I am deep in the Alaskan Arctic,  300 miles from the nearest road system, attempting to conduct the kind of science that usually requires a specialized laboratory. We rowed 30 miles of meandering flatwater today, bringing our total to 200 river miles in 12 days since we landed at a lonely gravel bar on the headwaters of Ambler River in Gates of the Arctic National Park.

Mosquitoes spangle the tent canopy arching over me. Backlit by summer solstice sun, the silhouettes of the insects make an inverted night sky of shifting constellations. The sun never sets on the banks of the Kobuk River this time of year. It hangs high above the horizon even now at 11 p.m., transforming my tent into a solar oven as I, ironically, work to uncover the secrets of a frog that can turn into ice.

… Read the rest of the article here.

The study:

One of the most consistent findings arising from 20 years of study in our lab is that wood frogs seem to adapt to life in cold, dark ponds. In general, cold-blooded animals like reptiles and amphibians are not suited for the cold and function much better in warmer conditions. So, wood frogs that live in colder ponds should have a harder time competing against their neighbors in warmer ponds.

In response, cold-pond wood frogs seem to have developed adaptations that level the playing field. In separate experiments, we’ve found that wood frog tadpoles in cold-ponds tend to seek out warmer water (like in sunflecks) and have lower tolerance to extremely warm temperatures. Most importantly, they can mature faster as eggs and larvae.

But I’ve always struggled with a lingering question: if cold pond frogs have adapted these beneficial adaptation to compete with warm-pond frogs, what is keeping those genes out of the warm-ponds? Shouldn’t cold-pond genes in a warm pond mean double the benefits? One would expect the extrinsic environmental influence and the intrinsically elevated growth rates to produce super tadpoles that metamorphose and leave the ponds long before all the others.

Kaija, who was an undergrad in the lab at the time, decide to tackle that question for her senior thesis.

We hypothesized that there might be a cost to developing too quickly. Studies in fish suggested that the trade-off could be between development and performance. The idea is that, like building Ikea furniture, if you build the tissue of a tadpole too quickly, the price is loss of performance.

So we collected eggs from 10 frog ponds that spanned the gradient from sunny and warm to dark and cold. We split clutches across two incubators that we set to bracket the warmest and coldest of the ponds.

Then we played parents to 400 tadpoles, feeding and changing water in 400 jars two to three times a week.

We reared the tadpoles to an appropriate age (Gosner stage 35ish). Those in the warm incubator developed about 68% faster than those in the cold incubator. In addition to our lab-reared tadpoles, we also captured tadpoles from the same ponds in the wild as a comparison. Development rates in the lab perfectly bounded those in the wild.

Once they reached an appropriate size, we put them to the test. We simulated a predator attack by a dragon fly naiad by poking them in the tale. Dragonfly naiads are fast, fierce, tadpole-eating machines and a tadpole’s fast-twitch flight response is a good indicator of their chance of evading their insect hunters. It’s a measure of performance that directly relates to a tadpole’s fitness.

Above the test arenas, we positioned highspeed cameras to capture the tadpoles’ burst responses. We recorded 1245 trials, to be exact—way more than we ever wanted to track by hand. Fortunately, Kaija is a wiz at coding; and with a bit of help, she was able to write a Matlab script that could identify the centroid of a tadpole and record its position 60 times per second.

We measured the tadpoles’ speed during the first half second of their burst response and looked for an association with their developmental rates. One complicating factor is that a tadpole’s fin and body shape can influence burst speeds. So, a weak tadpole with a giant fin might have a similar burst speed to a super fit tadpole with a small fin. To account for this, we took photos of each tadpole and ran a separate analysis mapping their morphometry and included body shape into our models.

As we had hypothesized, tadpoles reared at warmer temperatures show much slower burst speed than their genetic half-sibling reared in the cold incubator. We even saw a similar, but weaker effect for the tadpoles that were allowed to develop in their natal ponds. It seems that developing too fast reduces performance.

Thus, it certainly seems that the counter-gradient pattern we see of faster development in cold-pond populations, but not in warm-pond populations, is at least partially driven by the trade-off between development rate and performance.

