Investigating the Exposure to Wound Healing on the Rate of Regeneration in Dugesia tigrina Thomas Morey Results Abstract The regeneration of the planarian flatworm D. tigrina asexually by splitting into two exact replicas was used to helpdetermine whether specimens that use a single type of stem cell in the blastema called neoblasts to create cell division could have the rate of regeneration increased through different human wound healing solutions (Suzuki 2005). When human skin cells are damaged, the length and efficiency of the regeneration is determined by how quickly cellular proliferation takes place (Hiebert, Granville 2012). For a comparative study, the study of the regeneration process of was paralleled to human cell regeneration by applying three different wound healing solutions used by humans and a control to compare regeneration rates in the formation of a fully functional brain and head in the planarian. The results indicated that the solution that contained the turmeric solution was the fastest to regenerate the planarian, although not significantly. The growth of each planarian at 0 hours, 36 hours, and 72 hours after cutting were measured,. From these measurements, the amount of regenerative growth was calculated. This is presented in Table 1. Using the results found in Table 1, regression analysis was conducted in order to determine whether or not there is a linear relationship between regenerative growth and time. The test concluded that there is a linear relationship between growth and time, and the results are presented in Figure 5. From there, a T test was conducted to determine whether or not the variances between the average rates of regeneration of the four different groups of planaria were homogeneous. The test concluded that they were. The mean rates of regeneration of the four groups of planaria between three different time intervals—between 0 and 36 hours, between 36 and 72 hours, and between 0 and 72 hours—were calculated. The results are presented in Table 2. Three ANOVA tests were then conducted to investigate any differences between the means of the rates of regeneration of the four groups of D. tigrina at the three different time intervals. The process and results are presented in Table 3. Based on the ANOVA tests, there is not relationship between rates of regeneration of planaria and exposure to light or ultraviolet radiation. Introduction Materials and Methods Regeneration is the regrowth of an impaired or absent organ piece from the residual tissue to which humans as adults have limited regeneration capabilities in cells and organs such as those of the skin and liver (Ilowski 2011). Invertebrates, like planarians, can regenerate from either the head or the tail regenerating a fully functional segment no matter how big or small the missing segment was that was cut (Del Cerro, Decoste 2009). The study of the asexual regeneration process of D. tigrina can give us a better understanding of whether animals that use a single type of stem cell in the blastema can heal faster with the right solution which can benefit humans as well (Suzuki 2005). The standard “measurement” of regeneration will be evaluated by determining how long it takes for the photoreceptors to emerge which appear as black specs inside of the white circular eye (Del Cerro, Decoste 2009). Aloe vera promotes healing via the chemicals in the jelly-like substance found in the inner part of the leaf, which kills bacteria, increases circulation in skin blood vessels, reduces inflammation, and prevents cell death around a wound (Reynolds 1999). Turmeric, which contains the active ingredient curcumin, is used in Asia and India to protect the skin against diseases, reduce pain, and inflammation (Mukhopadhyay 1998). Airborne, which contains the antioxidants of vitamins A, C & E, is a good comparative candidate due to the effect begins in the dermal layer; unlike the previous two agents which promote primarily epidermal health to reach equilibrium (Hiebert, Granville 2012). Thirty-two small plastic petri dishes cleaned and sanitized in distilled water to prevent contamination. 