The Lizard Log

The Langkilde Lab in Action


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

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

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Obtaining adequate blood samples is vital for most studies involving immunology or physiology. Anurans (frogs and toads) present a particular challenge for obtaining adequate samples, largely because of their relatively small size compared to other vertebrates.

Here, we propose a new method for obtaining large amounts of blood from the ventral abdominal vein of euthanized frogs, which we call the lethal abdominal vein of anurans (LAVA) technique.

We tested this method on the locally common Wood Frog (Lithobates sylvaticus –  pictured above). Using the LAVA technique, we were able to collect blood from 100% of frogs. Each frog yielded an average of 0.09 mL (range: 0.03 to 0.17 mL) of blood, which contained an average of 40 µL (range: 15 to 100 µL) of plasma.

We also found that neither size, ambient temperature, nor site affected our blood yields. We show that the LAVA technique is an easy-to-use method that yields high amounts of blood from anurans, and could be potentially viable in other small vertebrates.

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A Langkilde lab #reviewforscience

While looking up ideas for how to run some of my immune assays in the field, I stumbled across the twitter hashtag, #reviewforscience, where scientists leave reviews for common, everyday items they use in their research. So, in the interest of sharing how our lab has repurposed some everyday items for science, I thought I’d do a few reviews of my own.

Dental flossdental floss

As has been noted by other esteemed colleagues, dental floss is excellent for making nooses when capturing lizards. Just tie the noose on the end of a cheap fishing pole to reach lizards high up in trees!

 

 

 

 

 

Plastic Sterilite tubs

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Work well for individual or group-housed adult fence lizards. Adding clean, damp sand substrate makes an ideal habitat for females ready to lay and bury their eggs. Smaller versions can be used for hatchlings and juvenile fence lizards. Easily sterilized in standard cage washes! Just remember to wrap in opaque paper before use to prevent adult males from seeing one another.

 

24 hour plug-in timerstimer

Work great for timing basking lights to match up with daylight hours outside, to maintain the circadian rhythms of captive-housed fence lizards. Make sure you get the type with 2 grounded outlets!

 

 

 

 

 

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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!

 

 

 

 

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

 

 

 

 

 

Bug vacuumbug vacuum

Works great for collecting leftover crickets in order to track how many were eaten. Fairly quiet, and less stressful to fence lizards than reaching in to hand-capture the crickets, as well as being much less time consuming! And crickets are undamaged, so can be used again in later feedings (hand capture of crickets often results in squished crickets). Long-lasting battery, can vacuum over 100 cages before needing to be recharged.

 

 

 

Honey

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Surprisingly effective organic glue for attaching deceased fire ants to live crickets for a fence lizard feeding study. I wanted the fence lizards to consume fire ants without being stung, which meant the fire ants needed to be euthanized first. But fence lizards won’t consume unmoving prey, so I had to attach the fire ants to a living insect. Using <5µl of honey, I was able to stably attach 10 fire ants to each cricket, and the cricket was still able to move about freely.

 

Tea strainertea strainer

Great container to hold isoflurane-soaked cottonballs for lizard anesthesia. The wire mesh allows the lizard to breath in the isoflurane, but holds in the cotton ball to avoid direct contact with the animal. Have not tried to use tea strainers with fire ants. Can also be used to strain particulate matter out of feces when performing fecal parasitology tests.

 

 

Plaster of Parisplaster of paris

Used in the production of fake lizard models for predation studies. Easy to use, and held up well to short-term exposure to the elements.

 

 

 

 

 

White duct tapeduct tape

Works well to make dry-erase labels for tubs, in order to quickly label and re-use for weighing crickets and lizards.

 

 

 

 

 

Sous vide cookersous vide cooker

I’m thinking of using one of these this field season as an inexpensive, travel-friendly alternative to a water bath. When in the field, just clip this onto the side of a container full of water, plug into a wall outlet or portable car charger, set the temperature you want the water at, and go! No need to transport an expensive, bulky water bath.

 

 


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Testing the environmental matching hypothesis – return to Alabama!

Another summer field season has now come and gone! This summer I returned to Solon Dixon Forestry Education Center in Alabama (one of my favourite spots on earth!) to continue my research on how stress during gestation influences the offspring of eastern fence lizards (Sceloporus undulatus).

