The Lizard Log

The Langkilde Lab in Action


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What’s in an egg?

Hello again everyone!

While most of my work has been on measuring hormones and metabolites from blood, or recording behaviors, I decided to try my hand at something new. I wanted to see if I could measure the contents of a lizard egg!

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As eggs can vary widely in the volume of water they contain, the first thing I had to do was dry the egg. Because I wanted to measure proteins and lipids, I wasn’t able to heat the egg up though, so instead I used a freeze-dryer.  Once dried, I carefully removed the shell (because shells are reaaaaally hard to grind) and then homogenized the yolk sample.

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Once the yolk was ground up, I needed a way to extract the proteins and lipids from the yolk. To do so, I weighed out a specific amount of the egg, added some dangerous chemicals, and then filtered that solution through an incredibly tiny filter. The size of the holes in a coffee filter are 20 microns, while the size of a bacteria is 0.6 microns. This filter had holes that were 0.2 microns!
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After filtering the solution, I could then try to measure the amount of proteins and lipids. To do so, I added a tiny drop of the solution to a piece of quick dry paper.

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Once the paper completely dried, I was able to shine a light through it and get an absorbance value.

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Stay tuned for the results of what I found!

Cheers,
David


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Switching fields

Hello, my name is Heather Engler. I have been working as a research assistant in the Langkilde lab since May 2016. And yet my background is not in biology. Instead, I have a B.S. degree in Accounting from Murray State University. So how did I wind up going from business to biology?

I began dating Dustin Owen, my boyfriend, while he was at Austin Peay State University. I was fascinated with his reptile research because I have always enjoyed learning about animals. So I naturally took an interest in his new eastern fence lizard research here at Penn State. I was really lucky that Tracy Langkilde didn’t mind me spending time with Dustin in her lab. I got to learn about all sorts of things from various lab members.

Last summer, Dustin and some of his new lab mates caught lots of eastern fence lizards to be used in their research projects. They were busy with their research, so they needed someone else to take care of the lizards on a daily basis. Since I had not landed an accounting job, Dustin put in a good word for me with Tracy. Luckily, she was willing to give me a chance.

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Female fence lizard digging a nest (photo: Heather Engler)

I absolutely loved taking care of those lizards last summer! Some of the females became gravid, and I got to help collect the eggs after they finished laying them. After all of the females had laid their eggs, it was time to incubate them in the lab. One random day in July, I was checking on the eggs and noticed some tiny bodies. The first of the eggs had hatched! It was so cool because the hatchlings were so little compared to the adults. Since we were short staffed at the time, I got to help record the morphology data on the hatchlings. I even learned how to toe clip them. I had never done this kind of work before, so it was a fun learning experience.

Since I had done such a good job with the adults, Tracy let me also take care of the hatchlings. And it has been a blast watching over those lizards. It won’t be too much longer until they’re fully matured adults. I’m even getting to help on a side project concerning them. Braulio Assis, one of the current grad students in the lab, has been taking photos of the juvenile lizards at 9 week intervals. One of the things he wants to know is if testosterone levels are related to the size of male cloaca scales. I’m helping to answer this question by measuring the area of the male cloaca scales in the photographs of the male juveniles. I get to use this really cool software, called ImageJ, to trace around the scales in order to get the measurements.

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Hatchling fence lizards (photo: Heather Engler)

If you had told me 5 years ago that I would go from working in the accounting department of an engineering firm to working in the lab of a world famous biologist, I wouldn’t have believed you. But here I am. I have moved from business to biology, and I couldn’t be happier.

 

 

[posted on behalf of Heather Engler]


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The Lone Master’s Student

The first year of my MSc will be coming to a close in May! After a semester of my first graduate school classes and my first experiences as a teaching assistant I was just starting to get the hang of it and the semester ended. This semester has been filled with research planning and manuscript writing. Just coming from my undergraduate degree, the thought of not taking any classes for an entire semester sounded insane, but so far, I have been very productive. I have one field season down, one coming up this spring, and one manuscript started.

Writing my first manuscript has been interesting to say the least. I have learned so much about statistical analysis of data, and the dreaded R. I am having a difficult time with the introduction section but the results and methods were a breeze. I look forward to getting back out into the field, that is why I got into this field after all.

My upcoming field season will begin sometime in March as the vernal pools begin to thaw and the wood frogs return to them to lay their eggs. I will be doing a transplant study that will follow up on research that I did in the lab for the first chapter of my thesis. I used a 3×3 full factorial design to look at how pH and UV-B affected developmental rates, mass, body condition, survival, and baseline and stressed CORT levels in wood frog tadpoles. The second chapter will take place within local vernal pools. Stay tuned for the results from the upcoming season!

