Better measures of tiny bits of living jelly

My student and I had a paper accepted last week. I like this paper a lot. First some official details, then a short story about how the paper came to be.

Forthcoming in "Marine and Freshwater Research" 

The consistent, non-destructive measurement of small proteiform aquatic animals with application to the size and growth of hydra 

Daniel Levitis and Josephine Goldstein

Abstract: Hydra (Cnidaria), the basal metazoan most often studied in cellular, molecular and developmental biology, is difficult to measure because it is small, proteiform and aquatic. To facilitate broader organismal and ecological study of Hydra, we evaluate three methods whereby a polyp's body column can be measured by means of photomicroscopy. The volume, cylindrical surface area and surface area corrected for changes in body shape are all highly repeatable methods (r=0.97) when shape varies little. However, shape changes alter volume and cylindrical surface area. Repeated corrected surface area measures of the same individuals in markedly different positions yield standard deviations that are less than 5% of the mean measured area. This easy, non-lethal means of individual size measurement explicitly accounts for the flexible morphology of a polyp's hydrostatic skeleton. It therefore allows for the elucidation of how growth and size vary over time, age and food intake. We find that hydra change size dramatically day to day, and that while food level influences adult size, it has little effect on the early growth of recently detached buds. Finally, we discuss ecological and biological applications of this method. 

Part of why I like this paper so much is that my student did most of the hard parts, and the reviewers didn't ask for a lot of changes, and the editor (Dr. Russell Death) moved things along quickly and efficiently, so it is the first paper I've published that didn't feel like pulling my own teeth. It is a solid methods paper. It says, "here is a method for doing something that many scientists may want to do, that we didn't know a good way to do before."

In particular, it is a simple way to accurately and easily measure a tiny transparent aquatic animal with no hard parts or consistent shape without harming it.  Take a picture of it and with a bit of simple math calculate its surface area. Simple, elegant, inexpensive, non-invasive, biologically meaningful, everything I hoped it would be.

But the method presented in the paper is a substantially different, and better, method than the one we started out with. In fact, we were almost ready to submit the paper when we changed the method drastically.

I started out just wanting to measure a hydra polyp because it was necessary for another research project. Nobody had a method that worked halfway decently without killing the hydra. A hydra's body, although it shortens and lengthens, bends and twists is almost always roughly a deformed cylinder. So I said, "Hey Josi" (that's my student), why don't we develop a method to measure a hydra by photographing it and estimating its volume as though it was cylinder?" She agreed, and off we went, taking photos of hydra during our whole research project, for almost two years. We finished gathering our data, and found that indeed we could measure hydra this way. It didn't work great, but it kinda worked. You could use it to tell the difference between a really huge hydra and one that was just kind normal. Viola, unimpressive but probably publishable methods paper.

We wrote it up and were pretty close to submitting when I decided to see what hydra focussed papers had come out recently, and found that someone else had just published almost the exact same method in a nice paper with an interesting biological point. Our formulation of that method didn't work any better than hers did, and we had no point beyond "here is how to measure." We couldn't publish the same method again, even if we came up with it independently. After some cursing and self-recriminations for being so slow, I decided to see if it was possible to salvage anything of the methods paper. Josi took another set of photos and I used them to repeatedly adjust my formula, with limited biological reasoning, until something worked better than the formula just published. In fact, one formula I came up with by eyeballing my graphs worked much better than the method just published. So much better that the calculated value hardly changed at all even when the shape of the hydra changed drastically.

Now you are probably saying to yourself that this is blatant cheating. Trying different formulas (perhaps 100 of them) until something gives you the result you want is a pretty sure way to get the result you want, if you are persistent enough. But two things combined to make this not just okay, but beautiful. First, the formula I stumbled upon made obvious biological sense, even before I knew it worked. This formula represents a hydra as a roughly cylindrical bag whose skin stretches as it elongates itself, or folds and ripples as it contracts. In other words, it describes a hydra accurately and reveals something I didn't previously know about the way a hydra moves. Secondly, and as importantly, when I applied the formula to the main data set, which I didn't use to develop the formula, it still gave a highly consistent measurement for any individual, even as the shape of the individual changed. The method in fact works for completely different populations of hydra. You could take two hydra that looked the same size under the microscope and conclusively decide that one was bigger than the other. You could tell how much a young hydra grew each day. You could really measure the buggers, eliminating most of the noise inherent to previous methods.

Still more lovely, the method Josi and I had just developed could make use of the photos and measurements we had already taken, so redoing all our calculations and figures required Josi to write only a few extra lines of code (Thank you Josi, thank you R). We did a little rewriting to compare our method favorably to our other method, blamed attributed the other method on to the person who had just published it, made our biological argument to explain why the method worked, and we had a drastically improved paper ready to submit. There is a lesson in this somewhere about the scientific method.

