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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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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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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All Good Things…

There is unrest in the Langkilde Lab. Several members have declared their intention to graduate and leave the Penn State University.

This graduatist movement, under the leadership of the mysterious Doctor Thawley, will make it difficult for the limited number of graduate students left to maintain scientific rigor and order in the fence lizard universe.

Principal Investigator Tracy, the Head of the Biology Department, returns to the field to orchestrate the critical succession of power and saurian expertise in the upcoming research season….

A More Dramatic Version…


The past few weeks have been eventful ones in the Langkilde Lab. Gail and I were both repping the lab at the inaugural Graduate Student Awards Luncheon. I received the Intercollege Graduate Student Outreach Achievement Award, an award close to my heart, for bringing research to the community and “commitment to advancing the welfare of the public through scholarly pursuits.” Gail received her highly prestigious Penn State Alumni Association Dissertation Award which included a very spiffy and heavy Distinguished Doctoral Scholar Medal. Previous lab members have received both of these awards, and we hope that our new lab members will continue this tradition of excellence and outreach!

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Gail receiving her totally legit medallion from Penn State President, Dr. Eric Barron.

Gail and I also both graduated this past Sunday. It was a great chance to hang out in the last row of the huge auditorium and crack jokes with Tracy, observe the diverse footwear of graduating graduate students, and attempt to determine the school with the most awesome PhD robes.

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We were totally serious throughout the entire two hour ceremony.

Unlike some other awards ceremonies (*cough* Awards Luncheon *cough*), we were allowed to sit and walk together which was great. None of us tripped while negotiating the steep, narrow stairs to the stage, and I even managed to shake the President’s hand with something approaching poise.

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I was the only graduate wearing formal Birkenstocks at the ceremony.

Afterwards, we gathered outside with our families and friends for a few pics to document our snazzy regalia and the fact that an institute of higher learning actually saw fit to give us advanced degrees.

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That’s a wrap!

Lab Undergraduate Extraordinaire, Mark Herr, graduated as well (though we have no photographic evidence of this). Mark has worked in the lab for four years and published several papers. In recognition of his work, Mark received several lab commendations, including a certificate of excellence for Use of the Word “Devastating” on a Daily Basis and the Award for Forcing the Creation of a Special SSAR Award for Undergraduates.

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Mark Herr is recognized for his efforts to reintroduce large mammalian predators to control overpopulation.

In spite of these losses, a new hope has arisen in the next generation of lab graduate students and post-doctoral scholars.

Tracy is leading the bulk of the lab force, including post-doc Kirsty and graduate students Cam, Dustin, David, and Michaleia, to Solon Dixon, where an old ally, Nicole Freidenfelds, will help them discover clues as to how maternal stress hormones influence behavior, stress physiology, and metabolism.

Gail has volunteered for one final mission in the field: training a team of lizard neophytes including Braulio and Caty to capture fence lizards, especially gravid females, at sites in Arkansas and Tennessee that remain uninvaded by fire ants. This team will return to the lab and allow these female lizards to lay their eggs. They hope to determine if ladies bearing typically male characteristics, in this case blue throat badges (“beards”), also have bearded offspring.

Stay tuned this summer for more exciting developments in the fence lizard universe!


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New lab member – Kirsty MacLeod!

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My hometown, Tobermory (Scotland)

 

I arrived in the Langkilde lab to take up a postdoc position (held jointly in the Sheriff lab) in March – and it’s been a brilliant first few weeks of getting to know not just a new lab and study system, but also a new country!

I grew up on a small island on the west coast of Scotland, which explains a lot about my love of the outdoors and exploring the natural world (also my love of whisky, bagpipes, and anything fried in batter). I was awarded a BSc in Zoology from the University of St Andrews in 2009, and then moved south of the border to take up a PhD position at the University of Cambridge.

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PhD fieldwork in South Africa – photo by Lyndsey Marris

For my PhD, I was interested in investigating how much helpers help in cooperative societies. Why might one individual provide help to another (for example, by assisting in rearing their offspring), and what determines how much they help? I looked particularly at allonursing behaviour, which is when a female nurses the offspring of another – in humans, we call this (now fairly rare!) practise “wet-nursing”. I had an amazing study system at my disposal to help me answer some of these research questions – the Kalahari meerkat!

After my PhD I moved to birds as model systems for researching maternal, rather than helper, investment in offspring. For my first postdoc project (with Dr Rose Thorogood), I investigated how food- and carotenoid-supplementation regimes in the endangered hihi in New Zealand might influence the outcomes of brood hierarchies and sex ratio variation. I was also awarded a research grant from the Association for the Study of Animal Behaviour to test whether maternal investment in offspring in a cooperatively breeding bird, the red-winged fairy wren, varies according to helper care. You can find out more about these projects, and my PhD research on my website.

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Catching jacanas in Zambia – photo by Claire Spottiswoode

 

I am really excited to join the fence lizard team and the Langkilde lab to work on unravelling the complex relationship between invasive fire ants and eastern fence lizards in the US. My projects will be looking at how stress influences fence lizard moms, and what the downstream consequences are for offspring survival and behaviour. We are heading to Alabama to start studying these guys in one month – so stay tuned for excitement from the field!

 

 


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Cam joins the lab!

Where do I begin? I guess I will start with a little background info. My name is Cameron Venable and I hail from the great state of Maryland. If that means nothing to you, then in a nutshell I really love the MD flag and crabs (which is a key part of a Marylander’s life).  Anyway, after high school, I went to Lebanon Valley College in Annville, PA. At LVC I majored in Biology and minored in Spanish.  As a side-note of my story, LVC was around 1500-1600 people… TOTAL. So coming to PSU was a huge jump! Not only were there exponentially more people, but also grad school is a completely different world.

I may be the odd ball of the current students because I am a second year. So how am I just now introducing myself? Well, that’s another long story, but basically I switched advisors last semester and joined the Langkilde Lab (for my lab-mates that may read this, thanks for making me feel welcomed). As a part of this lab, I’ve been endlessly brainstorming for future projects to begin. As of now the big plans for the near future are as follows:

1) Embryo nutrition in the fence lizard and fire ant system. The idea would be to investigate the influence of fire ant presence on the maternal input into offspring.

2) Looking forward to working with a past member of the Lab, Dr. Lindsey Swierk within her study system, which will focus on how urbanization affects wood frogs in various ways.

3) Lastly, becoming an expert on Bomb Calorimetry, which would be helpful for various projects in quantifying nutrition.

Those are the ideas for the upcoming months, but of course in this field things are subject to change. Which means… Stay tuned for the adventure!!!

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Cam on La Isla Mona, Puerto Rico with Anolis monensis