In fact, it may even be the case that we’ve been viewing the pattern backwards all along. Perhaps instead we should consider if warm-pond populations have developed adaptively slower development rates to avoid the performance cost. This especially makes sense given the range of wood frogs. Our populations are at the warm, southern end of the range. Maybe this tradeoff is also a factor constraining wood frogs to the cold north of the continent?

If warm weather and faster development are a real liability for wood frogs, it is only going to get worse in the future. We know from another recent study that our ponds have been warming quickly, especially during the late spring and early summer months. But climate change is also causing snow to fall later in the winter forcing frogs to breed later. The net result is that wood frogs may be forced to develop fast intrinsic developmental rates in response to a contracting developmental window, while at the same time, extrinsic forces drive development even faster. That’s a double whammy in the trade-off with performance. And might lead to too many “Ikea furniture mistakes” at the cellular level.

As a separate part of this study, we also measured metabolic rates in out tadpoles in hopes of understanding the relationship between developmental rates, performance, and cellular respiration. I’m in the process of analyzing those data, so stay tuned for more!

Now, five years, four blog posts, and uncountable hours later, I can say that measuring canopy structure with hemispherical photos is surprisingly difficult.

One of the biggest hurdles is understanding the equipment and deploying it properly in the field. For me, nothing is more tedious than standing waste deep in a mucky, mosquito-infested pond while I fiddle around with camera exposure settings and fine-tuning my leveling device. Add to that the constant fear of dropping a couple thousands of dollars of camera and specialized lens into the water, and you get a good sense of my summers.

So, it is with great pleasure that I offer an alternative method for capturing canopy photos that requires nothing but a cell phone, yet produces higher quality images than a traditional DSLR setup. This new method exploits the spherical panorama function available on most cameras (or in the free Google Street View app). Hemispherical photos can then be easily extracted a remapped from the spheres. You can check out the manuscript at Forestry here (a PDF is available on my Publications page) or continue reading while I walk through the paper below.

The old way

The most common way to measure canopy structure these days is with the use of hemispherical photographs. These images capture the entire canopy and sky from horizon to horizon. Assuming proper exposure, we can categorize individual pixels as either sky or canopy and run simple statistics to count the amount of sky pixels versus canopy or the number and size of the gaps between canopy pixels. We can also plot a sun path onto the image and estimate the amount of direct and indirect light that penetrated through the canopy. (You can follow my analysis pipeline in this post).

All of this analysis relies on good hemispherical images. But the problem is that there are many things that can go wrong when taking canopy photos, including poor lighting conditions, bad exposure settings, improperly oriented camera, etc. Another problem is that capturing images of high-enough quality requires a camera with a large sensor, typically a DSLR, a specialized lens, and a leveling device, which can cost a lot of money. Most labs only have one hemispherical photography setup (if any), which means that we sacrifice the number of photos we can take in favor of high-quality images.

The new way

In the past few years, researchers have tried to figure out ways to get around this equipment barrier. Folks have tried eschewing the leveling device, using clip-on hemispherical lenses for smartphones, or using non-hemispherical smartphone images. I even tested using a hemispherical lens attachment on a GoPro.

But, none of these methods really produce images that are comparable to the images from DSLRs, for three reasons:

1. Smartphone sensors are tiny compared to DSLR, so there is a huge reduction in quality.
2. Clip-on smartphone lenses are tiny compared to DSLR, so again, there is a huge reduction in optical quality.
3. Canopy estimates are sensitive to exposure settings and DLSRs allow for more control over exposure.

The method I developed gets around all of these issues by using multiple, individual cell phone images to stitch together a single hemispherical image. Thus, instead of relying on one tiny cell phone sensor, we are effectively using many tiny cell phone sensors to make up the difference.

Another advantage of creating a hemispherical image out of many images is that each individual image only has to be exposed for a portion of the sky. This avoids the problems of glare and variable sky conditions that plague traditional systems. An added benefit is that, smartphone cameras operate in a completely different way than DSLRs, so they are much less sensitive to exposure issues in general.

