The Lizard Log

The Langkilde Lab in Action


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Hormone assays in the lab this Fall

Now that our field seasons are (mostly) over, the members of the Langkilde lab have been busy processing the blood samples we collected over the summer.

As I wrote last time, most of us are looking in some way at the impacts of environmental stressors on an animal’s behaviour, and the characteristics of the offspring they produce. In order to test these questions, we need to be able to quantifiably measure the stress levels of the animals we study.

To do this, we took blood samples from our study species in the field to measure levels of “stress hormones” (glucocorticoids, factors produced by the adrenal glands in response to stress). These hormones circulate in the blood, and correlate with the baseline stress levels of an individual – the more glucocorticoids we find in the blood, the more stressed an animal is. We determine the concentration of glucocorticoids in our blood samples by first centrifuging the sample to separate red blood cells from the plasma (the clear fluid in the eppendorf tube below), and then running the sample through an enzyme immunoassay.

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Enzyme immunoassays work by using antibodies that bind to the factor of interest in a sample – in our case, the steroid hormone corticosterone. We add a known amount of plasma to each well of the plate (above right), and in each well, the corticosterone in the sample binds to the antibodies. The antibodies that aren’t bound by corticosterone are bound by a conjugate tracer, which gives off a colour. So, in the plate above, the more “yellow” the well appears, the less hormone it contains (meaning that more free antibody sites have been left to bind with the yellow tracer-bearing conjugate). We can compare the “yellowness” of each well with wells containing a known amount of hormone, and this allows us to calculate the concentration of hormone in each sample:

I’ve had a lot of fun learning these techniques this Fall, processing my lizard samples, and helping Chris process some of his rattlesnake samples! I’m looking forward to reporting back on the exciting results these data contribute to over the next few months.

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Preparing samples with Chris and Danielle!


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Basking Site Use by Timber Rattlesnake Morphotypes – By Shawn Snyder

My name is Shawn Snyder and I am currently a senior majoring in Wildlife and Fisheries Science.  This is my first and only year working in the Langkilde Lab.  During the summer of 2016, I worked under Dr. Chris Howey as a Research Technician studying the effects of prescribed fire on timber rattlesnake populations.  This position provided me the opportunity to radio-track timber rattlesnakes, record habitat data on tracked snakes, catch new snakes (extremely fun), learn how to safely tube a venomous snake (even more fun), and conduct vegetation surveys.  Also, this position provided me the opportunity to formulate my own scientific question to test! Together, Chris and I thought up a small side-project that I could conduct throughout the summer, which provided me the fantastic experience of going through the scientific process, collecting my own data, analyzing those data, and now writing a manuscript so that I can share those results with the scientific world.

When we first started collecting data for my side-project I was a little apprehensive.  Once the data was collected and analyzed I realized that this project was going to take time and a large amount of effort to complete.  As the process of analyzing the data and then coming up with a plan for the manuscript began to take shape, I started to feel challenged and nervous by this new task. But weekly meetings with Chris to discuss the process of writing a manuscript have helped immensely.  This is my first manuscript and yes it is challenging, but it will all be worth it once we have a finished product. I have ambitions to continue on to a Graduate program after I graduate and this manuscript will help me build my C.V. to apply to Grad schools.

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Two yellow morphs bask alongside three black morph timber rattlesnakes at a gestation site. Although we did not use gestating (i.e., pregnant) females as part of this project, this shows you the posture of a basking snake and the difference in color morphs.

My research is investigating if the two distinct morphotypes of timber rattlesnakes (a dark, black morph and a lighter, yellow morph; see above picture) use basking habitat with differing amounts of canopy openness and solar radiation. Previous research suggests that the dark morph evolved in response to thermal limitations in the northern parts of its range.  Darker snakes have more melanin in their skin, which allows them to absorb more solar radiation and maintain a higher body temperature than yellow morphs.  Yellow morphs having this thermal disadvantage, in theory would have to choose basking sites that receive more solar radiation to compensate for this limitation if they wanted to maintain a similar body temperature to the black morphs.  Specifically, I am testing the hypothesis that yellow morphs use basking habitat that has more canopy openness and receives more direct solar radiation (i.e., sun) than basking habitat used by black morphs.

 

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A black morph male timber rattlesnake is seen courting a basking yellow morph female.  Once again, the difference in color morphs is striking and has led many to ask what selective pressures are maintaining this polymorphism.

