The Lizard Log

The Langkilde Lab in Action


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Looking at brains and hormone receptors

Hi everyone!

Last time I talked about looking at how maternal stress effects egg nutrients and we have found that maternal stress increases yolk protein and decreases yolk lipid concentrations! With that out of the way, today I am going to talk about the next project I am working on which involves brains. We know that maternal stress can have many effects on animals, such as changing their hormone concentrations and behavior, however less is known about how maternal stress influences hormone receptors in the brain, so I wanted to explore this. First you have to preserve the brain and then slice it thin so you can eventually look at the cells.

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The white circles are brain slices, important to not let them dry out!

You also want to make some sort of barrier around the slices so fluid stays on the slides as they incubate in the different fluids. I used a barrier pen, but you can also use clear nail polish!

Next, through a series of washes and solutions, you want to punch holes in the cell membranes so your antibodies can get in and bind with the receptors.

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Carefully adding and removing solution can be done with pipettes

Because some of the solutions have to incubate for a long time, its best to have them in an enclosed container with a wet paper town so the fluids don’t evaporate. After the solutions are on, you want to let them incubate on a gently rotating surface so the solution moves and the antibodies can bind to the appropriate receptors. You can use different antibodies to bind to different receptors, so we used ones that bind to stress hormone receptors.

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Sometimes they incubate at room temperature….

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…and sometimes they incubate in a refrigerated room.

After you get the different antibodies attached to the receptors, you need to add something to differentiate those cells. For that we use a diaminobenzidine , or DAB for short, solution. However, you have to be really careful with this, as not only is DAB a hazardous material as a solid, to get it to dissolve into a fluid you need to use 10M hydrochloric acid, which has a pH of somewhere around -1.0 (measuring pH gets very weird when you get strong acids and are below 0)!

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Not only hazardous, but will also melt through organic things!

However, if we add this to our brain tissue without correcting the pH, that would be very bad! So by using a base (NaOH) and moving quickly (don’t want the DAB to come out of solution) we can correct the pH using a pH meter.

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pH meter along with solutions and protocol.

Once we bring the pH back to around neutral, we can add it to our brain slices so the dab will cause a color change in the cells with receptors for stress hormones.

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The thicker slices (50um) have more receptors so they are darker then the 15um slices.

Finally, after removing all fluids and sealing the slides with a cover slip, we can look at the slices to see what cells have receptors!

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The darker cells have stress hormone receptors compared to the tissue at the top.

The question I want to address with this technique is if maternal stress effects the amount of stress hormone receptors in the brains, so stay tuned!
Thanks,
David

 

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Testing the environmental matching hypothesis – return to Alabama!

Another summer field season has now come and gone! This summer I returned to Solon Dixon Forestry Education Center in Alabama (one of my favourite spots on earth!) to continue my research on how stress during gestation influences the offspring of eastern fence lizards (Sceloporus undulatus).

Last year, we investigated how physiological stress during gestation (at the level of a non-lethal predator encounter – for example, when a lizard encounters a couple of toxic fire ants, but isn’t killed by the ants) affects survival of mothers, and how many of their eggs successfully hatch. You can read more about this experiment, and the fieldwork that went into it, here (and stay posted for the published results soon!).

This year I wanted to build on these results and ideas to test how maternal stress influences the offspring that do hatch and make it out into the world. Do they themselves then cope better with a stressful environment, having been “primed” for it by their mothers (the “environmental matching” hypothesis)? Or are offspring born to stressed mothers poorer in quality, and less likely to survive in the wild, regardless of how stressful their environment is? In order to test these ideas, we first made the long trip south to collect gravid females from south Alabama early in the summer, and to build experimental enclosures in which to eventually release their offspring. I then repeated the maternal stress treatment from last year and once again became a lizard mama as I followed the females from laying their eggs, to incubating the eggs, and eventually seeing these bite-sized babies hatch out!

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Freshly hatched fence lizards – <1g!

Once they hatched, the offspring went into the enclosures that we’d built. These enclosures were designed to test whether maternal stress programs offspring to be able to better deal with a stressful environment. The enclosures either contained a key stressor (invasive fire ants), or were fire ant-free. Each day I conducted a mini-census, walking through enclosures to look for each lizard – as you can see in the video below, babies were marked so I could tell exactly who was present each day (and so, which lizards survived, and which didn’t). I also observed their behaviour, and how they used the habitat available to them (for example, did offspring from stressed/non-stressed mothers differ in whether they liked to be out in the open, like the lizards you see in the video – or did they hide more?).

After a great summer (if measuring 200+ baby lizards isn’t a metric of a great summer, I don’t know what is), I’m now back at Penn State with a box of data to work through. I’m excited to report back on what I found in the coming months – so stay tuned!

 

 

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Hanging out with an adult female Sceloporus at Solon Dixon

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Beautiful Solon Dixon


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It’s fieldwork season!

The lab is currently busy with a variety of field and lab-based projects. In April/May, Cam, David, Tom, and I were in Alabama collecting lizards. Here’s a glimpse of what the long trip to the South, and our work there, looks like! Check back later in the summer for more on the continuation of this project.


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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.