Novel molecular biopesticides targeting the potassium ion channels of the red flour beetle, Tribolium castaneum (Herbst.)

Abstract

The ion channels of insects are targets for several classes of chemical insecticides and, whilst they are very effective at controlling insect pest populations, they are often accompanied by undesirable effects on non-target organism. RNA interference technology is currently being developed as an alternative means of crop protection. In this study RNAi was used to target two potassium ion channel genes of the beetle Tribolium castaneum, namely the SK gene, which encodes the small conductance calcium-activated potassium channel and the SH gene, which encodes the voltage-gated potassium channel. The results demonstrate that specific double-stranded RNA (dsRNA) targeting these ion channels significantly reduced the levels of gene expression. The oral delivery of dsRNA for both target genes caused a dose-dependent effect, with 100% mortality of third instar larvae at 400 ng dsRNA/mg diet after 10 days. This was accompanied by a decrease in TcSK and TcSH gene expression of 98% and 83%, respectively, after 3 days continuous feeding. The injection of sixth instar larvae with dsRNAs (62.1 ng/larva) caused 100% and 57.8% mortality for SK and SH, respectively after 7 days. Moreover, the injection of early pupae, late pupae and adults showed significant (p < 0.05) down-regulation in gene expression for both genes, being 93%, 96% and 93% for SK and 94%, 92% and 72%, respectively, for SH. The toxicity of dsRNA targeting the T. castaneum SK and SH genes was tested against a beneficial insect, the honeybee Apis mellifera. The dsRNA for both targets had no significant effects on bee survival when fed 20 ng/μl of TcdsSK or TcdsSH. Furthermore, there were no effects on expression levels of SK or SH in the foraging bees, and neither were there any effects on the immunity of the honeybee, as measured by the effects on the titre of the deformed wing virus (DWV). One of the limitations of the applicability of RNAi-based approaches for insect control is that of cost and delivery. We report a technically cost-effective method for the production of dsRNA by the bacterium, Escherichia coli HT115. The results show that the toxic effects on larval mortality caused by dsRNA synthesised by in vitro transcription compared with that expressed in vivo by bacteria were similar. The amount of dsRNA via in vivo expression in bacteria was approximately four times that of in vitro transcription per reaction. Finally, this study demonstrated that the co-delivery of dsSK or dsSH increased the efficacy of two entomopathogenic fungi, Beauveria bassiana and Metarhizium anisopliae. With both larvae and adults of T. castaneum, the effects on mortality with dsSK were synergistic. Overall, this study demonstrates the efficacy of RNAi as a method of insect control and that the ion channels represent good targets. Furthermore, it demonstrates the potentiating effects of the technology with the use of BCAs such as entomopathogenic fungi.