Last year, we investigated how physiological stress during gestation (at the level of a non-lethal predator encounter – for example, when a lizard encounters a couple of toxic fire ants, but isn’t killed by the ants) affects survival of mothers, and how many of their eggs successfully hatch. You can read more about this experiment, and the fieldwork that went into it, here (and stay posted for the published results soon!).

This year I wanted to build on these results and ideas to test how maternal stress influences the offspring that do hatch and make it out into the world. Do they themselves then cope better with a stressful environment, having been “primed” for it by their mothers (the “environmental matching” hypothesis)? Or are offspring born to stressed mothers poorer in quality, and less likely to survive in the wild, regardless of how stressful their environment is? In order to test these ideas, we first made the long trip south to collect gravid females from south Alabama early in the summer, and to build experimental enclosures in which to eventually release their offspring. I then repeated the maternal stress treatment from last year and once again became a lizard mama as I followed the females from laying their eggs, to incubating the eggs, and eventually seeing these bite-sized babies hatch out!

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Freshly hatched fence lizards – <1g!

Once they hatched, the offspring went into the enclosures that we’d built. These enclosures were designed to test whether maternal stress programs offspring to be able to better deal with a stressful environment. The enclosures either contained a key stressor (invasive fire ants), or were fire ant-free. Each day I conducted a mini-census, walking through enclosures to look for each lizard – as you can see in the video below, babies were marked so I could tell exactly who was present each day (and so, which lizards survived, and which didn’t). I also observed their behaviour, and how they used the habitat available to them (for example, did offspring from stressed/non-stressed mothers differ in whether they liked to be out in the open, like the lizards you see in the video – or did they hide more?).

After a great summer (if measuring 200+ baby lizards isn’t a metric of a great summer, I don’t know what is), I’m now back at Penn State with a box of data to work through. I’m excited to report back on what I found in the coming months – so stay tuned!

 

 

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Hanging out with an adult female Sceloporus at Solon Dixon

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Beautiful Solon Dixon


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Immunapalooza

Not all of the lab went south this summer; Kristen (one of our awesome lab undergrads) and I stayed at Penn State most of the summer, working on immune assays.  Kristen was the recipient of the Erickson Discovery Grant, and spent much of her summer on an independent research project, which involved measuring the effects of corticosterone (CORT) on the cell-mediated immunity (i.e. one way the body responds to a toxic or foreign substance) of eastern fence lizard females. She was also trying to determine if the lizards’ life history (whether they were from sites with or without fire ants) affected their immune function or interacted with the CORT treatment. Kristen just recently gave an excellent talk on her research at the Three Rivers Evolution Event (TREE) on Sept. 9th, where she was one of the only undergraduates to present a talk.

We also spent a lot of time this summer developing, improving, and validating several different immune assays for use in fence lizards, including ELISA assays for measuring anti-fire ant antibodies (IgY and IgM), complement function, natural antibodies, and the activity levels of heterophils (a type of immune cell that kills bacteria). Work on the assays for IgY, complement function, and natural antibodies is ongoing, but the IgM and heterophil activity assays are ready to be used.

The IgM ELISA assay was developed to work with as little as 10μl of plasma, and accurately detected anti-fire ant antibodies in a pool of plasma of lizards from Alabama, where the lizards are regularly exposed to fire ants. It did not detect any antibodies in a pool of plasma of lizards from Tennessee, at sites which have not yet been invaded by fire ants. The next step is to test the plasma of individual lizards from different sites, to see what proportion of lizards in various invaded sites have actually developed IgM antibodies to fire ants. Once the IgY assay is working, we should be able to better characterize the antibody response of the lizards to fire ants, and see if this helps them recover faster from fire ant stings.

IgM in the plasma of Alabama lizards

The higher the proportion of plasma from invaded (Alabama) lizards, the higher the signal from the IgM antibody.

