When public health researchers want to know whether diseases such as COVID-19 or polio are circulating in a community, they can look for genetic evidence in wastewater.
Entomologist Dana Price wondered whether the same principle could be applied to mosquitoes. The question led to an unconventional experiment.
"What we did, colloquially, was build a mosquito toilet," said Price, an associate professor at the Center for Vector Biology, Department of Entomology in the School of Environmental and Biological Sciences and a lead author of a study published in Microbiology Spectrum.
The device, Price said, is deceptively simple: a 3D-printed funnel coated with a superhydrophobic (water-repelling) material that causes liquid to bead up and slide into a collection tube.
The tiny traces of mosquito waste it captured revealed a hidden world.
By sequencing genetic material in mosquito excrement, Price recovered a complete West Nile virus genome, found the first known evidence of a Hedwig-like virus in the Americas and detected several viruses that may be new to science. The approach also identified parasites and confirmed the mosquito species that deposited the waste droplets.
Findings from the study suggest mosquito waste could become a powerful surveillance tool, allowing scientists to search broadly for familiar pathogens as well as threats they don't yet know exist, Price said.
"If you don't look for it, you won't find it," Price said. "But what if you don't know what to look for?"
Mosquito surveillance historically involves collecting large numbers of insects, sorting them by species, combining them into pools and grinding them up for laboratory testing.
The labor-intensive process requires scientists to preserve the insects carefully, so their genetic material doesn't degrade. During an outbreak, those steps can delay information needed to direct mosquito-control efforts. Conventional tests also target specific pathogens, so researchers must know what they are looking for.
Mosquitoes excrete small amounts of liquid containing genetic traces of viruses and other organisms. Because the waste contains far less mosquito tissue, and therefore less mosquito DNA, than a crushed insect, the scientists reasoned that viral material might be easier to find.
The researchers collected two groups of 50 wild Culex mosquitoes from a yard in New Brunswick, N.J., in 2021 and 2022. They placed each group in a screened container over the funnel and fed them a sugar solution. The mosquitoes produced drops of waste, which slid into a tube.
The scientists used shotgun metagenomic sequencing – used to sequence all genetic material present in a mixed sample. Computers then assembled the millions of fragments generated and compared them with known sequences in scientific databases.
The 2022 sample contained a complete, high-quality genome of West Nile virus, which can cause serious neurological illness in people.
The researchers identified the virus as belonging to the NY07 genetic variant previously found in the region. Such detail eventually could help scientists track how genetic variants of a virus emerge,spread and change.
"What came out was an entire viral genome," Price said. "Rather than just positive or negative, we have genetic data."
The researchers were even more intrigued by evidence of a Hedwig-like virus, which hadn't previously been reported in the Americas.
Hedwig virus was first identified in Europe in snowy owl tissue, prompting scientists to name it after Harry Potter's famous white owl. Some of the infected birds also hadWest Nile virus .
Scientists don't know whether Hedwig-like viruses make birds sick, facilitate West Nile infections or simply coexist with other viruses. The Rutgers finding doesn't establish that the virus is a threat, Price said. It shows that it has been circulating where no one knew to search for it.
"We didn't even look for it specifically," Price said. "We just looked for everything, and we found Hedwig in there."
He currently is working with officials at the New Jersey Department of Environmental Protection's Division of Fish and Wildlife to screen organ and tissue samples for the virus. The samples are collected from deceased birds during a post-mortem examination.
A virus found for the first time in a region may have recently arrived or may have gone unnoticed for years. Because few broad mosquito-virus studies have been conducted in North America, organisms that seem geographically restricted may prove widespread.
"The more data we generate, the more we start to wonder if everything is everywhere," Price said. "Although we don't yet know how much of everything is everywhere, it may be a lot more than we realize."
The samples also contained sequences so different from known examples that the researchers classified the viruses as potentially new. They included a partiti-like virus, a picorna-like virus and a possible new virus associated with single-celled parasites infecting the mosquitoes. More study is needed to confirm they are previously undescribed species.
Most appear to infect mosquitoes, other insects or microorganisms living inside mosquitoes and are not known to cause human disease. Some insect-specific viruses may even interfere with mosquitoes' ability to carry West Nile, dengue or Zika viruses.
The waste also contained traces of trypanosomatids, a group of single-celled parasites known to infect mosquitoes. Mosquito DNA confirmed that the insects belonged primarily to the Culex pipiens group, a West Nile carrier.
Price said the research was an initial demonstration, not a replacement for existing surveillance. It examined 100 mosquitoes from one location, which were transferred to the funnel in a laboratory.
Researchers also cannot conclude from waste alone that a mosquito can transmit a virus, Price said. It could be infecting the mosquito or another organism inside it or simply passing through.
For now, the technique can answer a broader question: Is a pathogen circulating in this environment?
The team is working toward a field-ready trap that would collect mosquitoes and their waste. Price eventually envisions a system that could prepare samples, sequence genetic material, analyze the results remotely and alert scientists to a possible threat in real-time.
"The pie-in-the-sky idea is a device that we place in a forest somewhere, and it's constantly just beaming back data to us," Price said.
Such a system could be especially valuable in remote or resource-limited areas, he said. It could reveal known pathogens before they cause widespread illness and expose the ones no one yet knows to look for.
Other Rutgers researchers on the study include: Mehdi Javanmard from the Department of Electrical and Computer Engineering in the Rutgers School of Engineering; Taewon Han, an assistant research professor from the Department of Environmental Sciences in the School of Environmental and Biological Sciences; Nicole Wagner, a laboratory researcher; and former undergraduate students Miranda Barnes and Fiona Bezhani.
Explore more of the ways Rutgers research is shaping the future .
