29 June 2015

Crayfish feel the heat but not the burn (of chemicals)

Having a couple of papers accepted in the last few days (hooray!) reminded me that I haven’t told the story behind the most recent paper I co-authored, which came out a couple of months back.

This paper, co-authored with Sakshi Puri, was a long time coming. It’s a little hard to believe that our first crustacean nociception paper was released five years ago. When that paper came out, a lot of stuff done that just came out in the new paper was already done.

Here, for instance, is some video we shot testing one of our stimuli. The description of this in the paper ended up being written in typical academese:

One author (Z.F.) ate representative slices of the peppers, similar to those fed to the subjects, to confirm that there were noticeable differences in pungency, and the habanero slices were unpleasantly pungent.

But here’s what really happened:

Very early on in the process of working on this project, Sakshi and I were pretty confident that we were going to have two papers. We had plans to to behavioural and physiological experiments testing the responses of crayfish to acids and bases, and to chemical stimuli like capsaicin (the stuff that makes chilies hot).

Our first plan was that the first paper would be just the behaviour, and the second paper would be all the physiological recordings or neural activity.

Well, that plan didn’t survive contact with the enemy (a.k.a. reviewer #2). Journals kept rejecting our manuscripts that had only behavioural experiments. We decided that instead of dividing the experiments up by technique, into “behaviour” and “physiology” papers, we would divide them up by the stimuli, into “pH, with both behaviour and physiology” and “chemical stimuli, with both behaviour and physiology” papers. As soon as we did that, our first paper got accepted.

The experiments for capsaicin and isothiocyanate were locked down even before the first paper came out. You can see them in the poster at right, done for a conference just a few months after Puri and Faulkes (2010) came out.

What held up this new paper was experiments for high and low temperature.

We wanted to test high temperature, but it took us a while to figure out how to do it. We were concerned about the animals. We’re not in the business of torturing crayfish here. We wanted to give the animal a way of getting away from the stimulus if it wanted to.

Inspired by Dan Tracey’s paper on fly nociception (Tracey et al. 2003), we tried using a soldering iron as a noxious high temperature stimulus. Our original idea was to let the crayfish grab the tip of the soldering iron, and measure how long it was before it would let go. This turned out not to be practical, because crayfish don’t like grabbing hard things. But in our preliminary tests, it was clear that just touching the claw got a reaction from them when it was hot, much more so than when it was room temperature.

We have a plot of the behaviour in the paper, but the video is so clear:

A positive result was a first for this project. I’m always telling people that one of the advantages of working with crayfish are that “They’re cheap as dirt, and tough as nails.” Crayfish had been oblivious to everything else we’d thrown at them, and this was the first thing that bothered them. We were very curious as to whether a positive result  would make this paper have an easier ride than our first paper, which kept saying, “Nope, nothing here, either.”

While we continued the work, we presented this work at the 2011 Neuroscience meeting. Sakshi wrote about here experience here. I met lots of online people for first time, including Scicurious, Doc Becca, KatieSci, Namnezia, and too many more to mention at Doc Becca’s BANTER party.)

The feedback on the poster was good, but a key moment was when I saw another poster by Brenner and Gereau (whose work was later published in PLOS ONE, in 2012). They had come up with a simple way to test for low temperature nociception: use dry ice. Brilliant!

And the moral of this part of the story is: Don’t skip the poster sessions at conferences!

After the conference, we ran the behavioural tests for low temperatures.

We’re now around the start of 2012 with this project. And this is where we kind of got stuck, which was my fault. From the experience with our first paper, we were pretty sure this wouldn’t got anywhere without the physiology. But we couldn’t get enough physiology experiments in the can.

Unfortunately, moving from stimulating the antennae (which we’d used for acids and bases and chemicals) to the claws had created a problem. It turned out that recording from the nerve in the claw is trickier than the antennae. Trying to get what we considered the bare minimum number of recordings of the neuron turned out to be hard enough that we ultimately abandoned it. The Neruroscience poster is one of a couple of posters where we presented neural activity from the claw. The claw stuff just didn’t make it into the final paper.

We also had to change the stimulus when we tried to record action potentials. We couldn’t stick a soldering iron tip in water, but the tissue needed to be a saline solution. Plus, the soldering iron introduced electrical noise, making it impossible to see action potentials. We would have liked to have used the same stimulus, but it wasn’t possible.

We decided to move back to the antennae, because we knew we could get good neural recordings from those (we’d published a lot of them in the 2010 paper). If I remember right, I starting doing the physiological recordings there first, before doing the behavioural experiments. I knew that reviewers would savage the paper if I had behavioural and physiological data from two different parts of the crayfish. Fortunately, the antennae experiments were clear – the crayfish acted differently to hot and control – although not as interesting to watch as the claw experiments.

And Sakshi completed her bachelor’s degree along the way and got a job.

And I had a crazy few months of writing, and writing, and writing some more on projects that had deadlines. The papers from the parasite symposium I did with Kelly Weinersmith, a commissioned comment piece in Neuron, and writing and editing a crayfish book that will finally see the light of day next month, I hope.

So after many, many reminders from my co-author that we needed to get this out, submissions, rejections, workshopping it in journal club, resubmission, and final revisions, I was so pleased that this paper finally found a home.

To promote it on social media, I created some memes. These were fun to make. I think you’ll need to click this one to enlarge to read it:

I did one for hot, so I needed another for cold:

I’ll probably go to hell for this next meme, but I couldn’t resist:

The pre-print went up in March, and was the #2 more read article that month! This pleases me. I was hoping we’d top that, once the paper moves from “early edition” to the final volume.

Alas, “number one with a bullet” eluded us. But in May, we were still in the top ten!

I will admit that I thought we might get a little more media attention on this paper. “Does it hurt crustaceans when you boil them alive in the pot?” is such a common question, and this paper addresses that issue more directly than anything published before.

But there is little more satisfying than seeing a long project completed and published.

Related posts

Crustacean nociception: the embargo lifts
Crustacean nociception: Origins, part 1
Crustacean nociception: Origins, part 2
Crustacean nociception: The worry
Top ten again for crayfish nociception!

External links

Variations on a theme: crayfish nociception


Puri S, Faulkes Z. 2010. Do decapod crustaceans have nociceptors for extreme pH? PLoS ONE 5(4): e10244. http://dx.doi.org/10.1371/journal.pone.0010244

Puri S, Faulkes Z. 2015. Can crayfish take the heat? Procambarus clarkii show nociceptive behaviour to high temperature stimuli, but not low temperature or chemical stimuli. Biology Open 4(4): 441-448. http://dx.doi.org/10.1242/bio.20149654

Brenner DS, Golden JP, Gereau RWIV. 2012. A novel behavioral assay for measuring cold sensation in mice. PLOS ONE 7: e39765. http://dx.doi.org/10.1371/journal.pone.0039765

Tracey WD, Jr., Wilson RI, Laurent G, Benzer S (2003) painless, a Drosophila gene essential for nociception. Cell 113:261-273. http://dx.doi.org/10.1016/S0092-8674(03)00272-1

1 comment:

Mike Taylor said...

Many congrtulations on getting this work through the peer-review hoops ...

... but sheesh, WHAT a waste of your time and effort. You could have had the actual science published years ago and gone on to the new and better things. It's stories like this that me increasingly certain that the reviewer-as-gatekeeper concept has net negative value for scholarship.