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The Slime Mold Chronicles: Cracking the (Intelligent?) Behavior of the Brainless

y maze for slime molds
Our Y-maze with food on one side and nothing on the other. Guess where they went!

— Written by Amanda Putti & Milica Milosevic —

Well we made it! We’re at the final week of the BYB Fellowship! We faced many challenges throughout this project and had to pivot in order to get results, but we are happy where it ended.

To give updates on our progress, let’s first start where we left off 3 weeks ago. Using a blue light, we took one picture of the slime mold every minute over 24 hours. This allowed us to string the pictures together and make some great time lapse videos! After this, we were able to analyze the videos using a program in Python and create a special kind of video which converts yellow slime branches into lines of directed growth – skeletonized growth video. This was really helpful for understanding how the slime mold grows, explores new areas, and creates a network. But we also think that there’s more to it, and that behavioral analysis opens a lot of questions in the field of biophysics and is a project for itself.

We were very excited to get this imaging. However, we then made the decision to keep the rest of our experiments in the dark and in that manner reduce the amount of imaging done. Slime mold prefers to grow in the dark, and we wanted to make sure light wasn’t inhibiting growth in our experiments.

This means we were more focused on quantifying decision making rather than behavior. We set up a series of three types of experiments and ran many, many tests. (The order of explaining them isn’t coordinated with the chronological order of our work, but makes more sense this way!)

1. Solving Mazes

We wanted to test the ability of slime molds to choose a path that leads them to food, so we set up the easiest of mazes – a Y maze where there was food on one side of the Y and nothing on the other side of the Y (see photo above). Slime molds showed us that they have no intentions of staying hungry and that they’re doing just fine when it comes to finding the food source.

Then we wanted to make things more complicated for them, so we constructed a specific T maze – one side of the letter T was longer and had a food source, and the other was a lot shorter and had an object as a mechanical stimulus (we’ll get more into the mechanical stimulation in a bit). The idea was to check if they can see the difference between the food and something that isn’t food and if they are gonna choose the shorter path towards the no-food region. So, we tried to confuse them, but failed at it –  they knew where the food was and grew in that direction almost every time!

2. LED Light Experiment

This experiment was a negative stimulation test because we know that slime molds usually live and grow in the dark. We placed an LED light on one side of the petri dish in a dark box, and placed two food sources opposite from one another, but not opposite from the light source (see the picture, it’s a little confusing :)).

Then we tracked Slime Mold movement after a 24h growth. We ran 6 trials and in most of them, slime mold moved away from the light, completely ignoring the food! This means that growing in the dark means more to them than eating and also showed us that stopping the imaging experiments so we can keep them in the dark was a good decision! You can see our results in the pie chart below.

3. 3 vs. 1 Mechanical Stimulus Test

Okay, so we now know that slime mold likes the food AND knows how to recognize it AND doesn’t like the light AND knows to get away from it. I think it’s safe to say that slime molds make decisions that are crucial for their survival on a daily basis.

But what intrigued us in the first place and what we talked about in our first blog post was their answer to mechanical stimulation. We decided to test whether it would move toward a non-nutritive mechanical stimulus and also made some changes to the initial setup. Instead of the 3D blocks, we started using pieces of cotton gauze from the band-aid. The reason for that was the effect they have on agar – they are very light in weight and they could wick the agar by absorbing it.

Our hypothesis was that slime molds could feel that type of stimulus and grow towards it. We placed 3 cotton squares on 1 side of the agar dish and 1 cotton square on the opposite side, with slime mold being in the middle. We would leave the molds to grow overnight (or for a few days, depending on how stubborn that particular mold was) and then collect the data. The four outcomes could be observed: growth towards 1 side, towards 3 side, towards both or no growth. You can see our results in the charts below! 

So, all of this brings us to the final question – is it possible that an aneural (brainless) organism, or more precisely, a single cell shows complex intelligent behavior, such as decision making? Our results lead us to believe that it is – the percentage of the dishes in which slime mold chose the 3 side over 1 is significant! However, in the last month, slime molds showed us that there is still a lot we don’t understand about them and their complex behavior of decision making. 

Both of us believe that these experiments deserve the chance to be done in a lab, with more controlled conditions and the opportunity to test different things as well!

As we end this blog post and sign off for the summer, we want to add that we had a great time during this summer fellowship, learned a lot about working with living organisms, trying, failing and then trying again.

 Until next time!

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