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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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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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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Basking Site Use by Timber Rattlesnake Morphotypes – By Shawn Snyder

My name is Shawn Snyder and I am currently a senior majoring in Wildlife and Fisheries Science.  This is my first and only year working in the Langkilde Lab.  During the summer of 2016, I worked under Dr. Chris Howey as a Research Technician studying the effects of prescribed fire on timber rattlesnake populations.  This position provided me the opportunity to radio-track timber rattlesnakes, record habitat data on tracked snakes, catch new snakes (extremely fun), learn how to safely tube a venomous snake (even more fun), and conduct vegetation surveys.  Also, this position provided me the opportunity to formulate my own scientific question to test! Together, Chris and I thought up a small side-project that I could conduct throughout the summer, which provided me the fantastic experience of going through the scientific process, collecting my own data, analyzing those data, and now writing a manuscript so that I can share those results with the scientific world.

When we first started collecting data for my side-project I was a little apprehensive.  Once the data was collected and analyzed I realized that this project was going to take time and a large amount of effort to complete.  As the process of analyzing the data and then coming up with a plan for the manuscript began to take shape, I started to feel challenged and nervous by this new task. But weekly meetings with Chris to discuss the process of writing a manuscript have helped immensely.  This is my first manuscript and yes it is challenging, but it will all be worth it once we have a finished product. I have ambitions to continue on to a Graduate program after I graduate and this manuscript will help me build my C.V. to apply to Grad schools.

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Two yellow morphs bask alongside three black morph timber rattlesnakes at a gestation site. Although we did not use gestating (i.e., pregnant) females as part of this project, this shows you the posture of a basking snake and the difference in color morphs.

My research is investigating if the two distinct morphotypes of timber rattlesnakes (a dark, black morph and a lighter, yellow morph; see above picture) use basking habitat with differing amounts of canopy openness and solar radiation. Previous research suggests that the dark morph evolved in response to thermal limitations in the northern parts of its range.  Darker snakes have more melanin in their skin, which allows them to absorb more solar radiation and maintain a higher body temperature than yellow morphs.  Yellow morphs having this thermal disadvantage, in theory would have to choose basking sites that receive more solar radiation to compensate for this limitation if they wanted to maintain a similar body temperature to the black morphs.  Specifically, I am testing the hypothesis that yellow morphs use basking habitat that has more canopy openness and receives more direct solar radiation (i.e., sun) than basking habitat used by black morphs.

 

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A black morph male timber rattlesnake is seen courting a basking yellow morph female.  Once again, the difference in color morphs is striking and has led many to ask what selective pressures are maintaining this polymorphism.

To test this hypothesis, I measured canopy openness over basking yellow and black morphs. I used the timber rattlesnakes that are being radio-tracked for Dr. Howey’s main study as my sample population and placed a flag where a snake was found exhibiting basking behaviors (see picture below  for example).  We took a picture facing skyward directly over the snake using a camera with a fisheye lens.  This lens takes a picture of 180 degrees and captures an image of all of the canopy over the snake (see picture).  We can then analyze these hemispherical photographs using a computer program called Gap Light Analyzer to measure the percent canopy openness and the amount of direct solar radiation transmittance (i.e., rays of sunlight) for each basking site.  Direct solar radiation is when the sunlight reaches the forest floor with no obstructions from the canopy; as opposed to indirect solar radiation which may be radiation that is being reflected off of clouds, trees, or the ground itself.  Our study site is characterized as having a mature Oak/Maple forest with an abundance of closed canopy throughout the area.  Both morphotypes use this “closed canopy” forest throughout the summer as foraging grounds, and when they need to bask they must seek out areas where some sunlight is making its way through the canopy.  This is where my question becomes very important comparing the habitat used by each morph.

 

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A flag is placed next to a basking yellow morph.  An exact description of the habitat is recorded so that I can come back at a later time (when the snake is not there) and take a photo of the canopy directly over where the snake had been.

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Two examples of hemispherical photographs taken over two different basking timber rattlesnakes.  Both canopies actually have similar canopy openness, but the canopy on the left receives far more direct solar radiation based on the placement of those canopy openings.