To test this hypothesis, I measured canopy openness over basking yellow and black morphs. I used the timber rattlesnakes that are being radio-tracked for Dr. Howey’s main study as my sample population and placed a flag where a snake was found exhibiting basking behaviors (see picture below  for example).  We took a picture facing skyward directly over the snake using a camera with a fisheye lens.  This lens takes a picture of 180 degrees and captures an image of all of the canopy over the snake (see picture).  We can then analyze these hemispherical photographs using a computer program called Gap Light Analyzer to measure the percent canopy openness and the amount of direct solar radiation transmittance (i.e., rays of sunlight) for each basking site.  Direct solar radiation is when the sunlight reaches the forest floor with no obstructions from the canopy; as opposed to indirect solar radiation which may be radiation that is being reflected off of clouds, trees, or the ground itself.  Our study site is characterized as having a mature Oak/Maple forest with an abundance of closed canopy throughout the area.  Both morphotypes use this “closed canopy” forest throughout the summer as foraging grounds, and when they need to bask they must seek out areas where some sunlight is making its way through the canopy.  This is where my question becomes very important comparing the habitat used by each morph.

 

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A flag is placed next to a basking yellow morph.  An exact description of the habitat is recorded so that I can come back at a later time (when the snake is not there) and take a photo of the canopy directly over where the snake had been.

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Two examples of hemispherical photographs taken over two different basking timber rattlesnakes.  Both canopies actually have similar canopy openness, but the canopy on the left receives far more direct solar radiation based on the placement of those canopy openings.

So far, my results show that the two morphs use habitat that have similar percent canopy openness, however, there was a difference in the amount of UV transmittance between the basking sites used by the two morphs.  Canopy openness doesn’t necessarily designate a “warmer” site because the sun path may not go directly over the gaps in the canopy of that site, thus, the site wouldn’t receive large amounts of direct solar radiation.  Black morphs use basking sites that received lower amounts of direct sunlight.  They may be able to do this because the greater amount of melanin in their skin provides a greater ability to absorb whatever direct or indirect solar radiation is available more effectively. Yellow morphs use basking sites that received more direct solar radiation.  They could be forced to use these sites to compensate for their disadvantage in their thermal ability.  I am currently working on writing a manuscript for these data and hope to have it completed by the end of 2016.  Stay tuned for more on this manuscripts progress!

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Here is a picture of Shawn (holding a Hellbender!!) while on a break from collecting some amazing data.


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A million ways to die in the South (Or, maternal stress and offspring survival in eastern fence lizards)

When you tell people you are going to be studying lizards in Alabama for the summer, you get used to a raised eyebrow or two. The heat! The snakes! The bugs! When I told people I was going to be deliberately seeking out (ecological) public enemy number 1, fire ants, everyone made clear what I already knew – the theme of my summer was going to be STRESS.

In fact, stress (and in particular, maternal stress during gestation) is exactly what took me to Alabama. Stress during pregnancy can alter the characteristics of the resulting offspring, from morphology to behaviour. That’s assumed to be a bad thing. But could stress experienced by mothers during gestation actually program offspring for life in a stressful environment, giving them an advantage in the long run? This was the question I set out to test during my first, recently completed, field season.

For this study we focused on the eastern fence lizard, Sceloporus undulatus, a species well known to our lab. This lizard is particularly well suited to studying maternal stress because, as is the case for many reptile and amphibian species in the South East, it is subject to the considerable stress of coexisting with fire ants. We know a lot already about how fire ants change the behaviour and physiology of fence lizards – but what about the effects on the next generation through maternal stress effects?

A question this complex, with more than one generational level, required a number of steps. Step one was capturing gravid (carrying eggs) female fence lizards, which we did in May and early June. Having worked previously on mammals (meerkats) and birds (fairy wrens and hihi), this was a new and exciting experience for me! Safe to say, fence lizards (and herps in general!) quickly stole my heart.

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My first fence lizard!

Our next step was bringing the females into the lab, and subjecting them to a highly controlled “stress” treatment – a very low dose of a stress hormone every day, the equivalent of a single fire ant sting. It’s important to note that this is NOT a pain treatment – we use the hormone corticosterone, which is released as part of a lizard’s natural stress response, and which has a number of downstream effects including helping the body’s metabolic system turn amino acids into carbohydrates for use as fuel. In short, our treatment was tricking the lizards’ system (but not the lizard) into thinking they were in a stressful situation.

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A female in our lab housing facility. Note the non-toxic nail polish mark, denoting that this female was in the “no stress” treatment group.

The next step was waiting for the females to lay their eggs, at which point our stress treatment ceased, and females were ready to be returned to the wild. We incubated their eggs (incubation takes around 50 days) and waited for the babies to hatch to begin the next step of our experiment…

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Fence lizard eggs

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Teeny weeny fence lizard baby!

We hypothesised that if maternal stress was adaptively programming offspring to be be better suited to a stressful environment (for example, by making them more responsive to predators, or better able to cope with frequent stressors), then we should see offspring from stressed mothers surviving better in stressful environments than offspring from mothers that did not experience stress during gestation.