Smartphones are less sensitive to exposure issues because, unlike DSLRs that capture a single instance on the sensor when you hit the shutter button, smartphone cameras use computational photography techniques that blend the best parts of many images taken in short succession. You may not realize it, but your smartphone is constantly taking photos as soon as you turn it on (which makes sense since you can see the scene from the camera on your screen). The phone stores about 15 images at a time, constantly dumping the older versions out of temporary memory as updated images pour in. When you hit the button to take a picture, your phone then automatically blends the last few images with the next few images. The phone’s software selects the sharpest pixels with the most even contrast and color from each image and then composites those into the picture presented to you. With every new software update, the algorithms for processing images get better and better. That’s why modern cell phones are able to take photos that can compete with mid-range DSLRs despite the limitations of their tiny sensors.

So, if each phone photos is essentially a composite of 15 images, and then we take 18 of those composite images and stitch them into a hemispherical image, we are effectively comparing a sensor the size of 270 individual phone camera sensors to the DSLR sensor.

The best part is that there is already software that can do this for us via the spherical panorama feature included with most contemporary smartphone cameras. This feature was introduced in the Google Camera app back in 2012 and iOS users can access the feature via the Google StreetView app. It is incredibly simple to use.

Update: Check out my post on tips for taking spherical panoramas

Once you’ve taken a spherical panorama, it is stored in your phone as a 2D JPEG in equirectangular format. The best part about the photo sphere software is that it utilizes your phone’s gyroscope and spatial mapping abilities to automatically level the horizon. This is advantageous for two reasons. First, it means we can ditch the tedious leveling devices. Second, it means that the equirectangular image can be perfectly split between the upper and lower hemisphere. We simply have to crop the top half of the rectangular image and remap it to polar coordinates to get a circular hemispherical image.

How to extract hemispherical images from spherical panoramas

UPDATE: Please see my latest post to process spherical images with R.

Command line instructions

If you are proficient with the command line, the easiest way to extract hemispherical images from photo spheres is to use ImageMagick. After you download and install the program you can run the script below to convert all of your images with just a couple lines of code.

cd "YOUR_IMAGE_DIR"

magick mogrify -level 2%,98% -crop 8704x2176-0-0 -resize "8704x8704!" -virtual-pixel horizontal-tile -background black +distort Polar 0 -flop -flip *jpg

You may need to make a few modifications to the script for your own images. The -crop 8704x2176-0-0 flag crops the top half of the image (i.e. upper hemisphere). Be sure to adjust this to 1.00×0.25 the dimensions of your panorama dimensions. The -resize "8704x8704!" flag resizes the image into a square in order to apply a polar transformation. Be sure to adjust this to 1.00×1.00 the width of your panorama

Note that the code above will convert and overwrite all of the .jpg files in your folder to hemispherical images. I suggest that you practice on a folder of test images or a folder of duplicates to avoid any mishaps.

GUI instructions

If you are intimidated by the command line, extracting hemispherical images from photo spheres is also easy with GIMP (I used GIMP because it is free, but you can follow the same steps in Photoshop).

Update: You can also try out this cool web app developed by researchers in Helsinki which allows you to upload spherical panoramas from your computer or phone and automatically converts them to hemispherical images that you can download. However, I would not suggest using this tool for research purposes because the app fixes the output resolution at 1000p, so you lose all of the benefits of high-resolution spherical images.

First, crop the top half of the rectangular photo sphere.

Second, scale the image into a square. I do this by stretching the image so that the height is the same size as the width. I go into why I do this below.

Third, remap the image to a polar projection. Go to Filter > Distort > Polar Coordinates

Fourth, I found that increasing the contrast slightly helps the binarization algorithms find the correct threshold.

All of these steps can be automated in batch with BIMP plugin (a BIMP recipe is available in the supplemental files of the paper). This can also be automated from the command line with ImageMagick (see scripts above and in the supplemental materials of the paper).

