The Lizard Log

The Langkilde Lab in Action


1 Comment

Gearing up for SICB!

Happy New Year!

lizard

New year, new beginnings. And hats. Lots of hats.

As we start to wrap up our holiday break, Chris T. and I are preparing for a trip to Portland, Oregon for the 2016 annual meeting of the Society for Integrative and Comparative Biology (SICB). We will both be presenting talks on Monday.

I will be expanding on results I presented at ESA in August, which address how stress (fire ants) experienced in early life or in previous generations affect adult physiology and immune function in lizards. My talk is Monday, January 4th at 11:15 in room B110/111.

Gail_titleslide_SICB

Chris will be sharing his results from SSAR with a new audience. He will discuss how invasive fire ants have reversed geographical patterns in fence lizard ecology, including their behavior, stress responses, and morphology, across their range in less than 75 years. Chris’s talk is also on Monday, January 4th at 11:30 in room B114. A determined Langkilde Lab follower could attend both talks back to back in nearby rooms! 🙂

CThawley_SSAR2015_Title

Come say hello if you will be there!

Advertisements


2 Comments

What Makes Stress Stressful?

Stress is a familiar concept to most people. Paying the bills on time, entering a week of exams, caring for a sick loved one, or even sitting in heavy traffic on the way to work. When you get stressed out, your body goes through a series of changes to help you deal with that stress. This stress response includes both physiological and behavioral changes and is generally a good thing! For animals, the physiological stress response can mobilize energy and trigger important behavior, perhaps to get away from a predator. It can also enhance immune function in the short term to prepare for wounding or infection that might occur as a result of that stressful encounter.  Short term stress is typically called “acute,” and the resulting stress response is very similar across vertebrates—because it works!

Stressors come in a variety of forms.

Stressors come in a variety of forms.

If stress lasts for a long time, however, there can be costs to using so much energy on the stress response. If you have ever become sick after a week of exams or a particularly challenging week at work, you know what I’m talking about. Long term stress—typically called “chronic” stress—can suppress immune function as well as growth and reproduction.

Sometimes, however, these generalizations don’t hold up—short term acute stress may produce negative consequences or long term chronic stress may produce positive outcomes. This got us wondering—just what is it about stress that might lead to negative consequences? We discuss just that in our latest paper published in General & Comparative Endocrinology, which is now available online.

Now published in GCE!

Results published in GCE.

Stress is typically defined by duration—as acute or chronic— in the scientific literature as well as in veterinary and medical practices. I wanted to investigate not only stressor duration, but also other characteristics of the stressor, like frequency and intensity. There is some evidence that frequency and intensity affect the outcomes of stress, but few studies have attempted to look at how they might interact with each other or duration.

To test these ideas, I exposed fence lizards to different stress regimes. I did not want to use a physical stressor, so we instead manipulated a stress relevant hormone. When the stress response is activated, the glucocorticoid hormone cortisol (in humans) or corticosterone (in lizards) is secreted by the adrenal glands. We often measure CORT as a proxy for stress, and we can give a lizard CORT to replicate the increase in CORT that occurs in response to a stressor. After dissolving CORT in oil, one simply drops the solution onto the back of a lizard and it is quickly absorbed. One can also put the CORT-oil solution into a hormone patch for a slower release. These work a lot like a nicotine patch in humans, just with CORT and on a lizard.

A fence lizard with a slow release CORT patch.

A fence lizard with a slow release CORT patch. Stylish!

We used different regimes of CORT application to help determine how duration, frequency, and intensity affect immune outcomes in lizards. After the 9 days, we measured the innate immune system in two ways [similiar to  this post], both of which roughly measure the ability of lizard blood to deal with foreign particles. One of these assess hemagglutination, which is the ability of plasma to hold sheep red blood cells in suspension. Higher scores indicate greater ability, or better immune function.

The completed hemagglutination assay.

A completed hemagglutination assay.

