Seducing a mushroom pest with scent
Drive up to a mushroom farm, open the car door, and you’ll understand why facilities like this one operate in rural areas. An overwhelming odor of manure emanates from compost piles scattered around the farm and from inside mushroom houses — the long, squat, wood and concrete structures where mushrooms are grown.
“If there was a steak in that house, we wouldn’t be able to smell it, because all we would smell is poop,” says Kevin Cloonan, a doctoral student in Tom Baker’s lab. “But the fungus gnat’s sense of smell is so precise they can sort it all out.”
Fungus gnats, Lycoriella ingenua, are small enough to squeeze into a mushroom house through the tiniest of crevices. Although adult gnats don’t damage the crop directly, they can transfer a devastating fungal pathogen that lays waste to the tender young mushrooms. Even if a female isn’t carrying this pathogen, she spawns about 200 larvae that devour belowground mushroom parts and gobble up compost nutrients that the mushrooms need to grow. An infestation of fungus gnats can cut a mushroom harvest by 70 percent — a huge hit to an industry that contributes $2 billion to the Pennsylvania economy each year.
Insecticides that easily controlled these pests have been banned one by one over the years. “Now we’re starting to see a reemergence of these gnats, but we don’t know much about them,” says Cloonan. He is looking for a way to turn their acute sense of smell against them — to prevent their entry into mushroom houses or capture them if they do sneak in.
Luring male gnats
Cloonan, along with postdoctoral researcher Stefanos Andreadis, is working to identify chemicals that attract male gnats to female gnats. These chemicals, called pheromones, are produced and released by the female so the males can find her for mating. “If the female is producing something the male likes the smell of, we can use it to trap males and control the gnats,” he says. This tactic has been used successfully to control Mediterranean flour moths in flour mills and olive flies in olive groves.
In the lab, Cloonan uses a colony of fungus gnats collected from mushroom houses in Chester County, Pennsylvania. From that colony, he selects mature females and douses them in hexane, a solvent that washes off any pheromones clinging to them. Next, Cloonan feeds the pheromone-containing hexane into a gas chromatograph (GC) that separates the chemical blend into individual compounds.
The GC uses half of each compound for identification and loads the other half into an instrument called an electroantennograph that tests which compounds attract males. For this test, larger insects are commonly restrained with a straitjacket-like apparatus, but gnats are too tiny for that method. Instead, Cloonan sticks four male gnats to a base electrode with ultrasound gel that keeps them in place and helps current flow. Then, the antennae of the restrained males are gently placed across a recording electrode. One at a time, potential pheromones are puffed from the chromatograph across the males’ antennae. The recording electrode registers responses of the antennae in sharp peaks, like a heart monitor.
“We’re fairly confident that we’re about to identify the pheromone, but it’s a bit tricky,” says Cloonan. Each compound has potentially dozens of forms and he needs to find out which one attracts males. If he can identify the exact structure of this pheromone, it can be made in the lab in a pure, concentrated form.
“You can use a pheromone to trap all the males so they don’t mate with females,” Cloonan says. “Or, you can use mating disruption: You saturate an environment with the pheromone and the males get confused. They spend too much energy flying around and die before they mate.”
What do pregnant gnats crave?
In another approach, Cloonan identifies aromas that draw female gnats to mushroom houses. For a long time, scientists thought that female gnats were attracted to the mushroom crop itself. Cloonan read work from the 1960s that disputed that claim but no one had followed up on it — so he explored the old research for himself. In initial experiments, Cloonan found that female gnats were equally attracted to compost with mushrooms and compost without mushrooms. However, they weren’t interested at all in sterilized compost. That discovery led Cloonan to think that a microorganism in the compost was enticing the female gnats.
Females of other insects are attracted to microorganism smells, because they indicate good places for larvae to survive and grow. Cloonan didn’t know whether this was true for fungus gnats, so he did experiments to find out. He thought non-mushroom fungi in compost were the most likely candidates. Cloonan got several types of fungi from compost, larvae feces and adult fly bodies and tested which ones females preferred to lay their eggs in. Similar experiments with other insects, such as mosquitoes and moths, are typically done with a Y–tube olfactometer assay. Different odors are blown into the short ends of a Y–shaped glass tube. The insect is released at the long end of the Y, and she usually makes a choice and moves toward her preferred aroma. But the Y–tube assay didn’t work with Cloonan’s gnats.
He spent seven months trying all sorts of variations: at night, during the day and at different temperatures. Nothing worked. The females just sat there, not moving toward either end of the Y.
Scouring the scientific literature for other approaches to try, he finally found an assay called a static–flow, two–choice olfactometer. “When I found that paper, it changed my life!” he says.
A good test—and a way forward
This new assay is a round glass dish with two holes in the bottom, each connected to a glass tube that leads to a container holding a different fungus. Cloonan releases a female gnat into the dish and she flies down into the container with the fungus she likes. He records the choice the female makes and how many eggs she lays, then repeats the experiment with several hundred other females.
He has found they’re strongly attracted to a few fungi, includingTrichoderma aggressivum. As its name suggests, this fungus is an aggressive parasite that eats other fungi. “It’s like an army of dissolving warriors,” Cloonan says. Once a farmer notices the mushroom crop is infested, this “green mold” has probably already eaten most of the belowground parts and even worse, “If you try to get rid of it, the spores will be airborne and will go all over the place.”
Mushroom farmers have long believed the fungus gnats were responsible for bringing green mold into mushroom houses, but there was little experimental data to back them up. Fellow graduate student Maria Mazin has been able to show that not only are female gnats attracted to the green mold, but they probably bring it in the house and spread it around as they lay eggs. Then, when the larvae develop and emerge as adults from the compost, the fungi stick to their bodies and are carried further. “My lab is using science to validate what the farmers have seen,” says Cloonan. “When you talk to them and they respond ‘We knew it!’ that’s what cool — we can prove the anecdotal stories floating around.”
Cloonan’s research is a smelly, time-consuming business, but he says, “At this point in a research degree, you got here because you weren’t grossed out by stuff.” And he’s right where he wants to be, digging up old ways to answer his burning scientific questions.
Kevin Cloonan plans to graduate from Penn State in 2017 with a Ph.D. in entomology. He is supported by the United States Department of Agriculture. His advisor, Tom Baker, is a distinguished professor of entomology and chemical ecology.