The Lizard Log

The Langkilde Lab in Action


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Tree selection is linked to locomotor performance and associated noise production in fence lizards

New paper in Journal of Zoology featuring Kirsty, Tracy, and Nicole! The substrates on which animals spend their time can affect how they look, move, and sound. We found fence lizards more frequently on deciduous trees, on which they sprint faster and produce less noise relative to coniferous trees, which may affect their ability to catch prey or evade detection by predators. Noisiness and performance have received less attention in the context of substrate preferences than visual camouflage, but our results suggest they may also be important in determining the surfaces on which lizards prefer to be. (Check out the paper: Tree selection is linked to locomotor performance and associated noise production in a lizard.)

In the summer of 2016 I began a large-scale experiment investigating the effects of maternal stress on offspring characteristics in the eastern fence lizard, Sceloporus undulatus. My primary mission for the first part of that summer was simple: catch as many lizards as possible in the longleaf pine forests of southern Alabama. When you spend most of your day pursuing a small prey species, you quickly start to think like a predator. In what areas are you most likely to find them? At what times? On what surfaces? The cumulative years of experience of my fieldwork team (colleagues from the Langkilde lab) suggested that fence lizards were mostly found where there was a mix of hardwood deciduous trees and pines, and that they preferred the deciduous trees, like oaks and hickory, to the famous pines of the region. The longer I spent looking for lizards, the more I noticed that this observation held true. I didn’t put much thought into why until one day when I followed a lizard into a small stand of pine trees. I momentarily lost sight of the lizard, until I heard a loud scrabbling from a few metres away – there was the lizard, scuttling up a pine tree on the smooth, dry flakes of its bark. If the noise of the lizard’s claws moving on the pine bark alerted me so easily to its presence, I thought, perhaps the same was true for its real predators! Also – perhaps that noise was indication that this type of bark, with fewer crenulations and ridges on which to grip, was more difficult for the lizard to run on. Together, could these provide a reason that fence lizards seem to avoid pine trees despite their prevalence?

We decided to test this in the field. First, we quantified whether our anecdotal hunch that lizards prefer deciduous trees to conifers (pines) was really true by conducting thorough searches for fence lizards throughout our field sites, and noting the tree type we found them on, as well as the availability of trees in that area. This allowed us to test whether lizards were “choosing” deciduous trees in areas where they could also choose pines, as opposed to just being found in areas with only deciduous trees. As we expected, we found that even when availability of coniferous:deciduous trees was more or less 1:1, lizards were overwhelmingly found on deciduous trees, not pines.

Next, we tested our hypotheses that tree type changes how noisy lizards are when they move, and how quickly they are able to move. We did this by releasing wild lizards on either coniferous or deciduous trees, and then recording them as we stimulated them to run upwards on the tree by gently tickling their back legs. We then analysed these recordings and found, as we predicted, that the noise of lizards running (the sound level they produced when running compared to the background noise when they were still) was significantly higher when they were running on the smooth, flakier bark of coniferous trees. We also found that the sprint speed they attained on coniferous trees was lower than on deciduous trees. In other words: they are noisy and slow on pine bark compared to the bark of trees like oaks and hickorys.

Studies investigating where animals spend their time (either in terms of broader habitat preference, or more localised use of substrates) has often focused on coloration, and the camouflage it may or may not afford. Our study shows that other aspects of camouflage, such as acoustic camouflage, may also be important. It’s also important to consider how substrate affects performance, like sprinting speed: once you’re spotted by a predator, the speed at which you’re able to escape may be just as important as trying to remain hidden in the first place.

This was one of my favourite studies to be involved in, for a number of reasons! First, I love that we were able to find ways to test hypotheses based on a very simple natural history observation. Understanding the natural history of an organism is crucial for developing new ideas – and the “why does this happen?” questions are the bedrock of behavioural ecology. Second, this study was an opportunity to bring together friends and start new collaborations! Langkilde lab alum Nicole Freidenfelds brought her great knowledge and understanding of herpetofauna and natural history; local friends in Alabama helped me to identify tree species; I knew of Gavin’s prowess in acoustic analysis through Twitter, and asked him to help with this aspect of the project; and Tracy and I had a blast exploring these ideas with them!