Our heterophil activity assay is based off the assay described in Merchant, Williams, and Hardy (2009) for use in American alligators. To account for the much smaller blood volume of fence lizards, I altered the assay to work with 10μl of whole blood, and validated it in this species. This assay specifically tests for the presence of superoxide radicals, which are produced by heterophils as part of the oxidative burst used to kill bacteria and other organisms. When heterophils are more active (either because there are more heterophils or because the existing heterophils have been stimulated by something), the amount of superoxide in the blood increases. As part of the validation, we ran the assay with pools of blood treated with superoxide dismutase, which destroys superoxide, to test that the signal is actually caused by superoxide. We also ran blood with and without a stimulant of heterophil function, to determine if the signal reliably increases when heterophils are more active. The signal reliably decreases when inhibited by superoxide dismutase, and reliably increases when stimulant is added, indicating that this is a reliable test of heterophil function.

We also did a little bit of work optimizing the natural antibody test, increasing the sensitivity of the test so that it will work with less lizard plasma. And we also found a promising lead for testing alternative pathway complement function in fence lizards.

Aside from all the immunology work, we also got out into the field up here in Pennsylvania a little bit, although we didn’t find many lizards. All in all, it was a fun, productive summer.


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On a frog hunt

 

Earlier this Spring some explorers from the Langkilde Lab went on a trip to Connecticut to take part in some exciting field work with Lindsey Swierk, former lab member and current postdoc at Yale University.

Lindsey is investigating the possible impacts that urbanization and road noise might have on wood frogs’ (Lythobates sylvaticus) mate calling behavior. To do so, our team went out to collect male wood frogs from vernal ponds of different degrees of urbanization in the area of Madison, CT – from deep into the woods, to a friendly neighbor’s backyard.

Lindsey’s research is important especially because frogs are extremely dependent on acoustic signaling as a form of communication and mate attraction. Most likely, these animals are still not well adapted to urban environments with intense noise (think of heavy traffic and construction work), despite being exposed the them for a considerable amount of generations. If sound suddenly becomes an unreliable cue for mate selection and predator detection, the dynamics of natural and sexual selection could be altered, potentially removing adaptive traits from natural populations. We don’t know exactly how (or if) wood frogs cope with these changes in their surroundings. Amphibians are some of the organisms most sensitive to environmental change, and to protect them, it is crucial that we better understand these impacts.

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Hi there!

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This trip was an excellent experience: it was the first time some of us got to work with frogs. Also, the residents we came in contact with all seemed captivated by our work and science in general – a great opportunity for us to exercise our science communication skills. Special thanks to the folks at Field House Farm, LLC, we had a great time!

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There’s always time for a little posing

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Between behavioral trials, some lacrosse with the kids at Field House Farm

Finally, I was really happy to have my first contact with a real, colorful, living salamander! Being from Brazil, where these charismatic creatures sadly do not occur, I felt accomplished after having this much anticipated encounter.

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My first salamander – Ambystoma maculatum!

Besides all the field work, we also went out to explore the New Haven area, local restaurants (Frank Pepe Pizzeria Napoletana is highly recommended!) and part of Yale University’s campus. We also got to spend time with Lindsey’s family, two adorable dogs included, but most importantly, we learned about our mate Cam’s great culinary skills after a taste of his famous baked ziti – great company in the field indeed!

As you read this, Lindsey is processing the overwhelming amount of data obtained in this field season, so make sure to check her website to hear about her results in the near future.

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Team Swierk – a job well done!


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Even Mining Bees Do It

Animal life of all sorts was springing into action in Central PA this past week. The other day as I was prepping my bike to head into the lab, I noticed a fair bit of activity centered on the pair of dandelions (Taraxacum officinale) just outside my door. Dandelions are one of the first flowers to appear in the Spring and serve as important early sources of food for many pollen-oriented insects.

Upon further inspection, I noticed that a solitary female bee was feeding on one of the dandelion’s pollen and attracting attention from several suitors. Right as I returned with my camera, one of the male bees latched on to the female, and I settled in to observe the proceedings:

In the footage, you can see the male bee attempt multiple matings with the female (and seem to succeed several times). The female seems largely ambivalent to his attentions, preferring to devote herself to the delicious pollen repast in front of her and grooming her shapely antennae. At several points, other, less fortunate suitors make a quick pass at the lucky couple, but do not dislodge the primary male. The bees appear to be members of the genus Andrena, a diverse group of solitary mining bees found throughout the world (Thanks to Dr. Heather Hines for ID help!). These bees are a sign of Spring in many areas. Male bees often become active first and search for emerging females to mate with. Afterwards, they may conduct mate-guarding to prevent other suitors from fertilizing their mate’s eggs and increasing their offspring (and fitness). Happily, we had just talked about this in my Animal Behavior class (Biol 429), so I was able to use the footage you see above as an intro to my next class.

