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The experiments are starting to run into each other.

Here's an interesting thing about evolutionary experiments that you may not be aware of. It's what makes an "evolutionary experiment" what it is: replication. But what, exactly, does that mean? If we want to be able to tell a story about how some trait evolves or has evolved, we have to link that trait to changes in genes in a population. But it is insufficient to show this in a single population - doing so leads to one of many cases of "just so" stories in biology. Why did things turn out in a certain way in that certain population? Well, perhaps it had to do with something that the researcher studied, or perhaps not. It could have just been a fluke. So for those who are interested in getting at the mechanisms at the heart of things, it's necessary to repeat experiments, using more than one population.

In our case, we deal with *sets* of populations. Our starting point is thinking about "wing dimorphism" in wild insect populations. In a nutshell, "wing dimorphism" is where a given insect species consists of a mix of individuals who are able to fly and individuals who cannot fly. It's possible to take this simple notion and run with it in a hundred different directions (where are the flight-capable vs. flightless ones found - same population or different populations? What time of year? etc.).

In my case, the direction that has been taken is the direction of experimental evolution: around 25 years ago, TZ traveled to Florida and collected several hundred sand crickets, consisting of a mix of long-winged and short-winged individuals. He sorted those sand crickets into paired populations of long-winged-only and short-winged-only - three sets of pairs (aka three "blocks"), or six populations altogether. He allowed each population to breed, and then reared the offspring to adulthood. When the crickets reached adulthood, each of these second-generation populations contained a mixture of long-winged and short-winged individuals. For those that originated from long-winged-only parents, TZ culled out all of the short-winged individuals, and bred the second generation of long-winged adults. Vice-versa for the sets of short-winged populations. The result, 25 years later, is "selected lines" of long-winged and short-winged crickets that are fairly close to true-breeding for long wings or short wings.

We have to be careful about calling these "selected lines," because a lot of biologists tend to interpret that phrase as meaning "inbred lines," which means something completely different. TZ has been careful about maintaining large groups of breeding adult crickets, to ideally avoid any problems associated with inbreeding. In contrast, geneticists and people working in various health-related fields often specifically work with "inbred lines" that have intentionally or unintentionally reduced levels of genetic variability. But our goal is to study the effects of wing dimorphism in the context of the broad genetic variation that is present in natural populations, so we are trying to maintain as much genetic variability as possible, aside from the specific genes that generate long-winged vs. short-winged adults (and let me tell you, this is a polygenic trait).

One of the strengths of the entire nutritional research program I've been working on over the past three and a half years is its use of these selected lines to study how nutrition influences the physiological mechanisms that generate wing-dimorphism in these crickets. It's slightly more nuanced than that, but that's the big idea.

In practical terms, what it all means is that it's insufficient for me to just go and do a single set of experiments with a single block of crickets (one set of long-winged and one set of short-winged crickets). I need to be able to show that a given pattern is consistent across at least two of the three blocks.

Therefore, this spring, I started out by injecting the Block 2 crickets with radiolabeled glycine, to trace the metabolic fate of this one amino acid. That's after I fed each cricket one of 13 diets containing different total amounts and ratios of protein and carbohydrate, the idea being that the amounts of these two macronutrients could influence cricket metabolism. So, the math is: ~6 individuals each x 2 cricket morphs x 13 diets = 156 crickets. But wait, it's actually even more complicated. Even though they are close to true-breeding for long wings, the long-winged adults are actually a mixture of individuals who are flight-capable, and individuals with long wings who are _not_ flight-capable. So we have to add on extra crickets to ensure that we study an adequate number of the flight-capable long-winged crickets. That means the final number is closer to 250-300 crickets per experiment.

That's a hell of a lot of samples to crank through, which is why most evolutionary biologists study tiny insects, like aphids or fruit flies.

