The study that almost made me quit grad school

This is the second “story behind the paper” post from me. In the first, I focused on the paper that Ecology rejected that later won the Mercer award from ESA. In this one, I will focus on another study that involved a lot of angst at one point but that has a happy ending.

To set the scene: I was a third year graduate student. I had spent most of my first two years thinking that I was going to focus on hybridization for my dissertation. But it wasn’t proving to be that interesting, and, after learning more about parasites, I decided to switch to working on them. The summer before my third year of grad school and the fall of my third year were going to be my first big field season working on parasites. I planned a study where I would look, among other things, for effects of parasites on population dynamics. The plan was for me to do really intensive sampling of the population dynamics in a set of 6 lakes. My plan involved sampling each of the six lakes every 3 days (at least in the summer, when temperatures were warm and dynamics fast). On top of that, I was doing a larger survey of a set of 18 lakes, collecting data on selective fish predation, and monitoring habitat use via day/night Schindler series. A critical thing is that parasites cannot reliably be detected in Daphnia after preservation, so all samples (even the ones collected at 1 AM) needed to be counted live.

My original plan was to try to focus on Metschnikowia, a yeast parasite that we knew was in some of our lakes. While Metschnikowia has been a central focus of my research in the past 10 years, that first year happened to be a really bad year for Metschnikowia, with very low infection prevalences. But it didn’t matter, because there were tons and tons of bright red Daphnia, and I knew that A) there is a parasite, Spirobacillus cienkowskii, that turns Daphnia bright red, and B) Daphnia dentifera, my focal species, doesn’t produce hemoglobin (there is a paper somewhere that says this, though at this point I can’t remember which it is). So, red Daphnia must be sick Daphnia. And they were everywhere!

Except, it was more interesting than that. They weren’t truly everywhere: when I used a Schindler trap to collect samples at 1 meter intervals throughout the water column, I could see that the red Daphnia weren’t migrating vertically. Differential habitat use associated with infection – this was neat! Pretty much all the D. dentifera were deep during the day; at night, the red ones mostly stayed deep, while the clear ones migrated to the surface waters. I started forming lots of hypotheses: maybe the parasite was manipulating host behavior, perhaps to avoid fish predation? Maybe it was just that Daphnia that stayed deep were exposed to parasites in the sediments more – that is, maybe it was not that sick Daphnia don’t migrate, but that Daphnia that don’t migrate are more likely to get sick. I was trying to figure out how to test as many of these possibilities as possible, while doing all of the above work, too. And I was doing lots of day/night Schindler series to document the pattern really well.

One other thing I decided was important to do was to get data on the fitness impacts of infection. To do that, I set up flasks of red D. dentifera and control flasks of clear ones, so that I could see just how virulent this parasite was. I checked my flasks daily. For days. Then weeks. At some point, it seemed odd that none of the infected ones had died. I decided to pull them out of their flasks to see what was going on. They were all perfectly clear and healthy looking. Seeing them under the scope, realizing they were healthy, it became immediately apparent what was going on. They had never been infected. They were producing hemoglobin, despite the report that D. dentifera does not make hemoglobin. That explained why there was such a clear pattern of “infection” in the field – red animals were deep, because only animals living in the deep, poorly oxygenated habitat need to make hemoglobin.

I was devastated. I had spent the previous months exhausting myself and really excited about all the data I was generating for my thesis on parasitism. . . only to find out that I had almost no data on parasites. I did have a little data – there were several samples where it had seemed like there were two types of red things (in the end, these were truly infected animals and hemoglobin-producing animals) and I had counted those separately. But, really, I had very little data, and by this point I was most of the way through the field season in my third year of school.

I remember very clearly just leaving the lab and going to sit by Gull Lake. That morning, before I figured out what was going on, I had seen that Mike Lynch had posted an ad to evoldir, looking for a lab manager. I thought, “Well, that’s it. I’m in my third year, and I have no data. I’ll just quit school now and go and be Mike’s tech. That wouldn’t be so bad.” And, for a few days, that was my plan.

Fortunately, Alan Tessier, my advisor, happened to be visiting during this time. (He was a rotating program director at NSF at the time.) He did a good job of talking me off the proverbial ledge, helping me focus on the data that I did have that was useful, and helping me come up with a plan to salvage the field season. (In the end, it was salvaged. If it had taken me a few more weeks to figure out what was going on, it probably wouldn’t have been salvageable.)

It took a few months, but at some point I started to get curious about the data I had accidentally collected on hemoglobin production. I mean, it had been clear that there was a really strong pattern there in terms of habitat use. After playing around with the data for a bit, it became clear that there was something really neat going on: hemoglobin production was a marker for habitat use, and showed that there was intraspecific variation in habitat use in D. dentifera. Most strikingly, taking advantage of hemoglobin as a marker of habitat use, I could see that there was a clear negative relationship between the density of deep-living D. dentifera and their main competitor, D. pulicaria, which also occupies the deep part of lakes. That pattern was obscured if you just looked at overall D. dentifera density and D. pulicaria density. At some point, we started getting pretty excited about the data, and jokingly started to refer to it as Thesis 2. There were times when Thesis 2 seemed more promising than Thesis 1 (on parasitism).

It took me a long time to get around to writing it up, but, in the end, the hemoglobin study was published in Freshwater Biology. And, I’m proud of it. It’s a case where I did not have #overlyhonestmethods, and so it isn’t clear from reading the paper that it was an accidental study. But I find myself frequently telling the story behind this paper when I meet with grad students, because I think it’s important to recognize that things do not always go as planned, and that it’s possible to recover from what seem, at the time, to be pretty major setbacks.

Now that I’m back in the Midwest, it would be possible to follow up on this story. I hope we do. I think there’s a lot of potential to use this system to study the community consequences of intraspecific variation. But this time, we will study hemoglobin production and habitat use intentionally.