Something is changing the sex of Costa Rican crocodiles

If you want to know whether a crocodile is a male or a female, you have to catch it. Don't bring your good shoes. "It's a muddy, wet mess," says Chris Murray, who spent three dry seasons in and near Palo Verde National Park in Costa Rica, capturing American crocodiles (Crocodylus acutus) and determining their sex with a revolving team of helpers.

Even at night the heat is smothering, and a halo of bugs swirls around headlamps as the team motorboats down waterways or stalks the animals from shore. When Murray and his colleagues spot a croc, often half-submerged, they wade in after it or pursue it in the boat. In a typical catch, his friend Mike Easter uses a noose on a pole to snare the animal, which can be 2 meters long or more. As the croc thrashes and spins, Murray says, "everyone yells a bunch of stuff." Once it calms down a bit, they cover its eyes with a towel to reduce stress, close its jaw with tape, and lug it to the bank, stumbling through shoe-stealing mud.

With the animal restrained, telling its sex is straightforward, says Murray, who is a physiological ecologist at Tennessee Technological University in Cookeville. "You have to put a finger in its cloaca," the cavity at the base of the tail. "If there's a structure there, it's a boy. If there isn't a structure, it's a girl." Catching baby crocs is easier, but sexing them is trickier, so the researchers bring the animals back to the park's research station. Both sexes sport a tiny, red nub in the cloaca, but in males it tends to be longer, redder, and more complex, with an extra lobe.

After probing and peering at the genitalia of nearly 500 crocodiles in Palo Verde, Murray and his colleagues found something odd: The sex ratio was way out of whack, with males outnumbering females four to one among hatchling crocs. What's more, the animals' tissues were tainted with a synthetic steroid, which the researchers suspect was causing them to switch sex.

The hormone, 17α-methyltestosterone (MT), is sometimes prescribed for men with testosterone deficiencies and older women with breast cancer. Bodybuilders have been known to abuse it to bulk up. How could it end up in crocodiles from rural Costa Rica? A possible clue: Fish farms around the park raise tilapia on food laced with the hormone, which transforms females into faster-growing and more profitable males. Murray and his colleagues are now investigating whether MT from the farms has somehow contaminated the crocs.

The finding "has implications for the population and the broader ecological community," says physiologist Matthew Milnes of Mars Hill University in North Carolina, who was not connected to the research. Besides skewing the sex ratio, the hormone could be disrupting the animals' reproduction, a concern because American crocodiles are already listed as vulnerable, and this part of Costa Rica is a stronghold for the species. The contamination could also be altering the crocs' behavior, perhaps making them more belligerent. If so, conflict with humans—which Murray says is already a sore spot in Costa Rica—could increase. The substance could harm turtles, birds, fish, and other aquatic creatures as well. And because fish farms throughout the tropics are feeding chow that contains MT to their tilapia, the hormone may be causing problems elsewhere.

For more than 20 years, researchers have fretted about the effects of endocrine disruptors, molecules that meddle with the body's hormones. Crocodilians—the group that includes crocs and alligators—have furnished some of the most dramatic examples. In the 1990s, for instance, scientists reported that male alligators from Florida's Lake Apopka, which was fouled by a brew of hormone-mimicking chemicals, had shrunken genitalia and reduced testosterone levels.

Like many endocrine disruptors, those chemicals triggered the same effects as estrogens, or female sex hormones. Researchers have uncovered only a few cases of the opposite problem, masculinization caused by male hormones, or androgens. Molecular biologist Elizabeth Wilson of the University of North Carolina in Chapel Hill says that whereas "there are lots and lots of compounds that will activate the estrogen receptor," the cellular receptors that respond to androgens are choosy.

Of the few known environmental androgens, trenbolone acetate, a synthetic steroid implanted into cattle to speed their growth, has sparked the most concern. Studies found that a derivative excreted by juiced cattle reduces minnows' fertility, transforms female zebrafish into males, and induces other masculinizing effects. MT, however, "was not on the radar as an endocrine disruptor," says Christopher Martyniuk, an endocrinologist and toxicologist at the University of Florida (UF) in Gainesville.

When Murray arrived in Palo Verde in 2012 as a graduate student from Auburn University in Alabama, he was scouting for a juicy dissertation project. "You will work on crocodiles," said Mahmood Sasa of the University of Costa Rica, head of the research station in the park. He offered a topic ripe for further investigation: a recent study that claimed a three-to-one male-to-female sex ratio among the crocodiles in the area.

To test the claim, Murray teamed up with Easter, Sasa, and others to nab as many of the animals as possible and figure out what was going on. After identifying the sex of 474 crocs from seven sites, they discovered that the population was even more male biased than the previous study had indicated, with about 3.5 males for every female. The disparity held across ages. Males constituted almost 80% of hatchlings and 60% of the adults, the researchers reported in 2015.

