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!

Survival and reproductive costs of repeated acute glucocorticoid elevations – new paper in GCE

New paper out featuring Kirsty, David, Dustin, and Tracy!

You can read a summary of this work and why it’s important on Kirsty’s blog, here. Kirsty has now moved on to a Marie Curie fellowship that will take her to Lund University, Simon Fraser University, and the University of Tasmania! Follow her blog, and Twitter, for updates.

Maternal glucocorticoid effects across life stages in fence lizards – new paper!

“Anthropogenic disturbance is a growing threat, and the physiological consequences of exposure to such stressors is gaining increasing attention. A recent paper published in the Journal of Animal Ecology explores the consequences of stress-relevant hormones for mothers and their offspring…”

Read more in David’s new paper, and featured blogpost on the Journal of Animal Ecology blog!

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!

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

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

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.

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 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

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 timers

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 containers

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

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

 

 

 

 

 

Bug 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

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 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 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 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 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.

 

 

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!

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.

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

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.

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.

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.

Sometimes they incubate at room temperature….

…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)!

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.

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.

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!

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

 

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!

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!

 

 

Hanging out with an adult female Sceloporus at Solon Dixon

Beautiful Solon Dixon

It’s fieldwork season!

The lab is currently busy with a variety of field and lab-based projects. In April/May, Cam, David, Tom, and I were in Alabama collecting lizards. Here’s a glimpse of what the long trip to the South, and our work there, looks like! Check back later in the summer for more on the continuation of this project.