A new study in the Journal of Medical Entomology shows how electropenetrography (EPG) can quantify of the feeding behaviors of mosquitoes, specifically the species Aedes aegypti. Study leader Astri Wayadande (shown here) of Oklahoma State University hopes to encourage other researchers to try EPG to study the range of blood-sucking arthropods that transmit human and other diseases. Photo credit: Matt Bertone, North Carolina State University.

By Leslie Mertz

Studying the feeding behavior of mosquitoes is a tough job, especially when it involves allowing one mosquito after another to bite you. “It sure does itch. That’s the part that drives me nuts: I have to hold still and I just want to scratch,” says Astri Wayadande, a vector entomologist and professor of plant pathology at Oklahoma State University. Her sacrifice provided data for a just-published study showcasing a new approach for investigating mosquito behavior.

Called electropenetrography (EPG), the approach is a technology that Wayadande and other scientists have been employing for many years to deconstruct the plant-feeding behavior of aphids, leafhoppers, whiteflies, and other insects in the order Hemiptera. In particular, Wayadande is interested in the feeding behavior of leafhoppers, which use their mouthparts to pierce into tissues and suck up plant juices and can also transmit viruses or bacteria in the process.

The idea to expand the scope of EPG came from study co-author Elaine Backus. Backus is a research entomologist with the Agricultural Research Service of the U.S. Department of Agriculture (USDA-ARS) and has been involved in refining the technology and developing an EPG monitor. Wayadande recalled, “Elaine thought, why not apply EPG to blood-sucking arthropods? So she convinced a group of us to each take ticks, sandflies, or mosquitoes … and I got the mosquitoes.”

How EPG Works

EPG works by reading waveforms generated when an insect forms an electrical circuit with a host. In Wayadande’s study, the mosquito is outfitted with a small gold wire that serves as a recording electrode and short tether (the mosquito is in a glass pipette in A, B, and on a subject’s arm in C).

In EPG, researchers apply an electrode and a tiny electrical current to a host—plant or animal—and attach a small conductive wire to the insect. When the insect starts feeding, a circuit forms between the insect and host, and emits a series of different waveforms that appear on the EPG monitor as a line graph. The waveforms indicate different feeding behaviors, such as penetrating plant or human tissue, salivating, or sucking up plant juices or blood.

For the mosquito project, Wayadande looked specifically at Aedes aegypti, also known as yellow fever mosquitoes, from a “clean” lab-grown colony (so they didn’t carry any disease agents). The hope was that EPG would provide a faster and more precise way of collecting data than the old standby of video-recording mosquitoes during feeding and then interpreting the recording. She originally hoped to capture EPG waveforms as the insects fed on an artificial diet, but the insects didn’t cooperate. “I got tired of waiting for them to do something with the artificial diet, so I grabbed an electrode and put it in my hand, and I let a mosquito probe me. I could not believe it when I got the first waveforms from a mosquito feeding on my hand—they were gorgeous!” she recalled.

The waveforms clearly identified and measured the duration of four phases of feeding: pre-probing behavior; shallow penetration of the skin; deeper penetration to reach a blood vessel; and blood ingestion. “The beauty of EPG is that I can look at lots of different mosquitoes very rapidly and it is so quantifiable,” she said, noting that her study included data from more than 50 individual mosquitoes.

What EPG Can Show

EPG waveforms (shown here) are specific to different feeding behaviors. These graphs show five distinct waveform families: J is associated with pre-probing behavior; K with shallow penetration of the skin; L with deeper penetration to reach a blood vessel; and M with actual blood ingestion. The waveform family N had unknown behavioral meaning.

Although Wayadande wasn’t necessarily expecting EPG to reveal anything new about mosquitoes, it did. The EPG data showed that the species overall has a stereotypic approach to feeding, but some individuals are a bit slower or faster performing certain tasks, such as reaching a blood vessel. In addition, her research group followed up by examining whether a mosquito would adjust her feeding behavior when biting certain people. For instance, tests of individual mosquitoes showed they would take half as long to start feeding on a female vs. male subject. Duration is important with mosquitoes, she said, because it could mean the difference between getting noticed—and swatted—by a host, and living to bite another day.

Wayadande’s foray into mosquito research is over, but she hopes this study will encourage other mosquito researchers to try EPG. “You can use EPG to quantify whatever it is you’re looking at with respect to feeding behavior, whether it’s inoculation of pathogen, acquisition of pathogen, or just how mosquitoes behave on different hosts. For instance, do all mosquito species feed in the same way? I doubt it. What happens with different repellents, or if you eat garlic? Does either affect feeding behavior? You can actually test those things using this method and get quantifiable, measurable data.”

She added, “I think there’s a lot of potential here, so I’m hoping other people will get excited about taking their own bugs and their own questions, and then apply this technology to those questions.”

Leslie Mertz, Ph.D., teaches summer field-biology courses, writes about science, and runs an educational insect-identification website, www.knowyourinsects.org. She resides in northern Michigan.