Technology Solutions
The genetics behind environmental stress The U.S. EPA is investigating several DNA-based technologies for assessing the genetic diversity of animal and plant populations in response to ecological stresses. Proponents claim that the technologies can overcome some limitations of the methods currently used to evaluate ecosystem health. None of these technologies, which include allozymes, amplified fragment length polymorphisms (AFLPs), microsatellites, and random amplified polymorphic DNA (RAPD), is that new, but the use of most of mem for evaluating ecological health is fairly new, says Sheldon Guttman, professor of zoology at Miami University of Ohio. He is one of the researchers who has received funding from EPA's Science To Achieve Results (STAR) pro-
gram to evaluate the new technologies. All are sufficiendy affordable to be considered for use by EPA, as well as other agencies that conduct exposure assessment and monitoring programs such as the National Oceanic and Atmospheric Administration and the U.S. Geological Survey, according to Guttman. "Genetic indicators are cuttingedge science and may be the wave of the future," says Barbara Levinson, administrator of EPA's STAR grants. EPA currendy measures ecosystem health with two indices, the Index of Biological Integrity (IBI) and the Invertebrate Community Index (ICI). Both are determined by the presence (or absence) of animal species. "The problem with IBI and ICI, which have been used since the 1980s, is that you can calculate these Genetic diversity decreases as index values and say that an environment is pollution increases healthy, but it may be The RAPD profiles of crayfish collected from Lima, that the populations are OH, compare animals from a polluted site (the six genetically uniform," bands on the left side) with a population living in a Guttman explains. "We're pristine area. The blue bars highlight potentially proposing the use of significant PCR amplification products. analysis of genetic structure as an earlier warning of possible environmental damage. The preponderance of evidence suggests that if populations lose genetic diversity, they may be on the brink of extinction so that further perturbations like global warming or increased ultraviolet light
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due to the thinning ozone lciver C3u.sc them to be eliminated " All of the molecularbased techniques wing to redace the IBI and ICI rely nnon the Harriv— Weinberg principle of population genetics ex-
A • NOVEMBER 1, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY / NEWS
plains Dan Krane, an associate professor of biological sciences at Wright State University. That principle holds that the relative frequencies of different genes in a large population of complex organisms will not change unless new members of that species migrate into the area or the population is subjected to stresses, such as pollution, he says. Since the 1960s, Guttman has been studying how allozymes, structural variants of enzymes that can be detected by gel electrophoresis, can be used to assess the genetic diversity of populations. Allozymes were responsible for the first evidence that the genetic diversity of populations could be affected by exposure to pollution, he says. Together with Jim Oris, also of Miami University's zoology department, Guttman is using a STAR grant to determine what the best DNAbased ecological indicator may be. They are studying the genetic diversity of one fish, the Lahontan redside shiner, and one invertebrate, the signal crayfish, in Lake Tahoe, CA, and 13 nearby lakes in California and Nevada within three molecularbased methods: allozymes, microsatellites, and RAPD. "It's not clear that one [of these methods] is advantageous over the other," Oris says. The advantage of allozymes over DNA-based methods is that they have the potential to illuminate genetic variations that can confer a selective advantage when organisms are exposed to environmental stressors such as pollutants, Guttman says. But the problem with allozymes is that for a given population, only a relatively small number of the enzymes investigated may exhibit genetic variation—20 at best, and often less than 10, he explains. The advantage of microsatellites, stretches of DNA containing repeats of two, four, or six base pairs of nucleotides that can be analyzed using polymerase chain reaction (PCR) techniques, is that the method can locate © 2000 American Chemical Society
many more potential genetic variations than allozymes, Guttman says. It is not atypical to find 50-100 different mutations of a given gene in a study population, he says. AFLPs can bring to light a similar number of mutations by detecting DNA fragments through PCR amplification, Guttman says. Susan Anderson of University of California-Davis's Bodega Bay Marine Lab has a STAR grant to compare AFLPs and RAPDs as ecological indicators. RAPD techniques, which involve comparing PCR-amplified products of short, 10-nucleotide primers from different organisms, can also illuminate more variability in a population than allozymes, Guttman says. "You get a much faster payoff with RAPD PCR than allozymes," says Krane, who is using a STAR grant to study how the RAPD technique can be used to identify "sentinel species" for indicating ecological health. The Pacific herring has the potential to be a broad-based ecological indicator, Krane says. RAPD techniques also have the advantage of being less expensive than allozymes, easier to use than AFLPs, and easier to develop than microsatellites, according to scientists from the United Kingdom's University of Oxford (i). But there is some disagreement as to whether RAPD results are reproducible and heritable, Guttman says. And the technology has been faulted for failing to divulge what makes a given population diverse, he says. Because they require only a small amount of tissue, such as a swab from a fish's gill or a pill bug's leg, all of these molecular techniques can reduce the number of organisms used to conduct exposure assessments, the scientists interviewed for this piece agree. For all their promise, it remains to be seen whether any of these new technologies will ultimately be adopted, Levinson stresses. They need to be proven reliable, functional, and cost-effective for the state monitoring agencies, she says. —KELLYN S. BETTS (1) Gillet, E. M. Ed.; Which DNA Marker for Which Purpose? Molecular Tools for Biodiversity; Final C o m p e n d i u m of t h e Research Project Development, Optimisation a n d Validation of Molecular Tools for Assessment of Biodiversity in Forest Trees in t h e E u r o p e a n Union, DGXII Biotechnology FW IV Research P r o g r a m m e Molecular Tools for Biodiversity; Gottingen, Germany, 1999.
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