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A New Blood Sampling Method for Smaller Anurans that Preserves Critical Features of Specimens

Another new paper from Dustin In Herpetological Review! Summary below.

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Obtaining adequate blood samples is vital for most studies involving immunology or physiology. Anurans (frogs and toads) present a particular challenge for obtaining adequate samples, largely because of their relatively small size compared to other vertebrates.

Here, we propose a new method for obtaining large amounts of blood from the ventral abdominal vein of euthanized frogs, which we call the lethal abdominal vein of anurans (LAVA) technique.

We tested this method on the locally common Wood Frog (Lithobates sylvaticus –  pictured above). Using the LAVA technique, we were able to collect blood from 100% of frogs. Each frog yielded an average of 0.09 mL (range: 0.03 to 0.17 mL) of blood, which contained an average of 40 µL (range: 15 to 100 µL) of plasma.

We also found that neither size, ambient temperature, nor site affected our blood yields. We show that the LAVA technique is an easy-to-use method that yields high amounts of blood from anurans, and could be potentially viable in other small vertebrates.


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A Langkilde lab #reviewforscience

While looking up ideas for how to run some of my immune assays in the field, I stumbled across the twitter hashtag, #reviewforscience, where scientists leave reviews for common, everyday items they use in their research. So, in the interest of sharing how our lab has repurposed some everyday items for science, I thought I’d do a few reviews of my own.

Dental flossdental floss

As has been noted by other esteemed colleagues, dental floss is excellent for making nooses when capturing lizards. Just tie the noose on the end of a cheap fishing pole to reach lizards high up in trees!

 

 

 

 

 

Plastic Sterilite tubs

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Work well for individual or group-housed adult fence lizards. Adding clean, damp sand substrate makes an ideal habitat for females ready to lay and bury their eggs. Smaller versions can be used for hatchlings and juvenile fence lizards. Easily sterilized in standard cage washes! Just remember to wrap in opaque paper before use to prevent adult males from seeing one another.

 

24 hour plug-in timerstimer

Work great for timing basking lights to match up with daylight hours outside, to maintain the circadian rhythms of captive-housed fence lizards. Make sure you get the type with 2 grounded outlets!

 

 

 

 

 

Plastic deli containersIMG_20180112_111835361

So many uses in a lizard lab! Can be used to hold lizards, crickets, and eggs for weighing. Great for transporting hatchlings from the lab to the field! When filled with damp vermiculite, they make an excellent place to incubate fence lizard eggs!

 

 

 

 

Thermocouple thermometer img_20170221_131145

Like other scientists, we’ve found that a thermocoupler probe “fits neatly inside a lizard’s cloaca” for measuring body temperature.

 

 

 

 

 

Bug vacuumbug vacuum

Works great for collecting leftover crickets in order to track how many were eaten. Fairly quiet, and less stressful to fence lizards than reaching in to hand-capture the crickets, as well as being much less time consuming! And crickets are undamaged, so can be used again in later feedings (hand capture of crickets often results in squished crickets). Long-lasting battery, can vacuum over 100 cages before needing to be recharged.

 

 

 

Honey

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Surprisingly effective organic glue for attaching deceased fire ants to live crickets for a fence lizard feeding study. I wanted the fence lizards to consume fire ants without being stung, which meant the fire ants needed to be euthanized first. But fence lizards won’t consume unmoving prey, so I had to attach the fire ants to a living insect. Using <5µl of honey, I was able to stably attach 10 fire ants to each cricket, and the cricket was still able to move about freely.

 

Tea strainertea strainer

Great container to hold isoflurane-soaked cottonballs for lizard anesthesia. The wire mesh allows the lizard to breath in the isoflurane, but holds in the cotton ball to avoid direct contact with the animal. Have not tried to use tea strainers with fire ants. Can also be used to strain particulate matter out of feces when performing fecal parasitology tests.

 

 

Plaster of Parisplaster of paris

Used in the production of fake lizard models for predation studies. Easy to use, and held up well to short-term exposure to the elements.

 

 

 

 

 

White duct tapeduct tape

Works well to make dry-erase labels for tubs, in order to quickly label and re-use for weighing crickets and lizards.

