New Method Spots Gender-Bending Minnows

Exposure to endocrine-disrupting chemicals causes some fish species to look and behave like the opposite sex: Genetic males lay eggs, and females develop male gonads. Now researchers from the Environmental Protection Agency have designed a method to discriminate true male and female minnows from their gender-bending look-alikes (Environ. Sci. Technol., DOI: 10.1021/es103327r).

Endocrine-disrupting pollutants enter waterways from wastewater treatment plants and agricultural runoff. Endocrine disruptors, which include synthetic estrogens and androgens, mimic natural hormones in susceptible fish, sometimes turning on sex-related genes. Scientists worry that skewing the natural sex ratios could disrupt the health and population size of aquatic organisms.

To test for chemicals' endocrine activity, researchers often expose larvae of the fathead minnow (Pimephales promelas) to the chemicals and monitor their effects. They count apparent males and females, and then determine whether the male-to-female ratio deviates from the normal 50:50. But this approach requires large numbers of minnows to detect small changes in sex ratios.

Until now, researchers couldn't tell the difference between true females and feminized males, or between true males and masculinized females. "They look the same," says Allen Olmstead, a toxicologist at the EPA Mid-Continent Ecology Division in Duluth, Minnesota. And genetics was no help. Unlike human X and Y chromosomes, the sex chromosomes of minnows appear the same under the microscope. Specific sex-determining genes are unknown because scientists haven't sequenced the minnow genome.

So Olmstead and colleagues wanted to find a simple genetic marker that would report each fish's real sex. The team analyzed DNA sequences called amplified fragment length polymorphisms (AFLPs) in male and female offspring of a pair of fathead minnows bred in the EPA laboratory.

The researchers used restriction enzymes and the polymerase chain reaction to pinpoint and amplify AFLPs that were present exclusively in one gender or the other. They detected one of these sex-linked markers in all 56 male minnows tested but in none of their female siblings. However, the accuracy of this AFLP marker fell to 97% for other laboratory minnows that were not offspring of the same breeding pair.

Unfortunately, when they tested the marker on fish bred in other labs, they couldn't reliably identify the sex. Also, because wild minnows are genetically distinct from those bred in any laboratory, the sex-linked marker Olmstead identified probably couldn't be used to monitor endocrine-disrupting chemicals in the environment, he says.

Still, David Norris, an environmental endocrinologist at the University of Colorado, Boulder, is enthusiastic. "This new ability to determine genetic sex is very exciting to those of us who have been frustrated by the inability to do so," he says. The only downside is that researchers would need to identify a new marker for each population of minnows they examine, he says. Nevertheless, Norris predicts that the method will be an "excellent research tool." Olmstead agrees: "We've already incorporated this method into toxicity testing of an endocrine disruptor."