Immunapalooza

Not all of the lab went south this summer; Kristen (one of our awesome lab undergrads) and I stayed at Penn State most of the summer, working on immune assays.  Kristen was the recipient of the Erickson Discovery Grant, and spent much of her summer on an independent research project, which involved measuring the effects of corticosterone (CORT) on the cell-mediated immunity (i.e. one way the body responds to a toxic or foreign substance) of eastern fence lizard females. She was also trying to determine if the lizards’ life history (whether they were from sites with or without fire ants) affected their immune function or interacted with the CORT treatment. Kristen just recently gave an excellent talk on her research at the Three Rivers Evolution Event (TREE) on Sept. 9th, where she was one of the only undergraduates to present a talk.

Using a dial caliper to measure the foot thickness of a lizard.

We also spent a lot of time this summer developing, improving, and validating several different immune assays for use in fence lizards, including ELISA assays for measuring anti-fire ant antibodies (IgY and IgM), complement function, natural antibodies, and the activity levels of heterophils (a type of immune cell that kills bacteria). Work on the assays for IgY, complement function, and natural antibodies is ongoing, but the IgM and heterophil activity assays are ready to be used.

The IgM ELISA assay was developed to work with as little as 10μl of plasma, and accurately detected anti-fire ant antibodies in a pool of plasma of lizards from Alabama, where the lizards are regularly exposed to fire ants. It did not detect any antibodies in a pool of plasma of lizards from Tennessee, at sites which have not yet been invaded by fire ants. The next step is to test the plasma of individual lizards from different sites, to see what proportion of lizards in various invaded sites have actually developed IgM antibodies to fire ants. Once the IgY assay is working, we should be able to better characterize the antibody response of the lizards to fire ants, and see if this helps them recover faster from fire ant stings.

The higher the proportion of plasma from invaded (Alabama) lizards, the higher the signal from the IgM antibody.

Our heterophil activity assay is based off the assay described in Merchant, Williams, and Hardy (2009) for use in American alligators. To account for the much smaller blood volume of fence lizards, I altered the assay to work with 10μl of whole blood, and validated it in this species. This assay specifically tests for the presence of superoxide radicals, which are produced by heterophils as part of the oxidative burst used to kill bacteria and other organisms. When heterophils are more active (either because there are more heterophils or because the existing heterophils have been stimulated by something), the amount of superoxide in the blood increases. As part of the validation, we ran the assay with pools of blood treated with superoxide dismutase, which destroys superoxide, to test that the signal is actually caused by superoxide. We also ran blood with and without a stimulant of heterophil function, to determine if the signal reliably increases when heterophils are more active. The signal reliably decreases when inhibited by superoxide dismutase, and reliably increases when stimulant is added, indicating that this is a reliable test of heterophil function.

Inhibition of superoxide signal by superoxide dismutase.

Stimulation of heterophil function by bacterial stimulant.

We also did a little bit of work optimizing the natural antibody test, increasing the sensitivity of the test so that it will work with less lizard plasma. And we also found a promising lead for testing alternative pathway complement function in fence lizards.

Aside from all the immunology work, we also got out into the field up here in Pennsylvania a little bit, although we didn’t find many lizards. All in all, it was a fun, productive summer.

Thousand Steps has a lot of steps

Kristen found her first lizard!

Basking Site Use by Timber Rattlesnake Morphotypes – By Shawn Snyder

My name is Shawn Snyder and I am currently a senior majoring in Wildlife and Fisheries Science.  This is my first and only year working in the Langkilde Lab.  During the summer of 2016, I worked under Dr. Chris Howey as a Research Technician studying the effects of prescribed fire on timber rattlesnake populations.  This position provided me the opportunity to radio-track timber rattlesnakes, record habitat data on tracked snakes, catch new snakes (extremely fun), learn how to safely tube a venomous snake (even more fun), and conduct vegetation surveys.  Also, this position provided me the opportunity to formulate my own scientific question to test! Together, Chris and I thought up a small side-project that I could conduct throughout the summer, which provided me the fantastic experience of going through the scientific process, collecting my own data, analyzing those data, and now writing a manuscript so that I can share those results with the scientific world.

