Ever wonder how scientists collect much-needed DNA samples from endangered species? Their repertoire of non-invasive sampling methods is growing with leaps and bounds, but one of the most popular techniques involves scooping up their poop. (Um, the animals‘ poop that is.) Except, field biologists call it “scat” which somehow seems more palatable. (One animal’s trash is another animal’s treasure?) But to get DNA from scat requires a gifted touch. Just any old scat lying on the forest floor will not do. It is best fresh. And with a good smear of intestinal mucus. Or so says a new study out in the Journal of Wildlife Management.
Linda Rutledge from the Natural Resources DNA Profiling and Forensic Centre at Trent University set out to test the best way to swab scats in order to get the best DNA samples. Her goal was not an esoteric one, because scientists can easily spend oodles of money on DNA tests on scats they’ve swabbed in the field — with nary a clue for weeks as to whether they are actually capturing the double-stranded genetic material they are after. They must wait for a week or two to get their first samples back from a genetics lab to know with certainty if they are in fact collecting their target DNA, and if the material is quality. Wouldn’t it be better, Rutledge wondered, if there were a more detailed field method for swabbing scats that guaranteed a high return rate of quality, target DNA?
She and her colleagues combed the boreal forest west of Algonquin Provincial Park in Ontario, Canada for the scats of gray wolves (Canis lupus) and eastern wolves (C. l. lycaon). They found 20 using a systematic search, and they opportunistically collected an additional 20 in Chapleau Crown Game Preserve which they determined would have frozen immediately upon their deposit due to the ambient temperatures. They aged the scats qualitatively according to “travel history, scat moisture content, and appearance, and categorized them as ,12 hours, 12–24 hours, 24–36 hours, and >36 hours old.” Next, they rubbed a cotton swab all over the scat, and especially over areas that appeared to have the unique shine of mucus. (Ew, right? But somebody’s gotta do it…) For the frozen scats, the researchers allowed them to thaw back in the lab, and then they swabbed the fecal material. (Methinks swabbing thawed poop was a task assigned to a lowly grad student…)
They found that the freshest, unfrozen, field-swabbed scats yielded the best DNA samples with the most minimal genotyping errors and the lowest rates of “allelic dropout” — which basically means the lowest rate of failure in detecting the presence of an allele within a sample. These swabbed scats were the most likely to provide DNA that could be amplified in the lab using a multiplex reaction, which is a system that allows the researchers to copy and replicate multiple locations of the DNA strand in a single PCR test. Multiplex reactions can save vital study funds because they allow for multiple data points to be gathered in one test — but they only save money if the multiplex reaction actually captures the data from the multiple sites. They found that ten of the field-swabbed samples yielded amplification at all eight mitochondrial locations, whereas only three of the lab swabbed frozen and thawed samples yielded amplification at all eight sites. This has an important implication for someone setting out to do a scat study because if it is possible to collect scats in the warmer summer months, they will have a better chance of being able to do multiplex tests with a higher rate of success, versus sampling and swabbing frozen scats that are later thawed.
In order to test which swabbing method was best for obtaining the highest quality and quantity of DNA from scats, the researchers also amplified a specific region of the X and Y chromosomes (called the zinc intron finger) with contains really long base pairs. Whereas most PCR tests target locations that are 100 to 350 base pairs long, the region they targeted was 965 to 1,094 base pairs long. Successfully amplifying such a long DNA fragment would tell them if there was a difference in the swabbing methods in terms of the quality of the DNA that was collected and the quantity. Despite amplifying this long fragment successfully 12 times, they found only one allelic error, which they attributed to human error. Basically, this test was used to assess the trustworthiness of their method in producing results with low to no errors, and it did so.
Their study provides guidance, and perhaps cost-savings measures, for other scientists seeking to do scat studies. The authors concluded: “The main benefit of our method is that laboratory costs and time are substantially reduced because extraction of high quantity and quality target DNA allows reliable individual 8-locus genotypes to be obtained with only 2 PCRs as opposed to multiple single microsatellite amplifications that are typically required for noninvasive samples with low template.”
The trick of course, is that those fresh scats have to be discovered in less than 36 hours from when their owner deposits them, which increases the time and money spent on field surveillance and collection. One new way that scats are being found with a high reliability is the use of scat-sniffing dogs. (I am not making this up.) Dr. Samuel Wasser at the Center for Conservation Biology in the Pacific Northwest has been pioneering this field over the past decade or so. But more on that later…
NOTES:
Rutledge et al. 2009. Improved genotyping from wolf scat. Journal of Wildlife Management. (73):8, pg. 1430-1435