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Lizard poop and the parasites who love it

A lot of my work in the lab involves assessing the health and well-being of our fence lizards under different conditions, including their parasite burdens. Parasite infestation can vary with immune status, stress, or external factors such as predation (for example, lizards in fire ant invaded areas have fewer ectoparasites). Ectoparasite (ticks, mites, etc.) load is easy to assess, as we count them on each lizard shortly after capture. Internal parasites are a bit trickier, but one method commonly used in veterinary medicine is to collect their feces, and check it for intestinal parasites and eggs.

There are several different methods of testing for intestinal parasites, including direct smears, qualitative fecal flotations, and quantitative fecal egg counts. Direct smears are the simplest method, involving looking at fecal smears directly under a microscope, but they are also the least sensitive, and often don’t show any results. The most sensitive method is qualitative fecal flotations, the method of choice if you want to see all the possible parasites an organism may have in their feces.
The basic idea behind a fecal flotation is a feces sample is mixed with a solution denser than the parasite eggs you are looking for. The mixture is then spun in a swinging-bucket centrifuge. Due to the parasite eggs having a lower density than the solution, they float to the top of the tube while being centrifuged, and collect on a cover slip on the top of the tube. This results in most of the parasite eggs in the fecal sample being concentrated onto the cover slip for easy viewing.
Unfortunately, the fecal flotation method, while a great way to learn how many different types of parasites are in a fecal sample, does not tell you how many individual eggs are in each gram of feces. Such comparisons are important in veterinary medicine in order to tell if a treatment is working, and is important to us in the lab for comparing fecal egg loads between experimental groups. This is where quantitative fecal egg counts become useful. While less sensitive than fecal flotations (they may not identify lower-level infestations of parasites), fecal egg count methods can tell us how many eggs are in each gram of feces. To do this, we precisely dilute a set amount of feces into flotation solution, and mix it thoroughly. The mixture is then placed in a special slide, called a McMaster, and read after 5 minutes, using the grid on the slide.
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The McMaster slide we use

I’ve had some challenges adapting these methods to use in our fence lizards, as both fecal flotation and fecal egg counts require more feces than a lizard normally produces, but I have gotten some interesting results, mostly a variety of strongyle and coccidia eggs.

 


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Conference time for the Langkilde lab!

The Langkilde lab has recently returned from its annual pilgrimage to the SICB (Society for Integrative and Comparative Biology) meeting, which this year was held in beautiful New Orleans! All our lab members presented talks, and had a great time networking, catching up on top research, and telling people about our own.

Below are some of our thoughts on the meeting, summaries of what we presented – and some tips for conference-goers from all fields!

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Langkilde lab members past and present reunited at SICB (photo: Cate Pritchard)

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Beautiful New Orleans (photo: Kirsty MacLeod)

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Cam and Tracy hang out at the Data Blitz (photo: Cate Pritchard)

Kirsty MacLeod
“I talked about a paper I’ve been working on from our field season in Alabama last summer. Animals encounter environmental stressors daily; how does frequent, low-level stress influence survival and reproductive success? We show that, in Eastern fence lizards, a daily dose of low-concentration stress hormone led to increased adult mortality, and decreased hatching success of her eggs. This was the first conference I’ve been to where animal behaviour hasn’t been the primary focus – this reflects my broadening interests – I’m really excited by integrative research, so this meeting was a great way to see what other people are doing in more mechanistic fields (physiology, genetics, etc). It gave me lots of ideas for taking my own work forward!

My top conference tips are to contact people in advance that you want to talk to – that way you’ll be less likely to chicken out of approaching them! And – make use of Twitter before, during, and after the conference. It’s a great, informal networking tool. I met up with loads of top researchers that I’d first contacted on Twitter, and made lots of new friends!”

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Chris Howey

“I talked about the effect of temperature on the mode of locomotion a snake uses.  As a reptiles body temperature changes, so does its ability to perform specific tasks (digest a meal, sprint away from a predator, etc).  I was interested in how body temperature affected a snake’s ability to move, or slither, across its landscape.  I found that body temperature does affects how well they move, but also affects how they move.  As a snake warms up, it changes its mode of locomotion, uses different muscles, and performs differently.  One could compare this to a horse trotting at lower body temperatures and galloping at warmer body temperatures.  Obviously these are two different types of performances, and the question we raised with my talk is “Can we compare different performances across a single thermal performance curve?”  We argue that it depends on the question being asked.  Are you interested in the muscles, or the mechanisms behind the performance and how temperature affects those mechanisms?  Or, are you interested in the ecological ramifications of slithering across the landscape (i.e., escape a predator)?  Comparing different modes of locomotion along the same thermal performance curve may be flawed if your question is more the former, but may be justified if your question is more the later.
What I enjoyed most about SICB was talking with fellow colleagues and introducing myself to many new people.  SICB is a huge conference, but with a little effort, you can easily cross paths with someone conducting research you are interested in, someone whose research you’ve admired, someone you’ve only talked with on social media, and now you can see that person face-to-face, introduce yourself, and make a new connection.  And who knows, you may even grab a beer with a few of them.
This actually leads into my #1 tip for people going to conferences: Make an effort to introduce yourself to somebody new.  Once you do this, you will realize that it is nothing to be scared of, and you will find yourself talking with more and more new friends and colleagues.” 

