The Effect of Different Light Conditions on the Growth, Regeneration, and Reproduction of Brown Planaria

Dominic Cicconi and Justin Guider Mrs. Lounsbury December 2, 2013 Abstract We decided to test the effect of different light conditions on planarian growth, regeneration, and reproduction and how this could relate to the cell growth in humans. We hypothesized that if brown planaria were observed under sunlight, no light, and infrared light conditions in order to study their regenerative, reproductive, and growth properties, then the brown planaria under the infrared light would experience increased reproduction, regeneration, and growth because infrared light excites the mitochondria in cells, thus leading to increased ATP production and increased cell reproduction (through mitosis). We also hypothesized that sunlight would increase the reproduction, growth, and regeneration, but to a lesser degree (because sunlight includes infrared). Our hypothesis was partly supported. Neither sunlight nor infrared light affected the growth or reproduction of whole, adult planaria. However, infrared light did increase the regeneration of halved planaria. This is important because infrared light as a healing supplement to regrow human cells is a burgeoning practice in the medical field. Since planaria can regenerate using stem cells similar to those of humans, we wanted to investigate if planaria would grow, reproduce and regenerate faster when put under infrared light, and if there was a significant difference between pure infrared and the infrared light latent in sunlight. After

seeing that infrared effectively increased the regeneration of planaria, we hope that this may lead to a correlation to the growth of human cells as well.

For our junior research project, we wanted to investigate the effect of different light conditions (infrared light, sunlight, and no light) on planarian growth, regeneration, and reproduction and how these results could correlate to the cell growth in humans. We decided to pursue this topic due to the fact that infrared light is becoming a common healing practice for humans to treat cuts and other wounds (NASA, 2003). Since planaria can regenerate using stem cells similar to those of humans, we wanted to test whether or not planaria would grow, reproduce, and regenerate faster when put under infrared light. We also wanted to see whether or not there was a significant difference in the healing power of pure infrared light and the infrared light latent in sunlight. In order to conduct our experiment to its full potential, we decided to research planaria to enhance our knowledge of the organism. First, we studied the proper maintenance needed to sustain planarian survival in their normal environment. According to George Fox University (n.d.), planaria live in freshwater sources like ponds and streams. Planaria live a range of temperatures from 21-23 degrees Celsius, or roughly 70-73 degrees Fahrenheit. They feed on egg yolk (from hard boiled eggs) or liver, and their water must be changed at least once a week. Next, we researched the properties of planarian reproduction. When a planarian is cut in two, the epithelium, or tissue that forms on the outside of the body, covers the severed ends. After this occurs, a group of undifferentiated cells (similar to stem cells), called blastema, moves to these ends and begins to recreate the lost tissue. These cells are called neoblasts, and they eventually

evolve into specialized cells as the planarian regrows and needs different cells for its various functions. This type of regeneration is known as epimorphosis (University of Minnesota, n.d.). Planarian cells and human stem cells have a similar gene which controls regeneration. The planarian type of this gene is known as piwi, while the similar human gene is known as hiwi. Piwi is vital for planaria because it makes sure that the stem cells in planaria produce fullyfunctional offspring stem cells. These cells then can rebuild planaria that have been severed. Hiwi is shown in sperm and egg cells and stem cells that produce blood cells. While there is insufficient information to support it, it is hoped that this gene can cause stem cells to help regenerate body parts (Exploratorium, n.d.). Sometimes, doctors employ light therapy to treat medical conditions through the use of the visible and nonvisible light spectrums. According to Jerry Berg of NASA (2003), the light therapy is used in cancer treatment, in the form of visible light and UV rays. In treating wounds, LED and low-level laser lights are frequently used. These lights most notably treat cuts, burns, and injuries in muscles and tendons, as research has shown that they expedite cell growth (Eells et al., 2004). Near-infrared LED lights have been shown to excite the CCO (cytochrome c oxidase) in mitochondria in cells, thus causing them to produce more ATP, the energy source that fuels much of the operations of the cells, including reproduction, growth, and regeneration (Roberts, 2012). We chose infrared light based on scientific observations that concluded that infrared can enhance the growth and reproduction (through mitosis) of stem cells. Since planarian cells are strikingly similar to human stem cells, we wanted to see if planarian cell growth would be expedited (and if so, if this could open possibilities in the field of human stem cell growth). Our independent variable was the type of light and our dependent variables were the planarian

