Research

Disease Detectives

Student researchers partner with the county health department to track down mosquitoes

University of Notre Dame graduate student Michelle Huang and junior Joseph Afuso pulled on their wading boots and sprayed a cloud of mosquito repellent onto their skin before gathering their supplies, including some dry ice, which Huang dumped into a round thermos.

Mosquitoes are attracted to the carbon dioxide given off by the dry ice, and the two were hoping they had attracted some mosquitoes at three different styles of traps they had set at St. Joseph County’s Spicer Lake.

Two people walk a wooded trail carrying mosquito trap supplies.
A student pours a red thermos of dry ice into a hanging blue thermos.
Junior Joseph Afuso and graduate student Michelle Huang walk around Spicer Lake with their supplies, including some dry ice, which Huang dumped into a round thermos.

Of the 54 different species of mosquitoes in Indiana, around eight to 10 — there is not a firm number — are considered public health hazards. The University’s Department of Biological Sciences began working with the St. Joseph County Health Department this summer to boost vector surveillance efforts. Although the health department monitors mosquito traps around the county, lack of staff and the distance to Spicer Lake prevented technicians from surveilling that mosquito-rich location. The county needed a way to further monitor potential outbreaks of Eastern equine encephalitis (EEE) and West Nile virus, and Notre Dame wanted a new opportunity to engage undergraduate students in research specific to public health.

Notre Dame’s rich history in vector biology — the study of how certain organisms spread infections by picking up a pathogen from one host and infecting another with it — made the partnership a perfect fit.

The project started with training in June with adviser Jennifer Robichaud, associate teaching professor and director of undergraduate studies in the Department of Biological Sciences, and Huang and Afuso. By the end of August 2021, Robichaud invited three high school students to participate, and other new Notre Dame undergraduate students joined the project. All are excited to do public health research.

“There are certain projects that are much harder to manage, like going into clinics where you’re dealing with patient privacy,” said Mary Ann McDowell, professor of biology and member of the Eck Institute for Global Health. McDowell, who served as interim associate dean of research and graduate studies until September, advanced the idea for the project. “But with mosquitoes there are so many opportunities for modeling for things like climate change, disease … many other spinoffs to collect and analyze data.”

Two women, blurred, carrying mosquito trap supplies walk on a trail. A brown sign reads Wetland Trail.
During the summer, the team usually trekked out to Spicer Lake three days a week to set up traps and collect mosquitoes.
Illustration of a blue thermos hanging from a branch, alongside a mesh trap with a light.
Light trap: Carbon dioxide from a thermos filled with dry ice and light attracts mosquitoeds to this trap. A fan, suspended under a small metal dome, swooshes them into a round trap hanging beneath the light.

During the summer, Huang and Afuso usually trekked out to Spicer Lake three days a week, in blazing sun or misting rain. On Tuesdays and Wednesdays they set up traps, and on Wednesdays and Thursdays they collected mosquitoes. On a Wednesday in July, they walked at a blistering pace to reach the first light trap, hanging from a tree about a half-mile into the woods. Huang replaced the thermos filled with dry ice. Afuso changed out the portable battery on the forest floor that is connected to a light and a fan, suspended under a small metal dome. A round trap, ensconced in mesh, hangs beneath the light.

The principle of the contraption is simple: The combination of the light and the carbon dioxide woos the mosquitoes. But instead of landing a satisfying blood meal, they are swooshed into the trap by the power of a fan.

Afuso pulled out the net, which contained mosquitoes, beetles, moths and other insects. He tied it off in preparation for its trip back to the laboratory in 225 Jordan Hall, where the insects dry out for a bit before being frozen inside the lab’s standard white Frigidaire refrigerator. The team will collect, sort, identify and count all mosquitoes until the end of the season in mid-October, but they are particularly looking for the females of the species that may carry the viruses.

