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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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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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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Vernal Pool Macro-invertebrates in a Burnt Landscape

My name is Richard Novak and I am finishing up my freshman year. I am in the Schreyer Honors College and I am studying Wildlife and Fisheries Science, with the Fisheries option. This is my second semester working in the Langkilde Lab. In fall 2017, I began working under Dr. Chris Howey as a research assistant helping with rattlesnake gestation site video monitoring. Currently, Dr. Howey and I are working on a study with macroinvertebrate communities in vernal pools and how they are impacted by prescribed fire. I was fortunate to receive an Erickson Discovery Grant which will allow me to continue working on this project into the summer. So far, I have developed my research questions and data collection methods, and I have been gathering data throughout spring semester. This research experience has been valuable to me for several reasons. First, I have been able to get a first-hand look at the entire experimental design and execution process, something I can only read about in classes. Additionally, interacting with graduate students and other faculty has been very influential when thinking about my future ambitions and career path potentials.

Vernal Pool within a previously burnt landscape

Vernal Pool within a previously burnt landscape

The purpose of this study is to compare macroinvertebrates communities in vernal pools with varying fire histories.  Fire is being used as a forest management tool, which will create a more open landscape that some wildlife species may prefer.  Additionally, these prescribed fires may promote the growth of new vegetation and increase food for wildlife within the forest.  I am looking at water samples from 12 vernal pools; three that were burnt over once (in 2016), three were burnt and mowed over (in 2016; this is an additional disturbance to the landscape), three pools burnt over twice (in 2014 and 2016), and three vernal pools from a control group with no recent fire or disturbance history.  Specifically, I would like to answer the question, “do prescribed fire practices alter the macroinvertebrate communities of vernal pools?” This question has been relatively unexplored in previous research.  But preliminary data collected by Chris suggests that physiochemical (physical and chemical) characteristics of these pools are different, which could lead to differences in what macroinvertebrates are able to survive in these pools.  I will analyze water samples collected from these vernal pools for macro-invertebrates, identify all macroinvertebrates found to family, and determine abundance of each family. The water samples that I have been going through now were collected in 2016, and additional samples will be analyzed from 2017 that are currently being collected.  I look forward to getting out in the field this summer and assisting with measurements and collections.

Macroinv lab bench set up

This is what my lab bench typically looks like while I’m collecting data. My sorting tray with a sample spread out to the left, a hand-held magnifying glass, dissecting microscope, and the computer with my spreadsheet in the background. Note, there are also plenty of macroinvertebrate books to help me identify everything I find.

 

When I first began this project, I had to learn how to identify the macroinvertebrates to family. One of the reasons I am interested in macroinvertebrates is because of my interest in fly fishing, which requires basic knowledge of aquatic entomology, so I had some ID skills to bring to the table. I practiced using dichotomous keys to identify the specimens, a task I found time consuming but very learnable with practice. Now, I am very familiar with the families that I encounter most often. As of right now, I have identified the presence of over 20 families of macroinvertebrates among the vernal pools in the study. I find a lot of mosquito larvae (Culicidae), phantom midges (Chaoboridae), cased caddisflies (Limnephilidae and Odontoceridae), as well as several families of dragonfly and damselfly. To me, the coolest creatures that I find are fairy shrimp (Chirocephilidae) and water-boatmen (Corixidae) although I don’t come across either of those frequently.

Culicidae Pupae

Culicidae pupae. These will grow up to become the dreaded mosquito!

Chaoboridae Larvae

Chaoboridae larva. These are also known as phantom midges.

Chaoboridae Larvae

Limnephilidae larva. This is a type of caddisfly.  Caddisflies are known to build these ‘houses’ out of sticks, leaves, and rocks within their environment.  The actual larva is within this house made of sticks and you can see its head sticking out of the top.  Different species of caddisflies will use different substrates to build their houses, so you can tell species apart based on the house materials.

Limnephilidae

Odontoceridae larvae. These are another species of caddisfly. You can see that they use a different substrate material for their houses.

Chirocephilidae Larva

Chirocephilidae larva. This is also known as a fairy shrimp and can be very common in many of Pennsylvania’s vernal pools.

Corixidae

Corixidae adult. These are also known as water-boatmen. They are typically seen swimming across the surface of a vernal pool, but can dive to the bottom when foraging or escaping a would-be predator.

 

So far, I am finding more mosquito larvae (Culicidae) in unburned pools.  But among the burned pools, I am observing more mosquito larvae and caddisflies (Limnephilidae) in pools that were more disturbed (burned and mowed).  This trend among the vernal pools is interesting, because that mow was an extra disturbance on top of the burn, yet these two families appear to be doing better in these pools.  Please note though, these data are still being collected and these results may not accurately represent our final findings once we have analyzed all water samples.

Macroinv prelim data

Preliminary data for our macroinvertebrate communities within the four different treatments. In the future we will compare species diversity and richness among vernal pools. We will also see if there are any correlations between species presence/absence from vernal pools and the physiochemical characteristics of those pools.

