A Transformed Bacterium Expressing Double-Stranded RNA Specific to Integrin β1 Enhances Bt Toxin Efficacy against a Polyphagous Insect Pest, Spodoptera exigua

Abstract

BackgroundOral toxicity of double-stranded RNA (dsRNA) specific to integrin β1 subunit (SeINT) was known in a polyphagous insect pest, Spodoptera exigua. For an application of the dsRNA to control the insect pest, this study prepared a transformed Escherichia coli expressing dsRNA specific to SeINT.Principal FindingsThe dsRNA expression was driven by T7 RNA polymerase overexpressed by an inducer in the transformed E. coli. The produced dsRNA amount was proportional to the number of the cultured bacteria. The transformed bacteria gave a significant oral toxicity to S. exigua larvae with a significant reduction of the SeINT expression. The resulting insect mortality increased with the fed number of the bacteria. Pretreatment with an ultra-sonication to disrupt bacterial cell wall/membrane significantly increased the insecticidal activity of the transformed bacteria. The larvae treated with the transformed bacteria suffered tissue damage in the midgut epithelium, which exhibited a marked loss of cell-cell contacts and underwent a remarkable cell death. Moreover, these treated larvae became significantly susceptible to a Cry toxin derived from Bacillus thuringiensis (Bt).ConclusionsThis study provides a novel and highly efficient application technique to use dsRNA specific to an integrin gene by mixing with a biopesticide, Bt.

Alteration of insulin signaling to control insect pest by using transformed bacteria expressing dsRNA

“…Sonication treatment might impair the bacterial membrane through physical damage that results in leaks, through which the dsRNA in bacterial cells might be easily passed. Enhanced insecticidal activity by ultra‐sonication pretreatment has been observed in the application of dsRNA‐expressing bacteria to S. exigua . Thus, a dsRNA delivery system using recombinant bacteria pretreated with sonication can improve the insecticidal activity of dsRNA against lepidopteran insect pests.…”

“…To clone four IIS component genes, L4440 plasmid containing multiple cloning sites, which were flanked with two T7 promoters in an inverted orientation, was used. Restriction sites of Hind III and Xho I were chosen to clone InR, Akt, and FOXO of M. vitrata while Xho I and Sac I were chosen to clone TOR of M. vitrata .…”

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“…glycogen synthase, chitinase ). Other transcripts targeting locomotion, feeding methods, temperature control, growth, and development have also been successful …”

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“…The midgut was fixed with Bouin fixative, dehydrated with an ascending ethanol series, cleared with xylene, and infiltrated with paraffin; afterward, the midgut attached to the slide glass was sectioned, deparaffined, and stained (Kim et al 2015).…”