Some of our results were particularly interesting:

Two of our treatments would be considered “acute.”  Both were short in duration and differed only in the intensity of the dosage. Exposure to short duration low-doses of CORT  enhanced immune function (hemagglutination), while exposure to short duration high-doses suppressed immune function. This indicates that intensity is an an important factor when considering immune outcomes of stress.  This matches up with what we know about PTSD—short but intense stressors can have lasting effects in that context as well.

Additionally, while both of these treatments mimic “acute” stress, they produced opposite results. This demonstrates that the terms “acute” and “chronic” may not be enough to sufficiently characterize stress. These terms are also inconsistently used in the scientific literature, which only adds to the confusion.

Three of our treatments received the same average amount and total amount of CORT over each three day period and over the duration of the experiment but differed in how they were distributed–they varied in duration, intensity, and frequency. All three of these treatments, however, produced different outcomes—one enhanced immune function (frequent low doses), one suppressed immune function (infrequent high doses), and one was somewhere in the middle (slow release of the high dose). This suggests that average or total amount of stress (CORT) may not be comprehensive enough to characterize how the stress is experienced or accurately reflect its outcomes.

Although frequency and duration had lesser roles in this experiment, intensity was a major factor in altering the immune consequences of stress. We recommend that researchers consider and report aspects of stress other than duration, such as intensity and frequency, to aid our understanding of the consequences of stress. We should also move away from the terms “acute” and “chronic,” as they are inconsistency used and incompletely describe stress.

Because the environment is changing due to climate and human activities, wild animals will be exposed to new stressors or familiar ones more often. Determining what about stress leads to negative consequences is important to understand how species will respond to environmental change.

How will wild organisms respond to the stress of environmental change?

How will wild organisms respond to the stress of environmental change?

These results are published in General and Comparative Endocrinology. This research is also featured on the Penn State CIDD website, here.


Leave a comment

Measuring Stress Can be Stressful

In order to study stress, you need a good way to measure it. A common method to measure how “stressed out” an organism is at a particular time is to measure the concentration of “stress hormones” in their blood. In reptiles we measure corticosterone (CORT), which is similar to cortisol in humans and fish. Our lab uses an ELISA (enzyme-linked immunosorbent assay) to determine the concentration of CORT in our samples. These kits are commercially produced for a variety of hormones and make it very convenient to do so, though it takes a solid day of lab work.  Jenny, Chris, and I have each been running a number of CORT assays in the last months in order to work through our samples from last summer. One of the lab’s undergrads, Mark H, is also learning this technique to run some of his own samples.

DSC00149b

Gail teaching Mark how to prep a CORT plate.

Although these kits are pretty straightforward, you can’t help but be paranoid about doing it right. A mistake costs you not only the precious samples you are running but also the cost of the kit (which is not insignificant!). Because of this, we make sure any newcomers to the kit are trained up and very comfortable with the assay before we turn them loose.

photo 2

Completed plate!

After the day of lab work, the completed plate looks something like this (above). This plate is then placed in a micro-pate reader, which reads the absorbance of each well at various wavelengths. The result is a spreadsheet of numbers, which ultimately lets you determine the concentration of CORT in each of your samples. It’s always a little nerve-wracking at the end of the day when you walk away with just a flash drive, but the data usually turns out just fine!

The next step: data analysis!


1 Comment

Lizards don’t do math.

Being stressed out is something everybody can relate to. Driving in heavy traffic, taking an exam, interviewing for a job, or dealing with family issues could all produce a similar stress response. Humans aren’t the only ones to experience stress. Zebras and lizards may not do math problems, but they may flee from or respond to a dangerous predator. These types of short-duration stressors are called “acute” and often involve only a single event. In humans, that might involve driving in heavy traffic or doing some difficult mental math. In the ecological world, acute stressors can include fighting with a competitor for food or territory or an encounter with a predator. Stressors that are persistent or long-lasting are called “chronic.” In humans, this could include getting over a recent break-up, being unemployed, caring for sick relatives, or worrying about money problems. In other animals, chronic stress could include exposure to a long winter storm or drought or continued difficulty finding food.

