Culturing culture

I am supervising/mentoring/collaborating with two post-doctoral researchers (a.k.a. postdocs), and greatly enjoying the process. They are both smart, hardworking and enthusiastic, and, unlike me, talented experimentalists. I am good at proposing experimental designs, and at inventing or improving upon experimental apparatus, but I managed to get through all my years of training as a biologist without ever being taught the nitty-gritty of laboratory work. I like to make things up as I go along, and work on twelve projects at a time, paying sporadic attention to each, and you can't really do experiments that way. But these two post-docs actually know what they are doing in the lab. It is impressive to see how thoroughly they train the students, how carefully they document their doings, and how well they know their organisms. As a grad student I started working with rotifers with almost no prior knowledge of rotifers, and without any advisor who knew rotifers. I read the literature, but there is a deep cultural knowledge about how to care for aquatic lab animals, with lore and practice around each group, and I had none of that. These two know, and it is wonderful to watch and learn.

Fussy Hydra babies

Hydra babies come in two types, buds and hatchlings. Buds grow like tree branches out the side of the main trunk of their parent. Eventually the branch is almost as big as the trunk, and they separate, and you have two hydra. Eggs also grow on the sides of their mothers, but they need to be fertilized by free-swimming sperm that are released by male hydra. Then they make a hard shell, detach from the mother and settle down to wait some weeks or months before a tiny hatchling wiggles out. Where the buds are like small copies of their parents, the hatchlings are tiny and genetically novel individuals. They are too small to easily eat the crustaceans we usually feed the adults and buds (Artemia) so we feed them mashed Artemia, except they like their food alive. I have ordered rotifers to see if they will eat those, as they are smaller and softer than the Artemia.

I find myself looking forward to having rotifers in the lab again. They are just so familiar at this point.

Publication bias

Much of the culture and methodology of science has been specifically designed to help combat bias. The human brain takes sensory inputs, filters them through our biases before we even perceive them, assigns them whatever interpretation seems most reasonable given what we already think, stores them (or not) depending on how they fit into our preconceived notions, and then retrieves them when they seem to support the view we already have on the topic at hand. We are like perfect bias machines, and we have to bend ourselves into all sorts of intellectual contortions to achieve any measure of impartiality on almost anything. Most of the statistics scientists employ, rather than testing for support for our preferred hypotheses, are designed to test for support of the opposite of our hypothesis. Only if someone tries really hard to prove the opposite of their point and utterly fails to do so are we convinced that their data significantly support their point. The scientific review process, the sometimes painfully stilted manner in which science is written and even the reluctance of many scientists to have any polling based system of evaluating scientific consensus are all designed to help us keep our biases in check. The Spockian view that one should act as though emotions and unreason don't exist is clearly illogical.
Despite all this anti-bias fervor, scientists are still human, and still have biases. One such bias is that we want, as individuals, to be successful. We would rather study a topic that is going to win us praise, jobs and funding than something else that will take a long time to reach any conclusion, even if the payoff for society is much greater for the long term project. We are also far more likely to publish results that are going to advance our careers than those that won't. This leads to the title of a 2006 paper called, "Publication Bias: The Problem That Won't Go Away." To be clear, I am not referring to a reluctance to publish papers that challenge the dominant viewpoint. Name any truly successful scientist, and I will bet you that his or her career was built on a paper that challenged the dominant viewpoint. Scientists know this, and we are pretty much obsessed with finding holes in the dominant view. Publication bias rather is most commonly a tendency to publish clear positive results ("our data strongly support hypothesis X over hypothesis Y") over the less clear cut cases ("our data do not allow us to confidently support or refute the hypothesis we set out to test"). This kind of thing happens a lot.

Publication bias on the part of others, and by me, has been on my mind recently. In writing a paper on the evolution of paternal care in primates, I ran into the problem that almost nobody makes the statement, "in 72,000 hours of behavioral observation we did not observe males caring for their young." But if after 120,000 hours of observation they see a male pick a couple of burrs out of a juvenile's fur, they might well write a paper titled, "first observation of paternal care in species X." This results not only from the logical impossibility of proving the complete absence of a behavior, but because in many species where males don't care the assumption is that males don't care, and a failure to see counterexamples isn't that interesting to the people studying the species or the editors reviewing their papers. The result of this is that we have lots of publications stating the presence of care, even when that result is rare, but almost nobody stating the likelihood of its absence. I ended up having to make the rule that if multiple papers describe patterns of care in a species, and none of them say anything about male care, that counted as too little care to qualify. In my own studies of rotifers, I absolutely would have written a paper on parental care if I had observed any, but absolutely could not get a paper published in which I state that I didn't see any.
But my publication bias when it comes to the rotifers is much more profound than that. The results of one of my major experiments were negative, and as such not particularly interesting. I am writing it up anyway, because my dissertation committee wants me to, but I and they think it unlikely any journal will publish it. Of course if 20 different labs did similar experiments, and only one got a positive result, only the positive result would be published, creating a very false impression. I recognize this as a source of bias, I don't like it, and I don't see any way around it. The best I might be able to do is post the whole damn paper on my blog and hope that anyone interested in the topic stumbles upon it.

