Juggling flies, fish, and students all week long

farsidevet

Time for another reflection on my mundane week of teaching. I know this is unexciting, but I’m trying to be self-aware about what I’m doing in the class.

I’ve already summarized some of what I did this week: we explored the meaning of “epigenetics”, and I made a big push to get them to think critically about the papers we’re reading. They’re supposed to be developing a topic they’ll explore independently, so I’ve had them doing library work to find a line of research they find interesting, and master the skill of extracting the key questions the work is trying to address. I’ve got a small stack of short papers that I’m going to read this weekend and we’ll see how well they can do that.

We also discussed symbiotic interactions in development, and next week the topic is other environmental effects. They are getting much, much better at opening up and talking at the miserable hour of 8am.

The other regular highlight of my week is FlyDay, when I have to scrub dead maggots and pupae out of fly bottles. I had to postpone FlyDay this week! Yesterday I was scheduled to meet with students and parents visiting the university to confirm their plans to attend, and I was all spiffed up in a nice suit, which isn’t the best thing to wear when one is flicking bits of chitin and gooey medium around. I went in early this morning to scrub bottles and get them cooking in the autoclave.

By the way, at that student meeting I was the official biology representative, and although biology is currently the largest major on campus, almost no one stopped by to talk to me. It might have been my terrifying glare, or my sciencey reek, but no: it was because there was a separate table for the pre-professional programs (pre-med, pre-vet, pre-dental, etc.). This is a minor peeve of mine: this is not 19th century England. You do not graduate from your public school education and go straight into medical school — no, here in 21st century America you get a broad-based undergraduate education first, and then you apply to med school. You should be thinking about your liberal arts education first, and in a couple of years we’ll start coaching you on how to get into those professional programs.

Oh, well. They ignore me now, but I know that I’ll get my claws on most of them soon — they’ll want all those bio classes to prep them for the MCATs.

I should mention that I am teaching another course beyond ecological development — I’m teaching a lab course on transmission genetics. They’ve been doing crosses with flies all semester long, and we’re getting to an interesting point.

The first half semester we’re doing a mapping cross, using recombination to estimate the distances between a couple of genes on the X chromosome. We’re using flies that are mutant for eye color (white, w), wing length (miniature, m), and bristle morphology (forked, f), and I’ve also got a few groups mapping body color (yellow, y), wing veins (crossveinless, cv) and forked, f; the latter are doing a pilot test to see if I want to add that cross to our regular repertoire.

The way this works is that they are given wild type and triple mutant flies. I first have them raise a new generation of the purebred stock, simply to get a little practice in sexing flies and basic skills in growing them. So they first do these crosses:

♀w m f/w m f x ♂w m f/Y

which produces bottles full of homozygous white-eyed, miniature-winged, forked-bristled flies, and

♀w+ m+ f+/w+ m+ f+ x ♂w+ m+ f+/Y

which produces bottles full of homozygous wild type flies.

Then I have them do a reciprocal cross of flies from the two bottles. These are X-linked traits, so it matters which strain is the mother and which the father, and I want them to see that. That is, they cross wild type females to triple mutant males, like so:

♀w+ m+ f+/w+ m+ f+ x ♂w m f/Y,

which produces progeny that are all wild type, both male and female (they all inherit the dominant wild type allele at all loci from their mothers). After they’ve scored the flies from this cross, we dispose of them all and don’t think any further about them.

They also cross mutant females to wild type males, like this:

♀w m f/w m f x ♂w+ m+ f+/Y.

That has the useful result that all the sons inherit w m f from their mother and a Y chromosome from their father, so they all express the mutant phenotype. The daughters, however, are all heterozygous, inheriting the mutant alleles from their mother and a wild type chromosome from their father, so their genotype is:

♀w m f/w+ m+ f+

Now the fun begins. Meiotic recombination in those flies will rearrange the +’s and -‘s in those chromosomes with a frequency dependent on their distance from one another — you’ll get less recombination between genes that are close to one another.

This week, they completed the reciprocal cross and got their heterozygous females and mutant males. Yay! That worked. They are now setting up a test cross to assess recombination frequencies.

I just want to say that I think I planned everything perfectly. That test cross will be ready to score next week, which is the week before spring break, which means we’ll have the data for all the calculations before they leave, and when they get back, I’ll be able to lead them through all the theory. It also means I’ll be able to purge a lot of fly bottles and get them scrubbed up over the break (you can tell already that I have glamorous plans for my short vacation). Trust me, though, this is good — there have been semesters where, due to student error, the flies haven’t been ready, and then my spring break is spent maintaining 120 bottles of student flies.

