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New Study Points to Variability of RNAi Effect in Different Corn Rootworm Populations

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New research out of the University of Illinois shows that RNAi may be less effective in some populations of Western corn rootworms compared with others, adding a new wrinkle to ongoing efforts to use the gene-silencing technology for agricultural insect control.

As a result, those developing RNAi-based strategies to fight pests should consider the consistency of the technology in phenotypically different populations before commercializing them, according to the investigators.

Though it is more developed as a therapeutic modality, RNAi is becoming an area of focus for a number of ag-bio firms hoping to take advantage of the technology for improving the traits of crop plants, as well as dealing with pests that destroy them.

Perhaps farthest along in this effort is Monsanto, which has made significant investments in RNAi, including signing technology licensing deals with Alnylam Pharmaceuticals and Tekmira Pharmaceuticals, and is poised to introduce a strain of corn called Smart Stax Pro that expresses dsRNAs against a gene called Snf7 and a widely used Bt protein to kill Western corn rootworms.

Such a product would be the first of its kind, and the US Department of Environmental Protection is currently building a framework to assess the potential hazards of plants that express RNAi molecules, known as plant-incorporated protectants (PIPs).

But in addition to possible risks to humans and the environment, some RNAi PIPs may not be as effective in all populations of insects — including corn rootworms — in real-world situations as they are in the lab, according to a paper published last month in Pesticide Biochemistry and Physiology.

Corn rootworms are one of the most costly pests for the agricultural industry, with the mature insect feeding on the primary roots of the plants and larvae consuming the root hairs and small roots.

To deal with corn rootworms, Monsanto and others have developed transgenic corn varieties that express Bt proteins toxic to the insects and derived from the bacterium Bacillus thuringiensis, and farmers apply techniques such as crop rotation, swapping corn with soybeans each year so that larvae emerging in a field that no longer includes corn won't have access to their host plant.

However, there is evidence that corn rootworms are becoming resistant to Bt proteins. At the same time, that broad application of crop rotation has selected for a corn rootworm variant that is able to survive on soybean plants long enough to lay eggs, which hatch the next year when corn has been reintroduced to the field.

As a result, some companies have turned to RNAi as a new strategy to deal with corn rootworms that can no longer be controlled by traditional methods.

In terms of Smart Stax Pro, Monsanto has generated data showing that dsRNA against DvSnf7 — the rootworm ortholog of Snf7 and an essential component of key cellular machinery known as endosomal sorting complex required for transport — is lethal to the insects when ingested. It has also demonstrated that the spectrum of DvSnf7 silencing is narrow enough not to affect animals other than corn rootworms.

Yet much of this work has been conducted in a lab setting, and "after generations in the laboratory, insects gradually lose their natural diversity," Manfredo Seufferheld, senior author of the Pesticide Biochemistry and Physiology paper, said in a statement. "This makes it easier to control them, and may not accurately reflect actual insect responses in the field."

(Seufferheld was a professor of crop sciences at the University of Illinois when the study was conducted, but he has since taken a position in insect resistance management at Monsanto.)

To see whether RNAi effects observed in laboratory colonies of corn rootworms would be consistent across field populations with differing physiological characteristics, Seufferheld and his colleagues focused on two genes that are regulated differently in rotation-resistant and non-resistant rootworms: DvRS5, which produces the cysteine protease cathepsin L that enables the insects to digest plant proteins, and the immunity-associated gene att1.

Notably, Seufferheld previously reported that rotation-resistant corn rootworms express higher levels of cathepsin L. This allows the insects to overcome a soybean defense mechanism whereby the plant expresses protease inhibitors that impair insects' ability to digest plant proteins and, therefore, overcome the effects of crop rotation.

The latest study used corn rootworms from three previously characterized populations collected from two different sites in Illinois where crop rotation resistance has been reported, and one in Missouri where rotation resistance has not been identified.

After being fed the dsRNA, all three insect populations showed a decrease in DvRS5 and att1 transcript abundance. However, this effect was not consistent across the groups for DvRS5, with only one of the Illinois populations showing a significant decrease in transcript levels.

The removal of an outlier from this group increased the consistency of the effects, but the investigators wrote that they "could not rule out the possibility that natural variation may influence the effect of DvRS5-RNAi treatments at the transcript level."

As expected, treatment with DvRS5-targeting dsRNA caused a reduction in gut cysteine protease activities in all three corn rootworm populations. However, a greater level of protease activity was retained in insects from the areas with rotation resistance problems compared to those from Missouri where no such issue has been reported.

Noting that the variability of DvRS5 RNAi effects on transcript abundance cannot account for the substantial differences in the proportions of protease activity retained across the corn rootworm populations, the scientists speculated that gut microbia or other genes may be contributing to the effect.

"Bacteria could produce proteases, affect biochemical properties of their surrounding environments, or regulate host gene expression, all of which may influence the activity of digestive enzymes," they noted in their paper. "Our findings therefore suggest that physiological variation related to target pest trophic interactions might affect the outcome of RNAi treatments, a hypothesis with practical relevance for the application of RNAi to pest management."

Surprisingly, knocking down att1 appeared to slightly improve the survival of rotation-resistant corn rootworm populations on soybeans, while lowering the survival of non-resistant insects. The reasons behind the effect remain unclear, but the researchers speculated that att1 knockdown in different corn rootworm populations with differing immune backgrounds may lead to "differential disturbances in the immune system and gut physiology" that subsequently affect their tolerance to dietary stress.

Taken together, the findings indicate that different responses to RNAi may occur without differences at the sequence level of a targeted transcript, and imply that variability in corn rootworm populations' "physiology, trophic interactions, or functionally related genes/transcripts may be capable of influencing the effectiveness of RNAi in the field."

As such, it is important to consider testing the consistency of an RNAi trait's effect on phenotypically different populations "prior to wide deployment," they added. "Also, genes that are potentially subjected to differential selection should be avoided for such application."

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