Working on this project has been useful to me for many reasons. I have had a lot of fun sorting through samples and looking at the macroinvertebrates; it really never gets old to me which is good because I’ll be staring at trays a lot more this summer. It has been very satisfying to see my very own data begin to build on the spreadsheet as I work. Also, being around other lab members has given me a look into what school is like for graduate students. My freshman year is coming to a close, and I hope to take on new and exciting projects throughout the rest of my undergraduate career. When I came to college last fall, I did not expect to become involved in research right away, but I am very glad I took that step early and I have been fortunate in the opportunities presented to me. After graduating, I plan to pursue at least a master’s degree in a biology related field. I am interested in working for a natural resource management agency, although this experience has opened my eyes to the possibility of university research as a career. Whatever happens, my goal is to continue exploring more about biology and the organisms that fascinate me so much.

Richard Novak

Me looking hard at work keying out macroinvertebrates!


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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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Attracting Unwanted Attention

I’ve certainly attracted my share of unwanted attention: missing a key free throw in a basketball game when everyone in the crowd was watching, faceplanting while exiting stage right in A Midsummer Night’s Dream, or unconsciously ripping a huge burp at a fancy dinner. Fortunately, as a human, the stakes involved in these mistakes were fairly low: a little embarrassment and a good story once the shame had worn off. For many organisms, like lizards, however, attracting unwanted attention in the real world can have serious consequences…

From previous research in the Langkilde Lab, we know that invasive fire ants (Solenopsis invicta) can pose a serious danger to native fence lizards (Sceloporus undulatus), and that fence lizards from areas invaded by fire ants respond to encounters with these ants with a variety of twitches and scratches to remove ants as well as fleeing more often to escape them.

We know little, however, about the behavioral rules that govern fleeing and twitching in these lizards. Do they flee more from all predators? Do they flee from all ants? We know the benefit of twitching and fleeing (not getting stung!), but is there any cost to these behaviors? Because we’re ecologists, we looked to answer these questions by conducting a series of experiments and published them in Animal Behaviour.

So, do fence lizards from fire ant-invaded areas flee more from other predators? Because we couldn’t let actual predators attack our lizards, I borrowed a stuffed kestrel (Falco sparverius, a well-known predator of fence lizards), rigged it with a wire harness, attached some very serious and scary-looking eyes, and swung it at unprepared fence lizards to see what their reactions would be.

Taxidermied kestrels are mostly as scary as the real thing.

We found that fence lizards from sites with and without fire ants fled from simulated kestrel attacks the same proportion of the time, and with the same strength and latency (reaction time), suggesting that fence lizards exposed to fire ants don’t flee more from all predators.

We next tested our fence lizards’ reactions to fire ants (which we’ve done before) as well as two types of native ants which might annoy lizards by running on them, but lack the venom (and danger) of fire ants. In fact, these native ants are important sources of food for fence lizards under normal circumstances. In these tests, we found that fence lizards from sites with fire ants fled more from all types of ants, not just fire ants, indicating that this fleeing behavior is generalized to multiple types of ants that they encounter, including those that don’t pose a serious danger.

For the more visually oriented, this series of experiments was illustrated super-well by Tali Hammond, a behavioral ecologist, who was interested in the paper (check out that sweet Sceloporus!)

Check out more illustrated papers by Tali: @mammalLady

So what might the consequences of this generalization be for fence lizards? For one, it’s obviously not ideal to be running away from something (i.e., native ants) that isn’t a threat and should be a meal. This could result in lower food intake or time wasted running away from non-dangerous ants, though we haven’t tested for these effects. More dramatically, twitching and fleeing break crypsis, a lizard’s primary defense against its visually hunting predators, including snakes, and birds of prey like the kestrel. While fence lizards are usually quite well camouflaged, imagine how easy it might be for a predator to spot a lizard jerking around as in the video above. We looked for evidence of this cost in our lizards by quantifying the amount of injuries (broken tails, scars, missing limbs) to lizards at sites with and without fire ants.

A missing “hand” is an example of injuries many fence lizards have.

We found that lizards from sites with fire ants do indeed have more of these injuries than lizards at sites without fire ants. This result suggests that fleeing from fire ants might attract unwanted attention from other types of predators. And when we consider that these lizards also flee more from native ants, which are common in the environment, these antipredator behaviors might have a serious drawback. It is important to note that this evidence is circumstantial; we didn’t see predators preying on lizards running from ants (this would be very difficult), and there could be other explanations for this pattern, such as differences in predator communities, or perhaps differing skill levels of predators. However, this work suggests that lizard that twitch and flee in response to ants may be attacked more.

So why do we see this behavior if it has these drawbacks? My personal guess is that it comes down to consequences. As a human, the consequences of my unwanted attention were fairly minor (shame). The stakes for fence lizards are a bit higher: fleeing can lead to running from your own dinner or attracting attention from predators. BUT the costs of not fleeing when attacked by fire ants are likely even higher (serious injury or death). And in many areas fire ants are much more common and likely to interact with lizards more frequently than snakes and kestrels. In other words, the lizards are likely making the best choice available to them. In the future, perhaps, they will adapt to distinguish between dangerous and native ants, allowing them to make more optimal decisions, and reduce the costs of these antipredator behaviors. More broadly speaking, I believe this research shows that we need to consider a wide variety of potential costs as well as benefits when looking at organisms adapting to changes in their environments.

The full paper in Animal Behaviour can be found here.