regeneration, reproduction, and growth. We chose no light (darkness) as a control group because in the wild, planaria live in dark environments like under rocks and leaves (Wards Science Teacher Resources, n.d.) and because this would eliminate all sources of light, which would ensure that light would not affect growth results. We chose sunlight as a counterpoint to infrared light. We wanted to determine whether pure infrared light caused more growth than natural sunlight, which includes infrared wavelengths. For our experiment, we wanted to include a large sample size of planaria in order to make sure our data was as accurate as possible. We had 18 whole brown planaria in each light condition (sunlight, infrared, and darkness) to have a total of 54 whole brown planaria. We also included 5 anterior and posterior brown planaria (we obtained these through halving whole adults) in each light condition to have a total of 15 anterior planaria and 15 posterior planaria. The purpose of our study was to determine how near infrared LED light and sunlight would affect the reproduction, growth, and regeneration of brown planaria (Dugesia tigrina). This in turn was meant to provide an insight into how effective infrared light therapy is in healing wounds. We hypothesized that if brown planaria were observed under sunlight, no light, and infrared light conditions in order to study their regenerative, reproductive, and growth properties, then the brown planaria under the infrared light would experience increased reproduction, regeneration, and growth because infrared light excites the mitochondria in the cells, thus leading to increased ATP production and increased cell reproduction (through mitosis). We also hypothesized that sunlight would increase the reproduction, growth, and regeneration, but to a lesser degree (because sunlight includes a portion of infrared light). Methods and Materials

Our research subjects were 69 brown planaria (Dugesia tigrina). We ordered more than 69, as some were likely to die in transit. We built 3 wooden boxes with hinged lids in which to house our planaria. One was entirely closed off so as not to allow in any light. A second box was also almost entirely closed in, except for a small hole for the cord from the infrared light strip which ran along the inside of the box to pass through. Our third box had mesh wire on the lid so sunlight could filter through, but most foreign objects could not. Each of these boxes was about one foot long, half of a foot wide, and half of a foot tall. The planaria that we did not cut were kept in small glass jars that we filled about 2/3 of the way with water. We had 18 of these jars. The rest we kept in small petri dishes filled almost to capacity with water. We had 15 of these petri dishes. We had 3 plastic trays that we filled with water, and 3 floating thermometers, one for each plastic tray. We also had pH droplets and a reference sheet to determine the pH. We had 1 small scalpel to cut planaria in half, 2 flat plates that we cut the planaria on, and 2 small, clear rulers that measured millimeters. In addition, we had the infrared LED light strip that ran along the inside of one of the boxes, 1 dissecting microscope, 1 roll of scotch tape, 1 permanent marker, and 3 pipettes. For the water for the planaria, we used about of a gallon of stream water every feeding day. For the water in the plastic dishes (used just for taking temperature), we used about of a gallon of tap water each time the water needed to be replaced. The first step of our experiment, after planning, was to obtain the required materials and build the three boxes. We attached the infrared light strip to the inside of the appropriate box and kept it plugged in at all times. Then, we began the setup stage. We labeled 6 glass jars AS1 - AS6, A standing for adult and S standing for sunlight. Adult was simply a term we used for the more accurate whole planaria. We labeled another six AD1 - AD6, D standing for darkness. We labeled a third set of 6 AI1 - AI6, I standing for