The illuminated light bulb at the top of the light trap.
A woman in sunglasses wearing a Notre Dame t-shirt checks a mosquito light trap.
Jennifer Robichaud, associate teaching professor and director of undergraduate studies in the Department of Biological Sciences, checks a mosquito light trap.

There has been particular interest in hunting down mosquitoes that carry EEE locally, because it’s the disease that infected 14 horses in Indiana and killed an Elkhart County resident in 2019 — the first time the state had reported a death since 1964. There was another human case in 2020 in LaPorte County, along with four horse cases in the state. The wave of illnesses “came on suddenly, and people were caught flat-footed,” said Brett Davis, environmental health assistant director for the St. Joseph County Department of Health.

“At one point the county had a very large pest control program,” he said, describing how the West Nile virus scare had beefed up programs around the country in the early 2000s, after which programs were scaled back as a result of reduced funding. When EEE hit the area, “I had some traps from years past and I started doing a lot of trapping again.”

Mosquitoes become infected with EEE when they bite birds, which are plentiful at Spicer Lake. Humans, horses, llamas and a few other species become infected when they are bitten by an infected mosquito, but these mammals don’t spread the disease. In humans, symptoms begin with a fever, stiff neck, headache and lack of energy. Then they develop inflammation and swelling of the brain. About 30 percent of people infected with EEE die, and those who survive are usually permanently disabled. There is no treatment for the disease in humans.

A masked student wearing blue gloves holds up clear vial containing mosquitoes.
Afuso holds mosquitoes and other small insects collected in traps at Spicer Lake.

Davis’ work helped pave the way for funding to do aerial spraying to kill mosquitoes in at-risk areas in 2019, and the department assigned one of its interns to aid the vector surveillance efforts in 2020. EEE was identified in surrounding Indiana counties last year, and in Berrien County in Michigan. “We were in the center of a doughnut, which is not a good place to be; it proved that 2019 wasn’t an isolated year,” Davis said.

He was grateful for Notre Dame’s offer to assist, in particular because EEE infections seem to come in three-year cycles and he wanted to increase surveillance efforts. So far, a mosquito in Barry County, Michigan, has tested positive for EEE. “It’s a win-win situation, because while Spicer Lake is not a mapped bog, it is a similar environment and a great area to collect mosquitoes that have fed on birds,” Davis said. “This has been a big help to us.”

Shaded trees stand tall in the foreground, a sunny marsh area in the background.
Marsh area at Spicer Lake Nature Preserve in western St. Joseph County.
Illustration of a black box, open on one side to a red-painted interior on the forest floor.
Resting trap: Painted with red interiors, resting traps are meant to mimic pitcher plants that mosquitoes use as resting places after they have fed.

The light traps aren’t the only ones Huang and Afuso use at Spicer Lake. One series of eight traps, called resting traps, is located on the forest floor. Painted with red interiors, each box is meant to mimic pitcher plants that mosquitoes use as resting places after they have fed. In order to retrieve the mosquitoes from these, Huang and Afuso first slid N-95 masks onto their faces. Huang covered up the front of the box with an acrylic square, then sprayed triethylamine into a hole in the side. The triethylamine spray stunned the mosquitoes enough that Afuso was able to use a small aspirator to suck them out of the box. On this mission, the team retrieved only five mosquitoes, but can collect as many as 100.

A man and woman collect from bucket traps on the forest floor.
Afuso and Robichaud extract mosquitoes from gravid traps, where mosquitoes carrying West Nile virus like to lay their eggs.
Illustration of a black container with round mesh trap sitting on top.
Gravid trap: These traps are plastic containers filled with water and decaying alfalfa, and emit a scent that reeks of manure.

They hopped back into the van to drive to the forest edge, across from a field, to extract mosquitoes from a third set of traps. These gravid traps are plastic containers filled with water and decaying alfalfa, and emit a scent that reeks of manure. But mosquitoes carrying West Nile virus like to lay their eggs atop the stew, so collecting them is important for identifying the numbers of the Culex genus of mosquitoes, which are most likely to carry West Nile here.

