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New Research Links dsRNA Degradation to Lack of RNAi Response in Pea Aphid

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Researchers from Ghent University this month published new data pointing to the degradation of dsRNA as a factor behind the difficulty in obtaining an RNAi response in the pea aphid, a sap-sucking insect that is a key pest of legume crops.

As such, the study's authors see the use of chemical modifications and delivery vehicles pioneered by the RNAi therapeutics field as a potential solution to effective dsRNA-based gene silencing in pea aphids and other insects.

According to Olivier Christiaens, lead author of the study, the work stemmed from his interest in using RNAi as a functional genomics tool to study nuclear receptors in the pea aphid. Although there are studies from other groups showing RNAi gene knockdown in the insect, "we noticed a lack of any results," he said. "That's when we decided to have a look at some of the factors that could influence RNAi efficiency in this insect."

Given that the degradation of dsRNA in the gut and hemolymph of certain insects is a known issue, Christiaens and his colleagues focused their attention here, conducting microinjection and feeding experiments with dsRNAs against genes for two nuclear receptors with phenotypes known to affect molting.

In their study, which appeared in Peptides, they began by first seeing whether they could achieve RNAi in the insects by feeding them dsRNA against their target genes in an artificial diet, finding no differences between treated animals and controls not receiving the RNAi molecules. In vitro experimentation confirmed that the aphids' salivary secretions led to the breakdown of dsRNA.

The team also confirmed dsRNA degradation occurred in the insect's circulatory system, incubating GFP-tagged dsRNA ex vivo in collected hemolymph and observing a "rapid and strong" breakdown, according to the report.

Turning in vivo, the Ghent University group next used a qPCR approach to follow the degradation of GFP dsRNA injected into the aphid hemocoel, observing an almost complete elimination of the molecules after five hours.

To see if the RNAi machinery was engaged at any point after dsRNA was injected into the aphids, the researchers used qPCR to observe the expression of four components essential to the pathway: Dicer-2, Argonaute-2, R2D2, and Sid-1.

"None of these RNAi-related genes showed any upregulation after dsRNA introduction compared to the control, for which aphids were injected with water," they wrote.

Christiaens noted that he and the other scientists also tried to reproduce successful knockdown experiments described in the literature but were unable to do so, which could indicate that the pea aphid strain they used is insensitive to RNAi — something that has been observed in a variety of other insects.

He told Gene Silencing News that the presence of certain viruses in the insects could also have been responsible for his lack of success when following the protocols described in other papers.

Viral infections have been shown to upregulate RNAi-related gene in a number of species, he and his colleagues wrote in Peptides. "The presence of viruses could then not only activate the RNAi machinery but also have saturated the machinery resulting in a lack of RNAi response." This, he added, is one possibility that he is actively investigating.

Nonetheless, the data published this month confirm that dsRNA can "only stay intact in the hemocoel of the aphid body for a short period of time."

Although additional research is needed to fully understand the different factors that can influence dsRNA degradation in the insect, the delivery and modification methods used to overcome degradation in mammals could potentially offer a solution.

"Perhaps similar technologies could be developed for insects … in the future, which could then allow us to deliver siRNA efficiently in cells of insect species that possess a strong dsRNA-degrading capacity," the Ghent University group concluded in its paper. "Other methods, such as the expression in Escherichia coli cells and mixing these bacteria with the artificial diet could also have some potential in helping to avoid dsRNA degradation in the insect gut."

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