As the weather gets warmer (barring this past weekends’s snow (!) here), I’m looking forward to seeing many more species becoming active and all the neat behaviors they’ll be displaying.


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Here Be Dragons

In August, my two older brothers and I went to Komodo National Park in Indonesia for a week of scuba diving and backpacking.

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Departing the plane in Surabaya: this photo was actually taken midmorning. The hazy skies are due to smoke from the rampant slash and burn agriculture which is devastating virgin forests on Borneo and other islands.

Going to see Varanus komodoensis in Indonesia was most certainly on my herp bucket list, and so when my older brothers mentioned that they wanted to take a trip somewhere and Komodo was an option, I naturally jumped on it. My brothers wanted a place where we could backpack and also go scuba diving, and it turns out that Komodo has some of the most beautiful coral reefs in the world. Komodo National Park consists of a series of small islands (Including Komodo and Rinca, where one finds the dragons) between the relatively large islands of Flores and Sumbawa. It is a part of the Lesser Sunda Island chain. The Lesser Sundas are bio-geographically fascinating. They essentially form an arc of mid-sized islands directly to the east of Java – extending almost to Papua New Guinea. On the far western end of the arc is Bali – a popular vacation destination. Incidentally, we had a longer than expected layover in Bali on our way to Flores: Mt. Raung in eastern Java erupted while we were in the air and disrupted air traffic through the whole region. We had a mid-air reroute to Surabaya on Java and then barely made it into Bali where we missed our outgoing flight! We managed to get onto a standby flight early the next morning, though, and our schedule was left essentially intact.

Directly to the east of Bali is the Island of Lombok. Between these two islands runs the famous Wallace Line. The Wallace Line is the biogeographic barrier between the Asian and Australian Faunal regions. It was first identified by Alfred Russell Wallace – one of my professional heroes. Wallace collected zoological specimens throughout Indonesia, and I re-read his incredible travel memoir The Malay Archipelago during this trip.

Of all the Lesser Sunda Islands, only Bali is on the Asian side of the Wallace Line. However, the line is not a clean break. If it were, one would expect to go from seeing tigers and elephants in Bali to kangaroos and wombats on Lombok. This isn’t the case. While there actually are tigers on Bali (at least there were until they were hunted to extinction in the 20th century), crossing the Wallace Line instead takes you into a sort of transition zone between the true Asian and Australian faunal regions. A place where one can find a bizarre mix of species – like cockatoos flying over herds of native wild boar – as well as endemic species like the Komodo dragon. This region is referred to as Wallacea.

Incidentally, it isn’t just a transition point in terrestrial biogeography. The incredible biodiversity of Komodo’s reefs is due to the fact that the National Park lies at the interchange between the Pacific and Indian Oceans – resulting in an incredibly biodiverse marine ecosystem as well. The diving was incredible – but I’ll stick to the herp stuff for this blog entry.

The vacation was spent on a live-aboard dive boat, and we spent days alternating diving and trekking (really mostly just herping on the Islands!). We went on 2 big hikes, the first on Rinca island where we found the majority of the cool herps for the trip. The second was a 2-day hike to Mt. Satalibo, the high point of Komodo Island. We spent the night camping on the summit and then bushwhacked down to the tiny ranger station at Sebita where we were picked up by our dive boat’s zodiac.

Before either of those scheduled hikes, though, we had some downtime between dives. We were quite close to Komodo Island, and even though it was midday and blazing hot, I couldn’t help myself from getting on land. I asked Andrew and Daniel if they wanted to come with, and they said yes – so I directed one of the crew members to fire up the zodiac and drop us off for 30 minutes or so. He thought we were crazy to go on the island by ourselves but dropped us off just the same!