Immortaly Tomfoolery

Their is a saying among scientists that the more you know about the scientific subject a journalist is writing about, the less of what he writes makes any sense. There is a long new article in the New York Times magazine about a hydrozoan jellyfish, Turritopsis dohrnii, which the author claims holds the key to immortality. As someone who happens to work on hydrozoans, and on aging, I can assure you that not a bit of it makes any sense. The title, "Can a Jellyfish Unlock the Secret of Immortality?" should be a dead giveaway that this is magical thinking with a whitewash of pseudoscience. The argument behind the article, striped of its misunderstandings and untruths,  goes something like this:

1. There is this jellyfish that can develop back from the medusa phase, which we normally think of as the adult, to the polpy phase, which we normally think of as the juvenile. It can then develop into the medusa phase again.
2. We are going to assume that this is the only know case of an organism that does not show a human-like pattern of aging.
3. We are going to assume that this non-human like pattern is equivalent to immortality.
4. We are going to assume that if we understood the mechanisms behind this assumed immortality, we would know how to make humans immortal.
5. We would know by now what makes them immortal except that we are going to assume that the one researcher I talked to extensively for the article who studies the species is the only one doing so.
6. We are going to assume that this one researcher is unfunded and working alone not because he is considered a crackpot, but because the rest of science is just too blind and lazy to see the importance of this man and his work.
7. We are going to assume that when he has learned a little bit more, we will achieve immortality.

I do not recommend that you read it, and mention it only because I have been asked about it, and because I would like to speak briefly about the word immortality. Immortality is defined as immunity from death. Immortal beings cannot be killed. Turritopsis dohrnii can very easily be killed. Put one out of water for a few minutes, feed it to a predatory snail, heat it, freeze it, slice, dice or frappe it, and it will be dead. Ergo not immortal. However the journalists are not to blame for the misuse of the word. A very good recent paper in PNAS from a very good careful research group is titled, "FoxO is a critical regulator of stem cell maintenance in immortal Hydra." They use the word immortal as many people in bio-gerontology do, to mean that the risk of death does not increase with age. My general impression is that using the word in this way is misleading, but that it is a lot flashier than 'nonsenescing' and therefore widely used.

Hydra bud

To reproduce asexually, hydra bud. A bump grows on the lower stem of the adult, elongates, grows tenticles, develops a seperated body cavity, and finally releases from the mother to be a perfect little clone. This particular bud needs another few days of growing before it can detach, but its tenticles are already armed with poison stingers, and it can catch prey, or eat plankton passed to it by the tenticles of the mother. I've only seen this food-passing a couple of time, and it could even be coincidental, but it is nice to think that even cnidarians get tasty treats from their mommies.

Science picture of the month: Hydra hatchling

I've talked her before about hydra and their reproduction, so I thought I should post a few out of the thousands of pictures I have of them. I particularly like this one, and have a copy o up on the wall in the lab. It is a hydra hatchling, right out of the shell. The three bumps at the lower end of the picture are it's stubby little baby tentacles. The diameter of that shell is a bit under half a millimeter.

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.

More musings on individuality and Hydra

It was a trick question. I admit. Well not a trick exactly, but a question to which science does not have a right answer. Even when the question is defined fairly exactly, it isn't clear what unit we should be looking at.

I've already talked a little bit about the hyrda, but I want to give you more detail, because they are such an interesting and bizarre case. There are at least four levels at which we could define the individual in hydra. The smallest is the individual cell. Most cells in a hydra are capable of turning into any kind of hydra cell, producing a whole new hydra, and moving on their own. People have turned a whole hydra body inside out, and the cells that were on the outside just become inside digestive cells, and the cells that were on the outside become skin cells, and the community of cells goes on about its business. Second, the polyp, that thing with the tentacles and digestive system we classically think of as the individual animal. It looks like a little animal. It acts like a little animal (in most ways). It hunts, it reproduces itself, it has different cells doing different jobs. Third, there is the physically attached cluster of hydra. Through budding (growing a new hydra-shaped organism off the side of the old one) hydra reproduce asexually, but the buds get to be a fair portion of the size of the parent before separating, and are generally of almost full complexity while still physically and physiologically attached. One or two or occasionally more buds can be growing off the main polyp at the same time, and one could easily see this mass of genetically identical connected cells as one individual, despite the fact that it has multiple sets of tentacles feeding multiple digestive systems. Finally, one could consider that these genetically identical groups of cells remain part of the same individual even after physical separation. The genetic individual could after a short time encompass many thousands of polyps.