It also means we can launch into the next experiment as soon as they get back: we’re going to do a complementation cross between two eye color mutants, brown eye (bw) and scarlet eye (st). If I’ve got this one all timed out correctly, we’ll be getting F2 results of crosses between heterozygotes for both loci a week before the end of classes.

Now you know. I choreograph fly sex for my convenience.

Next up, I have to choreograph my schedule. It turns out I have been summoned to Howard Hughes headquarters on 8 March and 18 April, which punch big holes in my planned lessons, and which I hadn’t accounted for in my syllabi. I’m going to have to juggle lectures and exams and rearrange the order of various things in a big way this coming week.

Historical zebrafish!

Way back in the dim, distant past, before YouTube and publicly accessible digital media, two of my friends, Don Kane, now at Western Michigan University, and Rolf Karlstrom, now at Amherst, made a video of zebrafish development. This was in 1992. It was on VHS tape. (If you don’t know what that is, ask your grandparents).

Then in 1996, a whole issue of Development was dedicated to zebrafish development and genetics, and they translated that tape into modern technology: a flip book. The top right corner of the issue featured one frame of the video, so you could flip through it and see a nice little timelapse. Like this:

Isn’t that quaint?

Sadly, I have not been able to find a copy of the flip book transported to the convenient medium of youtube (maybe I can find my copy of the file and upload it, but that thing was over 20 freaking years ago, so it may take me a while to excavate it), but at least there’s a version available via facebook, as facebook reminded me today.

I routinely make better videos than that one now, but it’s because I’ve got hi-res digital video cameras and fancy software — just remember that historical flip book was made off of VHS tape and edited by hand frame by frame. It’s really a vast improvement over the prior version, which was chiseled on slabs of sandstone and mounted in a row, so you had to run past them very fast to get the animation effect.

Also, the subject didn’t get much reward or glory, and probably ended up going down a drain in Eugene, Oregon.

What the heckity-gosh-darn is epigenetics?

comfortable

Today in my class we talked for a while about epigenetics. I used it as an example of a term we’d encountered more than once in our ecological developmental biology course, but that has some complicated ambiguity and fuzziness that has led to all kinds of weird popular confusions about the subject. I was also using it as an example of critical analysis of a paper, as I discussed yesterday, and it was a lead-up to having the students discuss papers on relevant topics they were interested in — so we spent most of our time talking about other things.

But I’m going to talk now about just this one paper I read. You see, Larry Moran and I have been having this long-running disagreement about epigenetics — nothing hostile, just an occasional cocked eyebrow in each other’s direction — which you can see on display in this article by Larry on epigenetics, in which he disagrees with my definition of epigenetics, back in 2008. Here’s my definition:

Epigenetics is the study of heritable traits that are not dependent on the primary sequence of DNA.

And here’s the definition used in Gilbert’s text:

…molecular processes around DNA that regulate genome activity that are independent DNA sequence and are mitotically stable.

And here’s Larry’s objection:

Here’s the problem. If this is epigenetics then what’s the point? When I was growing up we had a perfectly good term for these phenomena—it was regulation of gene expression. Why is there a movement among animal developmental biologists to use “epigenetics” to refer to a well-understood phenomenon?

While I agree that “epigenetics” is a huge, broad, diverse category of phenomena, I think he’s overlooking a key point to claim it is synonymous with gene regulation. It is gene regulation that is heritable and mitotically stable. It’s still far too open-ended, but it’s not just any old example of gene regulation.

It’s also clear and consistent. Larry challenges us with eight instances of regulatory phenomena and asks which ones qualify as epigenetic. Easy. 1, 2, 6, 7, 8. Those are the ones where he specifically mentions multi-generational inheritance of a regulatory state. 3, 4, and 5 describe responses within a single cell in a single generation (5 is sneaky, though: Drosophila oocytes are having gene expression modified in ways that might be transmitted through multiple generations — it’s just that those cells are being loaded with bicoid RNA, not having their bicoid genes being set to a sex-specific state).

I am also comfortable with the idea that inheritance of the regulatory state of the lac operon is an example of epigenetics. It’s arguable whether that’s a useful category, but it does fit the definition.

