Frogz that Glowz

azandis@gmail.com

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.

This figure, from my poster at the Joint Meeting of Ichthyology and Herpetology, summarizes the limitations of current marking methods including calcein labeling.

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.

 

This is Fig. 1 from the paper and shows a living calcein-labeled larva within 24 hours of marking (A), a calcein-labeled metamorph approximately 10 d after marking (B), and ventral (C and D) and dorsal (E and F) views of a calcein-labeled (left) juvenile 63 days after marking and unmarked individual of the same age (right). Calcein fluoresces green in marked tissue when lit by a NIGHTSEA BlueStar handheld 440–460nm flashlight through a cancellation filter (A, B, D, F) but is not apparent in white light (C, E). In larval and metamorph stages the label is visible through the overlying tissue in the distal end of the tail along the notochord and in skeletal structures (arrows in B). In juveniles, the calcein label is most obvious from the ventral view in the bones of the limbs and feet (arrows in D) and from the dorsal view, in the parietal bones (arrow in F). Scale bar is approximate.

 

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

 

This is Fig S2 from the supplemental materials showing phalange cross-section (A), tibiofibular cross-section (B), and tibiofibula (C) from a wood frog marked with calcein at x12 (C) and x50 (A and B) magnification. In the end, I found that external observation of live animals was more reliable than post-mortem bone cross-sections in detecting labels.

 

Here is an example of the very simple administration setup I used. This could easily be scaled up to mark hundreds or thousands of animals and administered pond-side.

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.

Figure 2 from the paper. Predicted probabilities of detecting a calcein label 146 day after administration in juveniles of average initial mass within a given age class marked at an initial age from 0 to 30 days prior to metamorphosis. Predicted values estimated from the data with a repeated measures mixed effect model. Shading indicates 95% confidence interval.

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.

My brother, tax lawyer by day, mad scientist by night.

 

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.