infrared. The jars were labelled with scotch tape and permanent marker. We filled three plastic trays with tap water (the amount was unimportant as long as it was about equal in each). We placed a floating thermometer in each tray. We measured the temperature of the water in these trays in lieu of measuring the temperature of that in the glass jars. We obtained about of a gallon of stream water and measured its pH, and let it sit for about 30 minutes in order to let it reach room temperature, and did so each time we changed the water. Then, we filled each of the glass jars about of the way with the stream water, and each of the petri dishes almost to the top with water. After we had all of this set up, we made preliminary measurements and distributed the planaria. (Planaria move by stretching, and thus their length changes quite a bit as they wander. We measured each planarian when it was at its longest, right before it contracted again. To measure them, we held the ruler up against them to give them a straight edge to follow). Each planarian we intended to keep whole was placed on a flat plate and measured to the nearest millimeter prior to being placed in a glass jar. Three planaria were allocated to each jar: a small one, a medium-sized one, and a large one. These were all relative to the other two in the jar, and the only purpose of the distinctions was to be able to tell the three apart. Six jars were placed in each box. The planaria were labeled according to their size, the light condition they were in, and the number jar they were in. For example, jar AS1 contained three planaria: SS1 (small sunlight 1), MS1 (medium sunlight 1) and LS1 (large sunlight 1). Next we cut, measured and allocated the planaria that we intended to cut in half. We took 15 planaria and measured them to the nearest millimeter. We then took the scalpel and cut them in half horizontally, emulating a method of asexual reproduction called fragmentation. We divided the measurement of each in half to get the length of the anterior and posterior sections of each. (They were easily told apart as the anterior section has a head, while the posterior section does not. The heads that the

posterior planaria eventually grew were more translucent than those of the anterior sections.) Five petri dishes were placed in each box. The planaria were labeled according to which end they were, which light condition they were in, and which petri dish they were in. For example, the first petri dish in the light condition contained HS7 and TS7, H and T standing for head and tail, respectively. Head and tail were terms we used instead of anterior and posterior, which would have been more accurate. (We labeled the petri dishes starting at 7 and ending at 11, as 16 were taken up by the jars). Once this setup was completed, we began our actual experiment. The temperature of the water in the plastic trays, representative of the water in the dishes and jars, was taken and recorded twice a day, once between 6:30 AM and 9:30 AM, and again between 12:30 PM and 3:30 PM. Every other day the water was changed and the number of planaria in each contained was counted and recorded. To change the water, we moved the planaria using pipettes into temporary jars, dumped out the old water, cleaned the jar with a rag or a paper towel, refilled the jar with water, and moved the planaria back. We had to be careful to make sure that no planaria got lost during this process, and that we kept track of which planaria belonged where. We also took the pH of the water each time it was changed by siphoning a small amount of water from each container and putting it into the pH measuring vial. We then measured the pH with pH drops. The pH of the new stream water was also taken and recorded each time. We measured the planaria every 4 days, determining which planaria was which based on relative size. We did not measure the offspring, as that would have led to superfluous data. After the planaria in each jar were measured, we placed a small (half of a pea) sized piece of cooked egg yolk in the jar, and left it in for approximately 30 minutes. The halved planaria we did not feed, for according to an interview we conducted with Dr. Steve Bailey (2013), a flatworm specialist at Carolina

Biological, they do not eat while regenerating. After the 30 minutes, we changed the water, often having to dislodge the planaria from the egg with blasts of water from a pipette. On these days we still performed our regular duties, such as measuring the pH and the temperature. We conducted our experiment of the whole planaria for 24 days. The halved planaria were a late addition, and we conducted our experiment on them for 20 days. The last thing we needed to do was analyze our data. First, we calculated the mean and median of the temperatures of the water in each light condition. We then calculated the range and determined if there were any outliers. Next, we calculated the mean of the pH for each light condition. There were clearly no outliers, so we did not calculate the median, which is used to determine which numbers are outliers. Then, we measured the total change and rate of change in growth for every planarian, both whole and halved. We measured the means of the total changes and rates of change for the small, medium and large planaria in each light condition separately, as well as the means of the total changes and rates of change for the anterior and posterior planaria in each light condition. For example, we measured the mean of the total change of the growth in millimeters for all of the large planaria in sunlight, and for all of the medium-sized planaria in darkness. Next, we measured the total change and rate of change of the number of planaria in each jar (in other words, the reproduction). We did not do so for the petri dishes as there were so few offspring. We found the means of the rates of change and total changes of the number of planaria in the jars in each light condition separately. Finally, we did t-tests to determine the statistical significance of our data. We compared the means of sunlight and infrared against darkness, the control, for each group of planaria. We used .05 significance.

