BIO208/Rehnberg BOA Reproduction Lab Regeneration in the Segmented Worm Lumbriculus variegatus When most animals reproduce themselves, they usually adopt some form of sexual reproduction. This common mode of reproduction involves a male and a female and a lot of relevant anatomy and behavior that encourages the fusion of eggs and sperm. It appears very complicated and it often seems amazing that it works at all! Not all animals rely solely on sexual reproduction. Instead, they may reproduce sexually when the conditions are right, but at other times they may use an asexual process. What is asexual reproduction? For one thing, it s a type of reproduction that an individual can do by itself. There are different ways of doing it, but they all end up producing clones of the parent individual. In other words, the offspring are genetically identical to the parent. Asexual reproduction is found in certain invertebrate groups such as the Phylum Cnidaria (corals, anemones, jellyfish), Phylum Annelida (segmented worms), and Phylum Echinodermata (starfish). It is generally not used by vertebrate animals. Some animals can and do reproduce sexually and asexually (not at the same time). This is seemingly a very sound capability (why?). An animal known to use both modes of reproduction is the mudworm, Lumbriculus variegatus. The mudworm, like earthworms, is a segmented worm in the Phylum Annelida. Background on Mudworms Mudworms are aquatic animals, inhabiting the edges of ponds and lakes throughout North America. It prefers to burrow head down into mud that is rich in decaying vegetation. It feeds on the vegetation and all of the microorganisms attached to it. The tail end of the worm often projects up from the mud into the overlying water. Mudworms have no lungs, but oxygen is able to diffuse from the water directly into blood vessels in the tail. Mudworms are bilaterally symmetrical. The head end can be identified because it has darker pigmentation than the tail and is much more active in exploring its environment. Under some magnification you will clearly see a major blood vessel running the length of the worm and blood moving along in pulses. Look for blood cells. This large blood vessel can be considered the worm s heart since vessel contractions push blood in the tail-to-head direction. Mudworms have a well-developed nervous system consisting of an anterior brain, a large nerve cord extending from the brain towards the tail, and lots of nerves branching off of the nerve cord (draw parallels to the nervous systems of vertebrates). Reproduction in Mudworms Mudworms can reproduce sexually, although that process is thought to be relatively rare. Instead, the preferred mode of reproduction is a surprising process called fragmentation in which
the worm s body spontaneously breaks into two or more pieces!! Fortunately for the worm, the fragments have a wonderful capacity to regenerate the missing region thereby becoming whole again. While it s tempting to view this as a simple and primitive form of reproduction, there is nothing simple about a tail fragment somehow developing a new brain. That is a process that no vertebrate animal is capable of doing. The illustrations below show the process of regeneration for a 5-segment piece of worm taken from its posterior end. Note that the rebuilding of a whole, normal worm takes place on both ends of the 5-segment starting point. After 21 days, a normallooking whole worm has been produced. The regeneration capability of mudworms is not well understood at this time. You might be surprised to learn that your work in today s BOA lab is not far from being cutting edge research and could conceivably add to our knowledge of mudworm reproduction. Here are some basic questions: 1) Does regeneration require some minimum starting number of segments? 2) Do different body regions make better starting points for regeneration? For example, does the middle region have more or less regenerative potential than the head or tail end? 3) How much time is needed for complete regeneration to occur? 4) Does anatomical regeneration correlate with behavioral recovery? The following flowchart illustrates the experimental design that each group will use. The label n=20 refers to sample size and means that fragments from 20 different worms will be used. Worms will be cut to produce 20-segment pieces from the anterior (front), middle, and posterior (rear) regions. These 20-segment fragments will be the starting point for the regeneration process that will run for 21 days. One group of worms (n=20) will be the experimental Controls and will receive no cuts, but will otherwise be treated like all of the other worm fragments in the study.
