What experimental artifacts are you ignoring?


Collectively, as a scientific community, we have so many blind spots. I remember running into one of these blind spots about 15 years ago.

As a junior faculty member, I was joining a collaboration with a few other faculty in my department, to develop a full-factorial manipulative lab experiment to understand how environmental conditions affect animal decisions to behave more socially. Our C. elegans guy explained to us how these critters perform “social feeding” under certain conditions. Because C. elegans is a worm that lots of people raise in the lab, fitting large numbers of animals in a single petri dish, we thought this model system for development, neurobiology, cell biology, and such could be used to ask some questions about the fundamentals of animal behavior and the formation of social groups.

So, we wrote a grant. We designed a set of experiments to create groups of these worms, and place them in a variety of environmental conditions to test some great questions about the emergence and function of social behavior. At the time, I thought it was a cool use of a model system to ask questions in a field that doesn’t typically use that model system. Because everybody in C. elegans-land apparently knew that these worms behave socially but didn’t have a grand understanding about how this was shaped by the environment, other than as a response to food availability.

As we were finishing the grant, a metaphorical bomb dropped. (I honestly forget if it was before we submitted or after it went to review. This was 15 years ago, it’s all kind of blurry. Actually, come to think of it, I was taking care of a baby at the time. The lack of sleep probably has something to do with the fuzzy memory.) This was the bomb: A new paper came out and demonstrated that the “social feeding behavior” was essentially a physiological response to oxygen stress.

This worm simply wouldn’t perform its “social behavior” unless it was subjected to oxygen levels much higher than it would typically experienced in the wild. You see, before these worms became a model system for the lab, they were just known as worms that hang out in the soil. It turns out the soil they hang out in typically has much higher CO2 levels and much lower O2 levels than the air that you and I breathe. And they evolved to be used to it. So much that, it appears, ambient oxygen levels are higher than they prefer, and this “social behavior” is essentially a stress response. Which pretty much invalidated our set of experiments as planned.

While this was not a good day for me, can you imagine how all of the worm people must have felt?! Of course, this means that now all C. elegans labs are keeping their worms in incubators at the worms’ preferred O2 level (5-10% O2, instead of the ambient 21%). Right? Nope.

As far as I’m aware, the modus operandi for maintaining worms in typical C. elegans labs is to keep them at ambient oxygen levels, unless there’s a big reason for doing otherwise.

Which means that nearly every paper coming out about C. elegans is being done when the animal is experiencing hyperoxic stress. Are these experiments always designed with this in mind? Are they interpreted in this light? How many findings are less useful because of this experimental artifact?

I hadn’t thought about this worm oxygen thing for years. What reminded me? This:

I’m not a cell biologist and I’d be unwise to talk trash about research using cell cultures. But in general, clearly, it is reasonable to surmise that there are are a metric crap ton of experimental artifacts from growing cells in a lab, eh? And it must be super duper important to make sure that what we conclude from cell cultures also makes sense in vivo, right?

In my realm, this problem might be manifesting itself when folks study ant colonies in the lab. Some ant colonies, like groups of C. elegans worms, are kept in the lab in petri dishes. Bigger colonies of ants and colonies of bigger ants are typically kept in plastic tubs, with a few test tubes or vials for moisture and nesting. They might be in a light-controlled environment, but probably not. The spatial structure and physical properties of nests in the lab are radically different than in the field. Like in the C. elegans situation, might we expect ambient gradients of carbon dioxide must influence how ants use the nest and behave?

The more we learn about how ant colonies work collectively to perform tasks as a single unit, the more we realize that this is about the interactions between individuals and how they share information with one another (or don’t share information with one another if not interacting with one another). These relationships are heavily structured by the size and shape of the nest. So, then, what are the limits of applying what we know about lab colonies when trying to understand what happens outside the lab?

It turns out that some ant people are really good about thinking about these things.  It turns out, surprisingly enough, that carbon dioxide levels don’t influence nest use or construction. In one species at least. And while it is really hard to look at ants that are nesting underground, some folks who work in lab model systems have gone to substantial efforts to make sure that lab phenomena are recapitulated in the field, or go to the field to make sense of what happens in the lab.

Nevertheless, there are lots of scientists who are essentially in denial of the artifacts of laboratory manipulations, who don’t think much about them and don’t design experiments to take them into account as much as is needed.

In your field, what are the artifacts of manipulation and sampling that people are overlooking?

2 thoughts on “What experimental artifacts are you ignoring?

  1. One that immediately comes to mind in my field is the use of single visit deposition (SVD) to test pollinator effectiveness. Virgin flowers are exposed to a single visit by a pollinator then bagged to wait for fruit set or the stigma harvested to count pollen. We’ve all done it but that’s not what pollinators do: they visit flowers multiple times, sometimes dislodging previous pollen, etc. Also it can take more than the pollen from a single visit to initiate fruit set. Lots of issues with this approach.

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