Results

Mean Total Changes and Rate of Changes for Small, Medium, and Large Planaria in Three Types of Light Planaria Type Mean Total Change (mm) Mean Rate of Change (mm) Small Planaria in Sunlight 2.7 0.051 Small Planaria in Darkness 2.5 0.11 Small Planaria in Infrared 2 0.086 Medium Planaria in Sunlight -0.33 -0.015 Medium Planaria in Darkness 1 0.044 Medium Planaria in Infrared 0.17 0.007 Large Planaria in Sunlight -2.3 -0.1 Large Planaria in Darkness -1.5 -0.07 Large Planaria in Infrared -2.2 -0.1

Mean Total Change and Rate of Change in Reproduction of Whole Planaria in Sunlight, Darkness, and Infrared Planaria Type Mean Total Change Mean Rate of Change (planaria/23 days) Planaria in Sunlight 2.8 0.12 Planaria in Darkness 3 0.13 Planaria in Infrared 3.2 0.14

Mean Total Change and Rate of Change of Halved Planaria Under Three Kinds of Light Planaria Type Mean Total Change (mm) Mean Rate of Change (mm/day) Heads in Sunlight 1.2 0.06 Tails in Sunlight 1.2 0.06 Heads in Darkness 0.4 0.025 Tails in Darkness 2.1 0.12 Heads in Infrared 2 0.1 Tails in Infrared 1 0.05

Mean pH of Stream Water Water Type pH Level New Stream Water* 7.3 Sunlight Containers 7.4

Sunlight Petri Dishes 7.5 Darkness Containers 7.3 Darkness Petri Dishes 7.4 Infrared Containers 7.3 Infrared Petri Dishes 7.4 *Fresh stream water collected from White Clay Creek

Temperature Data Analysis for Sunlight, Darkness, and Infrared Conditions A.M. Light Mean Temperature Median Temperature Range of Temperature Number of Condition (in Celsius) (in Celsius) (in Celsius) Outliers A.M. 21.8 20.6 22.2 A.M. 22.7 21.7 23.2 A.M. 9.2 8.8 7.7 A.M. 1 4 0

Sunlight Darkness Infrared

P.M. Light Condition Mean Temperature (in Celsius) Median Temperature (in Celsius) Range of Temperature (in Celsius) P.M. 11.3 9.6 10 Number of Outliers

Sunlight Darkness Infrared

P.M. 26.5 24.6 25

P.M. 26.6 24.8 25.1

P.M. 0 0 1

Statistical Analysis (T-Test)

NOTE: For these calculations, darkness (no light) is the control group and the level of significance of 0.05. Our null hypothesis was that there would be no significant change in reproduction, growth, and regeneration in the whole and halved planaria when under sunlight, no light, and infrared light conditions.

