1. . Experimental Methods To test for the release of an alarm cue substance from the planarians, we used electric shock to serve as the initiating stimulus. This method left the planarians otherwise physically unharmed. Pilot experiments showed that the following parameters would subject the planarians to the most stimulation without leaving any physical signs of permanent damage: 200 pulses, 100Hz, 2 mA, duration = 2 ms. A planarian was placed in each of two wells, both filled with 0.5 ml of spring water and both with two conductive electrical wire tips submerged in the water (Fig. 1, b). One well served as a collection site of the control solution and thus was not electrically stimulated, while the other served as the test solution collection site and was connected to a Powerlab electric source (Fig. 1, a). Animals were given 10 min to acclimate, then the planarian in the test well was shocked using the parameters previous described. After the shock, both planarians sat for another 10 min to release possible alarm cues. For testing, a 6 cm long straw was cut to form a half-pipe shaped tunnel. Two small cotton balls were then placed at either end of the straw to create an enclosure (Fig. 2). Finally, the enclosure was filled with 1ml of spring water. A test planarian was then placed in the middle of the straw. Test stimuli were applied to the cotton in six volumes of 30μl each. One cotton ball received the test solution, the other the control. Fluorescein dye experiments showed that this method prevented turbulence inside the straw as the solutions were added (Jessica Winkler, personal communication). For half of the tests, the control solution was placed on the left and the test solution was on the right. Sides were reversed for the other half of the tests. Once all six increments were added, the planarian was timed for 10 min and scored on which side of the straw, test or control, it resided in at the end of each minute. Data were analyzed with a single-sample t test with apredicted sample mean of five.. Figure 3: Mean number of times planarians were found on the test solution side of the straw at the end of each minute. The dotted line represents the expected mean of five that would be found if the null hypothesis is correct. Results & Discussion Planarians were found on the side of the test substance an average of 6.2 times out of 10 (Fig 3). These results may be due to chance alone (t = 1.98, df = 19, 0.10 > P > 0.05).We cannot reject our null hypothesis as being a valid explanation for our findings. There is no convincing evidence for a preference between the two solutions. Our null hypothesis states that planarians do not release a chemical alarm cue substance when they are subject to this particular stimulation. Since our statistical analysis results prevent us from rejecting that notion, it is possible that planarians do not possess a chemical alarm cue system of communication at all. However, our particular experiment focused on only one form of stress-inducing stimulus in a laboratory setting. Our results most likely differ from Chivers & Smith due to the fact that they used an entire crushed planarian in their test solution, and some of their tests where performed in the presence of chemical cues from known planarian predators. For future experimentation, more than one stimulus should be tested to investigate the relevance of particular stimuli on the resultant test solutions. A more natural testing environment and greater sample size might also impact the statistical significance of the results. References Figure 2: The above apparatus was used to create a tunnel-like environment for the planarians, thus forcing them to chose between the side with the control solution or the side with the test solution. 1.Chivers, Douglas. Et al.(1998). Chemical alarm signallingin aquatic predator-prey systems: a review and prospectus. Orono, Maine. Department of Biological Sciences, University of Maine, 344-345. 2.Snyder, Noel and Hellen. (1970). Alarm response of diademaantillarum. American Association for the Advancement of Science, 276-278. 3.Wisenden, Brian D. and Millard, Melissa C. (2001). Aquatic flatworms use chemical cues from injured conspecifics to assess predation risk and to associate risk with novel cues. Biology Department, Minnesota State University Moorehead, 761-766 Introduction The use of both conspecific and heterospecific chemical alarm signaling in predation scenarios is common in many aquatic organisms (Chivers & Smith, 1998). Freshwater planarians make up one group of Platyhelminthes that are thought to share this system of chemical signaling. Wisenden and Millard (2001) recently conducted research exploring the nature of alarm cues within planarian species both with the presence of predator cues and without predator cues, however their experimental methods did not select for a specific alarm cue chemical from the planarians themselves. Noel and Hellen Snyder (1970) tested for similar alarm cues in Diadema antillarum, however they also used an entire crushed organism as their test solution rather than trying to select for a specific alarm cue. The purpose of this experiment was to determine if planarians are capable of releasing a specific chemical alarm cue when subject to stress-inducing stimuli without being physically traumatized and without the presence of natural predators. A. B. Figure 1: Apparatus used to electrically stimulate planarians to collect the test and control solutions: A. Entire Powerlab electrical circuit. B. Individual wells that housed the planarians and the dissecting microscope that was used to monitor the physical condition of the worms. Planaria Control Solution Test Solution Acknowledgments I would like to thank Dr. Frank Corotto for his guidance and consult throughout this entire project, The University of North Georgia’s Department of Biology for their materials, equipment, and funds, and Jessica Winkler for her help and support. Selecting for Conspecific Alarm Cue Substances in Planaria Species Using Electric Stimulation Cullen Winkler, University of North Georgia, Department of Biology; Dahlonega, Ga. 30533