So one approach that could be taken is to come up with a more specific or more practical definition.

Larry has a more recent article in which he agrees with a new paper by Deans and Maggert that tries to do exactly that. It also takes a much appreciated historical approach, giving the various definitions that have been wafting about since the 1930s. For instance, here’s Waddington’s ancient physiological definition:

the branch of biology that studies the causal interactions between genes and their products which bring the phenotype into being

Yes, I agree — that would simply be gene regulation nowadays. You can’t blame us wicked developmental biologists for promoting that one, though, because we don’t use it anymore.

Now we favor the Holliday definition:

the study of changes in gene function that are mitotically and/or meiotically heritable and that do not entail change in DNA sequence.

To me, “heritable” is the magic word that makes all the difference. This, however, is not enough for Deans and Maggert. They want to add more focus, often a good thing, and narrow the definition. I was not happy with their argument, and thought it poorly made, though. See if you can find what was objectionable in this section of their paper (I highlighted it to make it easy, an epigenetic modification that does not change the sequence of the letters in the text.)

We don’t feel that it is possible to reconcile Waddington’s focus on gene regulation with Holliday’s more specific criteria within one field and still maintain the level of clarity needed to produce a useful definition. The efforts to preserve a relationship between these two conceptualizations have been impaired by the fact that there are just too many phenomena, with too few mechanistic connections, to categorize into one field. Also, among the definitions that do maintain the requirement of heritability, we feel that many lack the detail to be functionally useful in directing the testing of specific hypotheses, particularly as it relates to the location or site (cytoplasm or nucleus) of epigenetic phenomena. To mitigate these shortcomings, we advocate defining epigenetics as “the study of phenomena and mechanisms that cause chromosome-bound, heritable changes to gene expression that are not dependent on changes to DNA sequence.”

We feel that this definition makes a strong distinction between gene regulation (Waddington’s definition) and epigenetic inheritance (Holliday’s definition), and also emphasizes that epigenetic phenomena must deal exclusively with chromosome-bound changes. By making these distinctions, we have efficiently separated expressional changes caused by cytoplasmic compounds, which are more closely tied to gene regulation, from those which occur on, or in close association to, the chromosome. Doing so makes the focus of the field much clearer and identifies epigenetic mechanisms more explicitly.

We feel that this definition touches on several important elements not encompassed by other definitions, yet commonly implied in most uses. To further explain the reasoning behind our definition, as well as its utility for improving epigenetic research, we would like to offer a clarification and a test.

Yeesh. I don’t feel that your personall feelings are a strong argument, and I cringed when I hit that page. At least edit it to remove the emphasis on your personal discomfort; just say that the old definitions lack detail, rather than that you feel they lack detail.

So let’s pull out their shiny new definition.

the study of phenomena and mechanisms that cause chromosome-bound, heritable changes to gene expression that are not dependent on changes to DNA sequence

Well. All this fuss for a single change, the addition of the phrase chromosome-bound. That’s it. I agree, it does narrow the topic, but it’s still covering an awful lot of territory. I’m not feelin’ it. I have the impression that the primary virtue of the new definition is that it reduces a class of phenomena to a subset that many people are comfortable studying already, and in part reinforces a gene-centered perspective on cellular behavior.

It also leaves me wondering…what about the inheritance of cytoplasmic or membrane-bound factors that induce consistent changes in gene expression in daughter cells? The gene regulation aspect may be mundane, but it’s the inheritance that is interesting. Under the Deans and Maggert definition, this is no longer under the umbrella of epigenetics — it’s something different for which we have no general name now.

It makes Larry happier, though.

I think this is a useful definition. Nobody cares if dividing E. coli cells inherit molecules of lac repressor and continue to repress the lac operon. That’s a trivial form of epigenetics that never posed a threat to our understanding of evolution.

That’s odd. I do care that the lac repressor is cytoplasmically inherited, but then my primary interests, in the most general form, would be in the patterns of stability and change in cellular properties, rather than the metabolism of sugar. Telling me that I should only pay attention to inherited proteins or methylation states that are directly bound to DNA seems arbitrary.

I also don’t consider “poses a threat to our understanding of evolution” to be a relevant criterion. I agree that lac repressors don’t challenge evolutionary theory, but neither do heritable histone modifications or methylation. I’m one of those people who think epigenetics (even under the old definition!) is important and interesting, but doesn’t affect evolutionary theory much at all.

