Wildlife population geneticists are increasingly using non-invasive sampling approaches to acquire data from their species of interest. Such approaches have proven to be useful to investigate questions related to animal behavior, population, demography and genetics, especially in rare and elusive animals, but remotely collecting biological materials such as hair researchers can study animals without disturbing, harming, or even observing their species of interest. While these techniques have mainly been applied to medium-sized to large carnivores such as grizzly bears, less effort has been focused on smaller herbivorous mammals to bridge this gap.
Novel, innovative approaches to sample small mammals are being developed. This video article describes a new and innovative way to unobtrusively obtain hair samples from small lagomorphs called American Pika. These small mammals are found throughout North American mountain ranges from New Mexico and California to central British Columbia.
They inhabit rocky slides called Tala Slopes, which provide shelter from predation as well as protection from environmental extremes. They're usually found at elevations exceeding 2, 500 meters above sea level in the southern part of their range, but may also be found at sea level in the northern most part of their distribution. Pikas do not hibernate.
They're active throughout the day in the summer, but concentrate their activity to mornings and evenings. When temperatures are cooler, they venture into meadows adjacent to the Tali slope to forge on neighboring vegetation. One particularity of the PIKA is that it doesn't just eat its food in the open.
It takes it back to its territory in the Tallis and stores it in hay piles. While the species is easily detectable, it is shy in nature and will often avoid live traps laid out by researchers. Furthermore, pikas have been found to be sensitive to handling, which may result in the death of the animal in order to study a range of ecological, behavioral, and evolutionary questions.
In this elusive small mammal while also minimizing impact, doctoral candidate Philippe Henry of the University of British Columbia's Okanagan campus has developed a non-invasive sampling technique that is well suited to the species. Before setting a trap, he seeks out evidence of active PIKA territories in the Tallis slope. This includes hay piles, which are vegetation caches that the animal collects in late summer for witcher survival and fresh scat.
That is usually found in specific lare sites. Once a potential peak of habitat is identified within the tlu, a hair snare is strategically placed over its entrance. Using a roll of packing tape, Philippe skillfully rolls strips of tape and arranges them in a web-like fashion to envelop.
The entrance of the hideaway traps are made in intricate designs that provide a hole that is not too big or too small for the animal to fit through. Hypothetically, a pika will then pass through the snare, leaving behind a sample of its hair while continuing its daily tasks.Undisturbed. The hair itself is very thin with a tiny root bulb.
In order to obtain a decent sample for analysis, a minimum of 25 hairs belonging to the same individual is desired. The impact locations must also be distinct along the tape. Any that are clustered independently are assumed to be separate individuals.
While those clustered together are assumed to be one and the same, the collected hair can then be carefully removed from the tape with sterile forceps without disturbing the structure of the sample, especially the root bulb that contains the highest concentration of nuclear DNA. Subsequently, the hair is carefully placed in a two milliliter cryogenic tube and stored in a liquid nitrogen doer until the sample reaches a laboratory for further analysis. Once in the laboratory, the hair sample is spun down in a micro centrifuge to avoid loss of hair opening the tube DNA is an extracted from the hair samples using the Promega DNA IQ system, the first step involves preparing the solution containing 100 microliters of incubation buffer, 1.8 milligrams per milliliter of proteinase K and 0.1 molar of DTT.
Next 100 microliters of the incubation solution is added to the hair and incubated at 56 degrees Celsius for one hour. The sample is then processed with 200 microliters of lysis buffer containing 0.01 molar of DTT and seven microliters of resuspended DNA IQ resin. The sample is then vortex at high speed for three seconds and incubated at room temperature for five to 10 minutes.
The digested sample is then vortex for two seconds at high speed and placed in a magnetic separation stent. At this point, the magnetic resin containing DNA fragments is instantly separated from the solution and formed into a pellet at the bottom of the tube. The remaining solution is then removed and discarded.
Next, the sample is treated again with 100 microliters of lysis buffer vortex and returned to the magnetic stand where the separation occurs. Again, this same step is then repeated three more times using a wash buffer. After the final wash, the sample is left to dry in the magnetic sand for 15 minutes.
Then 100 microliters of elution buffers added to the sample and incubated at 65 degrees Celsius for five minutes. Finally, the sample is vortex for two seconds and placed in the magnetic stand. The DNA solution is then transferred to a 1.5 milliliter einor tube and stored at negative 20 degrees Celsius.
Until further analysis, My name's Peter Bohi. I'm a PhD candidate in Dr.Michael Rosso's Ecological and Conservation Genomics lab at the University of British Columbia Okanagan campus. The DNA extracted from the hair we sampled this summer was then used to PCR, amplify various types of molecular markers.
We also used liver samples as a positive control and basis. For comparison of our method, we amplified nuclear microsatellite loci that will be used to study population, genetic structure in our animal, as well as A FLP genomic scans that will be used to study a genetic basis adaptation along an altitudinal gradient. We also amplified mitochondrial DNA sequences up to 800 base bears that can be used to study the phylo geography of the species, and we apply the molecular sexing tool based on zinc finger sequences and P-C-R-R-F-L-P in order to identify the sex of the individuals in our populations.
Across all markers tested DNA extracted from hair and liver samples produced comparable levels of DNA sequence and genotype quality. In this video, we demonstrated that this non-invasive sampling method, coupled with the DNA IQ extraction protocol is effective, that obtaining adequate amounts of hair that yields DNA of sufficient quality and quantity for PCR amplification. Overall, we anticipate that this method will be useful in facilitating data collection in support of population, genetic and behavioral studies of rare or elusive small mammals while minimizing impacts on the species under study.