When we first started collecting data for my side-project I was a little apprehensive.  Once the data was collected and analyzed I realized that this project was going to take time and a large amount of effort to complete.  As the process of analyzing the data and then coming up with a plan for the manuscript began to take shape, I started to feel challenged and nervous by this new task. But weekly meetings with Chris to discuss the process of writing a manuscript have helped immensely.  This is my first manuscript and yes it is challenging, but it will all be worth it once we have a finished product. I have ambitions to continue on to a Graduate program after I graduate and this manuscript will help me build my C.V. to apply to Grad schools.

Two yellow morphs bask alongside three black morph timber rattlesnakes at a gestation site. Although we did not use gestating (i.e., pregnant) females as part of this project, this shows you the posture of a basking snake and the difference in color morphs.

My research is investigating if the two distinct morphotypes of timber rattlesnakes (a dark, black morph and a lighter, yellow morph; see above picture) use basking habitat with differing amounts of canopy openness and solar radiation. Previous research suggests that the dark morph evolved in response to thermal limitations in the northern parts of its range.  Darker snakes have more melanin in their skin, which allows them to absorb more solar radiation and maintain a higher body temperature than yellow morphs.  Yellow morphs having this thermal disadvantage, in theory would have to choose basking sites that receive more solar radiation to compensate for this limitation if they wanted to maintain a similar body temperature to the black morphs.  Specifically, I am testing the hypothesis that yellow morphs use basking habitat that has more canopy openness and receives more direct solar radiation (i.e., sun) than basking habitat used by black morphs.

 

A black morph male timber rattlesnake is seen courting a basking yellow morph female.  Once again, the difference in color morphs is striking and has led many to ask what selective pressures are maintaining this polymorphism.

To test this hypothesis, I measured canopy openness over basking yellow and black morphs. I used the timber rattlesnakes that are being radio-tracked for Dr. Howey’s main study as my sample population and placed a flag where a snake was found exhibiting basking behaviors (see picture below  for example).  We took a picture facing skyward directly over the snake using a camera with a fisheye lens.  This lens takes a picture of 180 degrees and captures an image of all of the canopy over the snake (see picture).  We can then analyze these hemispherical photographs using a computer program called Gap Light Analyzer to measure the percent canopy openness and the amount of direct solar radiation transmittance (i.e., rays of sunlight) for each basking site.  Direct solar radiation is when the sunlight reaches the forest floor with no obstructions from the canopy; as opposed to indirect solar radiation which may be radiation that is being reflected off of clouds, trees, or the ground itself.  Our study site is characterized as having a mature Oak/Maple forest with an abundance of closed canopy throughout the area.  Both morphotypes use this “closed canopy” forest throughout the summer as foraging grounds, and when they need to bask they must seek out areas where some sunlight is making its way through the canopy.  This is where my question becomes very important comparing the habitat used by each morph.

 

A flag is placed next to a basking yellow morph.  An exact description of the habitat is recorded so that I can come back at a later time (when the snake is not there) and take a photo of the canopy directly over where the snake had been.

Two examples of hemispherical photographs taken over two different basking timber rattlesnakes.  Both canopies actually have similar canopy openness, but the canopy on the left receives far more direct solar radiation based on the placement of those canopy openings.

So far, my results show that the two morphs use habitat that have similar percent canopy openness, however, there was a difference in the amount of UV transmittance between the basking sites used by the two morphs.  Canopy openness doesn’t necessarily designate a “warmer” site because the sun path may not go directly over the gaps in the canopy of that site, thus, the site wouldn’t receive large amounts of direct solar radiation.  Black morphs use basking sites that received lower amounts of direct sunlight.  They may be able to do this because the greater amount of melanin in their skin provides a greater ability to absorb whatever direct or indirect solar radiation is available more effectively. Yellow morphs use basking sites that received more direct solar radiation.  They could be forced to use these sites to compensate for their disadvantage in their thermal ability.  I am currently working on writing a manuscript for these data and hope to have it completed by the end of 2016.  Stay tuned for more on this manuscripts progress!

Here is a picture of Shawn (holding a Hellbender!!) while on a break from collecting some amazing data.

