Wednesday, October 2, 2013

Bruce Means Q&A: Frog Science

National Geographic just published a story about the tepuis of the Guiana Shield* featuring my friend Dr. Bruce Means.

Pristine "Islands in the Sky" Are Window on Evolution: Unique plants and animals of South America's tepuis (mesas) reveal rich secrets.

Go read it now. Be sure to watch the video and then come back here. Pay attention to the part where he's holding a plastic bag with some pebble toads in it above the title Dr. Bruce Means: Herpetologist and National Gegographic (sic) Grantee.

At the end of the video Bruce says, "As in all science, when you investigate something you come up with a lot more questions than you can answers." Because there is a hierarchy of ignorance, though, I can get answers to my questions. I had plenty.

I emailed Bruce for more details and he sent me a PDF of a paper all about the DNA studies. I emailed him back to complain that there must be something in between the National Geographic fluff piece and this thing so full of italicized Latin and statistics-speak I can barely parse the sentences. I realized I needed to ask the questions directly and write it myself. Bruce said, "Stop by. I'm in."

Dr. Bruce Means, Herpetologist
Here's Bruce in his office in Tallahassee Tuesday afternoon. Over his left shoulder lives a beautiful black and white kingsnake with an articulated lamp shining on him. There are other snakes living there too. Unlike me, Bruce likes to capture and keep animals. It's this capturing and keeping I wanted to know about today. I tried to record our conversation but I did it wrong and got nothing. I'm going to write this in Question and Answer format anyway, but it's totally paraphrased from memory.

Q: In the video in the National Geographic story you're shown at the bottom of the chasm holding a ziplock bag with three frogs in it. Were they alive in there? 

A: Yes. That's how I collect frogs. I try to get about 5 males and 5 females. I put a few specimens in a quart size baggie and blow some air into it and zip it closed. Here I put it in a cooler to stabilize the temperature when I'm out collecting, but it's not necessary. In South America I can just put them in my pocket and they're fine for up to three days. I think those bags must be oxygen permeable.

Q: That would be an interesting experiment, to measure the gas permeability of the bags.

A: It would. You should do that. (I will)

Bruce said in the video it took him 36 hours at the bottom of that chasm to figure out how to find the pebble toads he was looking for. It took me under a minute with a flashlight to find three frogs on my lab so I could demonstrate this frog-in-a-baggie technique.
First one I found got away because I had a flashlight, my phone, and a baggie in my hands.
He disappeared inside my air conditioner. It wasn't on.
There were two frogs by this window.
I put my phone in my pocket after I took this picture and caught him in my hand.
Frog in a bag. Bruce swears they're fine like this for days at a time,
even three or four frogs in there together, inhaling and exhaling.
This one also peed. Bruce didn't mention them peeing.
He probably handles them until they get that out of their system,
as it were, before he puts them in the bag. I didn't like him in my
hand so I popped him straight in the bag.
This is where Bruce and I diverge, I just don't like messing with my neighbors.
I immediately put that frog back where I found him. Then I tested that true-tone
flash on my new camera phone. I'm way more technological than biological. 
Q: But surely you can't bring live frogs back with you. Don't you have to kill them and preserve them? How do you do that?

A: I usually collect for several days then process them all at once, but on the tepuis I have to do them every day.

Bruce in his wet lab. Specimens preserved in ethyl alcohol in jars. 
I get some of that stuff you use on a toothache, benzocaine, and squirt a little bit out into a bag, about an inch of it, and add some water and slosh it around. Then I put the frogs in and they immediately go limp.

 For frogs gently killed and lovingly preserved in ethanol;
over-the-counter preparation at my Publix on the way home.
Then I take the limp frogs and spread them out on a towel. I like the brown ones like in public restrooms. I use forceps to get them all arranged so their legs and feet are spread out. Then I put another towel over them and soak the whole thing in ethanol. That makes them get stiff. Then I can put the labels on and put them in jars of ethanol.