So far, my results show that the two morphs use habitat that have similar percent canopy openness, however, there was a difference in the amount of UV transmittance between the basking sites used by the two morphs.  Canopy openness doesn’t necessarily designate a “warmer” site because the sun path may not go directly over the gaps in the canopy of that site, thus, the site wouldn’t receive large amounts of direct solar radiation.  Black morphs use basking sites that received lower amounts of direct sunlight.  They may be able to do this because the greater amount of melanin in their skin provides a greater ability to absorb whatever direct or indirect solar radiation is available more effectively. Yellow morphs use basking sites that received more direct solar radiation.  They could be forced to use these sites to compensate for their disadvantage in their thermal ability.  I am currently working on writing a manuscript for these data and hope to have it completed by the end of 2016.  Stay tuned for more on this manuscripts progress!

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Here is a picture of Shawn (holding a Hellbender!!) while on a break from collecting some amazing data.


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Efficacy of Daylighting… Part I

Pregnant female rattlesnakes prefer to maintain an elevated body temperature (~32 °C), which allows for a more optimal development of embryos.  In Pennsylvania forests, however, these warm temperatures are not very abundant.  So, in order to achieve these elevated body temperatures, pregnant females seek out rare, open habitat (known as gestation sites) that receive a lot of sunlight.  Sometimes, females may travel up to a mile from their den sites just to gain access to openings within the forest canopy.

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Yellow morph female timber rattlesnake sits coiled under a small rock overhang.

However, not all open gestation sites are created equally.  Last year, Mark Herr, Michaleia Mead, and I uncovered a trade-off at timber rattlesnake gestation sites of various sizes.  Gestation sites that were very open provided pregnant females with more sunlight and warmer body temperatures for a longer duration of the day.  But, these same sites also came with an increased risk of predation!

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At a timber rattlesnake gestation site, a red-tailed hawk swoops in on top of a foam timber rattlesnake model (not seen). A yellow-morph timber rattlesnake model is pictured behind the hawk.

More predators visited these more open sites, predators like bobcats, fishers, and hawks.  But, smaller, more enclosed gestation sites were so thermally poor, we observed females returning to their den at the end of the field season still pregnant!  The use of thermally unfavorable gestation sites may provide the rattlesnake with a sanctuary from potential predators, but there are still terrible repercussions for choosing to use these sites.  We have found that snakes using thermally unfavorable sites tend to give birth at a later date.  The timing that an animal gives birth is very important.  If rattlesnakes give birth to their offspring too late in the year, the small neonate offspring will have little, to no time to complete their first shed and then obtain a small meal before entering the den for hibernation.  It is believed that survival for neonates unable to do these two things is close to zero.  Further, some pregnant rattlesnakes that use thermally unfavorable gestation sites are known to abort their entire litter toward the end of the summer if she decides that they are developing too slowly.

So why would pregnant females continue to use small, more enclosed, thermally poor gestation sites?  Possibly because there was a decreased risk of predation?  Possibly, however, because of strong site fidelity?  Possibly these sites were, at one time, thermally favorable, but over the years vegetation has encroached upon these open areas and shaded out the once warm, sunny rocks.  Due to the rattlesnake’s fidelic response to locating a favorable gestation site, they now find themselves sitting among the shade throughout much of the day.

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Timber rattlesnake taking advantage of a small break in the canopy and some sunlight reaching the forest floor.

 

We’ve addressed the problem, now how do we fix it?

In order to manage timber rattlesnake populations better, forest and wildlife managers have begun to open up gestation sites, un-shading these areas from vegetation, in a process called “daylighting”.  However, recall that more open sites have an increased risk of predation.  So can we open up these sites just enough to let the sun in, but keep the hawks out?  In an attempt to suppress the increased risk of predation that we observed at more open sites, I have begun to direct daylighting techniques to target specific trees that would increase the amount of solar radiation a site would see, without greatly increasing the risk of predation.  To do this, I will use hemispherical photography (see picture below), observe the path of the sun throughout the gestation period, and then target those trees that overlap with the path of the sun.  This way I can open up each of the sites just enough, but keep those trees that do not overlap with the path of the sun and could perhaps maintain some decreased risk of predation.

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Hemispherical photograph at one of our gestation sites. The path of the sun throughout the entire gestation period is shown by the yellow arc. Trees that overlap with this arc can be targeted to improve the thermal quality of the site.