The result is a large image with a diameter equal to the width of the equirectangular sphere. Because we are essentially taking columns of pixels from the rectangular image and mapping them into “wedges” of the circular image, we will always need to down sample pixels toward the center of the circular image. Remember that each step out from the center of the image is the same as each step down the rows of the rectangular image. So, the circumference of every ring of the circular image is generated from a row of pixels that is the width of the rectangular image.

With a bit of geometry, we can see that, the circumference matches the width of our rectangular image (i.e. 1:1 resolution) at zenith 57.3 degrees. Zenith rings below 57.3 will be downsampled and those above will be scaled up and new pixels will be interpolated into the gaps. Conveniently, 57.3 degrees is 1 radian. The area within 1 rad, from zenith 0° to 57°, is important for canopy estimates as gap fraction measurements in this portion of the hemisphere are insensitive to leaf inclination angle, allowing for estimated of leaf area index without accounting for leaf orientation.

Thus, we retain most of our original pixel information within this critical portion of the image, but it does mean that we are expanding the pixels (increasing the resolution) closer to the horizon. I tested the impact of resolution directly in my paper and found almost no difference in canopy estimates; so, it is probably okay to downscale images for ease of processing if high resolution is not needed.

The hemispherical image produced can be now be analyzed in any pipeline used to analyze DSLR hemispherical images. You can see the pipeline I uses in this post.

How do images from smartphone panoramas compare to DSLR

In my paper, I compared hemispherical photos taken with a DSLR against those extracted from a spherical panorama. I took consecutive photos at 72 sites. Overall, I found close concordance between measures of canopy structure (canopy openness) and light transmittance (global site factor) between the methods (R² > 0.9). However, the smartphone images were of much greater clarity and therefore retained more detailed canopy structure that was lost in the DSLR images.

Although the stitching process occasionally produces artifacts in the image, the benefits of this method far outweigh the minor problems. Care when taking the panorama images, as well as ever-improving software will help to minimize imperfect stitching.

Overall, this method is not only a good alternative, it is probably even more accurate than traditional methods because of the greater clarity and robustness to variable exposure. My hope is that this paper will help drive more studies in the use of smartphone spheres for forest research. For instance, 360 horizontal panoramas could be extracted for basal measurement or entire spheres could be used to spatially map tree stands. The lower hemisphere could also be extracted and used to assess understory plant communities or leaf litter composition. Researchers could even enter the sphere with a virtual reality headset in order to identify tree species at their field sites from the comfort of their home.

Mostly, I’m hopeful that the ease of this method will allow more citizen scientists and non-experts to collect data for large-scale projects. After all, this method requires no exposure settings, no additional lenses, and is automatically levelled. The geolocation and compass heading can even be extracted from the image metadata to automatically orient the hemispherical image and set the location parameters in analysis software. Really, anyone with a cell phone can capture research-grade spherical images!

Be sure to check out my other posts about canopy research that cover the theory, hardware, field sampling, and analysis pipelines for hemispherical photos, and my tips for taking spherical panoramas.

Our chapter tells the story of green frogs and the feral condition of their life with suburban human neighbors. We especially highlight the way that the human built-environment of lawns, pavement, sewers, and septic systems is infused into the biology of green frogs (a topic that Max and Dave have studied in depth). As a counter example, I told the story of the wood frog, a species that has escaped a feral fate by clinging to the remnants of wild space away from humans (a topic I study in depth).

I rarely get to write about wood frogs outside of academic articles, so it was a pleasure to contribute to this piece. I think it is some of my best natural history writing. I’ve excerpted my section below (or, read the chapter):

“To better understand why our housing patterns influence frogs, it is worth taking a frog’s-eye-view of suburbanization. Most frogs exhibit distinct life-stages. Like humans, frogs begin development as shell-less and fragile eggs, but while human embryos float within the protection of a womb, frog embryos are buoyed among the vegetation and flotsam of ponds. The embryos have an umbilical relationship to the water that surrounds them. Nutrients and oxygen easily pass through the transparent jelly and are consumed through delicately branching gills. Any contaminants in the water also suffuse the embryos.
Even before their eyes or mouths have formed, the developed embryos hatch as free-swimming larvae not much larger than a grain of rice. Hatchlings are vulnerable. Thus, frogs hedge their bets by producing hundreds of eggs per clutch, hoping that at least a few will win the lottery of life. Some species, like wood frogs, additionally safeguard their offspring by choosing impermanent pools that are devoid of fish as relatively safe nurseries.