Life is stressful...

Life is stressful…

Although the kinds of stressors are quite different, the way an animal’s (or human’s) body responds is actually quite similar in most vertebrates. This stress response includes a suite of physiological changes that happen inside the body to help deal with the stressor. Part of what helps encourage these responses is the production of “stress hormones,” like cortisol in humans and fish or corticosterone in reptiles, amphibians, and birds. This suite of changes is often thought of in terms of energy:  stress hormones help redistribute energy toward things that are immediately important, like escaping from a predator. This energy is taken away from functions like reproduction or growth, which are not as important in the short-term.

The stress response is very important for dealing with stressors, but what happens when the stressor doesn’t stop or is frequently repeated for long time (e.g. is chronic)?  Under conditions of chronic stress, growth, reproduction, and immune function are often suppressed. Perhaps frequently taking energy away from these processes is not the best idea if it means an animal won’t be able to become big enough to mate or successfully produce offspring.

Lizards get stressed too.

Lizards get stressed too. (Photo by Gail McCormick)

If you’ve ever been in the Southern US, I’m sure you can relate to the stressful experience of being bitten by a fire ant! Fire ants can bite, sting, and even kill a lizard, so it makes sense that they have elevated levels of stress hormones (corticosterone) after a fire ant attack. Because they are frequently attacked, these lizards experience chronic stress, which in theory should lead to immune suppression. This got Tracy and I thinking:  although they have some neat ways of dealing with fire ants, lizards from fire ant invaded sites may frequently get bitten, which breaks the skin, or stung (envenomated), which activates the immune system. Thus, it seems that the immune system is important to lizards at invaded (high-stress) sites and that suppressing the immune system under chronic stress would be a bad idea for these lizards. Perhaps populations from invaded high-stress sites have adapted so that they do not suppress their immune function under chronic stress.

Female fence lizard. (Photo by Gail McCormick)

Lady fence lizard. (Photo by Gail McCormick)

Last year (summer 2012), we designed a project to determine if this were the case. I simulated the elevation of stress hormones that occurs from a fire ant attack by directly applying corticosterone (CORT) to the backs of lizards. (Corticosterone is dissolved in sesame oil, which is easily absorbed through the lizard’s skin.) We applied CORT every day for 23 days to lizards from invaded high-stress and uninvaded low-stress sites. Some lizards received just oil each day, to act as a control and base of comparison. At the end of treatments, we measured immune function to see if lizards from invaded and uninvaded sites responded differently to this chronic stress.

We expected all lizards that received CORT treatments would have reduced immune function compared to lizards that received just oil (control lizards). Additionally, we expected that this reduction would be less in lizards from invaded high-stress sites versus those from uninvaded low-strew sites. The graph below demonstrates this expectation. Taller bars indicate “better” immune function.

We expected all lizards to have reduced immune function following 2 days of CORT-treatments, but that this decrease wouldn't be so dramatic in lizards from high-stress sites.

We expected all lizards to have reduced immune function following 23 days of CORT treatments, but that this decrease would not be so dramatic in lizards from high-stress sites.

We used two different measures of immune function, both of which roughly measure the ability of lizard blood to deal with foreign particles, in this case E. coli (not a virulent strain!) and sheep red blood cells. Surprisingly, lizards that received CORT treatments actually had higher immune function compared to lizards that received just oil. Additionally, lizards from both invaded high-stress and uninvaded low-stress sites responded the same way to treatments.

