Understanding insect response to popular insecticide in effort to develop new compounds

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Developed in the 1980s, insecticide-treated bed nets are estimated to be twice as effective as non-treated nets in preventing malaria, a deadly mosquito-borne disease that kills more than 600,000 people in Africa and around the world each year. Some studies have shown a protection rate as high as 70 percent compared with no nets.

Michigan State University (MSU) entomologist Ke Dong, who operates the MSU Insect Toxicology and Neurobiology Laboratory, has been studying various aspects of the one class of insecticides approved for use on mosquito bed nets — pyrethroids. The work has been ongoing for the past two decades with continous funding from the National Institutes of Health (NIH). She wants to understand the mechanism by which these compounds control various insects, including malaria-carrying mosquitoes. This large class of synthetic insecticides is derived from pyrethrum, a compound extracted from dried chrysanthemum flowers.

They work by binding to and forcing open the voltage-gated sodium channel in the nervous system, causing overstimulation of the nervous system and eventually death of the mosquito. In 2013, in collaboration with Boris Zhorov (McMaster University, Canada), Dong’s lab made a major discovery: there are two receptor sites, not just one, necessary to hold the sodium channel open.

Because of their low toxicity to humans and mammals, pyrethroids have been extensively used against insects, including agricultural pests. However, the widespread use has resulted in pyrethroid resistance. To better understand this problem, Dong’s lab, in collaboration with various other groups, has been looking into the genomes of mosquitoes, cockroaches, cattle ticks, tobacco budworms, bedbugs, varroa mites and fruit flies for the mutations that make insects and arachnids resistant to pyrethroids.

“It turns out that there are numerous mutations on the sodium channel causing the resistance,” Dong said. “Some mutations are common among many species, and others are detected only in particular species. Discovery of these mutations makes it possible to find solutions to monitor and to come up with new methods to manage pyrethroid resistance in the field.”

As part of an ongoing project, Dong plans to work with chemists to help develop new compounds that can bind to the mutant channel. She said that this is challenging because the receptor site was changed, as well as the structure of the sodium channel. They will need to ensure that the compound structure can fit into the mutated sodium channel. Some of these insecticidal compounds also repel insects. Because of technical challenges, however, little research has been done on how pyrethroids repel insects such as mosquitoes.

“Within the last couple of years, people really started paying attention to new chemistry to control resistant mosquitoes,” she said. “Because it’s very expensive and time-consuming to develop new pesticides, there was substantial interest in using pyrethroids as spatial repellents — to just keep mosquitoes out of houses where people sleep.”

Dong is now studying whether pyrethroid efficacy is due to physical contact with the compound, or if the insects are able to smell the insecticide.

“Mosquitoes have this behavior called excitorepellency, and we want to know if it’s spatial or if it’s contact,” she said. “We are working to determine whether the insects make contact with the nets and leave, or if they get near them, smell the compound and then leave.”

Postdoc Peng Xu has conducted an experiment in which fruit flies were put under the microscope with electrodes placed in front and in back of their antennae. Each antenna is covered with lots of hair, and at the base of each hair are sensory neurons. The odor can diffuse onto the antenna and bind to the olfactory receptors in the sensory neurons. Dong and her team wanted to determine which receptor/neuron was activated by the chemical.

In the study, various odors were puffed near the insect antennae while the researchers observed the neuron activity. Both the food odor and the pyrethroid caused activity, but there was none when air was puffed at the insect. Dong said this signified for the first time that the fruit fly could actually smell the pyrethroid. Next they released hungry fruit flies into a secure area with two chambers: one treated with a pyrethroid and the other without a pyrethroid (the control). Apple cider vinegar (which flies like) was placed at the bottom of each chamber. About 90 percent of the flies ended up in the chamber without the pyrethroid, indicating that the pyrethroid kept the flies out. Ultimately, the researchers want to determine which neurons respond to the pyrethroids and to be able to trace them back to a receptor.

“Without any odor, these neurons are spontaneously firing but with low frequency,” she said. “When certain compounds are puffed near the active receptor — this neuron just goes crazy.”

By doing this, Dong and her team have identified at least four olfactory receptors in the fruit fly that respond to pyrethroids. Next they plan to remove the pyrethroid receptor gene from the neuron and repeat the behavioral assay to determine if the insect can still smell. Graduate student Elizabeth Bandason is also working to better understand the molecular basis of the repellency, this time in mosquitoes. Her initial findings are very similar to those the group has observed in fruit flies.

“Mosquitoes basically have a similar phenomenon — there is a spatial repellency going on,” she said. “Now we’re looking into mosquito olfactory receptors — that will be electrophysiology, molecular work to figure out which olfactory receptor is responding and knock them out to see what happens with behavior and so on.”

Dong said there is much more work ahead of the team, but progress is being made. MSU research assistant professor Yuzhe Du is the co-principal investigator on this new NIH-funded project. MSU research specialist Yoshiko Nomura has also put in considerable effort on these projects.

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