To test this, we needed to create “stressful” and “non-stressful” environments in which to put the offspring. When we weren’t catching females or incubating eggs, we were building four 20x20m outdoor enclosures for this purpose! Thankfully, life in Alabama with fire ants everywhere is stressful enough, so we didn’t need to artificially create a stressful environment. To create a “non-stressful” environment, we removed fire ant mounds from two of the four enclosures. We hypothesised that in these enclosures, offspring from unstressed mothers should do best.

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One of our four hatchling enclosures

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Newly-hatched fence lizard enjoying life in an enclosure!

Once the offspring hatched, we put them into the enclosures and monitored their survival by checking them every day (not an easy task, they are small and wily!). I also recorded their habitat use, how far they were moving within the enclosures from their release spots, and how they responded to small, short-term stressors, like being picked up to be weighed. I’m now in the process of analysing this data, and am looking forward to seeing if our hypotheses hold true. Watch this space!

So, lizards aside, how did I cope with the stresses of a summer in Alabama? The heat – loved it! The snakes – try and keep me away from them! The bugs – who cares?! The people – a whole lot of new friends. There may be a million ways to die in the South, but there sure are a million and one things to love.

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Box turtle, Geneva State Forest

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Gray rat snake, Solon Dixon Forest Research Centre

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A devoted Alabama convert – can’t wait to be back!

 


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Efficacy of Daylighting… Part I

Pregnant female rattlesnakes prefer to maintain an elevated body temperature (~32 °C), which allows for a more optimal development of embryos.  In Pennsylvania forests, however, these warm temperatures are not very abundant.  So, in order to achieve these elevated body temperatures, pregnant females seek out rare, open habitat (known as gestation sites) that receive a lot of sunlight.  Sometimes, females may travel up to a mile from their den sites just to gain access to openings within the forest canopy.

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Yellow morph female timber rattlesnake sits coiled under a small rock overhang.

However, not all open gestation sites are created equally.  Last year, Mark Herr, Michaleia Mead, and I uncovered a trade-off at timber rattlesnake gestation sites of various sizes.  Gestation sites that were very open provided pregnant females with more sunlight and warmer body temperatures for a longer duration of the day.  But, these same sites also came with an increased risk of predation!

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At a timber rattlesnake gestation site, a red-tailed hawk swoops in on top of a foam timber rattlesnake model (not seen). A yellow-morph timber rattlesnake model is pictured behind the hawk.

More predators visited these more open sites, predators like bobcats, fishers, and hawks.  But, smaller, more enclosed gestation sites were so thermally poor, we observed females returning to their den at the end of the field season still pregnant!  The use of thermally unfavorable gestation sites may provide the rattlesnake with a sanctuary from potential predators, but there are still terrible repercussions for choosing to use these sites.  We have found that snakes using thermally unfavorable sites tend to give birth at a later date.  The timing that an animal gives birth is very important.  If rattlesnakes give birth to their offspring too late in the year, the small neonate offspring will have little, to no time to complete their first shed and then obtain a small meal before entering the den for hibernation.  It is believed that survival for neonates unable to do these two things is close to zero.  Further, some pregnant rattlesnakes that use thermally unfavorable gestation sites are known to abort their entire litter toward the end of the summer if she decides that they are developing too slowly.

So why would pregnant females continue to use small, more enclosed, thermally poor gestation sites?  Possibly because there was a decreased risk of predation?  Possibly, however, because of strong site fidelity?  Possibly these sites were, at one time, thermally favorable, but over the years vegetation has encroached upon these open areas and shaded out the once warm, sunny rocks.  Due to the rattlesnake’s fidelic response to locating a favorable gestation site, they now find themselves sitting among the shade throughout much of the day.

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Timber rattlesnake taking advantage of a small break in the canopy and some sunlight reaching the forest floor.

 

We’ve addressed the problem, now how do we fix it?

In order to manage timber rattlesnake populations better, forest and wildlife managers have begun to open up gestation sites, un-shading these areas from vegetation, in a process called “daylighting”.  However, recall that more open sites have an increased risk of predation.  So can we open up these sites just enough to let the sun in, but keep the hawks out?  In an attempt to suppress the increased risk of predation that we observed at more open sites, I have begun to direct daylighting techniques to target specific trees that would increase the amount of solar radiation a site would see, without greatly increasing the risk of predation.  To do this, I will use hemispherical photography (see picture below), observe the path of the sun throughout the gestation period, and then target those trees that overlap with the path of the sun.  This way I can open up each of the sites just enough, but keep those trees that do not overlap with the path of the sun and could perhaps maintain some decreased risk of predation.

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Hemispherical photograph at one of our gestation sites. The path of the sun throughout the entire gestation period is shown by the yellow arc. Trees that overlap with this arc can be targeted to improve the thermal quality of the site.

