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! ~TaylorWhat difference can one person really make in the fight against climate change? More than you might think! This week, leaders around the globe are meeting to address this very question at the United Nations Climate Change Conference. Global leaders and politicians from 190 countries have met in Paris to create a global climate treaty that will hold leaders responsible for halting the exponential warming trend and keep global temperatures below 2 degrees celsius. This conference will have an impact over the next several years on the amount of carbon emitted, and ultimately will require basic lifestyle changes regarding the use of clean energy. The impact of these efforts is promising, and may impact conservation efforts. These treaties directly intersect with the science of our laboratory. Specifically, salamander physiology, as global warming is an impending stressor for plethodontid species. Amphibian skin is vascularized and permeable, acting as an osmoregulatory and respiratory organ. In fact, he skin of a plethodontid salamanders serves as lungs, dictating survival through the exchange of gases across its skin. When temperatures rise, the ability of plethodontid salamanders to maintain the moist skin they need to breathe may be impaired. As a result, precipitation, humidity, and soil moisture, all influence water availability and can influence behavior and survival. Changes in temperature and precipitation influence the physiology, behavior, and ecology of these salamanders. Given the exponential trend of global temperatures, this lungless species could be at risk of future endangerment and range contractions. While this global meeting may not influence salamanders at a microhabitat level, the treaties and policies formed by our global leaders could drastically increase the use of clean energy and put a stop to warming trends. For more information about the Climate Change Summit or to keep updated on treaty progress, visit http://www.cop21.gouv.fr/en/ ~Molly Nielsen In the spring of 2014, I traveled to India to observe tigers at the Khana and Pench Tiger Reserves. Before this trip, tigers were just these large, striped cats that also happened to be the mascot of my school and favorite football team. But I soon gained an appreciation of just how incredible these animals truly are. Around the world, the tiger has been one of the most popular charismatic megafauna in ancient mythology and folklore and are often seen in modern media as well. Weighing around 850lbs, the tiger is the largest cat species on the planet. That’s bigger than 80 house cats combined! Not only are they large, they are also excellent nocturnal hunters. They feed mostly on deer or other ungulates that can weigh up to 200lbs and occasionally they eat other animals including wild buffalo, water buffalo, and wild boar. The tiger quickly became my favorite animal for all of these reasons, but I think the stripes are what make the tiger stand out. The TIGER is large, majestic, and an apex predator. Who wouldn’t fear the stripes? When I started this CI I became familiar with a different tiger! Way across the world, another striped animal has made a huge impact in science. The tiger salamander, also known as Ambystoma tigrinum, are useful organisms to study interactions between physiology and the environment. They are typically found in wetlands like marshes, lakes, and ponds and they can grow up to 14 inches in some cases. Just like the mammalian tiger, they are good nocturnal hunters. They eat insects, worms, and some adults may also consume small mice or frogs making tiger salamanders feistier than their close relatives A bit of a jump, I know, but what makes these two species similar? They seem completely different in almost every aspect. One is large and the other is small, one is a mammal the other is an amphibian, and they live in completely different areas of the world. However, both are very important organisms in their respective environments. They are both hardy, long-lived and in my opinion have earned their stripes!