The other option, which I won't take but is far more common than you might think, is to simply analyze my data until I do find something interesting in there, then write up the paper as though that was my main question all along. This is something that advisers have specifically told me to do in the past, although not on this project. It is grudgingly accepted that this happens, and like publication bias, isn't going away.

EvoDemo Poetry

A chimpess keeps the knack to breed
until she's not alive
But woman stops and then goes on
another forty-five

A hydra grows and buds and splits,
then does it all again
But man can live his life but once
before his certain end

A rotifer skips infanthood,
lays eggs on her first day
But humans grow and learn and find
all reasons for delay

You'll live your life as humans do
and never think it's odd
But redwoods still are saplings
when we're pushing up the sod.

Moving the last rotifer

For much of the last year my life and schedule have revolved around daily rotifer census. How often I go to campus, at what times, when I have time for anything else and the energy and time I have for anything else have all depended on lab work. When I could rely on my students to take care of it, I could do other things. Frequently, very frequently, my supply of dependable students was not up to the demands of taking data on and caring for several hundred animals each day. Even when my students are scheduled to do everything, it is rare for a day to go by without questions, problems or scheduling issues. If I am not in lab for a day or two both the quality of the data and the survival of the animals begins to decline.

So it feels like a big deal that my lab work will be done this week. Thursday. I've told my students that after that they are free to continue working on their side projects, but I'm not going to be in the lab. I'm not going to spend hours moving rotifers. I'm not going to be harassing them about keeping the lab organized and the rotifers' containers clean. I'm not going to be on campus six or seven days a week. I'm going to be at home, writing a thesis, and will come to campus on Wednesdays and Thursdays. And I'm taking my desktop (the lab's erstwhile main computer) home.

I like my students, and the rotifers are fascinating, and microscopes are fun. But I really like the idea of not needing to be in the lab every morning at 8. And the prospect of being able to have whole days to work on writing my thesis is positively thrilling.

Cephalic rotiferitis

I've been in the lab every day all day for the last week looking at rotifers. When I close my eyes, I not only see rotifers, I can count their eggs, see their teeth chewing and estimate their age.

Experts in a Lesser Known Phylum

Ask most people to name some phyla of animals and they will just look at you funny. Those who do know what you are talking about are likely to name Chordata, Arthropoda, Annelida, or maybe Mollusca. Most people will run out of Phyla long before getting to Rotifera. We humans tend not to pay a lot of attention to a Phylum whose members are mostly microscopic and don't cause any known disease. This is true not only among lay-folk, but among scientists as well. Web of Science, a catalogs of the scholaraly articles from about 8700 publications, lists fewer than 100 papers focusing on rotifers in the last year. Arthropoda, by comparison, gets more than 38,000 hits in the same period. So rotifers are not the best studied group in the world.
But those almost a hundred publications had to derive from somewhere. That somewhere is a scattering of experts across the globe. And it gets lonely being the only one in your city, state, country or continent with a strong interest in rotifers. (For example, I think I my lab is the only one in California which focuses on rotifers.) So what's a lonesome rotiferologist to do? Organize a conference, of course. Every two or three years there is a Rotifera conference somewhere in the world, and I have just found out that Rotifera XII is in Berlin, Germany next August. I very much plan on going, and hope to give a short talk on my work. They have about 60 slots open for presentations, which I think means almost everyone who studies rotifers will be there presenting. It should be interesting.

Team of Science

We attempted to get me and my entire team of undergraduate rotifer wranglers into our tiny lab space all at once. Two people couldn't make it, but 12 of us plus a photographer jammed in. The room is 12m^2 but about half of space that is occupied with counters, furniture and large equipment. Hopefully at my next job I will have a larger lab space, a smaller team, or both.

Population Doubling

As I am preparing for my talk, I am doing some intense demographic analysis of my rotifer data set. One interesting factoid I have calculated is that the population doubling time, assuming I could keep an infinite number of rotifers and didn't have to get rid of any, is 28 hours.

A related calculation: If I started with one newly hatched rotifer and let the population grow (with my average age-specific reproductive rates and death rates), after one month I would have 159 million rotifers.