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David Ensminger
“I presented on the impact of a stress treatment on maternal behavior and offspring physiology and morphology. The thing I enjoyed the most was getting to meet not only senior researchers but also new researchers and hearing both of their perspectives. 

My tip is to go to the socials and groups at night. They are fantastic places to talk with and meet people.

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Cam Venable
“We already know fire ants are an invasive predator to many organisms, including fence lizards. What I want to focus on is the interaction of Fence Lizards and Fire ants, but as a prey source. This is the first step in my research, by using this study system, to understand how native species adapt to invasive species. The academic side of me really enjoyed meeting other scientist and just chitchatting in informal ways. The 24 year old side of me loved the location of SICB, considering it was in New Orleans!

 Tips for conferences: Well this was my very first conference and I was worried about how to interact with so many bright and accomplished minds. The best and most cliché bit of advice I have, is simply be you. There is no point in putting on a different face, if you’re not even comfortable in it.”

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Michaleia Mead

“The water chemistry of vernal pools are often impacted by the environment. Changes in the pH and UVB impact the larval amphibians that live there. But how? Stay tuned for an upcoming publication! I LOVED meeting new people. I especially enjoyed meeting people who are working outside of my field of study. Their perspectives on my work are often very different than those within my field and I learn a lot from them.

My advice: TALK TO EVERYONE! You never know who you will meet. If you see a poster you don’t usually have an interest in, just stop and ask a question. If nothing else you may make a friend!

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Braulio Assis

“Going to SICB for the first time was fantastic, and New Orleans is a peculiar, very musical city, which I appreciated a lot. Being exposed to research from large variety of fields in biology certainly allowed me to appreciate other research areas better, so I definitely recommend attending talks that are out of your comfort zone. You never know what new ideas you might come up with!

Another valuable tip I have is, to never underestimate the power of a 25-minute nap during lunch break. The amount of information you receive over multiple days in a conference can be a bit overwhelming, so it’s important to rest whenever possible. It also helps you enjoy the nightlife better!”

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Dustin Owen

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Caty Tylan

“I presented info on validating the phytohemagglutinin (PHA) skin test in the green anole as a test of cell-mediated immune function. I also discussed how there are different types of PHA, and how the immune response to PHA differed in the anoles between two of these types (PHA-P and PHA-L). I most enjoyed going to talks, and meeting with researchers whose work I’ve been interested in. Also, the food was great.
My number one tip is to contact people to talk to ahead of time, because I certainly wouldn’t have been able to make myself do it during the conference. And take advantage of the lunch and  networking opportunities SICB sent out before the conference.”

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Tracy Langkilde
Many of you would have played with Mexican Jumping Beans as a child. Ever wondered why it is that they jump? I presented some undergraduate-led research revealing what motivates this fascinating behavior.

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Hormone assays in the lab this Fall

Now that our field seasons are (mostly) over, the members of the Langkilde lab have been busy processing the blood samples we collected over the summer.

As I wrote last time, most of us are looking in some way at the impacts of environmental stressors on an animal’s behaviour, and the characteristics of the offspring they produce. In order to test these questions, we need to be able to quantifiably measure the stress levels of the animals we study.

To do this, we took blood samples from our study species in the field to measure levels of “stress hormones” (glucocorticoids, factors produced by the adrenal glands in response to stress). These hormones circulate in the blood, and correlate with the baseline stress levels of an individual – the more glucocorticoids we find in the blood, the more stressed an animal is. We determine the concentration of glucocorticoids in our blood samples by first centrifuging the sample to separate red blood cells from the plasma (the clear fluid in the eppendorf tube below), and then running the sample through an enzyme immunoassay.