Those hatchlings that survive develop into recognizable tadpoles with bulbous bodies and slender tails. A pond’s version of cows, tadpoles graze along the bottom with scraper-like teeth. They consume algae and detritus along with any solid matter that washes into the pond basin. A long digestive tract allows the tadpoles to incorporate nutrients into a growing body. Where ponds neighbor septic systems, this means that human waste makes up a prodigious portion of a tadpole’s body.

The transition from a tadpole to a frog is a remarkable change. It makes the squeaking voice and acne of human puberty seem like a blessing. Every system in the tadpole’s body transforms. The tail gives way to bony limbs. The narrow, disc-shaped mouth morphs into a wide, insect-capturing, gape. The goggle eyes, so fine-tuned to underwater vision, mutate into something much like our own. Even the long and coiled digestive tract shortens and distends. At the end of this metamorphosis, the aquatic vegetarian leaves the water’s edge and becomes a terrestrial carnivore.

Green frogs are parochial and prefer a pond-side life. For a short time as juveniles they might range far and occupy any standing water from lakes and ponds to swimming pools and tire ruts. Upon adulthood though, they settle along freshwater shores where they patiently wait for dragonflies and other insects to approach within range of a lunging gulp. Since green frogs inhabit permanent ponds, they can breed throughout the summer, and without the threat of the pond drying out from beneath them, their tadpoles can be leisurely in development. When snow falls and the pond freezes, both adults and overwintering tadpoles take refuge deep in the insulating layer of pond muck. Because a green frog’s life is so reliant on a pond, they can survive in just about any permanent water with at least a narrow perimeter of vegetation. As long as a homeowner neglects the tufts of grass along the bank, green frogs are more than happy to remain neighbors.

Unlike green frogs, wood frogs become sylvan nomads after metamorphosis. As their home ponds dry up in the summer and fall, they wander the forest floor hunting among the leaves, only briefly returning to recently filled pools in early spring to breed. During the winter months, wood frogs burrow just under the blanket of leaves dropped in fall. This enables them to be the first out of hibernation as the forest thaws in spring. For these reasons, wood frogs rely on leaf-littered landscapes. Manicured lawns where leaves are regularly raked and bagged make inhospitable places for them. Where the balance of forest gives way to lawns, wood frogs disappear…”

Overall, this was a really fun project to work on that gave me a chance to switch up my writing style. It was also a lot of fun to be able to collaborate with my partner, Bayla, who painted the featured image for the chapter.

The online version of the book is a little counter-intuitive to navigate (I think this was intentionally designed as a rhetorical device), but if you can figure it out, it is worth checking out some of the other cool stories of our feral world!

Update (Nov. 2020): And, I’m especially thrilled that our piece will be the cover article for the journal, featuring a pair of breeding wood frogs from our population! My hands nearly froze trying to get this underwater shot, so I’m glad it was worth the effort!

Over the past 20+ years, our lab has been monitoring over 50 populations of wood frogs at Yale Myers Forest. Each year in early spring, we listen for the duck-like clucks of the male frogs which means that they have emerged from under the snow and moved into the breeding ponds. Shortly afterward, we head out into the freezing ponds to count the egg masses as a way to monitor population density over time.

In this study, we looked at how the oviposition date (the day on which frogs deposited eggs) has changed over time. As climates warm, we usually expect for the timing life-history events (like oviposition, emergence from hibernation, flowering time, etc.) called ‘phenology’ to advance in the year as winters get shorter. That’s just what most species do. And the trend of advancing phenology is strongest for amphibians.