At which of these four levels does senescence occur? We know from experimental evidence that the risk of death by individual cells increases with age, so we have senescence in level one. In level two, the polyp, the experimental evidence points to no senescence, and the same goes for level three. At level four we don't have experimental evidence, but Mueller's Ratchet implies that there would be slow senescence of the genetic individual. Without going into details, Mueller's Ratchet is a line of genetic reasoning which makes clear that the number of harmful mutations in an asexually producing population almost always increases with time, where the number could decrease with sexual reproduction. So as the genetic individual of the hydra keeps producing more polyps, the newer polyps on the average will always have more harmful mutations than those of earlier generations. And remember, just because the polpys don't age doesn't mean they are immortal. They still die, in large numbers, from causes such as being eaten. As the polyps are reproducing and dying, we end up with more polyps from more recent generations and fewer from older generations. The mutational load of the genetic individual increases, and over time this should lead to increased risk of the extinction of the genetic individual. So the genetic individuals, like the cells, senescence, but the two layers of organization in between, the polyps and the clusters, don't.

This is a real problem without a clear solution. Do hydra tell us something important about the evolution of aging, because unlike almost all other animals, they don't age, or are we just looking at the wrong scale?

A more useful way to phrase the question may be to ask why the cell and genetic individual age, but the polp and the cluster don't. The first answer that comes to mind is that both cells and genetic individuals accumulate damage in ways that they can't fully repair, while the polyp and the cluster can easily repair any damage that comes along because any one piece can completely rebuild the whole. In this context, aging occurs when organism are built in a way that doesn't allow for easy repair. At some point this idea will combine with some other idea to form something useful. Or it won't.

Individuality

The central question of interest here in the Laboratory for Evolutionary Biodemography is: how does evolution determine demographic patterns (usually individual lifespan) and, secondarily, how do demographic patterns (again usually individual lifespan) influence the evolution of other traits? This questions leads to all manner of difficult sub-questions. One of these, that comes up surprisingly often is, "what is an individual in this case?"

One colleague has been pondering this question in the context of eusocial insects. Eusocial means that some individuals do all the reproducing, and others don't reproduce at all, they just work to increase the survival and reproductive success of the breeders. Queen ants and their workers are a good example. It seems pretty easy to count ants, they have separate little bodies and they are genetically distinct "individuals" but because they don't breed (usually) from the viewpoint of propagating genetic material, their only role is to perform their appointed task within the colony, in order to aid the queen. This has led some ant experts to refer to the ant nest as a super organism, with the queen functioning as the reproductive organ, and the workers, like the cells in our intestines, as merely the body that supports this reproduction. In many organisms reproductive cells can last the whole lifetime, which intestinal cells are disposable, and frequently replaced. Likewise, queens live as long as the colony does, greater than 30 years in some species, while workers usually last only a few weeks or months. So is the colony a single organism, and therefore the workers its sub-parts, or is each worker an individual, and therefore the colony a multiplicity?

Another colleague is studying the demography of hydra, small mostly sessile cnidarians. Hydra are among the most demographically bizarre organisms. For starters, no one has been able to prove that hydra age at all, despite multiple long term attempts. Second, their primary means of reproduction is through budding, where a bump on the side of the organism gradually elongates, grows tentacles, forms a digestive cavity and takes on the form of a fully formed and functional (but somewhat small) hydra before detaching and becoming a separate individual. Add to this that if you mash them up to separate their cells from each other, each cell has the capacity to grow into a new hydra. Yesterday, I spent a few minutes watching through a microscope as a hydra with a large bud sticking off the side, about half the size of the main body, wiggled in a perti dish. Both sets of tentacles, both digestive systems worked, like conjoined twins. As I watched, I wondered if I was looking at one individual, or two, or hundreds. Each cell had the capacity to found a new colony, build a new hydra, and therefore each cell was in a sense an individual. Each stem could be called an individual, by the loose analogy to humans. Or the whole genetically identical, physically attached, coordinated being could be an organism. Depending on what unit we call the individual, we get very different answers as to the lifespan.

A final example I've been wondering about is the giant redwood tree. A single trunk of a redwood seems to the casual observer to be one huge individual. But redwoods bud prolifically from the base, and multiple trunks can grow out of the same stump, the same root system. Large groups of huge trees can be genetically identical, save for the mutations accumulated in their growing tissues over thousands of years of growth. If we consider one stem to be the individual, redwoods can live for thousands of years. But if we consider everything derived from one seed to be the individual, I don't know of any reason not to consider redwoods, like hydra, effectively immortal. Sequoia sempervirens indeed.

So I'm posing the question to you dear reader, what is an individual? What operational rule should be applied? How do we find the individual in a hydra, or in a redwood forest?