What we actually saw: lizards from both high- and low-stress sites had enhanced immune function after CORT-treatments.  (idealized data for better presentation)

What we actually saw: lizards from both high- and low-stress sites had enhanced immune function after CORT-treatments.
(idealized data for better presentation)

There are may reasons why me may have seen these results. Here are a few of our many ideas:

  • Our lizards were fed a healthy diet of tasty crickets–likely more than they would eat in the wild. Perhaps for immune suppression to occur under chronic stress, lizards need to be food (and thus energy) limited. (With a healthy supply of energy, there may have been no need to divert energy from other systems like the immune system to deal with the stressor.)
  • Perhaps some parts of the immune system are more important than others. Some components that we did not measure in this study may be “okay” to compromise under stress in this context, while the components we did measure may not.
  • Perhaps CORT levels were able to return to “normal” between treatments, which makes this method more of a “repeated acute” stressor than a “chronic” stressor. We suspect that frequency and duration of a stressor are important predictors for consequences of stress, and I performed a study this past August to investigate this idea. I’ll be sure to share the results of that experiment in a future update!

These results were presented at the 2013 SICB annual meeting.

 

UPDATE: These results are published in General and Comparative Endocrinology: read it here!


Leave a comment

Redemption in the Deep South

This undergraduate guest blog post has been dispatched by Mark Herr, a junior from Los Angeles who is majoring in Wildlife and Fisheries Science at Penn State. Mark is a member of the Penn State Presidential Leadership Academy, and is interested in continuing on to graduate school to study behavior, ecology, and, of course, reptiles & amphibians:

This past spring I applied for, and with the help of Dr. Langkilde received, a Penn State Discovery Summer Grant to conduct independent research. Initially, I planned to conduct two projects on similar systems. Unfortunately, only the second project (hence the title of this post) resulted in a successful field season. Which isn’t to say that the first project wasn’t valuable – it was perhaps the most valuable research experience I’ve had yet – but its value really lay in lessons learned rather than any publishable results!

Before I explain the project, I want to first give a shout-out to Drs. Langkilde and Sean Graham. Dr. Langkilde managed to let me know that my initially proposed projects were all rather grandiose and unfeasible, while convincing me to keep on firing away with ideas. Eventually, I sent her an idea that we both agreed was novel, and more importantly, possible. Sean went out in the field with me during every day of sampling and made the project possible. This is all the more impressive because he is a post-doc researcher and I am just an undergraduate – which says much both about him and the atmosphere that Dr. Langkilde fosters in her lab.

Dr. Sean Graham does his best to become an internet sensation by displaying a cottonmouth using the standard forced perspective method so that it appears to be of monstrous size. However, this snake is actually only a little over 3 ft. long. The camera really does add a few pounds.

Dr. Sean Graham does his best to become an internet sensation by holding a cottonmouth safely out of striking range while using the standard forced perspective method so that it appears to be of monstrous size. However, this snake is actually only a little over 3 ft. long. The camera really does add a few pounds.

I don’t want to delve into the specifics of my project idea in California, both because it will bring back the stinging memory of defeat (just joking!) and poison oak rashes so bad that they probably warranted hospital visits – both for Sean and I! Luckily, Sean and Dr. Langkilde had foreseen the fact that all might not go as planned with one of the projects – and so they had suggested that we conduct a second project as insurance. And so it was this “insurance policy” that became my last true hope for some publishable research during the summer break.

My project ran side by side with other work being conducted in Alabama by the Langkilde Lab, which allowed me to help out with other projects when I wasn’t out in the field with Sean collecting snake blood. Yes. Snake blood, and no, this didn’t involve door-to-door salesmen selling cure-alls or primitive rituals by witch doctors.

Sean and I investigated how the stress hormone (corticosterone) concentrations in Cottonmouths (Agkistrodon piscivorus) were related to their anti-predator behavior. Cottonmouths are large bodied, aquatic pit vipers native to the southeastern US. Most importantly, they are abundant. The snakes were honestly even more abundant than I had expected – even though we did have a period where we had trouble finding any, but more on that later.