Given that annual temperatures at our field site have increased by almost 0.6 C in the past two decades, we expected frogs to breed and lay eggs earlier. If our frogs were like other amphibians, we might expect oviposition to come around 6 days earlier.

Surprisingly, we found the opposite. Our frogs seem to be breeding 3 days LATER.

To figure out what might be going on with our frogs, we decided to look more closely at climate across the season, not just annual averages. It turns out that most of the increase in annual temperatures are felt later in the summer, but relatively less when frogs are breeding. Snowpack, on the other hand, is actually accumulating later and lasting longer. In the figure (Fig. 3 from the paper) below, you can see these trends. On the left are the comparisons between temperature, precipitation, and snowpack between 1980 and 2018. On the right, we plot only the difference in trends over time. At the top-right, we plot the oviposition dates to show how seasonal changes in climate line up with frog breeding.

We also looked at how the timing of oviposition correlated with climate across the season. We found that breeding occurs later when there is more snow at the beginning of the breeding window. Also colder temperatures just before breeding correlate with delayed oviposition (which makes sense if colder temps mean more snow and less melting).

So, we think that frogs may be kind of stuck. Persistent snowpack might be keeping them from breeding earlier. But at the same time, warmer summer temperatures might be drying up their ponds faster. If so, this could be a big problem for tadpoles that need to maximize their time for development. The figure below shows that frogs tend to breed earlier when winter and early spring air temperature are high. As we’d expect, more snowpack correlates with later breeding. High precipitation during the spring delays breeding (probably because it is falling as snow).

Twenty years is a long time to be collecting ecological data, but it is a pretty short window into the evolutionary history of wood frogs. And, we don’t know how long snow and temperatures may have been working against these frogs. So, as a final piece of our analysis, we used a machine learning technique called a random forest to predict oviposition dates backwards in time an additional 20 years. It doesn’t seem like much has changed over the past half-century or so. In one way, that could be good news in that at least things don’t seem to be getting any worse.

The big question is, how will frogs cope with these climate changes? If tadpoles are faced with an ever-shrinking window of time to develop into frogs, will they be able to keep up? Or, will they lose the race and end up as tadpole-shaped raisins in our ponds?

I won’t give away any spoilers, but I’m looking at our long-term larval datasets to ask that question next.

I found this population of toads on a tiny island during an expedition to the South Prince of Wales Wilderness Area. The toadlets were dispersing at edge of the tidal saltwater zone near a stream outlet.

There are three things that surprised me:

1. that this small island could retain water all spring (we had a really hard time finding water for ourselves to drink even on the larger islands),
2. that toad would have been able to disperse to the island in the first place given how far away it is from the mainland, and
3. that such a small island would be able to sustain a toad population.

The catalog entry (HERA.023177) and photo are available online through the Peabody Museum of Natural History collections.

Here is the full page to dowload.

Rats have traveled around the globe alongside humans. Where we make our homes, they are happy to make their own, too. Unfortunately, they can make terrible neighbors, especially when they become vectors for diseases.

The rats that live in the slums of Salvador, are to blame for the widespread of emergence of leptospirosis infections that poses a serious health risk. In response, the municipality planned a multi-year rat-eradication project.

The bad news is that it is nearly impossible to fully eradicate rats from large territories. After just a short time, the populations tend to rebound, either because some rats were missed and then repopulate from within or because new rats migrate from outside and colonize the area. Understanding how rats repopulate after eradication efforts is important for deciding how best to proceed with future management strategies.

To test which of these scenarios was at play in Salvador, my collaborators trapped rats before, during, and after the eradication campaign in three contiguous geographic regions within the slums. We looked at the genetic relatedness of the populations across the three time-points. If the rebound populations were more similar to each other post-eradication, it would suggest that the populations were recolonized from source populations outside. However, if the regions became more genetically distinct post-eradication, it would suggest that the rebound populations were seeded from the few local genetic lineages that persisted.

We found that the populations showed distinct genetic differences immediately after the eradication effort, suggesting that remnant rats from the original populations had repopulated from within. Those difference persisted after 7 months.

