Spiders can fend off ants by coating their web with the predators’ own alarm chemical, reports a new study of Singapore spiders and ants.

Only spiders whose webs are thick enough for ants to crawl on seem to use the chemical, suggesting that different spider species may have evolved targeted chemical weaponry.

The insect world is full of mysteries, and spiders and ants were the center of this one. Spider webs are made of silk, a biomaterial whose strength and elasticity make an efficient trap for catching prey. But staying on or near the web makes the spider a sitting duck for potential predators, including wasps as well as ants. Yet ants steer clear of webs.

Spiders are known for the defenses they have evolved for escaping wasps, their main flying predator: “leaf-refuges” for hiding; camouflage silk decorations to make the spider look threatening; “drag-lines” to drop from the web, and the ability to color-shift, as octopi sometimes do in corral reefs. But little was known about spider defenses against ants—or how a particular spider species’ weapons might adapt to its specific predators.

Orb-weaver spiders, a common spider family worldwide, are an attractive meal to ants, since they are large, meaty, sedentary, and easily immobilized by an ant army. Yet ants are rarely found on webs. Why not?

A group of biologists at the National University of Singapore published their answer this month in the Proceedings of the Royal Society B. The experiment began with a bug-hunt. Spider-expert Shichang Zhang and four Singaporean colleagues trekked around the University of Singapore campus and two parks in search of orb-web spiders and pharaoh ants. Pharaoh ants are a widespread pest in the forests where the spiders live, whose workers eat spiders.

They captured 21 adult and large juvenile spiders, and 20 small juveniles, as well as 2,000 ants of the native pharaoh species. In the lab, they also had colonies of two other ant species, to test whether any chemical in the webs repelled a broader range of ants.

The spiders spun fresh webs in cages in the lab, so the silk wouldn’t be contaminated by prey. The biologists then collected silk from each spider’s web and shook it in a vial of solvent for ten minutes to extract any chemicals from the silk surface. They used a technique called mass spectrometry to isolate whatever chemicals were on the threads and discovered one on the webs of adult and large juvenile spiders called 2-pyrrolidinone. Might this be the mystery ant-repellent? To find out, the scientists built “silk bridges” out of spider-web threads and used fruit-flies and house-flies as bait to attract the ants.

There were three kinds of bridges: the first, a “control” bridge, was a natural spider-silk thread with the 2-pyrrolidinone chemical on it; the second and third, labeled the “deterrent removed” and “deterrent added” bridges, were spider threads which had the chemical removed. Removal involved soaking the spider threads in a 70 percent alcohol solution for two hours, then baking the silk in an oven for two days (should you care to try this at home).

Next, the scientists gave pharaoh ants access to the bridges. In twenty-five trials, each with about 200 ants, they video-recorded for two hours to see which spider-webs the ants crossed to get to the flies. The ants avoided the web with 2-pyrrolidinone on it (the natural web), but crossed freely both bridges that had been stripped of the chemical. After two hours, between 150 and 200 pharaoh ants had swarmed across the two stripped bridges to feast on dead flies.

Now came the critical step: the scientists poured liquid onto all three bridges. On the natural and “deterrent removed” webs, they poured water; on the “deterrent added” bridge, they poured 2-pyrrolidinone. They video-recorded the ants for another two hours.

The change was dramatic and abrupt: as soon as 2-pyrrolidinone was added, ants avoided the “deterrent added” bridge as well as the natural web, and crossed only on the “deterrent removed” spider silk.

When they tried the same experiment with the other two species of ant, they found the same response; in nine of ten trials, these two ant species were also repelled by 2-pyrrolidinone, but did not avoid spider-silk when it was stripped of the chemical.

The spider-scientists wondered if the ant-repellent would lose its potency with time. So they repeated the experiment with 15-day-old and 1-month-old repellent. Ants avoided the old chemical too.

Importantly, the ant-repellent compound was found in the webs of all 21 adult and large-juvenile spiders, but none of the 20 small juveniles. The authors wondered whether the thin webs spun by small juvenile spiders were even accessible to ants, since the silk of the smallest spiders is four to seven times thinner than that of adults.

So they tried the spider-silk-bridge experiment again, this time varying bridge-thickness, and found that ants of all three species would not walk across webs of small spiders.

The fact that the ant-repellent is only produced by larger spiders suggests that it has evolved in response to ants, and is not a necessary part of web-building. But it is possible the smallest ants are just immature and not yet able to produce it.

So the solution of one insect mystery has opened up a new one: did ant repellent evolve in the spider’s thread as an ant-specific weapon, or is it an accidental by-product of web-building that happens to ward off ants?

Spider scientists need to scour the webs of the world’s 39,000 spider species to look for clues. The question is: is 2-pyrrolidinone produced only in the webs of spiders who live alongside ants and whose silk is thick enough to make ants a threat? says Mark Elgar, the Australian biologist who proposed the theory with the Singaporeans.

Hawaii might be a good place to start looking, suggests Jessica Garb, an expert on spider silk and venoms at the University of Massachusetts, Lowell, who has worked in Hawaii. Hawaii has many orb-web spiders but no native ants. “I’m not super convinced by the idea [that spiders produce ant-repellent] to the degree they are threatened by ants,” she says, but the theory could be tested by looking at Hawaiian spiders, who have evolved in a world with no ants. “Invasive ants are a big problem for Hawaiian orb-web spiders,” she adds, potentially because their silk lacks the defenses of their Singaporean cousins.

Not all who know about spider-silk structure are persuaded by the “silk-thickness” idea. Randy Lewis, a spider-silk expert at Utah State University who has produced thirty-six artificial spider silks, is skeptical. “I find it unlikely that even the smallest webs would not support an ant,” he explains. “Spider-web is highly over-engineered. It has to absorb movement of prey that fly into the web. Weight is not the limiting factor.”

Garb, the venom expert, adds that 2-pyrrolidinone is not the first chemical messenger discovered in spider-web glue. Female orb-web spiders, who are twenty times larger than males, put chemicals in their silk which attract males, even when there is no female on the web.

This new spider-web-pheromone, however, is novel in how it seems to speak to ants.