Our experiments revolve rather heavily around krill poo. You might be wondering why. And if you’re not, then hopefully reading this will make you want to know more about krill poo in the future.
So, why do we care so much about krill poo and why do we spend days carefully siphoning it out of 20-L buckets? Well, it all stems back to measuring growth. We have hypothesized that the different diets we are feeding our krill (i.e. plant food versus animal food) will affect krill growth in different ways. We expect that krill fed diatoms (single-celled microscopic plants) will grow more than those fed a mix of freeze-dried powdered zooplankton (other small drifting animals that live in the ocean). The reason we think this might happen has to do with the chemical make-up of diatoms versus that of zooplankton, and how this transfers to the krill.
You see, diatoms provide the krill with a type of fat called a polyunsaturated fatty acid (PUFA for short) and other zooplankton (specifically copepods) provide them with a different type of fat called a triacylglycerol (or TAG for short). When krill eat PUFAs they use this energy for growth. But when they eat TAGs, all of that energy gets stored as fat deposits. So, it makes sense that if a krill were to eat more diatoms, it might grow more than another krill eating zooplankton.
Okay, so how exactly does this lead to poop?
Well, we want to measure the growth of the krill in our feeding experiments throughout the winter. We don’t really expect that they’re growing a huge amount (at least not compared to summer time), but we hope to see some small difference between the krill fed diatoms and those fed zooplankton. Measuring growth in krill directly is a little bit tricky because it requires several hundred krill per experiment to get good results. We don’t have enough krill to do that as many times as we need to. But you can also estimate growth indirectly because it is part of an organisms energy budget.
If we think of the food we (or krill) eat as energy, then that energy that comes in must be balanced with the energy that goes out (as poop) or is used up by the body. Growth takes energy, so does metabolism - these are the parts that are used up by the body. Now, if we know how much energy is eaten (ingestion), how much is pooped out (egestion), and how much is used in metabolism (respiration), then the only remaining part of the energy budget the energy that is used for growth. Here is the simple equation:
Ingestion = Growth + Respiration + Egestion
Since we want to calculate growth, we can rearrange this equation as:
Growth = Ingestion – Respiration – Egestion
Every four weeks we run a bunch of different experiments to measure ingestion, respiration and egestion. Once we know these things, we can calculate growth. We combine the ingestion and egestion experiments into one, which is super handy, and I’ll tell you more about that here (Julia will fill you in on the respiration experiments at a later date). To start, we fill 20-L buckets with natural seawater and then add the food (either diatoms or freeze-dried zooplankton). Next, we put 10 krill into each bucket and leave them in an Environmental Room (a room where you can set the conditions, so they don’t change – ours is set to a temperature of -1˚C and there is constant darkness) for nearly 24 hours. We also have two 20-L buckets with natural seawater and food added but no krill – these are our experimental controls and allow us to estimate how much food the krill have eaten.
At the end of the experiment, we remove the krill from their 20-L buckets and measure their lengths and weights. Next, we take samples of seawater from each bucket (including those buckets that never had krill in them, the controls) so that we can measure the amount of food left over in each bucket. We know how much food each bucket should have had if there were no krill (we get this from the amount of food in the control buckets) and we know how much food is actually left over in each bucket after the krill have been eating it (we get this from the food in the krill buckets). The difference between the amount of food in the krill buckets and the amount of food in the control buckets tells us how much food the krill ate. It’s as easy as that.
Once we’ve taken out the samples of seawater for food measurements, we carefully siphon off the remaining water in each bucket so that we can collect all the krill poop lying on the bottom. This takes a little while because we don’t want to disturb the poop and have it sucked down the siphon. Once we get the water level in the buckets down as low as it can go without sucking up the poop, we pour the contents of each bucket into smaller beakers and do another round of siphoning. Eventually, we end up with a tiny amount of seawater and a lot of poop, which we pour into a glass bowl and examine under the microscope. We do this to ensure that there are no non-poop items in our poop sample. If we see any, we pick them out with a pair of forceps (the scientific name for tweezers).
From the glass bowl, we filter the contents onto a small glass-fiber filter (we use 25 mm diameter GF/F filters – Kirsten talked about them in the last blog), dry them in the oven at 60˚C overnight and then weigh them. Then the filters with the poop get placed into glass vials and stored in the -80˚C freezer for further analysis of their carbon content back in the States. We do this experiment multiple times to get enough replicates, this is why it takes us all week.