthesized in vitro (<em>Sl</em> <em>102</em> dsRNA-synt) and suspensions of sonicated bacteria expressing <em>Sl</em> <em>102</em> dsRNA (<em>Sl</em> <em>102</em> dsRNA-bac). Delivery with artificial diet showed a silencing response that was dose-dependent and more pronounced when bacteria were used as delivery vectors. <em>GFP</em> dsRNA synthesized in vitro and bacteria expressing <em>GFP</em> dsRNA were used in control experiments. The values reported are the mean ± standard errors (*<em>P </em>&lt; 0.0001, Student’s <em>t</em> test) <strong>Figure 3</strong> Encapsulation assay in <em>S. littoralis</em> 4th larvae treated for 3 days with <em>Sl 102</em> dsRNA synthesized in vitro (<em>Sl</em> <em>102</em> dsRNA-synt) or transformed HT115 <em>E. coli</em> expressing <em>Sl</em> <em>102</em> dsRNA (<em>Sl</em> <em>102</em> dsRNA-bac). Chromatography beads injected into the body cavity of control larvae were encapsulated and melanized (<strong>a</strong>). On the contrary, the efficiency of encapsulation was lower in silenced larvae, independently from the dsRNA administration method (gavage or with artificial diet) (<strong>b</strong>). The encapsulation index was affected by oral delivery method and, in the case of oral administration on artificial diet, by dsRNA quantity. <em>GFP</em> dsRNA synthesized in vitro and bacteria expressing <em>GFP</em> dsRNA were used in control experiments. The values reported are the mean ± standard errors (*<em>P </em>&lt; 0.0001, Student’s <em>t</em> test) <strong>Figure 4</strong> Bioassay with <em>S. littoralis</em> 4th instar larvae exposed to dsRNA before <em>Bt</em> treatment. Newly molted larvae were treated for 3 days with artificial diet layered with transformed HT115 <em>E. coli</em> expressing <em>Sl</em> <em>102</em> dsRNA (<em>Sl</em> <em>102</em> dsRNA-Bac, corresponding to 200 ng of dsRNA) and then with 12 µg/cm<sup>2</sup> of Xentari™ for 3 more days (see “Materials and methods” section for experimental details). Survival was monitored until day 8 (<strong>a</strong>), when the weight was assessed on the surviving experimental larvae (<strong>b</strong>). Bacteria expressing <em>GFP</em> dsRNA were used in control experiments. The timing of the treatments is indicated with arrows. The values reported are the mean ± standard errors (in <strong>a</strong> *<em>P </em>&lt; 0.0001 based on log-rank test; in <strong>b</strong> different letters denote statistical difference based on Kruskal–Wallis test, followed by Dunn’s multiple-comparison post hoc test) <strong>Fig. 5</strong> Bioassay with <em>S. littoralis</em> 4th instar larvae simultaneously exposed to dsRNA and <em>Bt</em>. Newly molted larvae were treated for 3 days with artificial diet layered with transformed HT115 <em>E. coli</em> expressing <em>Sl</em> <em>102</em> dsRNA (<em>Sl</em> <em>102</em> dsRNA-Bac, corresponding to 200 ng of dsRNA) and with 9 µg/cm<sup>2</sup> of Xentari (see “Materials and methods” section for experimental details). Survival was monitored until day 8 (<strong>a</strong>) when the weight was assessed on the surviving experimental larvae (<strong>b</strong>). Bacteria expressing <em>GFP</em> dsRNA were used in control experiments. The timing of the treatments is indicated by arrows The values reported are the mean ± standard errors (in <strong>a</strong> **<em>P </em>&lt; 0.0001 and *<em>P </em>&lt; 0.0046 based on log-rank test; in <strong>b</strong> different letters denote statistical difference based on Kruskal–Wallis, followed by Dunn’s multiple comparisons post hoc test) <strong>Fig. 6</strong> Bioassays with <em>S. littoralis</em> 5th instar larvae simultaneously exposed to dsRNA and <em>Bt</em>. Newly molted larvae were treated for 3 days with artificial diet layered with transformed HT115 <em>E. coli</em> expressing <em>Sl</em> <em>102</em> dsRNA (<em>Sl</em> <em>102</em> dsRNA-Bac, corresponding to 200 ng of dsRNA) and with 12 µg/cm<sup>2</sup> of Xentari (see “Materials and methods” section for experimental details). Survival was monitored until day 8 (<strong>a</strong>), when the weight was assessed on the surviving experimental larvae (<strong>b</strong>). Bacteria expressing <em>GFP</em> dsRNA were used in control experiments. The timing of the treatments is indicated by arrows. The values reported are the mean ± standard errors (in <strong>a</strong> *<em>P </em>&lt; 0.0001 based on log-rank test; in <strong>b</strong> different letters denote statistical difference based on Kruskal–Wallis test followed by Dunn’s multiple-comparison post hoc test)