Mean Total Change for Adult Planarian Reproduction T Statistic Significant? Sunlight vs. Darkness 0.18 No

Infrared vs. Darkness

0.14

No

Mean Total Change for Large Planarian Growth (mm) T Statistic Significant? Sunlight vs. Darkness 0.76 No Infrared vs. Darkness 0.14 No

Mean Total Change for Medium Planarian Growth (mm) T Statistic Significant? Sunlight vs. Darkness 2.3 No Infrared vs. Darkness 1.3 No

Mean Total Change for Small Planarian Growth (mm) T Statistic Significant? Sunlight vs. Darkness 0.15 No Infrared vs. Darkness 0.83 No

Mean Total Change for Anterior Planarian Growth (mm) T Statistic Significant? Sunlight vs. Darkness 1.7 No Infrared vs. Darkness 3.5 Yes

Mean Total Change for Posterior Planarian Growth (mm) T Statistic Significant? Sunlight vs. Darkness 3 Yes Infrared vs. Darkness 4.6 Yes

Mean Rate of Change for Anterior Planarian Growth (mm) T Statistic Significant? Infrared vs. Darkness 5.7 Yes

Mean Rate of Change for Posterior Planarian Growth (mm) T Statistic Significant? Sunlight vs. Darkness 2.3 No Infrared vs. Darkness 2.9 Yes

Discussion Our experiment went through many changes and setbacks before we settled on a design that worked and felt appropriate. Our original plan was to include ultraviolet light in our experiment along with infrared light; however, we decided that this would prove too logistically difficult, and was not relevant enough to be included. Furthermore, for quite some time, we had trouble deciding whether we would measure the weight or the length of the planaria. We vacillated between the two for some time before deciding to measure length, our rationale being that measuring weight would require too precise of an instrument, and that excess water would cause our data to become meaningless. We still had reservations about the accuracy of measuring the length of planaria, but we saw no alternative. First we attempted our original experiment, which involved only halved planaria. However, nearly all of the planaria died overnight after our initial measurements! We were uncertain as to the cause of their untimely demise, but we were told by a flatworm specialist that it might be because the planaria were in their sexual cycle. According to this specialist, planaria divert nearly all of their energy to reproduction during this time, and thus were not able to survive the stress of being cut in half (Bailey, 2013). Another hypothesis of ours was that the water we had been using was in some way unsuitable. We had been using Nice! brand bottled water, as we wanted to avoid any foreign substances that might be present in tap water or stream water. However, upon testing the pH of the Nice!, we discovered that it had a pH of 5.8, which is outside the ideal living conditions of planaria, not to mention extremely acidic for regular human drinking water. We had so few planaria left that we decided to act on the assumption that both reasons were partially responsible. Though we were reluctant to change our experiment to such a degree, we ultimately decided that we would use only whole planaria, and simply measure growth instead of

regeneration. In addition, we decided to use stream water, which had kept planaria alive in preliminary tests. We began our revised experiment, and all of the planaria survived the first night. We had some planaria that werent being used for the experiment, so we decided to cut some in half and measure their regeneration in addition to continuing to measure the growth of the whole planaria, working under the assumption that the Nice! water was the cause of death in previous attempts, not stress. These survived as well, and we concluded that the Nice! water was most likely responsible. This is how we arrived at our final experimental design of some whole planaria and some halved. After analyzing our data, we came to several conclusions. Neither the means of the growth nor the reproduction rates and total changes were statistically different between sunlight and darkness or infrared and darkness. From this, we can state that neither near infrared LED light nor sunlight affects the growth or reproduction of whole planaria, based on our experiment. Both the means of the rates of change and total change in the length of halved planaria between those subjected to infrared light and those subjected to no light were significantly different, for both anterior and posterior halves. From this, we can state that near infrared LED light increases the rate of regeneration in planaria, based on our experiment. The mean of the total change in growth for anterior planaria in the sunlight was significantly different from the mean total change of those in darkness, but the rate of change was not. Neither the means of the total change or the rate of change of the growth of posterior planaria in sunlight were significantly different from darkness. We can not conclude whether or not sunlight has an effect on the regeneration of planaria from this data; further testing would be required. However, since the effects of infrared were most certainly significant, we can conclude that infrared increases regeneration in planaria more than sunlight. A similar study done by scientists at Laurentian University measured the

