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!

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!

Sex-dependent effects of maternal stress in lizards

Check out the second chapter of Dustin’s thesis on the “Sex‐dependent effects of maternal stress: Stressed moms invest less in sons than daughters”!

Multigenerational effects can have important and sex‐dependent effects on offspring. Sex allocation theory predicts that females should differentially invest in sons and daughters depending on sex‐specific fitness returns and costs of investment. Maternal stress‐relevant (glucocorticoid) hormones may be one mechanism driving this effect. We investigated how maternal stress hormones differentially affected sons and daughters by manipulating levels of the glucocorticoid, corticosterone (CORT), in gravid female eastern fence lizards (Sceloporus undulatus) and quantifying reproductive investment and sex ratio of resulting clutches, and the mass, snout‐vent length, and body condition of sons versus daughters at hatching. We found no effect of maternal CORT‐treatment on the number or size of eggs laid or on the sex ratio of resulting offspring, but sons of CORT‐treated mothers were shorter, lighter, and of poorer body condition at hatching than were sons of control mothers. We found no difference in size or condition of daughters with maternal treatment. Our results suggest that maternal stress, mediated by elevations in maternal CORT concentrations, can have sex‐specific effects on offspring manifesting as lower investment in sons.

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.

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! 

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.

Using a dial caliper to measure the foot thickness of a lizard.

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.

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.

Inhibition of superoxide signal by superoxide dismutase.

Stimulation of heterophil function by bacterial stimulant.

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.

Thousand Steps has a lot of steps

Kristen found her first lizard!

Attracting Unwanted Attention

I’ve certainly attracted my share of unwanted attention: missing a key free throw in a basketball game when everyone in the crowd was watching, faceplanting while exiting stage right in A Midsummer Night’s Dream, or unconsciously ripping a huge burp at a fancy dinner. Fortunately, as a human, the stakes involved in these mistakes were fairly low: a little embarrassment and a good story once the shame had worn off. For many organisms, like lizards, however, attracting unwanted attention in the real world can have serious consequences…

From previous research in the Langkilde Lab, we know that invasive fire ants (Solenopsis invicta) can pose a serious danger to native fence lizards (Sceloporus undulatus), and that fence lizards from areas invaded by fire ants respond to encounters with these ants with a variety of twitches and scratches to remove ants as well as fleeing more often to escape them.

We know little, however, about the behavioral rules that govern fleeing and twitching in these lizards. Do they flee more from all predators? Do they flee from all ants? We know the benefit of twitching and fleeing (not getting stung!), but is there any cost to these behaviors? Because we’re ecologists, we looked to answer these questions by conducting a series of experiments and published them in Animal Behaviour.

So, do fence lizards from fire ant-invaded areas flee more from other predators? Because we couldn’t let actual predators attack our lizards, I borrowed a stuffed kestrel (Falco sparverius, a well-known predator of fence lizards), rigged it with a wire harness, attached some very serious and scary-looking eyes, and swung it at unprepared fence lizards to see what their reactions would be.

Taxidermied kestrels are mostly as scary as the real thing.

We found that fence lizards from sites with and without fire ants fled from simulated kestrel attacks the same proportion of the time, and with the same strength and latency (reaction time), suggesting that fence lizards exposed to fire ants don’t flee more from all predators.

We next tested our fence lizards’ reactions to fire ants (which we’ve done before) as well as two types of native ants which might annoy lizards by running on them, but lack the venom (and danger) of fire ants. In fact, these native ants are important sources of food for fence lizards under normal circumstances. In these tests, we found that fence lizards from sites with fire ants fled more from all types of ants, not just fire ants, indicating that this fleeing behavior is generalized to multiple types of ants that they encounter, including those that don’t pose a serious danger.

For the more visually oriented, this series of experiments was illustrated super-well by Tali Hammond, a behavioral ecologist, who was interested in the paper (check out that sweet Sceloporus!)

Check out more illustrated papers by Tali: @mammalLady

So what might the consequences of this generalization be for fence lizards? For one, it’s obviously not ideal to be running away from something (i.e., native ants) that isn’t a threat and should be a meal. This could result in lower food intake or time wasted running away from non-dangerous ants, though we haven’t tested for these effects. More dramatically, twitching and fleeing break crypsis, a lizard’s primary defense against its visually hunting predators, including snakes, and birds of prey like the kestrel. While fence lizards are usually quite well camouflaged, imagine how easy it might be for a predator to spot a lizard jerking around as in the video above. We looked for evidence of this cost in our lizards by quantifying the amount of injuries (broken tails, scars, missing limbs) to lizards at sites with and without fire ants.

A missing “hand” is an example of injuries many fence lizards have.

