We keep a varying amount of copepod species in cultures in our lab throughout the year, which we feed a mixture of the three algae species Isochrysis galbana, Dunaliella tertiolecta, and Rhodomonas salina. We may also use them as food in some of our experiments.
To know more about which algae adults prefer to eat, and which algae concentrations represent high and low levels of food, we have conducted a series of experiments to investigate what is known as functional response; that is, the foraging responses to increasing food concentrations. This knowledge will be of particular use for experiments later this year, were we plan to test effects of predation risk and copper exposure on copepod foraging and reproduction. Each of these experiments takes about three days to set up, conduct and take down.
On the first day, we need to sterile-filter about 5 L of seawater in order to eliminate contents in the water that might otherwise confound our data. This is for example organic particles from dead algae, or even tiny bacteria. We also have to pick and set aside about 100 female copepods to ensure that we have enough non-ovigerous females for the incubation; that is, females without egg sac.
On the second day, we initiate the experiment. We first have to measure the cell concentration in our algae stock solution. For this, we use a dreadful machine named CASY to count the number of cells in 10 mL samples of the stock. CASY gives good results but requires quite a lot of patience due to high error message rate. Knowing the algae stock cell concentration, we then prepare the food dilution series in which we incubate our copepods.
To make things extra interesting, we incubate our copepods individually instead of in groups, as is more common for small zooplankton species. Upon incubation, we place the well plates with the copepods on an automated imaging system (aka “The Robot”) that Jan developed in our previous project (LUMS), and leave them there for 24 hours to feast on the algae.
On the third day, we stop and take down the experiment. We first check the survival and egg sac status for all the incubated individuals. Next, we use three different methods to assess how much the copepods ate. We use the CASY to count cell numbers before and following incubation, and by comparing these, we can assess algae growth and consumption by copepods over the incubation period. We also use a plate reader to measure fluorescence in our replicates, prior to and following incubation. This gives us a relative number for chlorophyll in our sample that relates to the amount of living algae. By analyzing carbon content in all our food concentrations, we can back-calculate the fluorescence readings to carbon equivalents. Finally, we use the images from The Robot to count and measure the length of copepod faecal pellets; that is, to assess how much they poop.
We hope to get a good overview of the copepods feeding preferences and functional responses for these algae species by combining these three methods. This setup is much the same as we use in other experiments. The responses measured here for food type and concentration will thus be representable for many of our experiments in which we expose the copepods to natural and anthropogenic stressors.
It will be exciting to analyze these data and see what comes out of it!