concentration of stem cells in the same species we used, Dugesia tigrina, after being amputated and exposed to various frequencies of light. They found higher concentrations of stem cells in planaria exposed to near infrared light, which would seem to support our findings (Wu et al., 2011). There were various sources of error that may have contributed to problems with our data. One was the fact that when we halved the planarian, we assumed that the anterior and posterior halves were the same length (mm), even though our cuts were not perfect. Another possible source of error is that the amount of egg used to feed each glass container of adult planaria was not the same. We gave each container about a pea-sized portion of egg, but no exact measurement of egg was determined. This could have provided certain planaria with more energy than others. Moreover, some planaria received slightly less time to eat than others. Another source of error was the measurement process of both the adult and halved planaria. Planaria move by elongating, and though we tried to measure each at its longest point, there was definitely some error. Also, when we replenished the water in thes containers, we took all the planaria out of their light conditions onto a table in sunlight. This allowed for the presence of sunlight to affect the no light and infrared-exposed planaria. A very important problem is that planaria reproduction is affected by the conditions they live in. Due to unfavorable living conditions (like low population density, stagnant water, and a small living area), the planaria frequently used asexual reproduction to reproduce. According to Wards Natural Science (n.d.), when planaria reproduce asexually in favorable conditions they do so using a process called fragmentation, in which they perform a transverse constriction, and the two halves of the planaria move away from each other and regrow. However, in unfavorable conditions, planaria will undergo a method of asexual reproduction called dropping tails, in which they only drop

the very end of their body. This can result in stunted growth. We believe that this occurred in our experiment. This led to many planaria splitting in order to reproduce, thus dramatically decreasing the length and growth rate of that certain planarian. With improved living conditions, asexual reproduction may not have factored into our results. Lastly, the temperature was not exactly constant due to the different light conditions. There are multitudinous ways that we could mitigate these problems in future experiments in order to obtain more accurate results. For one, we could figure out a way to cease the movement of planaria when we try to dissect and measure them. In addition, we could control our constants more specifically, including the time of feeding for the planaria, the amount of food given, the temperature, and the amount of stream water supplied to each glass container, in an effort to limit the influence of the variables in our experiment. We could improve the living conditions of the planaria by providing a large space to live in, a constant flow of fresh water, and an increased population to prevent the problems that arise with asexual reproduction. Also, after halving the planaria, we could measure the posterior and anterior pieces instead of assuming that we had accurately cut the planaria in half. We did not do this in the first place because we were advised by Dr. Steve Bailey (2003) to limit the stress we put on the animal (because they were in their sexual cycle and additional stress might have killed them). Conclusion The purpose of our study was to determine how near infrared LED light and sunlight would affect the reproduction, growth, and regeneration of brown planaria (Dugesia tigrina). This in turn was meant to provide an insight into how effective infrared light therapy is in healing wounds. We found that infrared significantly increases the regeneration of planaria that have been divided, but that it does not significantly affect the regular growth of planaria, or the

rate at which they reproduce. We also found that sunlight does not significantly affect the growth or reproduction of brown planaria, but it may have some small effect on their regeneration. Our hypothesis was partially supported. Neither sunlight nor infrared light affected the growth or reproduction of the planaria, so that portion is rejected. However, infrared light did increase the regeneration, so that portion is supported by our data.

Bibliography Alvarado, A. (n.d.). Current Biology - Planarians. ScienceDirect. Retrieved April 8, 2013, from http://www.sciencedirect.com/science/article/pii/S0960982204006815 Bailey, Steve. Interview by Dominic Cicconi. Phone interview. 16 June 2013. Berg, J. (2003, November 5). Light emitting diodes bring relief to young cancer patients. NASA. Retrieved May 5, 2013, from http://www.nasa.gov/centers/marshall/news/news/releases/2003/03-199.html Carolina Protozoa and Invertebrates Manual. (n.d.). Carolina Biological. Retrieved November 25, 2013, from http://www.carolina.com/pdf/care-sheets/Protozoa-and-Invert-Manual.pdf Eels, J., Wong-Riley, M., Margolis, D., Whelan, H., VerHoeve, J., Henry, M., et al. (2004, September 4). Mitochondrial signal transduction in accelerated wound and retinal healing by near-infrared light therapy.. NCBI. Retrieved November 6, 2013, from http://www.ncbi.nlm.nih.gov/pubmed/16120414 Light Therapy. (2011, April 14).Cancer.org. Retrieved April 9, 2013, from http://www.cancer.org/treatment/treatmentsandsideeffects/complementaryandalternative medicine/manualhealingandphysicaltouch/light-therapy