Of course I want to address this management idea with as much scientific rigor as possible for an ecological study.  This past summer, I visited six historic gestation sites within Pennsylvania.  Four out of the six sites are pretty shaded over, and I consider thermally poor.  Two of the gestation sites are fairly open and should provide pregnant females with a plethora of sunlight.  This summer, I surgically implanted nine pregnant timber rattlesnakes with temperature-sensitive radio transmitters, which allowed me to track these individuals throughout the summer. I followed each snake and noted their behaviors, body temperatures, and the date that they gave birth to their young.  Additionally, I measured available body temperatures and risk of predation just like we did in the previous experiment that Mark, Michaleia, and I completed last summer.  The catch is, this winter I will go into three of the six sites (along with US Forestry personnel), and we will remove specific trees blocking out the path of the sun.  Then next summer I will repeat everything and determine if Daylighting improved these thermally poor sites.  Will I see warmer available temperatures within the Daylighted sites?  Will snakes within these sites maintain warmer body temperatures and for a longer duration of the day?  Will these snakes give birth at an earlier date, allowing their young to shed and get a first meal before hibernation?  Will I still see an increased risk of predation?

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Biophysical models laid out at a gestation site and measuring the potential body temperatures that a snake could achieve at that site.

Many of these questions I won’t be able to answer until next year.  But, I am collecting some interesting data thus far.  As expected, the two sites that were more open did have warmer available temperatures.  Snakes occupying these sites maintained warmer body temperatures, moved less often, and were the first to give birth to adorable baby rattlesnakes!

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Neonate rattlesnake coiled up next its mother.

But, these sites were also visited by more potential predators.  In fact, in one instance we observed a red-tailed hawk swoop down and nab a garter snake that was basking alongside our pregnant rattlesnakes!

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Red-tailed hawk grabbing a quick dinner at a gestation site. I’m fairly certain this was a garter snake that got nailed.

The more enclosed sites were indeed cooler and snakes at these sites maintained cooler body temperatures and moved more often.  It appeared that some of these pregnant females shuttled between nearby sites in order to track the path of the sun.  In the morning the snake may be at one site, and in the afternoon that same snake would move to a nearby site.

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Female at a thermally poor area was typically found in the morning at a cut tree stump. In the afternoon she typically moved to another location.

Other snakes continued to move throughout the entire summer from one site to the next; constantly searching for a thermally suitable site where she could continue to develop her babies.  Unfortunately, all of these movements brought one of our mommas too close to a nearby road where someone swerved into the shoulder in order to run her over.  Although the loss of this mom was a little tough on me, it did show me just how important it is to improve these historic gestation sites.

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Our big momma rattlesnake could never find a suitable gestation site. She moved from potential site to potential site for about a month before coming too close to a nearby road.

Currently, as I am writing this blog, we are still waiting on some of our snakes from the thermally poor sites to give birth.  As we find neonates (babies) at each of our sites, I will also collect data on each of them so that we can compare body condition (health) among sites.

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Collecting data on newborn rattlesnakes is a fantastic way to start anyone’s day. I’m collecting data on body length and mass so that I can determine the body condition (or health) of each individual.

 

Next year, I will continue to track snakes throughout these same gestation sites.  However, following our daylighting management, we hope that all of our pregnant snakes will give birth at early dates, move less, and stay clear of predators.  To be continued…

 


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Much Anole About Immunology

While most of the lab has been down in Alabama, I’ve spent a good part of this summer back at Penn State, working with a species I’ve never used before – the green anole (Anolis carolinensis).

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Isn’t he cute?

There is a test we’d like to use in our fence lizards, called the phytohemagglutinin (PHA) skin test. It involves injecting the pad of a rear foot with a small amount of PHA, which stimulates part of the immune system, and then measuring the swelling that occurs. This swelling is small, and temporary, abating in a few days with no lasting damage. But the level of swelling can provide information about the lizards’ immune function.

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A confused anole having its rear foot measured with calipers.

Unfortunately, while this test has been used in humans, birds, rats, and even amphibians, it has not yet been validated in any reptile species. Ideally I would validate the test in our species of interest, the eastern fence lizard, but I needed a larger number of lizards than we can reasonably catch. So, instead, we decided to purchase some green anoles for this project.

In addition to seeing if the PHA test works in reptiles, we’re also trying to determine if the type of PHA used makes a difference, as there are many different formulations of PHA used, and each formulation may have a different effect. I’m also determining exactly what the immune reaction to the different PHA formulations are, and how this evolves over time after the injection.


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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!

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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!

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

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Male fence lizard unamused with our attempts to catch him

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

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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!

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A barking tree frog tightly hugging my finger.

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

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

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A glass lizard!

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

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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!

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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.

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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!

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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

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

Cheers,
David

 


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One JMIH in the books

Destination: New Orleans, Louisiana

Purpose: 100th Joint Meeting of Ichthyologists and Herpetologists

Dates: July 6th- July 10th

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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?!

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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.

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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!