Tracking the culprit Chris Murray (right) and Michael Easter (left) draw blood from a crocodile to test for 17α-methyltestosterone. They have found the hormone in animals from Palo Verde National Park and the Tárcoles River, which may explain the park's excess males. CREDITS: (MAP) G. GRULLÓN/SCIENCE; (DATA) C. MURRAY ET AL., JOURNAL OF THERMAL BIOLOGY 60, 11 JUNE 2016 © 2016 ELSEVIER LTD.; C. MURRAY ET AL., COPEIA 103, 3, 24 JUNE 2015; (PHOTO) SERGIO PADILLA

The numbers are even more startling because a warming climate should be pushing croc sex ratios in the other direction. Unlike humans, crocodiles and alligators don't have sex chromosomes. Instead, whether an embryo becomes a male or a female depends on the nest temperature during incubation. The mean low temperature in Palo Verde has risen about 2.5°C in less than 20 years. To gauge what impact the increase should have had, Murray and his co-workers stashed temperature recorders inside plastic eggs and buried them in 25 croc nests. The team estimated, based on nest temperatures, that female hatchlings should outnumber males by nearly two to one, they reported last year. Something must be overriding the temperature effect, they concluded.

The researchers had heard that several tilapia farms around the park used MT, and they wondered whether it could bias the animals' sexual development. To test the idea, they dabbed three different concentrations of the hormone on American alligator (Alligator mississippiensis) eggs, which served as stand-ins for crocodile eggs. They then incubated the eggs at temperatures that should yield only females. About 60% of the eggs dosed with the two highest MT levels developed into males. "MT does have a masculinizing effect" on crocodilians, Murray says.

In April, another piece of the puzzle fell into place. The group reported finding the chemical in samples of blood and egg yolk from the Palo Verde crocs, confirming that they had been exposed to it. Contrary to earlier assumptions, "the hormone is not biodegrading under some field conditions," says Jeffrey McCrary, an environmental scientist at the National Autonomous University of Nicaragua in Managua.

Tilapia raised on MT-containing chow are safe for people to eat, regulatory agencies have concluded based on other studies. But Wilson says the evidence that MT is loose in the environment is worrying. "I can't tell you it's a human hazard," she says, but "low levels of androgens can be detrimental" in pregnant women.

The source of the hormone remains the big question. "MT does not occur normally in the environment," says reproductive toxicologist L. Earl Gray of the U.S. Environmental Protection Agency, who is currently an adjunct professor at North Carolina State University (NC State) in Raleigh. "It can only occur in the environment from human activity." Nearby tilapia farms are a natural suspect, Murray says, because they could provide an aquatic source of the hormone, although just how it might reach the crocs isn't clear. The animals occasionally slip into the farms and help themselves to a meal. But all of the crocs tested in the Palo Verde area harbored MT, and "they couldn't have all been to a fish farm," Murray says. Instead, he and his colleagues suspect that tilapia escape from the farms and get eaten by crocs, which absorb the MT and store it in fat. When females produce eggs, they pass the hormone on to their offspring.

Frank Chapman, an ecologist at UF who has worked with the tilapia industry in Latin America, calls the suggestion that MT originates on the farms "plausible." Kevin Fitzsimmons, a fisheries biologist at the University of Arizona in Tucson who has consulted with tilapia farms around the world, says it's unlikely but possible, "especially if [MT] was mishandled or accidentally released."

Murray acknowledges that he and his colleagues don't have any direct evidence implicating the farms, and they are investigating other potential sources. The compound has turned up far from Palo Verde, in blood and yolk from a croc population more than 100 kilometers away in the Tárcoles River, part of a different river system. Contaminated crocs from Palo Verde may be migrating to the Tárcoles area, Murray says. But MT could also be entering the environment through other routes, possibly in pollution that originates upstream in the country's capital, San José. Because people are taking the hormone, whether legally or illegally, it could enter the city's sewage. Fitzsimmons says that bodybuilders use so much MT that they are more likely than tilapia farms to be causing the contamination there and in the park.

If the farms are the source, however, he and Chapman say the problem is solvable. Fitzsimmons suggests that the farms may not be properly disposing of containers that held MT—an easy fix. Chapman notes that the finely ground feed prevalent in Latin America tends to accumulate at the bottom of tilapia ponds, where it can get swept into surrounding waterways or accidentally swallowed by crocs or other animals that enter the ponds. Producing larger pellets like those available in the United States could increase the chances that the food will be eaten before the hormone leaks into the environment, he says. Chapman predicts that if the farms are at fault, the industry will be willing to take steps to protect the crocs. "This kind of thing gives aquaculture a black eye."

Not all scientists think the case against MT adds up. Environmental and endocrine toxicologist Gerald LeBlanc of NC State notes that female and male crocs harbor the same levels of MT, suggesting it may not be the cause of the male excess. "The information is just too weak at this time to say, ‘Yes, this is the cause,’" he says.

But other researchers think Murray and colleagues are on to something, and are eager for data on just how MT affects the crocs. A key question, says Milnes, is, "are they fully functional males?" A population overburdened with males isn't necessarily at risk, Martyniuk says, but the animals' numbers could dwindle if MT hinders their reproduction. Murray's lab is now studying alligators to find out whether early exposure to MT changes the number of androgen receptors in their brains and makes them more aggressive. He and his colleagues also hope to examine troublemaker crocs that have been euthanized near the park to find out whether the animals are fertile.

In future work, the team wants to determine whether MT is having an impact on crocodilians elsewhere. In the United States, where use of the hormone-laced tilapia food is limited, "We see no evidence of MT-related problems," Murray says. But tilapia are raised in more than 80 countries, and in many of them, crocodilians live alongside farms that dole out MT-containing chow. He and his colleagues have already collected blood samples from other sites in Costa Rica, and they plan to check for altered sex ratios and MT contamination among crocs in Indonesia and South Africa as well.

For Murray, more croc-wrangling lies ahead.