We found that lizards from sites with fire ants do indeed have more of these injuries than lizards at sites without fire ants. This result suggests that fleeing from fire ants might attract unwanted attention from other types of predators. And when we consider that these lizards also flee more from native ants, which are common in the environment, these antipredator behaviors might have a serious drawback. It is important to note that this evidence is circumstantial; we didn’t see predators preying on lizards running from ants (this would be very difficult), and there could be other explanations for this pattern, such as differences in predator communities, or perhaps differing skill levels of predators. However, this work suggests that lizard that twitch and flee in response to ants may be attacked more.

So why do we see this behavior if it has these drawbacks? My personal guess is that it comes down to consequences. As a human, the consequences of my unwanted attention were fairly minor (shame). The stakes for fence lizards are a bit higher: fleeing can lead to running from your own dinner or attracting attention from predators. BUT the costs of not fleeing when attacked by fire ants are likely even higher (serious injury or death). And in many areas fire ants are much more common and likely to interact with lizards more frequently than snakes and kestrels. In other words, the lizards are likely making the best choice available to them. In the future, perhaps, they will adapt to distinguish between dangerous and native ants, allowing them to make more optimal decisions, and reduce the costs of these antipredator behaviors. More broadly speaking, I believe this research shows that we need to consider a wide variety of potential costs as well as benefits when looking at organisms adapting to changes in their environments.

The full paper in Animal Behaviour can be found here.

Conference time for the Langkilde lab!

The Langkilde lab has recently returned from its annual pilgrimage to the SICB (Society for Integrative and Comparative Biology) meeting, which this year was held in beautiful New Orleans! All our lab members presented talks, and had a great time networking, catching up on top research, and telling people about our own.

Below are some of our thoughts on the meeting, summaries of what we presented – and some tips for conference-goers from all fields!

Langkilde lab members past and present reunited at SICB (photo: Cate Pritchard)

Beautiful New Orleans (photo: Kirsty MacLeod)

Cam and Tracy hang out at the Data Blitz (photo: Cate Pritchard)

Kirsty MacLeod
“I talked about a paper I’ve been working on from our field season in Alabama last summer. Animals encounter environmental stressors daily; how does frequent, low-level stress influence survival and reproductive success? We show that, in Eastern fence lizards, a daily dose of low-concentration stress hormone led to increased adult mortality, and decreased hatching success of her eggs. This was the first conference I’ve been to where animal behaviour hasn’t been the primary focus – this reflects my broadening interests – I’m really excited by integrative research, so this meeting was a great way to see what other people are doing in more mechanistic fields (physiology, genetics, etc). It gave me lots of ideas for taking my own work forward!

My top conference tips are to contact people in advance that you want to talk to – that way you’ll be less likely to chicken out of approaching them! And – make use of Twitter before, during, and after the conference. It’s a great, informal networking tool. I met up with loads of top researchers that I’d first contacted on Twitter, and made lots of new friends!”

Chris Howey

“I talked about the effect of temperature on the mode of locomotion a snake uses.  As a reptiles body temperature changes, so does its ability to perform specific tasks (digest a meal, sprint away from a predator, etc).  I was interested in how body temperature affected a snake’s ability to move, or slither, across its landscape.  I found that body temperature does affects how well they move, but also affects how they move.  As a snake warms up, it changes its mode of locomotion, uses different muscles, and performs differently.  One could compare this to a horse trotting at lower body temperatures and galloping at warmer body temperatures.  Obviously these are two different types of performances, and the question we raised with my talk is “Can we compare different performances across a single thermal performance curve?”  We argue that it depends on the question being asked.  Are you interested in the muscles, or the mechanisms behind the performance and how temperature affects those mechanisms?  Or, are you interested in the ecological ramifications of slithering across the landscape (i.e., escape a predator)?  Comparing different modes of locomotion along the same thermal performance curve may be flawed if your question is more the former, but may be justified if your question is more the later.

What I enjoyed most about SICB was talking with fellow colleagues and introducing myself to many new people.  SICB is a huge conference, but with a little effort, you can easily cross paths with someone conducting research you are interested in, someone whose research you’ve admired, someone you’ve only talked with on social media, and now you can see that person face-to-face, introduce yourself, and make a new connection.  And who knows, you may even grab a beer with a few of them.

This actually leads into my #1 tip for people going to conferences: Make an effort to introduce yourself to somebody new.  Once you do this, you will realize that it is nothing to be scared of, and you will find yourself talking with more and more new friends and colleagues.” 

David Ensminger
“I presented on the impact of a stress treatment on maternal behavior and offspring physiology and morphology. The thing I enjoyed the most was getting to meet not only senior researchers but also new researchers and hearing both of their perspectives. 

My tip is to go to the socials and groups at night. They are fantastic places to talk with and meet people.”