There are three main fates for the radiolabeled glycine: it can be used for energy and respired in carbon dioxide, it can be stored as lipids for later use as energy, or it can be used as a building block to construct proteins. I have to measure the captured carbon dioxide immediately, so the first step is already finished for the Block 2 crickets. After that, I dissect out the crickets' ovaries (we're working with all females), and then I have an undergraduate help me to extract and quantify and "count" the crickets' lipids. Quantifying tells me the total mass of lipids in a cricket, while "counting" tells me how much of the radiolabeled glycine has been converted into fat. But wait, there's more. Then I take the fat-free cricket tissues, wash out the unbound amino acids (with tricholoracetic acid), dissolve all the protein, and "count" the protein solution, to quantify how much of the radiolabeled glycine has been synthesized into protein.

Anyway. We aren't finished with all of that work yet. It takes me two days and change to measure a given set of protein samples. The undergraduate can get through 12 crickets in a day. And now, the next Block is nearing adulthood, so I am starting to spend some time every day sorting out the new adults (day 0) so I can put them on one of the 13 diets and repeat the whole glycine-injection business with another batch of 250-300 crickets.

If that was all that was going on, it actually wouldn't be that bad.

But it isn't.

Something happens when you keep and breed animals in captivity over a span of 25 years. It's called "lab selection." Whether you like it or not, gradually, the captive population becomes adapted to the posh conditions provided in the laboratory. These crickets don't have to deal with predators, and they receive abundant, high-quality food. Those things will tend to favor the survival and reproduction of certain individuals in a way that differs from which individuals might survive and reproduce in the wild. Eventually, and especially after 25 years, you will reach a point where it becomes really difficult to extrapolate back to what's happening in wild populations.

Therefore, last year, TZ went back to Florida and collected another set of crickets. He's now maintaining this set of crickets as a "mixed" population of long-winged and short-winged individuals, who all get to freely interbreed.

And so, a second undergraduate is repeating one of the main experiments that I conducted in Texas, quantifying the feeding patterns of these "field" crickets to compare against the feeding patterns of the selected Block crickets. And I'm also injecting the field crickets from a subset of five of the thirteen diets, because I'm not completely and utterly batshit insane. Only partially so. TZ and I both find the "field" crickets more intellectually interesting at this stage.

On the one hand, the intellectual context is really interesting and rewarding.

On the other hand, a lot of this work is highly finicky and demanding, every single day. Every day I wake up with my mind buzzing with the day's to do list, which I have to write down so I can figure out how to fit all of the puzzle pieces together and get things done at the appropriate time (e.g. a round of injections can take 4-6 hours, I have to do periodic prep work for those, plus coordinate time at the dissecting scope to dissect out ovaries, plus coordinate time on the balances to weigh crickets, plus help my helper get set up with lipid samples, plus help with rearing the thousands of crickets used in the experiments, et cetera). There are a lot of little steps that can go wrong, so everything needs to be done carefully, meaning I have to go to work well-rested and not try to rush through anything.

And you know, writing all of this out is at least a small comfort in the face of the fact that I can't seem to ever leave work on time. I once heard the rule of thumb that an ideal job requires about 80% of one's full intellectual capacity. To a large extent, this job fits that bill, in that I am much happier trying to juggle all the stuff above than I am when I am underworked. On the other hand, when I finally do manage to leave work, I'm tired to the point where it takes me a long time to transition into doing anything other than just sitting and drooling at the internet. Eventually I would like to get back to a point where I am making more progress on miscellaneous small projects, like the quilt sitting on the coffee table.

Ah well. Time for bed.


( 2 remarks — Remark )
Jun. 12th, 2015 03:46 am (UTC)
That really helps explain what you're doing. I remembered you talking about flight muscles, and again about long/short, and again yet about metabolism, but I didn't really have a feel for how the whole set was related.
Jun. 12th, 2015 01:59 pm (UTC)
Yeah, I think this entry helps illustrate why I've only talked about bits and pieces up until now - it's a complex project and there's a lot of background information to cover before the details start to make sense!
( 2 remarks — Remark )

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