The Lizard Log

The Langkilde Lab in Action


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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.

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.

IgM in the plasma of Alabama lizards

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.

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.

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Much Anole About Immunology

While most of the lab has been down in Alabama, I’ve spent a good part of this summer back at Penn State, working with a species I’ve never used before – the green anole (Anolis carolinensis).

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Isn’t he cute?

There is a test we’d like to use in our fence lizards, called the phytohemagglutinin (PHA) skin test. It involves injecting the pad of a rear foot with a small amount of PHA, which stimulates part of the immune system, and then measuring the swelling that occurs. This swelling is small, and temporary, abating in a few days with no lasting damage. But the level of swelling can provide information about the lizards’ immune function.

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A confused anole having its rear foot measured with calipers.

Unfortunately, while this test has been used in humans, birds, rats, and even amphibians, it has not yet been validated in any reptile species. Ideally I would validate the test in our species of interest, the eastern fence lizard, but I needed a larger number of lizards than we can reasonably catch. So, instead, we decided to purchase some green anoles for this project.

In addition to seeing if the PHA test works in reptiles, we’re also trying to determine if the type of PHA used makes a difference, as there are many different formulations of PHA used, and each formulation may have a different effect. I’m also determining exactly what the immune reaction to the different PHA formulations are, and how this evolves over time after the injection.


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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.