They are also famous for being aggressive, or, at least that is what the man on the street will say – the consensus among scientists and in peer-reviewed research is that they aren’t anything close to the bloodthirsty mankillers they are made out to be. Cottonmouths do, however, have an extensive suite of anti-predator behaviors. They vibrate their tails (even though they have no rattle), they hiss, they can strike, and they can open their mouth wide in what is called a ‘gape’ in order to convince a possible predator that they aren’t worth the trouble. Actually, this gaping behavior is what gives them their common name – the inside of their mouths has a white lining that is highly visible (especially in their often dark habitats).

A Cottonmouth from the Everglades showing the display for which it is named.

A Cottonmouth from the Everglades showing the display for which it is named.

The fact that this species has so many different anti-predator behaviors means that I was able to formulate a point system that would rank each individual snake based on how “defensive” it was acting. The procedure was to approach the snake and stand in close (but safe) proximity to it for 15 seconds and then grasp it mid-body with a set of snake tongs for another 15 seconds – all the while taking note of every behavior that the snake exhibited. We would then use a snake tube to restrain the animal and take a blood sample.

As I write this I am laughing out loud at how simple that last sentence makes the blood drawing seem.

The problem, as you might imagine, is that often times (read: every time) the already defensive snake wants nothing more than to avoid slithering up a tight clear plastic tube so that we might get our blood sample safely. For obvious safety reasons, the entire tubing maneuver has to be completed using nothing but a set of snake tongs and a tremendous amount of patience – tubing is the safest way to handle venomous snakes – by far (both for the snakes and the researcher). It’s the industry standard technique.

The process goes something like this:

  1. Using a pair of snake tongs, the snake is grasped firmly enough at the midbody to prevent the snake from escaping but not so tightly as to injure the animal.
  2. The tube is maneuvered such that it fits over the snake’s head, and ideally the snake will crawl up the tube such that its body is half inside and half outside the tube, with the snake’s posterior body portion and tail hanging out of the end.
  3. Swiftly and steadily, the snake is grasped at midbody at the exact point where it hangs out of the tube – with the hand holding the snake firmly grasping both the animal itself and the tube to prevent the snake from either moving further forward or backing out.

Step #2 is sometimes easy, as the snake will cooperate and crawl up the tube as soon as it is able. Usually, though, it is not so easy. More often it would rather keep dodging and moving its head away. Even more often it would rather just strike the tube repeatedly. This process was further complicated by the fact that we were on a tight time schedule –we needed to obtain blood from the snake before its hormone levels had risen significantly (which takes only a few minutes). Did I mention the fact that we were unable to physically touch the snakes until they were in the tube?

A safely and successfully tubed Cottonmouth on display after obtaining a blood sample.

A safely and successfully tubed Cottonmouth on display after obtaining a blood sample.

We went out during the day and at night searching for snakes, usually spending at least 4-5 hours at a stretch slogging through swamps in waist deep swamp water, and we managed to get to within about 5 snakes of our sample size when we hit a wall. No. More. Snakes.  I can’t even remember how many times Sean and I ventured forth without finding even a single snake. It was as though cottonmouths had gone from being the most common snake in the Conecuh National Forest to almost nonexistent. A change in the weather may have been the culprit, but I wasn’t nearly as concerned with the source of the problem as with the prospect that I might not find enough snakes to complete the research.

Luckily, we had a solution. Sean had a location that he knew would guarantee snakes en masse – unfortunately, it was too far away for just a day trip. Our solution was to hit this magical spot on the way back to Pennsylvania on our very last day.

I can’t tell you how nervous I was when we went out that final night.

To set the stage:

  1. We didn’t have enough snakes. More importantly, we really needed to find a specific subset of snakes, adult males, which are significantly more difficult to locate than adult females or juveniles.
  2. We absolutely had to leave for Penn State the next morning, making this the absolute last possible time to get enough snakes to complete the project.
  3. The previous four days hadn’t yielded even a single snake.
  4. Just for dramatic effect, there was an absolutely MASSIVE electrical storm just prior to our sojourn, shaking the ground and lighting up the sky like a child repeatedly turning the lights on and off inside an otherwise pitch black room.

