The Lizard Log

The Langkilde Lab in Action


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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.


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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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Racing baby lizards (for science)!

In the latest chapter of the “bearded lady” saga (female fence lizards bearing ornamentation that is typical of males), we continue to investigate what potential advantages these “unattractive” females could have that allow them to persist in high numbers.

We know that in many species, colorful, conspicuous ornaments have a tight relationship with levels of particular hormones (such as testosterone), which themselves are related to physical performance. One of our current guesses is that even though females bearing male-like ornamentation are not prime sexual partners in the lizard world, their offspring might be more physically competitive than offspring of the more desirable females (read more here). The costs and benefits of both strategies could be responsible for the coexistence of the two!

A good way of measuring the physical performance of an animal is by how fast they can run. With the help of two enthusiastic undergraduate students, Maggie Zemanek and Sean Dailey, I am recording slow-motion videos of juvenile lizards running on a race track. This will help us calculate how fast each of them can run, and compare that to what their moms looked like: are the faster runners offspring of ornamented females?

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Do your best!

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Body temperature greatly influences performance in reptiles, so Sean makes sure we record how warm each lizard is

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Maggie sets a contestant on its marks

Maggie, Sean and I still have a lot of juvenile lizards to race, but hopefully we’ll find some interesting patterns in our experiment. Stay tuned!


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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!

woodfrog


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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.