What if I told you, I could breathe through my skin? While this may seem strange as a human, amphibians do just that! What is cutaneous respiration? Amphibians, like frogs and salamanders, have a specialized way of breathing that drastically contrasts the respiration of humans. They rely on their skin for some of their oxygen intake, and especially for their carbon dioxide output. Blood flowing on the inner surface of the skin runs in a countercurrent fashion, flowing in the opposite direction of the absorbing oxygen or surrounding water. This allows for the diffusion of oxygen molecules across the concentration gradient between the blood vessels and water droplets resting on the outer surface of their skin. How does this affect Plethodontid salamanders? The salamander species that we study, Plethodon metcalfi, uses cutaneous respiration for nearly all of their gas exchange needs. This is a pivotal physiological hurdle these salamanders face because they must maintain moisture on their skin, either by expending energy reserves or by travelling to a moist environment. Plethodon salamanders are commonly seen burrowing in dirt to find moisture! Over an evolutionary time scale, this species may adapt to a dry and warm climates, but the energy required to maintain tolerance to drying out and desiccating may create other physiological constricts! But what about climate? If the air becomes too dry, or too warm, the moisture on a salamander's skin essentially becomes “whisked away” into the atmosphere. As the air warms, it takes in moisture, extracting water molecules from the ground and organisms. This phenomenon is also known as vapor pressure deficit (VPD), which is a pivotal factor affecting cutaneous respiration in the Plethodon species we study. Every decade, the earth seems to warm about 0.1 degrees Fahrenheit more than the prior decade. This climate trend directly impacts VPD by increasing the capacity of water that air can hold, decreasing the amount of moisture available to salamanders. In the coming decades, it will be interesting to continue to research Plethodon physiology as they will face a choice of moving to colder and wetter ground, or develop a desiccation tolerance to warmer climates. -Molly Nielsen
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Since I first got involved in this lab over a year ago in August 2014, I have had many great and rewarding experiences. Not the least of these experienced occurred over the summer when I was able to get hands-on working with salamander fecal samples! Yay! OK, so it’s not as gross as it sounds, as they were dried and frozen samples we had taken earlier in the year. My job was to prepare these samples to be measured for their concentrations of corticosterone (a stress hormone) to cross-check the stress-levels measured through Leukocyte ratios last year. Flashback to anybody's awkward visit to the doctor where they ask for a stool sample. Suddenly I was on the other side of this less-than-pleasant exchange! It turns out that working with salamander stool samples can be more exciting than I thought, however. The main steps involved weighing the samples, adding methanol to extract the corticosterone, and then drying and reconstituting the methanol extractions in a buffer solution before running through a Enzyme-Linked Immunoassay (ELISA). Things looked bleak when, after drying of the first sample took 2.5 hours, we realized that processing the other 300 or so samples would take us over 700 man-hours! Determined to improve this prognosis, we had to come up with a more time-efficient method for processing our samples. So with a goal of finishing the samples by the end of my summer term, we modified our procedure and found ways to reduce our time spent per sample without compromising on the accuracy of the data. One way we “oiled the gears” so to speak, was to aliquot 50 micro-liters to dry rather than attempting to process the entire methanol extraction. This helped us to speed up the drying time to less than 30 minutes each, and another step took that even further. We then set out to create an apparatus that would allow us to dry multiple extractions (up to 8!) at a time by splitting the output of a Nitrogen tank into eight smaller tubes to dry the samples simultaneously. After overcoming this hurdle, we were able to start running and processing the fecal extractions at a much greater pace, and ended up finishing most of them by the end of the summer. Corticosterone MoleculeWhile certainly not the most glamorous experience, I was able to learn a lot about different lab techniques such as ELISA and I got to see the more hands-on side of research where we had to problem-solve to overcome a specific hurdle. In the end we were able to process all 300 or so extractions and are working on processing the data now! ~Taylor |
The Slime Times is written by the Stress After Dark Creative Inquiry TeamScience isn't all white lab coats and ivory pillars, sometimes you just need to get slimy. Archives
August 2016
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