16 solutions each of Aloe Vera, turmeric, and Airborne© were made and a control containing just spring water. Lids of the petri dishes were labeled with the solution and individual planarian numbers. Planarian were cut into two pieces with a scalpel Head fragments were put into a large petri dish while the tail fragments weretransferred into the corresponding labeled petri dish using the plastic transfer pipette. Each subject was checked through a disceting scope at the same time daily at 11:00 a.m., except on days 3-6 when they were checked at 8:00 am and 8:00 p.m. when regeneration is the most plausible (Del Cerro, Decoste 2009). Regeneration was assessed by monitoring how long it tookfor the photoreceptors to appear. A single-factor ANOVA was used to correlate significance. Figure 1: A photograph of a bisected planarian, with the anterior end being measured. Using CellSens Dimension, the initial length of the planarian’s anterior end was measured. Magnification is 0.7X. Figure 2: One 24-well tray with bisected planaria. The anterior and posterior ends of each bisected planarian from each group were organized in four 24-well trays. Figure 3: The 48 planaria were placed in four 24-well trays. Each of the four 24-well plastic dishes were labeled according to what condition the planaria were subject to: “L” means “light”, “D” means “dark”, “N” means “no ultraviolet”, and “UV” means “ultraviolet”. Figure 4: The setup of the experiment. The two 24-well plastic dishes outside the box housed the planaria that were exposed to light-and-dark cycles. The box covers the other two 24-well dishes, which housed the planaria kept in the dark. The bottle is full of chemical-free spring water for the planaria. Time (hours) Light, UV Light, No UV Dark, UV Dark, No UV 36 131380.88 138694.06 132714.58 125872.26 72 122863.23 137028.00 157398.38 128810.80 Time (hours) Light, UV Light, No UV Dark, UV Dark, No UV 0-36 3649.4688 3852.6128 3686.5161 3496.4517 36-72 631.2627 1017.7313 567.9869 452.7618 0-72 1706.4337 1903.1667 2186.0886 1789.0389 Table 1: The average regenerative growths, in micrometers, of brown planaria under four different treatments, measured across 36-hour intervals after bisection. Subjects showed no measurable amounts of growth immediately after bisection across all four treatments. The measurements under 36 hours represent the average growths from 0 hours to 36 hours. The measurements under 72 hours represent the average growths from 0 hours to 72 hours Table 2: The average rates of regeneration, in μm/h, of brown planaria under four different treatments, measured across 36-hour intervals after bisection. The average rates of regeneration were measured between 0 and 36 hours, between 36 and 72 hours, and between 0 and 72 hours. Figure 5:. Discussion ANOVA 0-36 Hours 36-72 Hours 0-72 Hours Sum(x) 326082.3294 47161.17099 131590.6646 Sum(x2) 1166972425.00 125294606.90 296306573.70 SST -27743143.50 92586135.62 41657999.87 SSB 1401833.259 3114926.775 1353425.394 SSW -29144976.76 89471208.84 40304574.48 VB 467277.7531 1038308.925 451141.798 VW -342882.0795 1397987.638 629758.9762 Fcalc -1.363 0.743 0.716 Fcrit 2.71 2.75 Literature Cited Based on regression analysis, there is a linear relationship between regenerative growth and time. Based on the three ANOVA tests, exposure to light and ultraviolet radiation has no affect on the rate of regeneration in D. tigrina. Difficulties in measuring the exact lengths of planaria may have affected the results. Different wavelengths of light may produce more significant results. Exposing planaria to UV after bisection may have different results. Carolina protozoa and invertebrates manual. (2006). Burlington, NC: Carolina Biological Supply Company. Dasheiff, B. D., & Dasheiff, R. M. (2002). Photonegative response in brown planaria (dugesia tigrina) following regeneration. Ecotoxicology and Environmental Safety, 53, 196-99. Kalafatic, M., Kovacevic, G., & Franjevic, D. (2006). Resistance of two planarian species to uv-irradiation. Folia Biol (Krakow), 54(3-4), 103-8. Reynierse, J. H. (1966). Some effects of light on the formation of aggregations in planaria phagocata gracilis. Animal Behaviour, 14(2-3), 246-50. Wenemoser, D., & Reddien, P. W. (2010). Planarian regeneration involves distinct stem cell responses to wounds and tissue absence. Developmental Biology, 344, 979-91. Table 3: Three ANOVA tests to find any differences between the average rates of regeneration of the four groups of planaria at three different time intervals. SSt is the total sum of the squares present. SSb is the sum of the squares between groups or treatments. SSw is the sum of the squares within groups or treatments. Vb is the variance between groups or treatments. Vw is the variance within groups or treatments. Fcalc is the calculated F value based on Vb and Vw. Fcrit is the critical F value based on degrees of freedom. Acknowledgements I would like to thank Dr. Evans-Anderson and Pooja Pardhanani for assistance.