Background

Canola, cabbage, broccoli, and other cruciferous crops are attacked by a variety of insect pests, but flea beetles (Coleoptera: Chrysomelidae: Galerucinae: Alticini), are the most damaging, causing millions of dollars damage annually in those countries that grow these crops extensively. Neonicotinoid insecticide seed coatings, and pyrethroid, carbamate, and organophosphate foliar insecticides are used to control flea beetles, but these broad-spectrum insecticides not only kill the pest insects, but also kill non-target beneficial insects and can also adversely affect vertebrate species.

To reduce reliance on these broad-spectrum chemical insecticides and their associated problems, reduced-risk insecticides are needed. A technology that offers the promise of a reduced risk approach to insect pest control is RNA interference (RNAi). RNAi is a method of reducing or silencing a single gene’s expression by applying double-stranded RNA (dsRNA) to the cells of most eukaryotic organisms. To mediate RNAi in invertebrates such as insects, long dsRNAs are preferred, as they can generate a mixture of siRNAs that can bind to more of the target mRNA, and they are readily taken into the insects’ cells. For example, some investigations have observed that dsRNAs less than 60 nt failed to enter the gut cells efficiently, but in some instances, 21 nt siRNAs were also effective at inducing systemic (body-wide) knockdown of the targeted mRNA.

Here, an alternative set of pesticides, based on double-stranded RNA (dsRNA), is shown to be effective at killing flea beetles when the dsRNAs are applied to a leaf surface and fed to the insects. Insects that fed on leaves sprayed with the dsRNAs died within 8 days of first exposure, but also showed reduced feeding activity within several days, thereby reducing feeding damage to the plants. Importantly, the dsRNAs were designed to be specific for the flea beetles, and when delivered to non-target beetles, no adverse effects were observed, illustrating the specificity of the this new type of pesticide.

Technology Overview

The utility of RNAi technologies to kill flea beetles of the genus Phyllotreta was assessed, with the aim of developing topically-applied dsRNAs as an alternative to transgenic-mediated protection of the plants.

First, it was demonstrated that dsRNA can be applied to canola leaves and when ingested by flea beetles, gene-specific knockdown of mRNA transcripts occurs.

Secondly, a set of flea beetle of the genus Phyllotreta genes expressed in both larvae and adults was identified using transcriptomic analyses, and a subset of genes with no shared 21-nt lengths with other genes in NCBI’s GenBank was subsequently identified. dsRNAs were prepared to >50 different flea beetle genes, and using feeding bioassays, a smaller subset of dsRNAs was identified that killed the flea beetles within 8 days. While this time-to-kill period is longer than that of conventional neurotoxin chemical pesticides, the species-specificity of the dsRNAs is a distinct advantage over the broad-spectrum activity of our currently used chemical pesticides. Furthermore, the feeding activity of the dsRNA-fed flea beetles was considerably reduced, relative to untreated controls, indicating that the crop plants will still be protected even before the insects die. A dose-dependency was observed for many of the dsRNAs, and some failed to kill the insects at even the highest doses tested, which indicates that not all dsRNAs are effective insecticides. dsRNAs were then mixed together, and some combinations provided improved efficacy, using less dsRNA in total to kill flea beetles.

Figure 1 - dsRNA-targeted genes

Stage of Development

Technology Readiness Level (TRL): 4

Benefits

Broad-spectrum insecticides not only kill the pest insects, but also kill non-target beneficial insects and can also adversely affect vertebrate species. With the continued use of these insecticides, there have been growing public concerns about the long-term adverse effects on non-target species and the increased risk of insecticide resistance developing in the pest populations, the latter of which results in increased frequencies of pesticide applications and ultimately reduced efficacy and increased damage. The development of foliar RNAi technologies will reduce excessive chemical inputs and promote agroecological health. Implementation of RNAi technologies that prevent flea beetle outbreaks will be accessible to researchers and producers, thus enhancing the resiliency of the agriculture sector.

Applications

Alternative insecticide

Opportunity

The University is actively seeking a licensee to license the patent to move this technology to the market.

The University of Manitoba would also readily welcomes interactions with any investor or entrepreneur that would like to license the patent to create of a start‑up based in Manitoba, or outside of the province.

Flea Beetle Specific RNAi-Based Pesticides

Patents

IP Status

Seeking