Resistance Movement

Tequila is rarely touted for playing a role in good decision-making, but a wacky idea Jessica Hua hit on over margaritas a few years back turned out to be a stroke of genius.

At the time, Hua was a PhD student at the University of Pittsburgh, working with Rick Relyea, a biologist who has extensively studied amphibians and pesticides (the toxins are often implicated in the decline of amphibian populations worldwide). When it comes to pests, resistance to the chemicals intended to kill them is a well-known problem. But it’s not just weeds and insects that are able to withstand the poisons—Relyea’s work had shown that lethal doses of pesticides didn’t always kill all frogs, either.

The standard explanation would be that over many, many generations, some hoppers had developed the ability to tolerate the toxin, and their offspring were born with immunity. But that night, Hua had a different thought: What if exposure to a little bit of pesticide at a young age made the creatures better able to withstand doses of the toxic stuff later in life? In other words, what if any frog could become pesticide-resistant?

Her lab mate encouraged her to pitch the idea to Relyea, so she did—later, in the light of day. “I said, ‘That’s crazy,’ ” laughs Relyea, who is now at Rensselaer Polytechnic Institute. “I’d never heard of any animal that could induce tolerance. But she came right back and said there was one example: a mosquito.”

With that, Relyea gave her the go-ahead, so Hua’s very first step was to collect eggs from wild frogs as soon as they were laid. “They’re explosive breeders,” says Hua, who would go out and listen for wood frogs every day. That means they all get it on at the same time—signaled by a crescendo of their duck-like calls. Miss the orgy and she’d be out of test subjects. Her persistence paid off, and she collected fresh eggs from four ponds, twice at night, by herself. “That’s the fun, outdoors, kind-of scary part,” she says, explaining how she’d don a headlamp and chest waders, venture into the water after the adults had departed, slice open a sac packed with embryos, and bring them back to the lab. Then the not-so-fun part: painstakingly separating thousands of eggs with a pipette.

Hua bathed different batches of embryos in various concentrations of pesticides until they hatched. They lived in clean water until they developed into tadpoles, when she then exposed them all to a lethal dose of the common insecticide carbaryl. In a complementary experiment, she also exposed some hatchlings to insecticide-tainted water, and then later exposed all hatchlings to lethal doses when they became tadpoles.

“We found that tadpoles that had been previously exposed actually survived the lethal dose better,” says Hua. “It was very unexpected, very exciting.”

“It was completely contrary to what we thought,” says Relyea. “Nobody knew you could induce tolerance in just a couple of days.”

As for how the amphibians induce tolerance, it has to do with a key enzyme in their nervous system called acetylcholinesterase (AchE). Carbaryl kills frogs by binding to AChE. “The heart can’t beat well, breathing becomes difficult, and they die,” says Relyea. The team found that sublethal exposure to the insecticide stimulated the tadpoles to produce greater amounts of AChE —which made them more tolerant to the chemical later in life (to a point—a large dose of carbaryl still killed all the frogs).

Subsequent work revealed that tadpoles resistant to carbaryl are also resistant to other insecticides that kill in a similar way.

The team has also discovered recently that wood frogs living closer to agriculture are naturally more tolerant to carbaryl than those farther from farms, and that hoppers that live farther away have the greatest ability to increase tolerance. In other words, there’s a limit to the frog’s resistance to pesticides, and the amphibians that live right next to farm fields—and thus experience greater exposure to the chemicals—are more likely to be at that maximum level, says Hua, who is now a biologist at Binghamton University.

And they’ve found that the type of pesticide makes a difference: Wood frogs are more tolerant to older insecticides, like carbaryl, than newer neonicotinoid pesticides (the ones linked to bee declines).

These findings are coming at the same time that government agencies are starting to take a closer look at the effects of pesticides on wildlife. The U.S. Environmental Protection Agency recently announced that, as part of a court-ordered settlement, over the next five years it will study the effects of four common pesticides, including glyphosate (the main ingredient in Roundup) and atrazine (which is known to cause sex changes in amphibians), on 1,500 endangered species. And last year the U.S. Forest and Wildlife Services agreed to investigate the risks five insecticides pose to endangered species.

Hua hopes her work might encourage other scientists who are assessing the threat of pesticides to consider where they get their test subjects. “Our work suggests that if you study populations farther from agriculture, you have a better chance of overestimating the toxicity of a chemical, but if you use populations close to agriculture that are more tolerant, you might underestimate the toxicity.”

Hua is continuing to dig into how, exactly, tolerance is induced, and whether it comes at a price, making frogs less capable of mounting an attack on pathogens. Relyea, meanwhile is zooming out from his focus on frogs to look at how pesticides affect the larger pond ecosystem, setting up a series of 300-gallon tanks to study everything from zooplankton to fish to insects. “If resistance only protects one species, that’s good for that species, but of limited benefit,” he says. “If it protects the whole food web to some degree, that’s another matter entirely.”