The Lizard Log

The Langkilde Lab in Action


Leave a comment

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!
Untitled

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.

9911821

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

Advertisements


Leave a comment

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.

20171019_102901

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.

20171019_111745

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.

20171019_104850

Sometimes they incubate at room temperature….

20171019_124801

…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)!

20171020_170907c

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.

20171020_170854

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.

20171020_172226

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!

20171022_094746.jpg

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

 


Leave a comment

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!

19260419_10101017500547912_5189633714969866181_n

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!

 

 

18447626_10100989138525632_2985881622888526129_n-2

Hanging out with an adult female Sceloporus at Solon Dixon

18010043_10100974225167122_2322507596788552366_n

Beautiful Solon Dixon


Leave a comment

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.


1 Comment

Solon Dixon!

Hey everyone!

Even though I am continuing the same project from last year (how maternal stress affects the offspring in fence lizards), there are still some striking differences. One of the biggest is that there are fellow grad students and a post doc this summer!

20160509_192753

From left to right: Cameron (PhD), myself (PhD), Kirsty (post-doc), and Dustin (PhD).

Also, last year we made the drive in one day, however this year we broke the drive up over 2 days. This gave us an excellent opportunity to experience different parts of the USA on our drive. For the night we stopped in Knoxville, TN and had dinner at an amazing place called Calhoun’s On The River. True to its namesake, it had a beautiful view of the Tennessee River!

20160509_213747

After all the driving, we finally made it back to Solon Dixon and started catching lizards. As usual, the lizards’ personalities were very evident.

20160529_144657

Male fence lizard unamused with our attempts to catch him

20160603_085045

Apparently the female lizards found that corner of the tub to be very interesting.

20160606_185626

As I went to put this female back in her tub, she refused to let go of my fingers!

On top of finding many fence lizards, we were also about to see many other reptiles and amphibians!

20160523_195354

A barking tree frog tightly hugging my finger.

20160523_212139

An American alligator, at a very reasonable size to handle.

20160529_095725

A yellow bellied slider who found a little bit of water to sit in.

eastern glass lizard

A glass lizard!

As I spend more time down here, I find it rubbing off on me more and more.

20160628_131938

Very tempted to get a cowboy hat.

After catching the females, our first trip came to an end. However, we were quickly back down to release the females and run experiments with the hatchlings. With us this time we had an undergraduate researcher, Jen!

IMG_7213

The Bayfront Park, overlooking Mobile Bay. Located right next to one of our field sites, Blakeley State Park.

As we wait for more hatchlings to emerge, we have been focusing on removing fire ants from some of the enclosures we built. As fire ants are highest in the mounds earlier in the day, this means some early mornings. On the up side, it also means we always get to see the sunrise.

20160721_060424

Sunrise right near the enclosures.

Most things have gone well, with only one piece of equipment starting to show signs of wear, but this just gave me an excuse to do some handywork!

20160714_121230

Used some steel epoxy to seal a leak in the pot we boil water in for fire ants.

Things have started to pick up in terms of hatchling, so soon you should be able to hear about how things are going with them. Until then, here is a pic from right here at Solon Dixon

20160726_093444

With the drier weather they are finally able to do prescribed burns.

Cheers,
David

 


1 Comment

What’s all the fuss about Alabama?

Since joining the lab, I’ve heard nothing else more than field seasons in Alabama; from the heat, lizards, those darn fire ants, and the wonderful people they come in contact with. So of course, I was anxious as ever to finally embark on my first field season in the Langkilde lab. After months of preparation, designing projects and all the logistics involved, May 9th had finally arrived and it was time to head south.

The first round of people in the Alabama crew this year had 3 newbies to the lab; Dustin Owen (our personal herpetology specialist), Dr. Kirsty Macleod (our Scottish Post Doc who should’ve been born in the southern US) and Myself aka Frog stallion (long story). The last member of the crew was David Ensminger. This was his third year there, sort of making him our expert of all things and everything that we needed to know (in other words he was our ear to ask a million questions).

The wonderful staff of The Solon Dixon Forestry Education Center welcomed us to the 5 starred field station (in my opinion – I don’t know who could possibly refute that). There were also lots of Auburn University students that were super interested in our work and simply cool people to hang out with. Essentially, everyone we met made sure we left knowing that southern hospitality is 100% real. I now understand why everyone in the lab loves and talks about it so much.

img_3945

Some Auburn students were successful on their first lizard hunt. It was Herp week in their class, luckily for them, they had some experienced catchers around

Now on to the fun science! In case you’re reading this and don’t know, the Langkilde lab is well invested into the Fence Lizard and Fire Ant system, but I’m just going to focus on my specific part for now. In the broadest of terms, I am interested in the diet of the Fence Lizards, but of course I can’t help but wonder about other aspects of this creature. A former member of our lab (the undergrad king Mark Herr) published a paper that suggests fence lizards seemingly build an addiction to the fire ants. My first thought was the possibility that the fire ants may be more nutritious. In Alabama, I collected loads of ants to quantify carbs, lipids, and proteins in comparison to fire ants, hoping for something to support a risk-reward relationship. My next thought was, if the lizards are presented with a second option, what will they pick? To test this, The Lizard Queen (Dr. Tracy Langkilde) and I ran food preference trials. We used the fire ants and “Dory ants” (still waiting for a true identification, but we call them Dory ants) in tubs with one lizard to see the choices they made. Now back in State College, I’m going through and analyzing all the data. I won’t spoil the surprise, which will hopefully be published, so stay tuned in the near future.

 

 

IMG_3927

My first Catch of the trip. Gotta love when they just pose and hug your thumb!

 


Leave a comment

The Natural State

Happy Memorial Day!

I am happy to report that the “uninvaded” team has returned to PA, lizards in hand–or bag as it were. Our team, consisting of Braulio,  Caty, and myself, traveled to Tennessee and Arkansas. Arkansas prides itself on being “The Natural State” for its “natural scenic beauty, clear lakes and streams, and abundant wildlife.” I can’t speak to most of that, but it does have lizards!

Fence lizard with a regenerating tail.

Fence lizard with a regenerating tail.

Rainy and overcast days slowed us down a bit. As ectotherms, lizards rely on external sources of heat, which means they like to bask in sunny spots in order to warm up. The thick clouds didn’t provide many good basking opportunities, but thankfully a few lizards made an appearance in the brief moments of sun.

Sometimes fence lizards like rocky habitat.

Sometimes fence lizards like rocky habitat.

2blendinMany other lizards like to bask on trees.

We did see a few sunny days, which gave Braulio and Caty the opportunity to catch their first lizards.

1BraulioLR

Braulio with a Tennessee lizard.

1CatyLR

One of Caty’s first catches!

1Gail

Lizard selfies are the best selfies.

We even managed some “expert” catches, on more than one occasion slowly driving by a basking lizard and noosing it through the open car window.

Because we were looking for females, we of course became experts at catching males. One male lizard really hoped I was a tree. We tried to return him to his log, but on two separate occasions he ran up my leg. Sorry little guy!

Nope, not a tree.

Nope, not a tree.

Another male, pictured below, really surprised me. Lizards vary in coloration, but not usually by much. I’ve never seen a fence lizard so dark!

3darkmorph

A very dark fence lizard. His chest badges were impressive as well!

After two weeks of catching, we headed back to the lab. Our females are now happily housed in their nesting boxes, and one has laid her first clutch of the season. We’re all excited to see the resulting hatchlings!

Check back soon for more stories and photos from the field as well as updates on the specific research projects happening this summer.

We even spotted a fence lizard on a fence. So satisfying. A fence lizard on a fence. So satisfying.