McIntosh, L., Sassanfar, M., & Synder, J. (n.d.). Regeneration: Animal Growth and Development. Massachusetts Institute of Technology. Retrieved April 7, 2013, from https://biology.mit.edu/sites/default/files/planarialab.pdf NASA technology for growing plants in space brings relief to young cancer patients. (2003, November 11). NASA . Retrieved April 9, 2013, from http://www.nasa.gov/centers/marshall/multimedia/photos/2003/photos03-199.html Newmark, P., & Alvarado, A. (n.d.). Regeneration in Planaria. Neuro. Retrieved April 9, 2013, from http://planaria.neuro.utah.edu/publications/PN_ELS01.pdf Planaria. (n.d.). Ward's Science Teacher Resources. Retrieved April 7, 2013, from https://www.wardsci.com/www.wardsci.com/images/Planaria.pdf Planaria: A window on regeneration. (n.d.). Exploratorium. Retrieved April 9, 2013, from http://www.exploratorium.edu/imaging-station/research/planaria/story_planaria3.php Planaria Care Instructions. (n.d.). George Fox University. Retrieved April 6, 2013, from http://www.georgefox.edu/academics/undergrad/departments/psychology/ckoch/research/ pl ancare.pdf Planarian. (n.d.). Animal Planet. Retrieved April 7, 2013, from http://animal.discovery.com/worms/planarian-info.htm Planarian Regeneration. (n.d.). UMN. Retrieved April 9, 2013, from https://wiki.umn.edu/pub/Biol4361/LabMaterials/13_Planarian_Regeneration.pdf Roberts, S. (n.d.). LED Light Therapy.LED Light Therapy. Retrieved November 6, 2013, from http://heelspurs.com/led.html

Washington, I. (n.d.). LED Lights & Healing. eHow. Retrieved April 9, 2013, from http://www.ehow.com/way_5678838_led-lights-healing.html Wu, H., & Persinger, M. (2011, February 7). Increased mobility and stem-cell proliferation rate in Dugesia tigrina induced by 880nm light emitting diode. NCBI. Retrieved November 6, 2013, from http://www.ncbi.nlm.nih.gov/pubmed/21146998

Acknowledgements We would like to thank our mentor Mrs. Lounsbury for her great advice and encouragement, as well as for lending us a dissecting microscope. We would also like to thank Mr. Guider for his assistance in building the boxes for the planaria and for locating several essential materials. We would like to thank Mr. and Mrs. Cicconi for providing us a place to perform our experiments. Finally, we would like to thank the staff at Carolina Biological for their advice, particularly a Dr. Steve Bailey.

Appendix Temperature Readings (C) for Sunlight D ate 6/ 17/2013 6/ 18/2013 6/ 19/2013 6/ 20/2013 6/ 21/2013 6/ 22/2013 6/ 23/2013 6/ 24/2013 6/ 25/2013 6/ 26/2013 6/ 27/2013 6/ 28/2013 6/ 29/2013 6/ 30/2013 7/ 1/2013 7/ 2/2013 7/ 3/2013 7/ 4/2013 7/ 5/2013 7/ 6/2013 A.M. (6:30-9:30) *N/A 20.1C 17.7C 15.9C 15.9C 18.8C 20.3C 21.7C 21.1C 21.0C 22.7C 22.5C 22.7C 23.5C 24.2C 23.7C 22.9C 21.3C 23.5C 24.5C P.M. (12:30-3:30) 26.5C 20.6C 23.6C 23.8C 21.0C 25.8C 26.0C 27.0C 30.1C 29.0C 29.0C 27.4C 26.4C 24.2C 24.1C 26.9C 27.3C 28.9C 28.0C 30.6C