Larry and I agree.

Methylation is trivial.

Well then, if inheritance of the lac operon is such a trivial form of epigenetics that it should be excluded from the definition, then we apparently need yet another definition that excludes the triviality of methylation.

Or, really, we should recognize that “trivial” is not a good reason to exclude something.

I will still second Larry’s argument that none of this stuff overthrows modern evolutionary theory in any way. It would require extremely persistent inheritance of an epigenetic state over many generations to have those kinds of repercussions.

(The Gilbert text does mention one significant effect: the toadflax plant, Linaria vulgaris, has a radically different flower morph, Peloria, that Linnaeus himself classified as a different species. As it turns out, they only differ in the methylation state of the cycloidea gene, but the DNA sequence is identical. This is a case of an epigenetic change persisting for hundreds of generations. It’s a rare case, though, and also…would still definitely fall under the Deans and Maggert definition.)

Ecological Development: Getting critical

far_side_good_hands

In class last week, we continued our discussion of developmental plasticity and began to talk about epigenetics, and in particular, the underlying molecular mechanisms for epigenetic inheritance. In addition, students had to discuss papers on plasticity that they’d researched. Some of the topics covered were: sneaker males and alternative reproductive strategies; aggression in dog breeds, how much is genetic and how much is training; temperature-dependent and behavior-dependent sex determination in reptiles and fish; and physiological responses to variations in gravity (someone has put pregnant rats in a centrifuge and looked at the effects of 2 gravities on development). It was all fun stuff, and I made the students do all the work. Perfect!

This week they have another assignment. There’s a common problem in student writing about science: they tend to describe what a paper says. That makes for very boring reading, I’m sorry to say — if I just wanted to know what was in the paper, I could read it myself, after all. So this week they’ve been asked to write a critical analysis of a science paper relevant to the course. This is a routine skill that needs to be cultivated and practiced.

What’s involved? You first have to identify a key question or assertion in the paper, or even in a short section of the paper, and ask yourself if the authors have adequately defended the claim. Even if you agree with the claim, and think it’s eminently reasonable, you have to approach it as a critic and try to tear it down.

I’m going to try to lead by example, so I have given them a couple of papers to read ahead of time. One is “Novelty and Innovation in the History of Life” by Douglas Erwin, which makes an argument that should be familiar to the students, because we’ve already talked about some of the concepts. Here’s the abstract:

The history of life as documented by the fossil record encompasses evolutionary diversifications at scales ranging from the Ediacaran–Cambrian explosion of animal life and the invasion of land by vascular plants, insects and vertebrates to the diversification of flowering plants over the past 100 million years and the radiation of horses. Morphological novelty and innovation has been a recurrent theme. The architects of the modern synthesis of evolutionary theory made three claims about evolutionary novelty and innovation: first, that all diversifications in the history of life represent adaptive radiations; second, that adaptive radiations are driven principally by ecological opportunity rather than by the supply of new morphological novelties, thus the primary questions about novelty and innovation focus on their ecological and evolutionary success; and third, that the rate of morphological divergence between taxa was more rapid early in the history of a clade but slowed over time as ecological opportunities declined. These claims have strongly influenced subsequent generations of evolutionary biologists, yet over the past two decades each has been challenged by data from the fossil record, by the results of comparative phylogenetic analyses and through insights from evolutionary developmental biology. Consequently a broader view of novelty and innovation is required. An outstanding issue for future work is identifying the circumstances associated with different styles of diversification and whether their frequency has changed through the history of life.

Let’s take that apart. Erwin is saying that there are some long-held assumptions in evolutionary biology that he is going to suggest are possibly invalid. Those assumptions are:

  1. Diversity is the product of adaptive radiations;

  2. Radiations are driven by ecological opportunities; and

  3. Most morphological variants emerge early, in the process of filling open niches.

He’s going to propose alternative processes.

  1. Initially non-adaptive variants are going to generate morphological diversity;

  2. Novel forms construct the niches that they will fill; and

  3. Variation is a constant event in a lineage.

I am predisposed to like those new perspectives, and I’m also biased by the evidence we’ve discussed in class, that ecology and development are in a constant state of reciprocal feedback. But rather than reporting and describing this paper as something that reinforces my views, I need to examine it critically. Does Erwin adequately support his claims? Are there significant questions he does not address? He’s also given us a list of sources of evidence that he’ll use to challenge orthodoxy: “the fossil record, by the results of comparative phylogenetic analyses and through insights from evolutionary developmental biology”. Does he succeed?