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Enzyme immunoassays work by using antibodies that bind to the factor of interest in a sample – in our case, the steroid hormone corticosterone. We add a known amount of plasma to each well of the plate (above right), and in each well, the corticosterone in the sample binds to the antibodies. The antibodies that aren’t bound by corticosterone are bound by a conjugate tracer, which gives off a colour. So, in the plate above, the more “yellow” the well appears, the less hormone it contains (meaning that more free antibody sites have been left to bind with the yellow tracer-bearing conjugate). We can compare the “yellowness” of each well with wells containing a known amount of hormone, and this allows us to calculate the concentration of hormone in each sample:

I’ve had a lot of fun learning these techniques this Fall, processing my lizard samples, and helping Chris process some of his rattlesnake samples! I’m looking forward to reporting back on the exciting results these data contribute to over the next few months.

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Preparing samples with Chris and Danielle!


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A million ways to die in the South (Or, maternal stress and offspring survival in eastern fence lizards)

When you tell people you are going to be studying lizards in Alabama for the summer, you get used to a raised eyebrow or two. The heat! The snakes! The bugs! When I told people I was going to be deliberately seeking out (ecological) public enemy number 1, fire ants, everyone made clear what I already knew – the theme of my summer was going to be STRESS.

In fact, stress (and in particular, maternal stress during gestation) is exactly what took me to Alabama. Stress during pregnancy can alter the characteristics of the resulting offspring, from morphology to behaviour. That’s assumed to be a bad thing. But could stress experienced by mothers during gestation actually program offspring for life in a stressful environment, giving them an advantage in the long run? This was the question I set out to test during my first, recently completed, field season.

For this study we focused on the eastern fence lizard, Sceloporus undulatus, a species well known to our lab. This lizard is particularly well suited to studying maternal stress because, as is the case for many reptile and amphibian species in the South East, it is subject to the considerable stress of coexisting with fire ants. We know a lot already about how fire ants change the behaviour and physiology of fence lizards – but what about the effects on the next generation through maternal stress effects?

A question this complex, with more than one generational level, required a number of steps. Step one was capturing gravid (carrying eggs) female fence lizards, which we did in May and early June. Having worked previously on mammals (meerkats) and birds (fairy wrens and hihi), this was a new and exciting experience for me! Safe to say, fence lizards (and herps in general!) quickly stole my heart.

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My first fence lizard!

Our next step was bringing the females into the lab, and subjecting them to a highly controlled “stress” treatment – a very low dose of a stress hormone every day, the equivalent of a single fire ant sting. It’s important to note that this is NOT a pain treatment – we use the hormone corticosterone, which is released as part of a lizard’s natural stress response, and which has a number of downstream effects including helping the body’s metabolic system turn amino acids into carbohydrates for use as fuel. In short, our treatment was tricking the lizards’ system (but not the lizard) into thinking they were in a stressful situation.

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A female in our lab housing facility. Note the non-toxic nail polish mark, denoting that this female was in the “no stress” treatment group.

The next step was waiting for the females to lay their eggs, at which point our stress treatment ceased, and females were ready to be returned to the wild. We incubated their eggs (incubation takes around 50 days) and waited for the babies to hatch to begin the next step of our experiment…

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Fence lizard eggs

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Teeny weeny fence lizard baby!

We hypothesised that if maternal stress was adaptively programming offspring to be be better suited to a stressful environment (for example, by making them more responsive to predators, or better able to cope with frequent stressors), then we should see offspring from stressed mothers surviving better in stressful environments than offspring from mothers that did not experience stress during gestation.

To test this, we needed to create “stressful” and “non-stressful” environments in which to put the offspring. When we weren’t catching females or incubating eggs, we were building four 20x20m outdoor enclosures for this purpose! Thankfully, life in Alabama with fire ants everywhere is stressful enough, so we didn’t need to artificially create a stressful environment. To create a “non-stressful” environment, we removed fire ant mounds from two of the four enclosures. We hypothesised that in these enclosures, offspring from unstressed mothers should do best.

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One of our four hatchling enclosures

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Newly-hatched fence lizard enjoying life in an enclosure!

Once the offspring hatched, we put them into the enclosures and monitored their survival by checking them every day (not an easy task, they are small and wily!). I also recorded their habitat use, how far they were moving within the enclosures from their release spots, and how they responded to small, short-term stressors, like being picked up to be weighed. I’m now in the process of analysing this data, and am looking forward to seeing if our hypotheses hold true. Watch this space!

So, lizards aside, how did I cope with the stresses of a summer in Alabama? The heat – loved it! The snakes – try and keep me away from them! The bugs – who cares?! The people – a whole lot of new friends. There may be a million ways to die in the South, but there sure are a million and one things to love.

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Box turtle, Geneva State Forest

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Gray rat snake, Solon Dixon Forest Research Centre

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A devoted Alabama convert – can’t wait to be back!