Cam Venable
“We already know fire ants are an invasive predator to many organisms, including fence lizards. What I want to focus on is the interaction of Fence Lizards and Fire ants, but as a prey source. This is the first step in my research, by using this study system, to understand how native species adapt to invasive species. The academic side of me really enjoyed meeting other scientist and just chitchatting in informal ways. The 24 year old side of me loved the location of SICB, considering it was in New Orleans!

 Tips for conferences: Well this was my very first conference and I was worried about how to interact with so many bright and accomplished minds. The best and most cliché bit of advice I have, is simply be you. There is no point in putting on a different face, if you’re not even comfortable in it.”

 

Michaleia Mead

“The water chemistry of vernal pools are often impacted by the environment. Changes in the pH and UVB impact the larval amphibians that live there. But how? Stay tuned for an upcoming publication! I LOVED meeting new people. I especially enjoyed meeting people who are working outside of my field of study. Their perspectives on my work are often very different than those within my field and I learn a lot from them.

My advice: TALK TO EVERYONE! You never know who you will meet. If you see a poster you don’t usually have an interest in, just stop and ask a question. If nothing else you may make a friend!”

Braulio Assis

“Going to SICB for the first time was fantastic, and New Orleans is a peculiar, very musical city, which I appreciated a lot. Being exposed to research from large variety of fields in biology certainly allowed me to appreciate other research areas better, so I definitely recommend attending talks that are out of your comfort zone. You never know what new ideas you might come up with!

Another valuable tip I have is, to never underestimate the power of a 25-minute nap during lunch break. The amount of information you receive over multiple days in a conference can be a bit overwhelming, so it’s important to rest whenever possible. It also helps you enjoy the nightlife better!”

Dustin Owen

Caty Tylan

“I presented info on validating the phytohemagglutinin (PHA) skin test in the green anole as a test of cell-mediated immune function. I also discussed how there are different types of PHA, and how the immune response to PHA differed in the anoles between two of these types (PHA-P and PHA-L). I most enjoyed going to talks, and meeting with researchers whose work I’ve been interested in. Also, the food was great.

My number one tip is to contact people to talk to ahead of time, because I certainly wouldn’t have been able to make myself do it during the conference. And take advantage of the lunch and  networking opportunities SICB sent out before the conference.”

Tracy Langkilde
Many of you would have played with Mexican Jumping Beans as a child. Ever wondered why it is that they jump? I presented some undergraduate-led research revealing what motivates this fascinating behavior.

One JMIH in the books

Destination: New Orleans, Louisiana

Purpose: 100th Joint Meeting of Ichthyologists and Herpetologists

Dates: July 6th- July 10th

Early morning flight out of State College and I couldn’t resist a cloud picture.

Flying has never been my favorite thing but I was really excited for this trip. To say I was also nervous would be the understatement of the century. I had been told about all of the important and, in my mind, “big name” people I would meet. These same people would potentially be at my talk. No pressure though, right? Wrong! I booked a practice room the morning of my talk (the 7th) and had a half an hour to work out all of the bug in my talk. I was certain I would never be ready but I had no choice. After weeks of preparation for my first talk, it was finally time to get up in front of a room full of strangers, important strangers. The whole thing was a blur but everyone clapped and a few of the observers even came up to talk to me after. It was a huge relief for me to be done with my talk because that meant I could go to other talks and meetings and enjoy myself. I met some of those “big name people” and believe it or not, they were people just like you and I. Who would have thought?!

Seeing your name in print for everyone else to see is a pretty great feeling.

Throughout the conference I got to see quite a bit of the city of New Orleans. I saw live music, street performers, and the place where the Mardi Gras floats are made. I had some amazing food, some a little too spicy for my tastes but, when in Rome.. I made friends from all over the country and learned a lot about what I wanted to do with the rest of my life. I had no idea there were so many options in this field. There was a live concert put on by those members of the conference who brought instruments, an unlimited Rock ‘N’ Bowl, and so much free food. I cannot put into words how rewarding and fun this trip was for me.

To wrap up the conference, there was a live auction with a lot of interesting things to bid on. The first thing I could work up the courage to bid on was a “The Book of Frogs”. Lucky for me it was a student only item so only other poor graduate students could bid against me. The auctioneers were so entertaining that I didn’t mind that I wasn’t bidding. The night ended a little later then anticipated but I think it was worth it.

I was irrationally excited about this win!

The whole trip made me grateful to be studying wood frogs and to be a part of this scientific community. I would recommend that every new graduate student attend a meeting/conference of some sort as early as they can. The networking possibilities are endless, communicating your work to others is priceless, plus it is a blast! If giving a talk seems like too much, poster presentations are another great option. You truly never know who you are going to meet! Stay tuned for more updates from the Lizard Lab!