The Lizard Log

The Langkilde Lab in Action


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Undergraduate research in the spotlight!

The Langkilde lab was well-represented at this year’s Undergraduate Research Symposium by three of our lab researchers! It’s been an excellent year for undergraduate research. Congratulations to Richard Novak, Kristen Sprayberry, and Andrea Racic on their poster presentations! Not pictured is Jennifer Heppner, who also completed a brilliant thesis in the Langkilde lab this year. It’s been a pleasure having you all in the lab, and we will be sorry to see you go – but look forward to hearing about your future endeavours!

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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.

frogs

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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Offspring influenced by their evolutionary history more than their own experience in fence lizards

Check out the first chapter of Dustin’s thesis on the “Trans-generational but not early life exposure to stressors influences offspring morphology and survival”, recently published in Oecologia!
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Environmental changes, such as the introduction of non-native species, can impose novel selective pressures. This can result in changes in fitness-relevant traits within an individual’s lifetime or across multiple generations. We investigated the effects of early life versus trans-generational exposure to a predatory invasive insect stressor, the red imported fire ant (Solenopsis invicta), on the morphology and survival of the eastern fence lizard (Sceloporus undulatus). We captured gravid lizards from high-stress populations with long histories of invasion by fire ants and from uninvaded sites. Resulting hatchlings were exposed weekly to one of the three treatments until they reached maturity (42 weeks): (1) sub-lethal attack by fire ants; (2) topical application of the stress-relevant hormone, corticosterone (CORT), to mimic the stress of fire ant attack; or (3) control handling. Exposure to post-natal early life stress (fire ants or CORT) did not interact with a population’s evolutionary history of stress to affect morphology or survival and early life stress did not affect these fitness-relevant traits. However, morphology and survival were associated with the lizards’ evolutionary history of exposure to fire ants. Offspring of lizards from fire ant invaded sites had longer and faster growing hind-limbs, gained body length and lost condition more slowly in the first 16 weeks, and had lower in-lab survival to 42 weeks, compared to lizards from uninvaded sites. These results suggest that a population’s history of stress/invasion caused by fire ants during ca. 38 generations may be more important in driving survival-relevant traits than are the early life experiences of an organism.

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You can read more about Dustin’s research here and here! 


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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.