 

 

 

 

 

Sous vide cookersous vide cooker

I’m thinking of using one of these this field season as an inexpensive, travel-friendly alternative to a water bath. When in the field, just clip this onto the side of a container full of water, plug into a wall outlet or portable car charger, set the temperature you want the water at, and go! No need to transport an expensive, bulky water bath.

 

 


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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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On a frog hunt

 

Earlier this Spring some explorers from the Langkilde Lab went on a trip to Connecticut to take part in some exciting field work with Lindsey Swierk, former lab member and current postdoc at Yale University.

Lindsey is investigating the possible impacts that urbanization and road noise might have on wood frogs’ (Lythobates sylvaticus) mate calling behavior. To do so, our team went out to collect male wood frogs from vernal ponds of different degrees of urbanization in the area of Madison, CT – from deep into the woods, to a friendly neighbor’s backyard.

Lindsey’s research is important especially because frogs are extremely dependent on acoustic signaling as a form of communication and mate attraction. Most likely, these animals are still not well adapted to urban environments with intense noise (think of heavy traffic and construction work), despite being exposed the them for a considerable amount of generations. If sound suddenly becomes an unreliable cue for mate selection and predator detection, the dynamics of natural and sexual selection could be altered, potentially removing adaptive traits from natural populations. We don’t know exactly how (or if) wood frogs cope with these changes in their surroundings. Amphibians are some of the organisms most sensitive to environmental change, and to protect them, it is crucial that we better understand these impacts.

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Hi there!

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This trip was an excellent experience: it was the first time some of us got to work with frogs. Also, the residents we came in contact with all seemed captivated by our work and science in general – a great opportunity for us to exercise our science communication skills. Special thanks to the folks at Field House Farm, LLC, we had a great time!

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There’s always time for a little posing

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Between behavioral trials, some lacrosse with the kids at Field House Farm

Finally, I was really happy to have my first contact with a real, colorful, living salamander! Being from Brazil, where these charismatic creatures sadly do not occur, I felt accomplished after having this much anticipated encounter.

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My first salamander – Ambystoma maculatum!

Besides all the field work, we also went out to explore the New Haven area, local restaurants (Frank Pepe Pizzeria Napoletana is highly recommended!) and part of Yale University’s campus. We also got to spend time with Lindsey’s family, two adorable dogs included, but most importantly, we learned about our mate Cam’s great culinary skills after a taste of his famous baked ziti – great company in the field indeed!

As you read this, Lindsey is processing the overwhelming amount of data obtained in this field season, so make sure to check her website to hear about her results in the near future.

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Team Swierk – a job well done!


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Even Mining Bees Do It

Animal life of all sorts was springing into action in Central PA this past week. The other day as I was prepping my bike to head into the lab, I noticed a fair bit of activity centered on the pair of dandelions (Taraxacum officinale) just outside my door. Dandelions are one of the first flowers to appear in the Spring and serve as important early sources of food for many pollen-oriented insects.

Upon further inspection, I noticed that a solitary female bee was feeding on one of the dandelion’s pollen and attracting attention from several suitors. Right as I returned with my camera, one of the male bees latched on to the female, and I settled in to observe the proceedings:

In the footage, you can see the male bee attempt multiple matings with the female (and seem to succeed several times). The female seems largely ambivalent to his attentions, preferring to devote herself to the delicious pollen repast in front of her and grooming her shapely antennae. At several points, other, less fortunate suitors make a quick pass at the lucky couple, but do not dislodge the primary male. The bees appear to be members of the genus Andrena, a diverse group of solitary mining bees found throughout the world (Thanks to Dr. Heather Hines for ID help!). These bees are a sign of Spring in many areas. Male bees often become active first and search for emerging females to mate with. Afterwards, they may conduct mate-guarding to prevent other suitors from fertilizing their mate’s eggs and increasing their offspring (and fitness). Happily, we had just talked about this in my Animal Behavior class (Biol 429), so I was able to use the footage you see above as an intro to my next class.

As the weather gets warmer (barring this past weekends’s snow (!) here), I’m looking forward to seeing many more species becoming active and all the neat behaviors they’ll be displaying.