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28 January, 1997

Subject: Class questions

For todays journal I want to share with you some of the questions sent by studnets working with me on the experimental design for the rotifer experiments

Student question: on whether the small rotifers are the same species: isolate the small rotifers and see if you can raise them to a larger size, then compare them to the large ones.

Reply: How long should I wait? Is there a way you would recommend a long range experiment - like maybe over winter?

The parthenogenic rotifers do have genetic variation due to random mutations.

Reply: Quite true - good!

One student believes that the best set up would be to run trials on a single rotifer at a time, then repeat it hundreds of times to get some representative results.

This would be better than putting dozens of rotifers in together because they might change their feeding by interacting with each other.

Reply: They live in dense populations of about 100 per square centimeter. Will putting them on a slide or chamber as a single influence water currents or populations of food?

Student suggestion: You should use filtered lake water. Everyone intuitively believes that therotifers would prefer to eat bacteria or "natural" food rather than microbeads.

Reply: I have tried that which you suggest. Thanks. There does seem to be a higher feeding rate on beads when there is no other food available. Still, when there are several rotifers in a chamber at once, there is no pattern of feeding. Results range from 0 to 34 in a given period of time for rotifers in the same chamber. Also I have to count the beads in live rotifers rather than preserving them because they ball up into a tight little wad when preservative is added.

Student: Otherwise the rotifers will spit out the microbeads and eat bacteria or smallprotists instead. There seems to be some feeling that rotifers might "fill up"on non-bead food and this would cause them to slow down their filtering rate.

Reply: How can I tell what the retention time is for the beads in the rotifer gut? Does this maybe explain why some rotifer bodies are glowing green with fluorescence but have no beads inside of them?

Several students wanted to know if ingesting beads would stuff the rotifers and eventually kill them or if they could pass them through. If they don't clogup, but are living in otherwise empty lake water, won't they starve and die,would their filtering rate vary as they beging to starve?

Reply: I think the beads must just pass through the rotifers. When undisturbed, the rotifers seem to have their filtering mechanisms going full time. When watching undisturbed under the scope for a while, you can get some sense of the regularity with which they feed.

This discussion raised the specter that the rotifers might be able to taste the beads and reject them as non-food items. Are rotifers selective feeders. It is clear that in the Biology class, Paramecium were selective ( in fact we had to cook the dyed yeast to get them to eat them).

Reply: I agree with you. Some of the rotifers appeared to reject the beads once taken into the first chamber of their anatomy. That is why I decided to try the same experiment with phytoplankton that has been fluorescently labeled. They seem to eat those FLOs at the same rate as the FLMs.

One student wonders if the rotifer's exposure to microbeadsis representative of the concentration of beads in the water, or if the beads mightn't be "clumped up" in the water. We agreed as a class that the beads probably will be unevenly distributed in the water and either the number of rotifers, number of beads or time of filtering shiould be sufficiently long to average aout irregularities caused by randon distribution of parcticles or adhesion of parcticles to each other.

Reply: Good question. I have considered this and use the same concentration of beads or FLOs as is naturally found in their lake water. The exact concentration is stated in an earlier journal entry. I also sonicate the mixture of FLMs and FLOs to make sure they are not clumped up before I micropipette then into the rotifer chamber.

A student: wonders if it is possible to knock the rotifers out instead of killing them to subsequently count the beads.

Reply: One can count the beads while the rotifers are alive. However, once removed from the lake, they can not be returned. They become part of the collected "grey water"!

She also wants to know if one of the two rotifer species came into existence before the other and, assuming that such a scenario is true, whether the later evolved rotifer would necessarily be better adapted than the other one.

Reply: I don't know. At the moment, I suspect that they might be the same species because I have seen so many rotifers with live babies inside. I have not seen any being born, but the babies are much smaller that their Mom and do not have as much of that bright orange pigment at their Mom either. It would be interesting to do some comparative studies of the two types and see if they really are different or the same in their live styles. Others here think that they are different species because we see no intermediate sizes as one would expect if the smaller ones are growing. I have not been here long enough to find out.

No one in this AP class came up with the idea that you should use a concentration of beads and rotifers that mirrors natural concentration, but instead focused on the practical: as Ann wrote, "You should isolate one rotifer in a small volume of water with a COUNTABLE number of beads.

Reply: The beads are definately countable. I have tried two ways of counting. First I simply took some sample with feeding rotifers and counted the beads in the gut. One can also count the remaining beads on the slide. The numbers are quite reasonable. But, it is difficult to make sure you have counted all the beads in the gut and not those clinging to the outside of the rotifer bodies.

Ann distingishes between this technique to find rate of feeding from a separate method to determine how well rotifers filter water. For that she suggestsputting a countable number of rotifers into a large volume of water (~10liters!) with a known concentration of beads, then subsequently sample thewater daily and measure the concentration of beads remaining.

Reply: This is a good idea! Can I be sure that once the beads are taken in that they will stay there? Maybe so if there are no glowing rotifers. This suggestion generated comments that it would not work if the rotifers passed undigested beads through their guts in less time than you ran the experiment.

There was some concern that parcticle removal might not be a true proxy for filtering rate. Several students suggest devising a system to "tag" the water so it would appear different once it had passed through a rotifer. However, none of us could think of a way to do this.

Reply: This sounds like a good idea. I wil;l discuss it with the scientists here and let you know what they thing.

Students: The ambiguity of whether the two types of rotifers were part of a single population raised the specter that you will have to adjust grazing rates oflarge adult rotifers by the size distribution of rotifers in the population. A census of rotifers, and a size distribution would be needed as well as a measure for relating filtering rate to size.

Reply: Thanks! These are great questions and your help is appreciated. Have you found any rotifers there yet?

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