Well, that night could have shared a title with this post: Redemption in the Deep South. After working from 8:00 pm to 2:00 am, slogging through waist deep (read: sometimes neck deep) blackwater while exhausting our drinkable water, getting lost, and receiving (conservatively, of course) 1,000,000 mosquito bites, we found our 5 snakes. Three of the five were adult males. Our goals were complete. Victory.

I am still in the process of inputting data and running samples, so I can’t yet tell you how stress hormone levels in cottonmouths relate to their behavior. What I can tell you is that working through the trials and tribulations of this summer’s research has made me a better scientist by far. I have many to thank for making this experience possible, but my highest gratitude extends to Tracy Langkilde, Sean Graham, and the cottonmouths of south Alabama. Thanks guys.

Adapted from Mark Herr’s Presidential Leadership Academy Blog


Leave a comment

To the lab cave!

(Originally posted on our 2012 Field Blog —  by Renee Rosier)

Field season is a magical time of the year when we set up experiments, record videos, collect blood samples, measure animals, and generally spend each precious moment gathering data. As animal breeding and activity seasons come to a close, we wrap up the experiments and are faced with the next phase: figuring out what happened.

Brad_science4-thumb-319x239-339395

All of those preserved specimens must be identified, dissected, and organized. Other samples need to be analyzed for content, which can involve long periods of time at a stereoscope separating out ant heads from lizard poop for diet analysis, or counting zooplankton in water samples. Time spent at the bench can feel a bit tedious after the controlled chaos of field season but this is when we really begin to get information from the experiments. Sometimes there are great discoveries, too! What seems mundane can be quite shocking under magnification (just look at Brad‘s reaction to zooplankton). If you’re ever in front of astereo/dissecting microscope, take a chance to look at your finger under high magnification – you might find some surprises.

CORT_assay-thumb-319x239-339396

Since we are predominantly an ecology lab, our actual lab space is often a collection of everyday objects, like child swimming pools, plastic storage bins, nail polish, rulers, flagging tape, and the like. This past week it has been transformed into something closer to what you might imagine for a laboratory – namely, a place of eppendorf tubes, nitrile gloves, pipettes, and people with serious looks on their faces. Several studies in our lab are investigating the effects of some potentially stressful factor (a stressor), such as a fire ant attack, on hormone levels and behavior, or the effects of the stress hormone corticosterone (CORT) on behavior. This research involves collection of blood samples as quickly as possible, which are then spun in a centrifuge to separate the red blood cells from the plasma (the plasma portion contains the CORT).

The plasma samples are kept in the freezer until there are enough samples to analyze through a common laboratory technique called enzyme immunoassay (similar to ELISA, if you’d like more details). In a series of steps, wells on a microtiter plate are prepared so that the hormone contained in the plasma sample will attach to the walls of the plate. After some incubation and wash steps, an enzyme substrate is added, which changes the color of the liquid in the wells. The plate is then inserted into a microplate reader that passes light through the liquid and measures the absorbance of the light. There are controls on the plate with known concentrations of the hormone; we use the absorbance from these wells to make a standard curve. We’re then able to use the slope of this curve to determine the concentration of CORT in our samples. Voila, numbers from blood samples!

Sean using the multichannel pipette for the CORT assay.

Last week Sean, Gail, and Jenny were running CORT assays for lizards and amphibians. I was able to catch them in the act – the act of science! As you can see, real scientists don’t always wear lab coats. The photos shown here are not staged, but are actual snapshots of scientists in their natural laboratory environment. The last photo shows the final step of the analysis: the number crunching in a spreadsheet to make the standard curve and calculate the CORT concentrations.

Jenny_CORT2-thumb-600x800-339402

Jenny prepping her wood frog plasma samples for CORT assay.

Gail_CORT9-thumb-600x800-339400

Gail adding her samples to the microtiter plate.

Jenny, Gail, and Sean crunching the numbers after running CORT assays last week.