The average volume of a rotifer is about .001 cubic millimeters. A million of them pressed together makes one milliliter. A billion makes a liter. 10^27 would be a cubic kilometer. Earth's oceans have a total volume of 1.347*10^9 cu km, meaning I would need 1.347*10^36 rotifers to fill them completely with no space between rotifers. At the demographic rates they maintain in my lab, assuming I didn't cull any, this would take 138 days.

I only have time, container space and staff to keep track of 450 rotifers at a time, so I end up culling a significant portion of my population every day.

Rotifer Demography Talk Wednesday

I'm a biology grad student, but my funding and my fellowship are all through the Demography department. One service I return to the Demography department is to attend their weekly seminar and who ever the speaker is, suggest biological literature relevant to her topic of study. Some demographers take better to this than others. Most seem to appreciate the new perspective, even if they are not really interested in thinking about humans in a biological context. (For the record, I also go to biology talks and bring up demographic concerns.)

The next speaker I will have to deal with differently, because the speaker this coming week is me. I'll be presenting on demographic aspects of my rotifer research. Age specific mortality and reproduction. Effect of food supply on longevity. Infant mortality. I'll get into the biology a bit too, but mostly they'll want to hear about the demographics. If I was in my audience, I would suggest more of a focus on the biology.

I wiggle my eyebrows ~1000 times a day.

I spend about eight hours a day in front of a microscope. I generally have a student on either side of me. I look at the first rotifer in our population and report how many eggs and juveniles it has, whether it is alive, and anything else notable about it. "Two forty six dash bee five is alive has three eggs, two juveniles and extended foot syndrome. The largest juvenile has one egg."
The student on my left, at the computer, enters all of this into the spreadsheet and tells me what to do with the juveniles, based on our established culling rules. "Put the biggest juvi in two forty eight dash a one, cull the other two."
I pick up the mom rotifer in a specially bent glass pipette and wiggle my eyebrows such that my glasses slide off my forehead and onto my nose so that I can see the student to my right. She uses her pipette to point at the hole where the rotifer is going. I squeeze the bulb at the end of my pipette to eject the rotifer into that hole. She looks through a second microscope to make sure the rotifer is actually there. I push my glasses back up, look through my microscope, pick up the juvenile, wiggle my eyebrows again, move it to the well where it needs to go, then move on. All of this takes 15 seconds to one minute, depending on the complexity and which students are working with me. We repeat this process 450 more times each day. By the end of each day we have gathered more demographic data than many field studies of long-lived vertebrates do in several decades. By the end of a month we can see significant evolutionary changes based on the selective pressures we apply through our decisions about who to cull and how much to feed them. It is not glamorous, but it is effective.

Eek!

This semester I am taking on the biggest and most complicated experiment I have ever attempted. Three populations of 150 rotifers, each population with different rules about how each individual is fed and which ones I cull. Frightening complexity just in planning the experiment, not to mention hiring an training enough undergraduates to staff 150 person-hours per week. This is not going to be an easy time, but I think it could produce a really significant paper. Lucky for me, my advisors think so too, and I should have enough funds to pull it all off.

Biodemography poem

http://www.demog.berkeley.edu/~dlevitis/213ex1

Rotifer talk

I went into lab meeting this morning feeling like I didn't have much to say, and rather unprepared. After an hour of presenting one not quite done project after another, my professor asked, "Well, did you know rotifers were going to be such a rich and productive system when you started?" I realized everyone there but me was impressed by how much progress I've made on so many fronts. That was okay.

Down to the wire

One of my professors, RL has this habit of being uncannily right most of the time. My student, LZ, and I were working on analyzing our data this afternoon. The first draft of her senior thesis is due tomorrow, and we decided we may as well try one last thing, calculating a variable that RL had suggested. We plugged it into our model and BAM! highly statistically significant, much more so than anything else. And, in retrospect, it makes beautiful biological sense. Of course it all depends on cumulative lay order across generations! Any brilliant biodemographer would have seen that at once.

Now LZ has at least 12 hours to rewrite her paper.