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Daniel and Andrew in the grassland on Komodo with their sticks for defense! I had assumed – correctly – that we wouldn’t encounter any dragons with the midday sun beating down on us like it was.

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Daniel and Andrew with the dinghy in the background. The tan and gold colors of the islands contrasted beautifully with the blues and greens of the surrounding ocean.

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That night we went to a spot frequented by the dive boats in the area. The mangroves are packed with flying foxes – and there were thousands of them! Each one of the dark flecks in the sky in this photo is a fruit bat leaving the mangroves and headed to forage in the moist forests on nearby Flores.

The next day we began our first hike on Rinca Island. Rinca and Komodo are home to the majority of the dragons in the world. I was excited to finally get in some serious herping. Our first spotting was a mammal though: a semi-aquatic crab-eating macaque in the mangroves by the dock.

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First dragon of the trip! This was a juvenile dragon basking in the morning sun by the ranger station. Like deer by any National Park headquarters in the US, the dragons near the ranger stations on both Rinca and Komodo were habituated to humans. Once we got off the common tourist trails we found that the lizards acted much differently.

We waited for our guides and then set of on a hike across the island. Although we’d set everything up ahead of time, when we actually got there none of the professional guides wanted to go on the hike that we’d scheduled – it was too long! It took some negotiating, but we finally got some guides that were willing to take us on the planned route.

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A swarm of bees making a hive in the monsoon forest.

I started flipping logs and rocks along the trail looking for herps. Our guides were laughing at us and told us that they see snakes only very rarely. That might be true, but it only took us a few minutes on the trail before we’d found our first snake.

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I glimpsed a snake slithering in the cracks of a dried-up buffalo wallow. I grabbed it with the tongs and there it was – my first ever live Elapid!

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A Javan Spitting Cobra – Naja sputatrix

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I quickly tubed the snake (wearing sunglasses!) and showed the guides – who at this point were past the “Americans are INSANE” stage and into the “Wow this is actually really cool!” stage. This was their first time touching a cobra, and they loved it! Showing people that snakes are something to be respected and admired, rather than feared, feels awesome.

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The spitting cobra lived up to its name and doused my go-pro while I was getting some close up footage!

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Water buffalo spending time in a pool during the heat of the day. Our guides told us that this pool was one of the few that didn’t dry up completely in the dry season, making it a favorite ambush spot for hungry dragons.

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Next one of our guides spotted this Island pit-viper Trimeresurus insularis high up in a tree.

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I flipped this gecko underneath driftwood. I’m totally unsure about the species, but I think it’s a species in the widespread genus Cyrtodactylus.

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This giant centipede was under the same piece of driftwood as the gecko! Unfortunately I don’t have anything good to show the scale but the centipede must have been 9-10 inches long. Easily more terrifying than the cobra – and quite capable of making a meal of the gecko!

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Our guides asked if we’d be interested in seeing a bat cave that they knew of. Of course we were!

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The day after Rinca: more diving!

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After a full day of diving my brothers retired to eat some dinner. I still had energy, and we had anchored off of Komodo Island – where we would begin our hike early the next morning. I hopped on the dinghy and went to see what I could find at night and found this gecko foraging in the intertidal zone! I think that it’s a Hemidactylus of some sort.

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After returning to the boat with a single herp for the night, I was ready to eat some food and drink some beers and get ready for the following day. Incredibly, I would get one more herp for the night back at the boat!

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With tongs at the ready, we managed to get this banded sea krait! She may have been attracted to the boat’s lights.

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She sure looks like she’s ready to lay eggs!

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The crew told us that the local lore said that it was good luck to have a sea krait swim up to a boat. This boat was new and it hadn’t happened yet – so the captain was particularly excited! It was his first time touching a snake of any sort, and definitely the first time one had been on his boat!

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The next day we began our trip to the top of Mt. Satalibo. For reference, almost nobody does the hike we did. The head ranger managed to scrounge a tent that they give to film crews to use on the porch of the ranger station to keep bugs away. What we did is definitely not part of the normal menu – and by the time we made it back to the boat the next day we were well aware of why that is.

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A dragon that was NOT habituated to humans.