After each collection is complete, students bring their haul back to campus, where the identification begins in Jordan 225. The laboratory room is decked out with microscopes and boxes containing records of mosquito surveys. Many were completed by George Craig, Notre Dame’s first entomologist and vector biologist who completed population studies on mosquitoes for most of his 38 years at Notre Dame, until his death in 1995. Students will be logging Craig’s data into a new digital database that can be used for mathematical modeling.

Robichaud, the faculty adviser for the project, makes sure the mosquitoes and other insects are deposited into petri dishes. Students pick through them with tweezers to find all of the female mosquitoes, and separate all the different genera and species. Some of the work can be completed without the aid of a microscope, but other work has to be done under magnification.

“It’s very easy to tell the difference between the males and females without a microscope,” said Huang, describing how male mosquitoes have what appear to be fuzzy faces because of their bushy antennae.

Robichaud described several of the different types of mosquitoes to Oliver Munn, a senior at Adams High School who has joined the project. As he separated the mosquitoes in a dish from other bugs, he described the process as “almost relaxing.”

“And I’m pretty excited to participate in science that will have a positive impact on the community,” Munn said.

Boxes of manilla folders cover tables in the lab. Microscopes and mosquito trap supplies are on the other tables.
The laboratory is decked out with microscopes and boxes containing records of mosquito surveys, many completed by George Craig.
Black and white photo of George Craig with an illustration of a mosquito.
George Craig, Notre Dame’s first entomologist and vector biologist, completed population studies on mosquitoes for most of his 38 years at Notre Dame.

All of the mosquitoes are identified and counted, Robichaud said, as part of the abundance portion of the project. After counting, the males are discarded because they do not bite, and therefore cannot be vectors of disease. Robichaud has taught the students how to distinguish between the different species of mosquitoes under a microscope, and mosquitoes in the few genera that can carry disease are sent to Davis at the St. Joseph County Health Department.

He combines these mosquitoes with the samples collected from the rest of the county. Although he and Notre Dame researchers can identify the different types of disease-carrying mosquitoes, they cannot tell whether the mosquitoes actually harbor West Nile or EEE. To find out, Davis ships the insects each Wednesday through priority mail (so they can arrive on Friday and won’t degrade in a hot mailbox over the weekend) to the Indiana Department of Health, where the insects can be examined using a laboratory technique called reverse transcription polymerase chain reaction (RT-PCR). The process amplifies RNA sequences to identify the presence of viruses, and has become better known during the past year because of testing during the COVID-19 pandemic.

The process isn’t pretty for the mosquitoes. Bryan Price, senior medical entomologist for the Indiana Department of Health, supervises three other entomologists who work in the northern, central and southern part of the state. Samples received from county health departments or towns that assist in sampling, and those collected by the IDOH regional entomologists, are taken to the State Department of Health Virology Lab in 2-milliliter microcentrifuge tubes, loaded with sterile ball bearings. The laboratory workers add reagents, and using a vortex mixer, spin the samples to pulverize the mosquitoes into a macabre cocktail.

After the samples are tested, Price reports the findings to the counties with positive samples.

A student looks through a microscope at a tray with many mosquitoes, sorting through them with tweezers.
A close up of many mosquitoes on a tray, being sorted through with tweezers.
The mosquitoes are deposited into petri dishes, and students pick through them with tweezers to find all of the female mosquitoes, and separate all the different genera and species.

A sample can have as many as 100 mosquitoes, and as few as three or four. It’s not useful or possible to complete the process with a single mosquito, said Price, who is based in Indianapolis. The turnaround time can vary between four and seven days, though during the COVID-19 pandemic, some analysis is taking longer.

“It’s a constant battle with mosquitoes,” said Price, whose mother was a student at Saint Mary’s College and whose predecessor, Michael Sinkso, was a student in George Craig’s laboratory. “When I was growing up, the only mosquito-borne disease anyone paid attention to was St. Louis encephalitis, which was the most prevalent in the ’60s, ’70s and ’80s.”