Cleaning Data

My studies of rotifer demography and evolution would not be possible without extensive student participation. I need three people working together for four hours every day just to get the census done. Most of the time I have six or seven students on my team. Only one student, LZ, has been working with me the whole time. She started last year as an Undergraduate Research Apprentice and this spring and summer has been working on her Senior Thesis. She decided, quite independently, that she was going to study which demographic factors affect the probability of a rotifer reproducing sexually, rather than asexually. She is a fabulous student, has become a talented researcher, and I hate to see her go.
A rough draft of her thesis is due this Friday, and as we have been trying to analyze our data, we have instead been spending most of our time fixing typos and errors in the data. We've been finding mistakes ranging from recording a rotifer as being in plate 1111 instead of 111 to the same information recorded quite differently in two different places. LZ and I spent ten hours yesterday going through our data line by line and cleaning up errors. Some sections were perfect, others awful, and we couldn't help but wonder which students had taken the clean data and which had gotten sloppy, lazy or confused.
Doing this has brought into sharp relief both the good and the bad of having such student-powered research. Relying on students is extremely useful and motivating and inspiring, but can also be frustrating and time-munching. Which way it goes depends enormously on the students I choose. I am very shortly going to be needing to find another couple of assistants, and will have this experience in mind.

Question Creep

If a billion scientists studied life on earth for a billion years, they would leave more unanswered questions than we now know exist.

Every project I have ever been involved with, or heard of, has generated far more questions than it has answered, not because the researchers were doing a bad job, but because one can't study any organism in detail without realizing how little one knows about it. Humans are the favorite research subject of humans, and we have published literally billions of articles on us, every aspect of our biology and behavior has been the topic of intense research, and yet there is still a huge amount we don't understand about ourselves, neurologically, chemically, socially, medically, you-name-it-ly.
And after all this research, we have succeeded in creating whole new fields of study to generate more questions. Genomics being an obvious example of a new field that is question rich.

That level of research (at least on the biological and chemical sides) could be applied to any of the hundreds of millions of life forms out there, and we would still be generating questions about that species. And then their are all the questions about the differences and similarities between species, the way that life has changed over time, the evolutionary, ecological and social interactions between species, and so on and so forth, ad infinitum.

This plays out quite clearly in my own little lab. As a grad student, I am lucky to have a very small lab space that I share with another student and my ~$25K worth of borrowed equipment. Oh but the use that tiny little room gets. Any day of the year there are at least three people in there looking at rotifers for four hours. I got into studying rotifers to answer a question about humans, why our females have such a long post-reproductive lifespan. But now that I need data on rotifer demographics, and have a horde of undergraduates studying them, my students and I have raised a huge range of related questions. To give you a sense, here is a short list of the projects currently under way, or in planning:

1. I started with the question, will rotifers evolve a post-reproductive lifespan if exposed to selective pressures similar to those thought to have caused human females to survive well past menopause?

2. I brought on LZ as an assistant, and she wondered what demographic factors influence when a rotifer reproduces sexually as opposed to asexually?

3. I hired PB as a work-study student, and she is working on an assigned side project, looking at how the size of a rotifer's eggs vary based on her size and age. This arose out of discussion with LZ in which we wondered if egg size could be affecting mode of reproduction.

4. Another student, NN, came on as a volunteer and got fascinated with rotifer biomechanics. Her senior honors thesis will ask how rotifers use their cilia for movement and feeding, and how does this vary with size and between males and females?

5. We noticed that when we do get sexually competent rotifers, they generally won't mate with their own sons. I asked a former student of mine, SM, to join the rotifer lab and investigate incest avoidance in rotifers. Now she is in the lab most afternoons taking video of rotifers mating, or choosing not to mate.

6. In studying the demography, we found that we could often visually identify when a rotifer was 'sick.' We wondered if we could categorize these 'sick' behaviors, and use our demographic data set to test whether our perceptions of sickness consistently presage death. HC, who came on as a volunteer, is working on this.

7. With all these projects looking at rotifer behavior, I decided it was important to have a unified terminology to describe the behaviors we were seeing. So I have two students, LF and HL, working on an ethogram, a list of behaviors with a description and diagram of each.

8. One of my lab mates in the Moritz lab is studying how water filtration affects the progression of the Chytrid disease that is killing off large numbers of amphibian species. It occurred to me that rotifers live in the same habitats as amphibians, and eat things the size of the Chytrid zoospores, the mobile cells that cause infections and reinfections. A former rotifer rangler, CW, is planning experiments to ask whether rotifers will eat chytrid zoospores, and if so whether that information can be applied to amphibian conservation. (Chytrid Experiment).

Every one of these projects is doable, and all of them will generate far more questions than they answer. I have my billion years work cut out for me.

Sigh

I've spent the afternoon helping two of my best assistants move on to research that more closely matches their interests than what they have been doing with me. I'll still be helping to advise them, but I'll no longer have them on my project. They will be hard to replace, but after all the work they have done for me, I owed it to them to help advance their careers.
NN will be studying rotifer biomechanics in another lab (with me acting as rotifer expert), and MR will (if all goes as planned) be doing coral censuses in PNG (with me helping to urgently prepare her for her trip). My other long-term lab assistant, LZ, who is totally irreplaceable, is graduating in a week and moving to India two weeks after that to work in public health. Time to recruit more assistants.