This was the first non-habituated dragon that we encountered. I was amazed by how curious it was. Curious in a predatory sort of way. I absolutely don’t want to be sensationalist, but the fact is that the dragon saw the group of us and immediately began a slow and steady walk towards us. When it got particularly close, the guides repeatedly hit it in the head with their forked sticks. In response, the dragon backed up and then tried to circle around and come from a different angle. It was a fascinating experience. The dragon wasn’t sprinting at us – it was merely walking. However, I have no doubt that if we were to stand still and allow it to approach, that it would have sunk its teeth into one of our legs. It wasn’t in a rush like we normally see predatory encounters, so it was an alien experience – but I am quite certain that that dragon wanted to eat us. That obviously doesn’t make it malicious, but the experience is one that I cannot get out of my mind. It simply wanted to eat us if we didn’t cause much trouble. As it was, the guides hit it in the head several times (which I am led to believe is standard procedure), and eventually it retreated and stared as we walked away.

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Soon after encountering that dragon, the bushwhacking became incredibly difficult. I have had the privilege of going through some thick and thorny vegetation during my research in the lab. The “Blue-creek hell hole” that lab alumnus Sean Graham and I went through to collect Cottonmouths comes to mind. I can say that without a doubt, the bushwhacking on Komodo Island was the worst I’ve ever been through. I want to come up with a way to describe the horrible-ness of the Thorn Forest on that island, but I cannot. Anything that I could write about it would detract from the reality of how bad it was.

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After the “Thorn Forest” we arrived the highest elevation forest type. The late Walter Auffenberg wrote several lengthy monographs about Komodo dragons and the herpetofauna of Komodo Island. While he was curator of Herpetology at the Florida Museum of Natural History, he decided that he wanted to research Komodo dragons. To accomplish this, he simply moved with his wife and small children to Komodo Island and began research! His writings made up the bulk of my pre-trip research. He categorized the vegetation communities on the Island and referred to this semi-moist forest as Quasi-Cloud Forest. It was clearly distinct from the lower elevation forests – with more evergreens and bromeliads indicating a wetter environment.

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We reached the top of Mt. Satalibo and set up camp. Backcountry camping on Komodo is something that almost nobody does. Once we set up camp and got a fire going, I actually asked the guides how frequently they camp on the island with tourists. The answer was something like “We did it one time with a crazy Dutch guy like 5 years ago.”

At about 1am I gave up sleeping. Naturally, I hadn’t brought any cold weather gear on our Komodo Island hike, and it was amazingly cold up at the top. First I, then Daniel, surrendered and just spent the rest of the night huddled with our guides by the fire. Somehow Andrew managed to sleep through the whole night. Once it got light enough we began our hike down. The plan was to hike from where we had camped on Satalibo down to the small ranger station of Sebita. We had scheduled things rather tightly and had a flight to catch in the afternoon but figured that we would be able to make it down to Sebita relatively quickly.

I’ll just say up front that we did not make our flight. The bushwhacking the day before had just been a taste. I knew we were toast early on, when I checked my GPS and found that 2 hours into our hike we had made it a grand total of 750 meters. We spent hour after hour going through a thorn forest that – like before – I won’t try to describe based on principle. Everything had thorns. Every branch, every tree trunk, every vine. There was one single tree species that had no thorns; I remember it vividly: at even the lightest touch it would hemorrhage black ants that would cover everything. I was stung early on in the hike, and hours later my arm still throbbed.

We spent hour after hour hiking through an endless thorn forest, going prone for significant stretches when it was too thick to crouch or crawl on our hands and knees. Turns out that when the guides had “done this before” with the Dutch gentleman that they had gone back down the way they came to the main ranger station – not to Sebita. This was everyone’s first time doing this bushwhack. At the utter mercy of the Island, I started laughing at the hilarious helplessness of our situation. There was nothing to be done but push through the thorns and the ants until the end. It was the most difficult day of hiking in my life so far, but I am glad we did it. My respect for the ecology of that island is utterly cemented.

After making it to the ocean we got on our boat and figured out flights for the next day,  I even managed to make it home in time for one final week of field work in Pennsylvania before the semester started!