No cut Controls Large Pool of Worms C (n=20) Retain anterior Retain middle Retain posterior 20 segments 20 segments 20 segments AS MS PS (n=20) (n=20) (n=20) Work in groups of 3 or 4 classmates. The lab on September 16 will be devoted to sectioning the worms. At the end of the regeneration process (October 7), each lab group will evaluate mudworm behavior and count segments on all regenerated worms in the study. Handling the Worms Work with healthy, normal-looking individuals that are roughly equal in size. The worms are fragile and should not be moved around by mechanical means. In fact, we ll try and never touch the worms except when sectioning them. To move a worm, use suction to pull some fluid and the specimen up into a wide-mouth plastic pipette. Gently eject the fluid and worm to the desired location. Fragmenting the Worms Place a worm on a Petri dish containing filter paper moistened with creek water. To cut the worms into fragments it is important that they lie still. By placing the Petri dish on ice, we can slow them down well enough to work on them. Tilt the Petri dish and drain excess water by using your pipette. To make cuts, use a new single-edge razor blade held at 90 o to the worm s body. Push the blade straight down against the paper and hold it there for a few seconds. Ideally there will be no bleeding and the regeneration process will begin. Worm Storage Worm regeneration will take place in small capped plastic tubes. Each tube should have a small piece of wet paper towel and some creek water. After creating worm fragments, add water back to the Petri dish and then pull the desired fragment into the pipette. Eject the fragment into an appropriately labeled storage tube. At the end of the day, all of your tubes will be placed together
in a dark cool location. 3 Weeks Later (October 7) Retrieve your animals and begin making observations and collecting data. If a fragment didn t survive, you ll see nothing in the tube other than water and paper towel. If, however, you see a worm (look carefully), the fragment survived and probably did some regeneration. Use a pipette to transfer the worm into a Petri dish as described above. Assessing the presence or absence of behavioral regeneration should be the first task with each of the worms. Designate one group member to do all of the behavioral assessments in a single blind manner. NOTE: The first five worms tested for behavior should be Control worms. After the first five have been tested, the order of testing worms should be randomized. The other three group members should put a worm in a dish, noting its identity, and then give it to the behavior observer for a behavior test. The test should involve gently touching the worm on its head end and ranking the strength of the response on a scale of 0 (= no response) to 3 (strongest response). After 10 sec, touch the worm on its tail end and rank the response. Sum the two response estimates to produce a Total Response Score (0-6) and record that on the spreadsheet. Finally, immobilize each of your regenerated (and control) worms with Protoslo and then count all segments. Record, if possible, the number of old (dark) segments and new (lighter colored) segments. LAB REPORT IMRAD Style Remember to cite the sources of all information taken from the internet or other literature. Strongly paraphrase all cited information. Know the meaning of all technical words that appear in your report. Introduction In this section you should provide some background information on mudworms and the process of asexual reproduction through body fragmentation. Web sites are acceptable as sources, but they should be cited where used and then listed at the end of the report in Literature Cited. It would be interesting to know if any other animals reproduce in a similar way. At the end of the Introduction, state your hypotheses. Methods Indicate how you tested your hypothesis. Describe the overall process and then provide the details of your approach. How were your worms handled? How were cuts made? Talk about
your fragment lengths and locations. Provide a specific description of how you determined the location of the middle 20 segments. Indicate sample sizes for your groups. Were controls part of your design? Describe the storage conditions for all fragments. How were tasks divided up among group members? Provide information on how you assessed worm behavior. At the end of the regeneration period, what were the steps taken to record data? What types of data were recorded? Results and Discussion Present your data in a reader-friendly format. Raw data are usually not readily viewed or easily grasped. Do not present raw data. Instead, provide data that have been condensed or summarized to some degree. Summaries involving means (= averages) of group data are often more effective at providing a sense of trends. Graphs and summary tables are great ways to visually convey your information use them. Provide rational interpretations of the numbers. Point out any interesting trends, but also address the issue of data variability. In retrospect, would you redesign your study? Were there problems that you recognized, but couldn t fix? What happened that is unexplainable? Always end this section with a final paragraph that sums up your work and provides the reader with a take-home message. Literature Cited List all of the sources that you cited in your report.