“If we can continually improve our predictions with more and more surveillance data, the vector control department can do more targeted, efficient sampling and abatement efforts.” —Jenna Coalson

West Nile came into the United States like a speeding train in 1999, and was first detected in Indiana in 2002. “Now we can graph the increase in West Nile each year to the point where you can somewhat predict when you might see human disease,” Price said. West Nile fever runs the gamut in humans from no symptoms to death, which occurs in 10 percent of cases that progress to the more serious neuroinvasive form.

Of course, Price says, because control and avoidance of mosquito bites is more accessible to most in this country than it is in other parts of the world that have even more mosquitoes and different types of potential diseases, he doesn’t recommend people avoid areas like Spicer Lake just because they may encounter a mosquito. He advises people to wear proper clothes and use spray-on mosquito repellents to avoid being bitten.

“I have friends on Facebook and Instagram who talk about how they were ‘eaten alive’ when they went out fishing, and this doesn’t have to happen. … I want to say, ‘You weren’t wearing repellent,’” said Price, who has worked as one of the state entomologists for 14 years. “People should take every step to avoid mosquito bites — make sure you dump water out of tires and other things, make sure your gutters are flowing and do what you can around your own property.”

Some disease-carrying mosquitoes

A mosquito with white markings on it's body and legs sucks on skin against a leafy background.

CDC

Aedes albopictus

Habitat
Small, shaded bodies of water surrounded by vegetation, as well as tree holes and pitcher plants. Also breeds in manmade containers like tires and bird baths. Found primarily in the Eastern and central parts of the country, as far north as northern Indiana.

Diseases it may carry
West Nile virus, Eastern equine encephalitis virus, dog heartworm, Zika, chikungunya, dengue fever, and possibly Saint Louis encephalitis virus and La Crosse encephalitis virus

Top view of brown mosquito.

Jedesto

Culex pipiens

Habitat
Natural or artificial bodies of standing or stagnant water in the northern United States and southern Canada.

Diseases it may carry
West Nile virus and Saint Louis encephalitis virus

A brown mosquito sits on skin.

Sean McCann

Coquillettidia perturbans

Habitat
Freshwater marshes and lake edges with heavy aquatic vegetation. Found throughout North America, especially in the eastern half of the United States.

Diseases it may carry
West Nile virus and Eastern equine encephalitis virus

His advice doesn’t mean that further surveillance and modeling shouldn’t be part of the plan, he and the researchers say. Jenna Coalson, former assistant professor of the practice in the Department of Biological Sciences and the Eck Institute for Global Health, has been advising on options for modeling and spatial analysis. Researchers will link the mosquito surveillance data with other sources of spatial data that are available for St. Joseph County, which could include human population data from the U.S. census or for locally specific health records, weather information and data from satellite images that show land cover.

“We would be especially concerned if the mosquito abundance occurs in areas with high human population density or with vulnerable groups,” Coalson said. “If we can continually improve our predictions with more and more surveillance data, the vector control department can do more targeted, efficient sampling and abatement efforts.”

Afuso has been interested in the geographical aspects of the research, too. “I’ve been considering a career in epidemiology, so this project gave me great exposure to how public health programs work and what effects they have,” he said.

The ongoing research, in all of its different forms from mosquito population counting to modeling, might someday expand to ticks, Davis and McDowell said. But for now, the work done this season, which is being funded by the College of Science and the Eck Institute, should help set up the rest of the research. It will bring the county a step closer to safety from mosquito-borne diseases, and the eventual outcome will be improved safety for those living in northern Indiana and southwest lower Michigan.

“We hope to continue growing these relationships into the next collection seasons, and hopefully play a larger role in vector surveillance activities to free up county health resources,” Huang said.

Davis agreed.

“The sky’s the limit,” he said.