Studying and Staying Sane

The researchers in our lab have many interests, not all of which are related to science. In this post, undergraduate Tommy Cerri describes his preparation for the MCAT…as well as other actives that help him stay sane. 

Spring 2015 has been one of the most hectic semesters for myself. My time is split between working in the lab, being a full time student, and studying for the MCAT. I plan on taking the MCAT exam this summer in June, and the studying process so far has been grueling. Here is a picture of my study materials I got in the mail earlier this semester. A total of 9 books that I have to get through in only a few short months.

As of April this year, the format for the medical school admissions test has been changed. Most importantly, it is no longer a test of about 4 hours, it is a test that runs about 7 hours. This means they added biochemistry, sociology, psychology, as well as a few other topics that will be tested on the exam. Either way my studying has been going swimmingly, and I continue to study on a daily basis hoping for the best (fingers crossed)!

Due to this huge time commitment, my participation in the lab has not been as big as I would have hoped. I do help out though! As of late, I have picked back up where I left off last semester assisting Gail with lizard behavioral videos. I do enjoy these videos: the lizards are extremely active, which can be quite amusing. A few other miscellaneous tasks have come up within the lab and I love helping out with whatever needs done when I have the time.

Outside of lab I have given a handful of tours for prospective Penn State students. Don’t worry, I always give the Langkilde Lab a shoutout on each of the tours. Many times the parents are very interested about the research we do in the lab and I am always happy to fill them in on all the amazing things we are all doing! Parents of prospective science students are always intrigued by the potential research opportunities we have to offer here at Penn State, and I love to talk about some of my awesome experiences I have been presented with. The students always get a good laugh when I tell them I fed crickets to lizards once a week when I was a freshman.

In other news I did score 2 goals in my most recent intramural soccer game last Monday, and my team won our IM basketball game as well! Club sports might be on the horizon of my senior year, who knows…

THON 2015

by undergraduate Cecilia Zemenak

This semester, I have been working on my first manuscript from my research with Mexican Jumping beans. The paper will be focus on the variation amongst individual bean behaviors.

This past weekend though, I took a break from my first draft to take part in the largest student-run philanthropy event in the world, THON!

For those who don’t know, THON is a yearlong effort to raise money for the Four Diamonds Fund, which assists pediatric cancer patients at the Penn State Hershey Medical Hospital. It is a 46 hour dance marathon for the kids and their families to forget about the illness for one weekend of games, performances, and fun with a supportive Penn State student family.

The whole event takes place in the Bryce Jordan Center, with the dancers on the floor and everyone else cheering them on in the stands. I take part in Atlas, which is a special interest group organized solely for the purpose of raising funds for THON. We had 10 dancers on the floor this year, and a huge group of Atlas members supporting them from the bleachers.

Every year, a different logo is chosen that encompasses why it is Penn State Students dance for the kids and for a cure. This year’s motto was to “Empower the Dreamers.”

A few phrases of the line dance, which is performed every hour by every person in the BJC, really explains the message behind the logo chosen:

Paint a face within the moon

Look closely and you’ll see yours too

Dreams grow in these small hours

Make them real, BE EMPOWERED.

I stood in the bleachers from Friday afternoon until midnight, and then I went back to my apartment to sleep while the dancers danced on. Saturday afternoon I excitedly went back and stood 25 hours until the very end Sunday afternoon. At the end of those 46 hours, the grand total raised by everyone was revealed: a whopping 13 million!

I am so proud to be a Penn State student because of this extremely worthy cause. 13 million dollars will go towards cancer research, providing care to pediatric cancer patients, and paying for treatment not covered by insurance. I am very fortunate to be a part of such an amazing student body at such a prestigious research university. WE ARE… PENN STATE!

The Transition to Veterinary School: Mastering the Art of “Suffering Happily”

by former undergraduate Courtney Norjen

After four fantastic years at Penn State, I packed up all of my stuff in August and moved to Columbus, Ohio to start veterinary school at the Ohio State University (but don’t worry, I will always truly be a Nittany Lion!)

