NEW YORK (GenomeWeb) – Researchers from the China Agricultural University (CAU) this month reported new data demonstrating the utility of RNAi for genetic analysis in the cotton aphid, or (Aphis gossypii Glover. Their work also revealed a gene involved in xenobiotic compound metabolism as a promising target for pesticide resistance management.
Although the agricultural damage caused by the cotton aphid is far lower than that caused by other pests such as the corn rootworm, the insects can cause significant economic losses under certain conditions and, importantly, they are capable of developing resistance to a spectrum of insecticides including commonly used organophosphorates (OPs).
Carboxylesterases (CarE) have previously been identified as playing key roles in the metabolism of xenobiotics in many insects, and various groups have linked elevations in esterase activity to a degree of insecticide resistance in some species. In fact, overexpression of esterases has become a dominant criterion in identifying the development of resistance to organophosphorus insecticides, the CAU team wrote in a paper appearing in PLOS One.
In their own studies, the CAU scientists have found that CarE was more highly expressed in apterous adults of Aphis gossypii strains resistant to both OP and malathion as compared to apterous adults from susceptible strains.
Aiming to confirm the involvement of CarE in OP resistance, the researchers turned to RNAi given the success other groups have had using the technology to elucidate gene function in various insect species. And while the small size of cotton aphids prevents the use of microinjections to deliver dsRNA, they are susceptible to environmental RNAi via diet.
Using the same strain of cotton aphids in which they previously identified CarE overexpression, the CAU team transferred third instar larval stage insects grown on cotton leaves onto artificial diet devices containing either CarE-targeting dsRNA at 50, 100, or 500 nanogram per nanoliter concentrations, or a control diet lacking the RNAi molecules. After feeding for 24 hours, the aphids were collected or transferred onto cotton leaves for examination.
The survival rate for dsRNA-treated cotton aphids was greater than 90 percent, the scientists noted, indicating that delivering the RNAi molecules via feeding is a suitable approach for these insects. Meanwhile, qRT-PCR analyses showed that CarE transcript levels were decreased by up to 33 percent after 72 hours at the 100 nanogram concentration versus control insects.
This, the team noted in its paper, marks the first example of RNAi in the cotton aphid.
Transcript levels decreased gradually, suggesting that it takes some time for dsRNA to enter midgut cells and induce RNAi. Additionally, the gene silencing observed was incomplete — a result that may indicate dsRNA degradation inside the insect prior to the molecules reaching cells. Such an effect was recently described by US Department of Agriculture investigators, who showed that the saliva of the tarnished plant bug, or Lygus lineolaris, rapidly digests dsRNA.
Still, the CarE knockdown achieved was significant and had a measurable effect on the cotton aphids' OP resistance. Upon exposure to the pesticide, mortality of dsRNA-fed aphids was 68.44 percent versus 50.78 percent in control aphids, an effect the scientists chalked up to a reduction in detoxification caused by reduced CarE activity.
In light of these data, "it will be appealing to use dsRNA targeting of the CarE gene for controlling the OP resistant aphids, and possibly even other pest insects that have similar resistance mechanisms," the scientists wrote in PLOS One.
"The plant-mediated RNAi method has been used to effectively silence genes of Lepidopteran, Coleopteran, and Hemipteran insect species," they added. "It would be a revolution in plant protection if plant-mediated RNAi can be used extensively to protect plants from sucking insect pests."
Through the work of companies in the ag-bio space, RNAi-based pest control methods are moving closer to reality. Currently, Monsanto is preparing to launch in the next few years a pest-resistant strain of corn that expresses dsRNA and pesticidal proteins. Meanwhile, the US Environmental Protection Agency is looking to create a framework for the evaluation of these kinds of products.