Of course I want to address this management idea with as much scientific rigor as possible for an ecological study.  This past summer, I visited six historic gestation sites within Pennsylvania.  Four out of the six sites are pretty shaded over, and I consider thermally poor.  Two of the gestation sites are fairly open and should provide pregnant females with a plethora of sunlight.  This summer, I surgically implanted nine pregnant timber rattlesnakes with temperature-sensitive radio transmitters, which allowed me to track these individuals throughout the summer. I followed each snake and noted their behaviors, body temperatures, and the date that they gave birth to their young.  Additionally, I measured available body temperatures and risk of predation just like we did in the previous experiment that Mark, Michaleia, and I completed last summer.  The catch is, this winter I will go into three of the six sites (along with US Forestry personnel), and we will remove specific trees blocking out the path of the sun.  Then next summer I will repeat everything and determine if Daylighting improved these thermally poor sites.  Will I see warmer available temperatures within the Daylighted sites?  Will snakes within these sites maintain warmer body temperatures and for a longer duration of the day?  Will these snakes give birth at an earlier date, allowing their young to shed and get a first meal before hibernation?  Will I still see an increased risk of predation?

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Biophysical models laid out at a gestation site and measuring the potential body temperatures that a snake could achieve at that site.

Many of these questions I won’t be able to answer until next year.  But, I am collecting some interesting data thus far.  As expected, the two sites that were more open did have warmer available temperatures.  Snakes occupying these sites maintained warmer body temperatures, moved less often, and were the first to give birth to adorable baby rattlesnakes!

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Neonate rattlesnake coiled up next its mother.

But, these sites were also visited by more potential predators.  In fact, in one instance we observed a red-tailed hawk swoop down and nab a garter snake that was basking alongside our pregnant rattlesnakes!

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Red-tailed hawk grabbing a quick dinner at a gestation site. I’m fairly certain this was a garter snake that got nailed.

The more enclosed sites were indeed cooler and snakes at these sites maintained cooler body temperatures and moved more often.  It appeared that some of these pregnant females shuttled between nearby sites in order to track the path of the sun.  In the morning the snake may be at one site, and in the afternoon that same snake would move to a nearby site.

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Female at a thermally poor area was typically found in the morning at a cut tree stump. In the afternoon she typically moved to another location.

Other snakes continued to move throughout the entire summer from one site to the next; constantly searching for a thermally suitable site where she could continue to develop her babies.  Unfortunately, all of these movements brought one of our mommas too close to a nearby road where someone swerved into the shoulder in order to run her over.  Although the loss of this mom was a little tough on me, it did show me just how important it is to improve these historic gestation sites.

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Our big momma rattlesnake could never find a suitable gestation site. She moved from potential site to potential site for about a month before coming too close to a nearby road.

Currently, as I am writing this blog, we are still waiting on some of our snakes from the thermally poor sites to give birth.  As we find neonates (babies) at each of our sites, I will also collect data on each of them so that we can compare body condition (health) among sites.

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Collecting data on newborn rattlesnakes is a fantastic way to start anyone’s day. I’m collecting data on body length and mass so that I can determine the body condition (or health) of each individual.

 

Next year, I will continue to track snakes throughout these same gestation sites.  However, following our daylighting management, we hope that all of our pregnant snakes will give birth at early dates, move less, and stay clear of predators.  To be continued…

 


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Much Anole About Immunology

While most of the lab has been down in Alabama, I’ve spent a good part of this summer back at Penn State, working with a species I’ve never used before – the green anole (Anolis carolinensis).

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Isn’t he cute?

There is a test we’d like to use in our fence lizards, called the phytohemagglutinin (PHA) skin test. It involves injecting the pad of a rear foot with a small amount of PHA, which stimulates part of the immune system, and then measuring the swelling that occurs. This swelling is small, and temporary, abating in a few days with no lasting damage. But the level of swelling can provide information about the lizards’ immune function.

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A confused anole having its rear foot measured with calipers.

Unfortunately, while this test has been used in humans, birds, rats, and even amphibians, it has not yet been validated in any reptile species. Ideally I would validate the test in our species of interest, the eastern fence lizard, but I needed a larger number of lizards than we can reasonably catch. So, instead, we decided to purchase some green anoles for this project.

In addition to seeing if the PHA test works in reptiles, we’re also trying to determine if the type of PHA used makes a difference, as there are many different formulations of PHA used, and each formulation may have a different effect. I’m also determining exactly what the immune reaction to the different PHA formulations are, and how this evolves over time after the injection.