In addition, we looked at the genetic signatures of population expansion and contraction. We found that post-eradication populations had pronounced reductions in genetic diversity. As animals were removed from the population during eradication efforts, it created a bottleneck. Since the new populations arose from the few remaining rats, only those few genetic variants left over persisted.

If enough genetic diversity is lost, and is not replaced by migrant mice from outside the original population, it might eventually lead to inbreeding and reduced fitness. If that is the case, it means that creating serial bottlenecks in the population through eradication efforts could be an effective way to weaken the rat populations to manageable levels, even if the overall population number rebounds in the short term.

I think my collaborator Dr. Jonathan Richardson (@JRichardson_44) did a nice job of succinctly outlining our findings in this Twitter thread:

I'm very excited about our new paper out today on the significant genetic impacts that accompany an urban eradication campaign of rats in 3 areas of Salvador, Brazil, w/ @georgisil_ & many Twitterless colleagues. The TL,DR version is in short thread below:https://t.co/WLqmQ1UJkD

— Jonathan Richardson (@JRichardson_44) June 6, 2019

I’m excited to announce that my paper “A new, noninvasive method for batch marking amphibians across developmental stages” is now published at Herpetological Conservation and Biology.

This project originally grew out of frustration that no methods existed to be able to track amphibian larvae through metamorphosis into adulthood. This is a key bit of information needed in understanding amphibian ecology and asking questions like “How many tadpoles survive to adulthood?” “What percentage of frogs return to the same pond?” “Do tadpoles tend to school with kin or strangers?” etc. And ultimately this information is integral to estimating dispersal kernels and defining microgeographic variation.

Originally, I was looking into using radioactive isotopes to mark tadpoles, but just as my digging made that method seem intractable, I came upon a paper (Mohler 2003) that used a calcein fluorochrome solution to mark salmon.

Calcein binds to calcifying tissues like bones and scales. Although it had been tested in fish and bivalves, it had never been trialed on amphibians or any terrestrial applications. My study demonstrates that this technique is extremely promising for herps and solves a major limitation of marking amphibians. Below are the pertinent figures from the paper and supplemental materials, and also a couple extras.

Video of a wood frog tadpol approximately 24 hours after administration of calcein label.

This technique allows for both short and long term labeling. Short-term marking is detectable throughout the entire integument for 3-4 days and is visible in internal structures for up to 20 day in tadpoles marked within 28 days of metamorphosis. Labels are most useful for long-term marking (over 146 days) across metamorphosis when applied within 10 days of metamorphosis with 99% detection rate. If marked within 16 days of metamorphosis, the detection rate falls to 90% and sharply declines if tadpoles are marked earlier in development.

Check out the paper for more info. And also check out my poster on the project.

Not GFP!

A lot of folks ask me if this is technique is similar to the GFP (green fluorescent protein) that Shimomura, Chalfie, and Tsien discovered in the 60s (Tsien 1998). The answer is, no. GFP is a gene that can be introduced to animal genomes to induce production of a growing protein originally derived from jellyfish genomes. GFP is a genetic technique and so must introduced in germline or other stem cells. In contrast, calcein is a molecule that binds chemically to calcium. This means that calcein can be administered to any tissue for immediate fluorescence without interacting with the genome.

Thanks

This project required keeping lots of tiny frogs in the lab for almost 8 months, which turned out to be a major cleaning and feeding project a couple times a week. I couldn’t have done this work without loads of help from our lab manager and undergrad researcher–they deserve glowing medals for their service.

I especially would like to thank my brother Wes for spending part of his vacation playing scientist and helping me set up the experiment.

References:

Mohler, J. W. (2003). Producing fluorescent marks on Atlantic Salmon fin rays and scales with calcein via osmotic induction. N. Am. J. Fish. Manage. 23, 1108–1113.

Tsien, R. Y. (1998). The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544.

An article I wrote along with Lisa Ronald and Bob Dvorak about perspectives on the future of Wilderness stewardship is out in the International Journal of Wilderness. This piece is a sort of wrap-up for the Millenial track at the 2016 National Wilderness Workshop that I helped to organize.