I’m just going to consider his first point, whether it is adaptive variation that drives radiations (lesson for students: focus. Better to do one thing well than 3 things poorly). His evidence in this section comes primarily from analysis of the fossil record, which is going to raise some objections.

Erwin does not deny the existence of adaptive radiations, and wisely begins by discussing known examples. He cites the work on Galapagos finches, where we have strong evidence of morphology being shaped by adaptive necessity. He also discusses cichlids, where variations in the environment have clearly played a role in, for instance, feeding adaptations. To then argue for alternative mechanisms using the fossil record is problematic: adaptive radiations are seen in cases where you’ve got close-up, fine-grained observations of single clades, but the evidence for adaptation fades when you use a more coarse-grained, less well-sampled method?

One piece of evidence presented is basically an absence-of-evidence argument. There is a lack of evidence of character displacement in the fossil record. Character displacement is the shift in morphology away from each other from two similar species competing in an overlapping range; it ought to be seen if two populations are adapting to avoid competition.

His argument that solutions to adaptive problems can exist for milllions of years without a radiation occurring is more interesting. He points to Anolis lizards in the Caribbean that converge on similar strategies when they evolve on different islands as an indication that the potential for particular morphologies is present in the species before they find themself with fresh opportunities on a new island. The carnivore fossil record shows a limited repertoire of optimal feeding strategies, which canids exploited repeatedly. Sea urchins have been evolving to follow similar feeding patterns repeatedly, as well.

It’s a somewhat frustrating argument, though. He’s trying to show that a radiation can’t have been driven by the acquisition of an adaptation if the adaptation had existed for long periods previously without a radiation. I can see the point, but one could argue that the radiation depended on both the prior potential in the organism and ecological circumstance, which is part of his second point…which makes point #1 and point #2 codependent on one another.

I’d have to say that I wasn’t entirely satisfied that he’d supported his first conclusion to my satisfaction. It’s also the case that he’s arguing that both adaptive and non-adaptive radiations occur, meaning it’s a quantitative question of which of the two is most important under what conditions, and he hasn’t done anything to measure that balance. I don’t reject the hypothesis, but I also don’t think the work has been done to confirm it — yet. He concludes the whole paper by predicting that gene regulatory networks are characterized by stability, so morphological novelties may be based on features established outside the core GRNs, and are thus more flexible. I don’t know. That’s definitely well outside anything you could figure out with fossils, so it’s going to require a different approach.

That’s how I’m going to talk about a paper I enjoyed with my students (and you Pharyngula readers think I’m harsh with my mere movie reviews). I’m also going to discuss a second paper on epigenetics that I didn’t care much for — the heart of the paper is a painful exercise in writing about how they feel about certain definitions of epigenetics which made me snarl — but I still think it made some valid points.

That’s really the purpose of the whole exercise. Stop treating science papers as holy writ that you can’t challenge; think critically about everything, and try to find logical holes that can be plugged with better evidence. That’s how science gets better and better. These are smart students and they just need to learn that they can actually disagree with Famous Scientists.

The real challenge, too, is that my plan is to talk about these examples for maybe 15 minutes, and then put the students into groups to discuss the papers they’ll have brought with them, trying to punch holes in them. It might be fun. It might be difficult and frustrating. As mentioned, one of the annoyances of student writing is that too often they think of it as reporting, describing what’s in it rather than engaging with the ideas with their very own brain and questioning what the paper says.

Hey, maybe I shouldn’t call it “reporting” since that’s also what journalists should be doing, but too often aren’t. Reciting summaries credulously shouldn’t be what either scientists or journalists do.


Erwin DH (2015) Novelty and Innovation in the History of Life. Curr Biol 5;25(19):R930-40.

The resurrection of the mammoth will not occur in two years

Not even in the next decade. For a good debunking of the claim of cloning a mammoth that’s in all the news, John Hawks has you covered.

When I heard the story came from George Church, I admit that I rolled my eyes and moved on. Church is a very smart guy, but he also tends to start babbling far out science fiction when he’s got an audience. As Hawks points out, he’s made 45 edits to elephant cells in a dish; that’s an awful long way from the thousands he’d need to begin to re-engineer an extinct animal, and a single cell is even further from a healthy, functioning large mammal.