Looking for the forceps.
We're not so different. I have forceps in my lab too,
but I keep them in this bag with my soldering iron for
rework of tiny components on surface mount circuit boards.
Bruce demonstrates how he would arrange the limp frogs
on toweling with forceps
Q: What about the DNA analysis? You can collect the material later from the preserved frogs?

A: No, I do it in the field. For the specimens I want for DNA analysis I cut their stomach skin open with scissors and collect a tiny bit of heart muscle and liver tissue and put it in one of these.
Specimen containers for heart and liver tissue for DNA analysis
I label the specimen container with the same identifier as the tag I attach to the frog. I put some ethanol in the container to preserve the tissue. You can't use formaldehyde because it fixes the DNA.
Photograph of frog specimens arranged on a brown paper towel, stiffened with ethanol.
Live frogs are very colorful. This is Pristimantis dedrobatoides. It's bright red underneath.
Ethanol dissolves all the red and blue pigment from the preserved frogs leaving only melanin.
(These are not the same frog as above.)
Preserved frog showing identifying tag attached.
If you've ever been in a silkscreen shop you know the smell of ethanol. They use denatured alcohol as a solvent for the dyes they press through the tiny holes in the silkscreens onto whatever they're printing. When the alcohol evaporates the color is left behind. It makes perfect sense to me that the color would be removed from frog specimens preserved in ethanol.

(Regarding the colorful red and brown frog above: I called it the Louboutin frog because it's bright red underneath. Bruce didn't get the reference.** He calls everything by the real scientific name. My grandmother did that too. If I hear it enough I pick it up, which is why I say gambusias instead of mosquito fish, but Bruce has an ear for Latin and easily remembers Pristimantis. He doesn't need a nickname to call the image to mind.)

Q: What information do you record about a new kind of frog when it's still alive? 

A: I photograph it to record the color. I handle it to get it to release its defensive excretions, hold it right up to my face to smell it, and then taste it.

Q: Do you just dab a bit on your bottom lip like my grandmother used to do to test mushrooms? If her lip went numb after ten minutes she wouldn't cook that one.

A: No, I just lick it like any other predator would. I try not to lick it a lot in case it is really bad. This one just tasted very bitter. (The Louboutin frog)

Q: And then when you've got it arranged on the paper towel and stiffened you take all your measurements?

A: Right. I measure it with dial calipers and a ruler and write that all down in a form I make up ahead of time.

Q: How do you tell the males from the females so you can be sure you got some of each?

A: Some of them I can't tell. I have to dissect them when I get back to the lab to be sure. Most frogs have some external difference, like the males have the throat pouch for calling.

Then all this information ends up in these three ring binders that you see all over his office. It's quite impressive organization. He can find anything he's looking for immediately.

It's a lot more paper than I like myself. I'm so space limited I tend to keep everything electronic. I'm trying to talk Bruce into letting me do an iBook of his Eastern Diamondback Rattlesnake research, but he really likes the printed and bound version of things. At the end of our conversation I came away with a dropped hard drive that wouldn't turn on and a note to bring him a Firewire 800 to Firewire 400 cable so he can hook his slide scanner to his new Macbook Pro. I might not be inclined to handle animals as much as Bruce, but I can contribute to the advancement of natural science in my own physical science way.

** History of the Guiana Shield and Guyana the country, and Guayana, Land of Many Waters. Venezuelans originally called the region the Guayana Shield, pronounced "gwah-yana." Then Columbus came along and they called the region Guiana, pronounce it like it was spelled Guyana, and then read like a Frenchman would say the man's name Guy, "gee." Then in 1970 they became an independent English speaking republic and changed the name to Guyana, pronounced "Gye-yana" (see Wikipedia for more.)
** Louboutin reference explained. Pigalle Ada $650
Bruce's website. You can buy a book and support science! Stalking the Plumed Serpent is a great read.

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