The Velige People

One of the great pleasures of working with really talented undergraduates is that they know stuff I don't and make me learn stuff I would never otherwise have gotten to learn. Take NN. I met NN when I went to the Invertebrate Zoology class she was taking to ask if any of the students wanted to help me with my rotifer experiments. A couple of days later I got a resume from NN, and quickly realized that in addition to being a great assistant, she knows enormously more about aquatic invertebrates, and a bunch of other biological topics, than I do. A good start.

One of the topics she knows and I don't is biomechanics. She quickly developed an interest in rotifer fluid mechanics. So yesterday afternoon NN and I went up to the lab of Dr. Mimi Koehl, aquatic invertebrate biomechanician extraordinar and talked to her grad-student and my friend, Lindsay.

It turned out Lindsay and NN have a lot in common. They both are fascinated by aquatic invertebrates, biomechanics, fluids, and the interactions of these concepts. And they both know what a veliger is. In fact, they had quite a long discussion about veligers of different types, and how many of them have a range of similarities to rotifers. I, throughout this entire conversation, played the game of trying to take part in the discussion while also attempting to deduce what a veliger was.

I figured out this much: veligers are a subset of the larvae of aquatic invertebrates. They are motile plankton whose motive force and/or feeding is supplied by rings of compoud cilia, the same as one would find in a rotifer. They often grow up into forms that are not moved by cilia, like muscles and snails. When said quickly, veligers sounds a lot like villagers, which renders funny any conversation about what sorts of creatures eat veligers.

Today I looked up what a veliger actually is. I wasn't too far off the mark. A veliger is a young mollusk in a developmental stage provided with a velum, a membrane edged with clilia used for locomotion and feeding. Cilia are only a reasonable way to get around if one is small (a couple of millimeters at most) so once a veliger grows that big it generally moves on to other means of locomotion, losing its velum and thereby its veliger status. Veligers are found in the bivalves (clams, muscles etc) and the marine gastropods (snails and sea-slugs).

Lindsay says that several people have wanted to study how veligers use their cilia to feed and move, but that little progress has been made because of the difficulty of finding and keeping them. They die, they grow up, they don't reproduce quickly. So since I know how to provide a large number of the animals that most closely resemble veligers in their use of cilia, rotifers, and Nicole and Lindsay know about biomechanics and veligers and all that, it seems we have a good collaboration.

P.S. Here is a photo of a Fusitriton veliger that Lindsay sent me. I Photoshopped it to increase the contrast, to make the cilia more visible.

Rotifer fluid mechanics

One of my best students, NN, has become fascinated with the ways in which rotifers use their cilia, and wants to do a senior honors thesis on the fluid mechanics of rotifer cilia. She is interested in issues of scaling, how they use their cilia in feeding and locomotion, and the differences in the cilia between males and females. Males use their cilia only for locomotion, while females use them for both locomotion and feeding, leading to some fascinating questions about the trade-offs females make to have their cilia serve these two very different, and very important roles.

I very much want to encourage her in this, but started by explaining to her that as an evolutionary demographer, I know squat, make that squat/2 about biomechanics. And of all the parts of biomechanics I don't understand, Low Reynolds Number Fluid Mechanics may just be the part I understand least. I understand it so little I don't even know what it means. The Reynolds number has something to do with the ratio of the size of the object to the viscosity of the fluid, or something like that. This helpful Wikipedia article describes it in terms I only vaguely understand. What I understand it to mean is that the smaller an organism or piece of an organisms is, the more viscous the fluid effectively is. A whale moving through water experiences an environment in which viscosity is much less important than a rotifer moving through the same, equally viscous water. The rotifer has a very low Reynolds number, and its cilia have even lower Reynolds numbers. The viscosity of the water they move is so high, it is like a human arm through tar. Or something like that.
But don't quote me on any of that because it is probably wrong.

So I told NN that I was glad to work with her on that, but she would need an adviser from a biomechanics lab, like that of Mimi Koehl. Luckily, one of my best friends in my department is one of Mimi's students, and is interested in ciliary feeding. And this morning we went to the grocery store together, and by the time the groceries were in the fridge, my friend had agreed to meet with my student and help her figure out how to frame and answer her question. My only claims on the project will be that I helped generate the questions, am brining the collaboration together, and am providing the study organisms. In other words, I may end up co-advising a senior thesis on a topic I know nothing about. Hopefully by the end I will at least know what a Reynold's number is.