I was asked to write about what starting veterinary school is like, and I have been struggling to come up with an accurate description. So to procrastinate, I flipped through a Tumblr blog called “Shoulders Deep in Vet School” (it’s hilarious if you’ve never seen it). As I was scrolling, I came across a perfect GIF for describing what veterinary school is really like. It is a quote from Harry Potter, when Ron is reading Harry’s tea leaves and claims “you’re gonna suffer…but you’re gonna be happy about it.” Vet school is incredibly challenging and the workload is massive, but it’s also unbelievably rewarding and I could not be happier to be here.

There were two major educational culture shocks when I started vet school. First, there is no “syllabus week”. There isn’t even a “syllabus day.”   My classmates and I walked into class the first day excited to start school and figure out what our classes would be like. And suddenly it was like trying to drink out of a fire hose. No one could take notes fast enough and everyone was looking around in panic, wondering if they were the only ones who couldn’t keep up. The second shock was getting used to a schedule that was more like high school than college. We typically have class from 8 am to 4 pm, and we are in the same auditorium all day except for when we have laboratories. But unlike a normal 8-hour workday, we have to study after school to keep up with the material.

It took about a week to get used to the sheer volume of material that we cover in class daily, and to be mentally prepared to sit in lecture for most of the day. But once I was adjusted, school became much easier (or if not “easier”, at least more manageable). I found that there is actually plenty of time during the week to do things other than study, and I think that having a good balance of school and fun is absolutely vital to success in veterinary school. Outside of school, I work at a small animal emergency hospital on Sundays, volunteer for community outreach activities through the vet school, and make time to explore all Columbus has to offer with my friends and classmates.

Vet school has definitely been a huge transition, and it is a lot of work, but I wouldn’t trade it for anything. I still wake up every day excited to go to class and learn the information that I’ll need to be a veterinarian in just 3.5 short years. So I will continue to happily “suffer” through the insane hours of studying to keep getting closer to my dream job.

A Rattlesnake Summer

This past summer I began putting the pieces together for my 4 year project investigating the effects of prescribed fire on Timber Rattlesnakes.  The objectives of this project will be to determine how Timber Rattlesnakes are directly impacted by the fire and to determine how rattlesnakes are affected by changes within their environment which are caused by the fire.  This past summer’s objectives, however, were to basically get my feet wet, get to know the areas that may serve as potential study sites, get to know the focal species, and attempt to understand how to work with state agencies (SPOILER ALERT => this last objective would prove to be the most difficult).  It took awhile to get permits and all other paper work in line for the project, but all-in-all the summer was a great success because I identified 2 potential study sites (and possibly 2 more), I learned a great deal about these wonderful little creatures, and with the help of 2 great technicians (Rex Everett and Mark Herr) we came up with a couple great side projects.

Male Timber Rattlesnake post-data collection. Note the painted rattle which allows us to identify it as a previous capture if we happen to stumble upon him later in the day. For all rattlesnakes we recorded basic morphometrics, sex, reproductive status, and marked the individual with a PIT tag which will allow us to identify it if captured much further down the road.

To date, we have identified two great study sites (and possibly more).  The first is located in Forbes State Forest and will be a rather large burn (a little over 600 acres!).  We’ve identified at least three potential gestation sites in this area, and although we were not able to spend a great deal of time down in Forbes, we know through talking with state foresters that there is a healthy population of Timber Rattlesnakes in the immediate area.  The actual area was nicknamed the “Snake Pit” by the district forester.  Aside from being a beautiful landscape, it is also the highest elevation within the state!  The “Snake Pit” is scheduled to burn in 2016 via an aerial ignition burn (dropping fire from helicopters!), which will provide me with a full pre-burn year to collect data in 2015 (Fantastic!).  In addition to Forbes, we also have a smaller prescribed burn planned for Rothrock State Forest.  We have visited this area quite frequently throughout the summer and we’ve captured a handful of Timber Rattlesnakes within the area as well (including one individual that we’ve radio-tracked to a den site).  The odd thing about this area is that all of the rattlesnakes we’ve captured thus far… have all been males of various ages.  Where are the females?!?  The presence of 1 year old males, however, suggests that the females are hanging out somewhere near by but we have not found where….  But, it leads us to question why we did not find any gravid females hanging out on the fantastic rocky slopes within the area?  Was this just a bad year for gravid females? Could the females be down in the valleys hunting chipmunks and gathering the energy needed for future reproductive bouts?