Someone needs to start a Journal of Pizza Quality Research, stat

We need somewhere to bury sloppy research on fast food, after all. Brian Wansink gets interviewed on Retraction Watch (y’all remember Wansink, the fellow who ground his data exceedingly fine to extract four papers from a null result), and he does himself no favors.

Well, we weren’t testing a registered hypothesis, so there’d be no way for us to try to massage the data to meet it. From what I understand, that’s one definition of p-hacking. Originally, we were testing a hypothesis – we thought the more expensive the pizza, the more you’d eat. And that was a null result.

But we set up this two-month study so that we could look at a whole bunch of totally unanswered empirical questions that we thought would be interesting for people who like to eat in restaurants. For example, if you’re eating a meal, what part influences how much like the meal? The first part, the middle part, or the last part? We had no prior hypothesis to think anything would predominate. We didn’t know anybody who had looked at this in a restaurant, so it was a totally empirical question. We asked people to rate the first, middle, and last piece of pizza – for those who ate 3 or more pieces – and asked them to rate and the quality of the entire meal. We plotted out the data to find out which piece was most linked to the rating of the overall meal, and saw ‘Oh, it looks like this happens.’ It was total empiricism. This is why we state the purpose of these papers is ‘to explore the answer to x.’ It’s not like testing Prospect Theory or a cognitive dissonance hypothesis. There’s no theoretical precedent, like the Journal of Pizza Quality Research. Not yet.

That last bit sounds like a threat.

Here’s the thing: we all do what he describes. An experiment failed (yes, it’s happened to me a lot). OK, let’s look at the data we’ve got very carefully and see if there’s anything potentially interesting in it, any ideas that might be extractable. The results are a set of observations, after all, and we should use them to try and figure out what’s going on, and in a perfect world, there’d be public place to store negative results so they aren’t just buried in a file drawer somewhere. There’s nothing wrong with analyzing your data out the wazoo.

The problem is that he then published it all under the guise of papers testing different hypotheses. Most of us don’t do that at all. We see a hint of something interesting buried in the data for a null result, and we say, “Hmm, let’s do an experiment to test this hypothesis”, or “Maybe I should include this suggestive bit of information in a grant proposal to test this hypothesis.” Just churning out low-quality papers to plump up the CV is why I said this is a systemic problem in science — we reward volume rather than quality. It doesn’t make scientists particularly happy to be drowning in drivel, but Elsevier is probably drooling at the idea of a Journal of Pizza Quality Research — another crap specialized journal that earns them an unwarranted amount of money and provides another dumping ground for said drivel being spewed out.

Wansink seems to be dimly aware of this situation.

These sorts of studies are either first steps, or sometimes they’re real-world demonstrations of existing lab findings. They aren’t intended to be the first and last word about a social science issue. Social science isn’t definitive like chemistry. Like Jim Morrison said, “People are strange.” In a good way.

Yes. First steps. Maybe you shouldn’t publish first steps. Maybe you should hold off until you’re a little more certain you’re on solid ground.

No one expects social science to be just like chemistry, but this idea that you don’t need robust observations with solid methodology might be one reason there is a replicability crisis. Rather than repeating and engaging in some healthy self-criticism of your results, you’re haring off to publish the first thing that breaches an arbitrary p-value criterion.

There really are significant problems with the data he did publish, too. Take a look at this criticism of one of his papers. The numbers don’t add up. The stats don’t make sense. His tables don’t even seem to be appropriately labeled. You could not replicate the experiment from the report he published. This stuff is incredibly sloppy, and he doesn’t address their failings in the interview, except inadequately and in ways that don’t solve the problems with the work.

Again, I’m trying to be generous in interpreting the purpose of this research — often, interdisciplinary criticism can completely miss the point of the work (see also how physicists sometimes fail to comprehend biology, and inappropriately apply expectations from one field to another) — but I’m also seeing a lack of explanation of the context and relevance of the work. I mean, when he says, “For example, if you’re eating a meal, what part influences how much like the meal? The first part, the middle part, or the last part?”, I’m just wondering why. Why would it matter, what are all the variables here (not just the food, but in the consumer), and what do you learn from the fact that Subject X liked dessert, but not the appetizer?

It sounds like something a restaraunteur or a food chain might want to know, or that might might appeal to an audience at a daytime talk show, but otherwise, I’m not seeing the goal…or how their methods can possibly sort out the multitude of variables that have to be present in this research.