7/ 7/2013 25.1C 7/ 8/2013 22.7C 7/ 9/2013 23.1C 7/ 10/2013 23.6C 7/ 11/2013 24.3C *Project started in the afternoon

31.9C 25.9C 26.8C 26.6C 24.0C

Temperature Readings (C) for Infrared Date A.M. (6:30-9:30) P.M. (12:30-3:30) 6/17/2013 *N/A 23.1C 6/18/2013 21.1C 21.4C 6/19/2013 19.0C 21.5C 6/20/2013 17.1C 22.3C 6/21/2013 17.1C 19.9C 6/22/2013 18.4C 25.0C 6/23/2013 20.5C 25.0C 6/24/2013 22.3C 25.0C 6/25/2013 21.1C 26.9C 6/26/2013 22.0C 26.9C 6/27/2013 24.0C 27.0C 6/28/2013 23.2C 26.0C 6/29/2013 22.2C 25.5C 6/30/2013 23.2C 24.1C 7/1/2013 24.0C 23.9C 7/2/2013 23.9C 25.1C 7/3/2013 23.4C 25.1C 7/4/2013 20.7C 27.2C 7/5/2013 23.9C 26.8C 7/6/2013 24.8C 29.9C 7/ 7/2013 25.0C 28.0C 7/ 8/2013 23.2C 25.2C 7/ 9/2013 23.2C 25.0C 7/ 10/2013 24.2C 25.9C 7/ 11/2013 24.8C 24.2C

Temperature Readings (C) for Darkness Date A.M. (6:30-9:30) P.M. (12:30-3:30) 6/17/2013 *N/A 25.0C 6/18/2013 19.0C 19.3C 6/19/2013 16.9C 20.8C 6/20/2013 15.0C 21.9C 6/21/2013 14.9C 18.8C 6/22/2013 17.0C 24.5C 6/23/2013 18.9C 24.4C 6/24/2013 20.6C 24.3C 6/25/2013 19.8C 27.3C 6/26/2013 20.0C 27.0C 6/27/2013 21.8C 27.0C 6/28/2013 21.6C 25.6C 6/29/2013 20.9C 25.5C 6/30/2013 21.9C 22.9C 7/1/2013 22.9C 23.0C 7/2/2013 22.4C 24.6C 7/3/2013 21.8C 25.7C 7/4/2013 19.5C 27.0C 7/5/2013 22.1C 26.2C 7/6/2013 23.1C 28.4C 7/7/2013 23.7C 28.0C 7/8/2013 21.8C 24.5C 7/9/2013 21.9C 24.8C 7/10/2013 22.8C 24.9C 7/11/2013 23.4C 22.9C

6/17/2013 6/19/2013 6/21/2013 6/23/2013 6/25/2013 6/27/2013 6/28/2013 6/29/2013 7/1/2013 7/3/2013

Planaria Observation Schedule Start of Experiment Water Change and Count Feeding, Measurement, Count* Water Change and Count Feeding, Measurement, Count Bad Weather (NO RECORDING) Water Change and Count Feeding, Measurement, Count Water Change and Count Feeding, Measurement, Count

7/5/2013 Water Change and Count 7/7/2013 Feeding, Measurement, Count 7/9/2013 Water Change and Count 7/11/2013 Final Measurement and Count (END) *A new batch of planaria were dissected into anterior and posterior parts for observation. **The temperature of the containers (sunlight, darkness, and infrared) was taken twice each day, in the time slots 6:30-9:30 a.m. and 12:30-3:30 p.m. in degrees Celsius.