On the left, rising out of the thick ocean of Mt. Laural is a beautiful rock slab bathed in sunlight. This is fantastic gestation site. The large cap rock atop of the rock slab provides cover for the gravid females and refuge from high temperatures during the mid-afternoon and low temperatures at night. On the right, another type of gestation site is the large rock fields strewn out among Pennsylvania’s mountain sides. These gestation sites may be 100 m long and very open. Although this provides good thermal habitat for longer periods each day, would snakes be detected by predators more in this largely open area?

Throughout the summer we located close to 50 Timber Rattlesnakes.  Although we did not find gravid rattlesnakes at some potential study sites, we found many gravid rattlesnakes at other sites.  Sometimes these gravid rattlesnakes were in small areas that were about 10 m X 10 m (top left picture).  These smaller areas had all the essential habitat characteristics for a gestation site: an open canopy, rocks to lay out on, and rocks to hide underneath.  Other times, gravid rattlesnakes were found in large rock fields (top right picture).  Obviously the thermal environment would differ between these gestation sites.  Sun light would hit the more open rock fields early in the morning and last till much later in the day as compared to the smaller gestation sites surrounded by towering trees.  This may translate into the gravid Timber Rattlesnakes being able to maintain a preferred, optimal body temperature for a longer period of time each day within the more open rock fields.  Maintaining this optimal body temperature for a longer period of time each day may translate into more energy available for developing offspring and shorter gestation periods.  However, these more open areas may provide an advantage to visual predators such as hawks.  So would it be more beneficial for a gravid Timber Rattlesnake to gestate within a large open rock field despite the potential increased risk of encountering a predator?  To answer this question, a Penn State undergrad, Mark Herr, and I are putting together a side project for next summer where we will measure the thermal properties and predation intensity of these different types of gestation sites.  Additionally, we will radio-track gravid females at each gestation site in order to determine body temperatures maintained by individuals, duration of time spent at gestation sites, and date when females birthed young.  This should be a fantastic study! This past summer we began collecting preliminary data at a few gestation sites (using biophysical models) and found that operative temperatures at smaller gestation sites averaged 6 degrees C less than operative temperatures at large rock fields.  Additionally, this was a cooler summer than years past and only the gravid females at large rock fields birthed young at those gestation sites.  Gravid rattlesnakes from smaller gestation sites remained gravid as they traveled back to their den sites.  Most of these individuals appeared to return to their dens gravid, which likely means bad things for those potential young (i.e., mortality).

On the left, Penn State undergrad, Mark Herr, collects habitat data at a smaller gestation site. Note the biophysical model in the foreground next to the backpack. These models will measure the operative temperatures for Timber Rattlesnakes, which are basically the potential body temperature of an individual if it was not thermoregulating (i.e., if it were conforming to the environment). On the right, a neonate Timber Rattlesnake hiding under a birch leaf (… that is a small leaf). Only at large, open gestation sites that maintained warmer operative temperature did we find neonates this summer.

Gravid females from smaller gestation sites returned to their dens still gravid.  This likely spells disaster for the potential young.  Can you find the Timber Rattlesnake at this den?

Next spring we are ready to hit the ground running as snakes begin to emerge from their den sites.  In addition to the project’s main objectives, we are also prepping for a few side projects.  In addition to the project described above, I am also trying to understand why Timber Rattlesnakes choose one potential gestation site over another.  I will also be surveying and monitoring vernal pools within burn sites to determine how amphibians are affected by prescribed burning.  Stay tuned as we continue this project.  In addition to Mark, I also have one of the best Timber Rattlesnake expert in the state assisting me with this project: Rex Everett.  Together Rex, Mark, and myself are determined to make this project a success.  Additionally, we will be taking on at least two more technicians for next summer and any other undergrad interested in reptile ecology, thermal biology, and conservation.  For more information feel free to contact me at cah62@psu.edu.

The fearless leader collecting data on a Timber Rattlesnake found crossing the road at Rothrock State Forest.

Rex Everett, snake expert, with a large Rat Snake found adjacent to the planned burn area at Rothrock State Forest