~Michael Baker As college students, we are very familiar with attempting to manage copious amounts of stress. From studying, to taking exams, to finding time to eat lunch, it seems as though we can never catch a break. However, we are definitely not the only animals that experience this horrible feeling. In fact, the mechanisms behind stress responses are similar among all vertebrates. The Human Stress Response It is Sunday night. You have a lab report due tomorrow, a Biology exam on Tuesday, and a research paper for English due Thursday. You are stressed to the max. On the outside, you try your best to stay cool, calm, and collected, but on the inside, hormones are surging. The hypothalamic-pituitary-adrenal (HPA) axis has been activated, signaling neurons in the hypothalamus to release corticotropin-releasing hormone (CRH). Another hormone, adrenocorticotropin (ACTH), is released from the pituitary gland into the bloodstream, where it reaches the adrenal glands that are located above the kidneys. This triggers the secretion of “stress hormones”: cortisol and the catecholamines (epinephrine and norepinephrine). These hormones signal the fight-or-flight response, in which we experience the all-too-familiar increase in heart rate and blood pressure. It is possible to quantify amounts of these hormones using blood, urine, and saliva. Salamanders get stressed out, too!?! Salamanders may not have exams to worry about, but they experience stress regularly. As the most abundant vertebrate group in the southeastern United States, salamanders play an important role in the ecosystem. However, increases in temperature, decreases in moisture levels, predicted habitat loss, and the possibility of disease threaten the wellbeing of salamanders. The ultimate goal of this project is to understand how these stressors affect the physiological stress response of the Southern gray-cheeked salamander, Plethodon metcalfi. When an amphibian is exposed to a stimulus that disrupts homeostasis, such as high temperature, its body reacts by adjusting metabolic rates. This may be a result of the activation of the hypothalamus-pituitary-interrenal (HPI) axis, which signals the release of glucocorticoid molecules and corticosterone. Corticosterone is a stress hormone that circulates through the bloodstream and increases in concentration as the amphibian faces a stressful event. Subsequently, blood glucose concentrations also increase, allowing the amphibian to change its behavior and immune response, and therefore, alleviate the effects of the stressor. So...next time you're stressed out, just remember that you're in very good company. ~Samantha Okay, so last semester didn’t actually involve any tears, but I spent a lot of time looking at salamander blood smears under a microscope, and it wasn’t always easy. The majority of time I spend in lab was dedicated to counting the different cells that I saw. As I peered through the microscope, I identified different types of white blood cells. By counting these cells and looking at differences among the ratios of different cell types we can estimate how stressed a salamander is. However, identifying cells under a microscope after learning what they look like from pictures takes some getting used to. It is complicated to learn how to identify cells under a microscope from a reference pictures because the cells under the microscope don’t necessarily look the same as in a picture. Intracellular variation can be high! Therefore, there is a lot of trial and error in learning to identify cells under a microscope until you get the hang of it. Practice, practice, practice! The types of cells that I identified in the blood smear included erythrocytes (red blood cells) and leukocytes (white blood cells). When looking at slide, the erythrocytes are abundant and easy to identify. It is interesting that they are nucleated in amphibians, which is different than our own red blood cells. Since erythrocytes are easy to spot, the catch is not finding them, but counting them as there can be dozens in one field of view! To identify a lymphocyte I looked for a round agranular cell with the nucleus taking up more of the cytoplasm than an erythrocyte. Neutrophils have multi-lobed nuclei and were easy to spot since they stood out from all the other cells with their amorphous nuclei. Eosinophils had a slightly pinkish color to the cytoplasm when being observed under the microscope and were a little more granulated but also had a lobed nuclei. Basophils are very rare to spot, but they are very dark and granulated. I was able to find monocytes by looking for a nucleus that was less rounded and sometimes had a dimple in it making it appear like a kidney bean (although not always). The picture above is from one of the slides that I counted. The arrow is pointing to an eosinophil and the circle is around a neutrophil. It's not just these easy to identify cells that are on the slides! When counting the slides you occasionally come across thrombocytes, cells that were crushed, or dividing cells. I was careful not to accidentally include them in my count, by mistaking them for something else. I don't count every single cell that I see, only the ones that I am supposed to identify. Some of other cells visible are not relevant to the counts I do. Taking a look at the picture below you can see some of the challenges I encountered with the slides. Just look at that mess! Not every field of view is countable — Imagine trying to figure out how many cells are on that field of view, let alone distinguishing what they are. Spoiler alert, it's not going to happen, but that's ok. I just skipped over these views and moved to more countable ones. When I first started counting slides I thought that I was never going to get the hang of it as it seemed overwhelming and confusing! Luckily, the more time I spent doing it the easier and more natural it became. By the end of the semester I was able to count the slides much faster and with much more confidence! ~Alexa DeMaio You may have learned a little about what makes me tick, but now it is time to meet the Stress After Dark Creative Inquiry Team. This group of undergrads keeps the lab running, while gaining valuable training in the scientific process. We will be hearing more from them in the future, but let's stick to introductions for now.
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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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