Anti-Oxidant Remediation of UV-Stressed Earthworms By: Pat Ebbert Pittsburgh Central Catholic PJAS 2010

Introduction Vitamin C acts as an anti-oxidant to slow the cell-crippling effects of free radicals in organisms. UV exposure can promote free radical production.

Vitamin C Exists as ascorbic acid in its reduced form and dehydroascorbic acid in its oxidized form. Acts as a reducing agent (anti-oxidant) by neutralizing oxidative species such as hydrogen peroxide. Reduced Form Oxidized Form

Ultra-Violet Radiation Electromagnetic radiation ranging in wavelength from about 10nm-400nm. Photolysis of molecules can cause the production of free radicals in cells. Direct UV effects, as well as free radicals can disrupt DNA synthesis and production of proteins needed for tissue growth and regeneration.

Anti-Oxidant Role in Tissue Regeneration Because anti-oxidants remove free radicals that can have damaging cell effects, including disruption of protein production, they may play a role in promoting the re-growth of lost tissue. Practical Application: Administration of anti-oxidants could improve recovery rates in humans suffering from tissue damage.

Lumbricus terrestris Commonly known as the “Canadian Night- crawler”, abundant in Europe and North America. Have the ability of regenerating segments after severe tissue damage has occurred. UV exposure may disrupt tissue regeneration ability, and introduction of Vitamin C may help to lessen these effects.

Purpose To determine the difference in regeneration ability between earthworm heads and tails. To determine the effect of UV radiation exposure on the regeneration ability of earthworms. To determine the effect of UV radiation and ascorbic acid on the regeneration ability of earthworms exposed to UV radiation.

Hypothesis #1 Null: There will be NO significant variation between the regeneration abilities of the heads of the earthworms and the tails of the earthworms. Alternative: There WILL be significant variation between the regeneration abilities of the heads of the earthworms and the tails of the earthworms.

Hypothesis #2 Null: UV radiation exposure will have NO significant effect on the regeneration abilities of the earthworms, and introduction of ascorbic acid will have NO significant effect on either the UV or no-UV groups. Alternative: UV radiation exposure WILL have a significant effect on the regeneration abilities of the earthworms, and introduction of ascorbic acid WILL have a significant effect on both the UV and no-UV groups.

Materials 32 live Canadian night-crawler earthworms 48 paper cups Large screen to fit over cups (prevent worms from escaping) Large supply of topsoil (enough to fill 48 cups half- way) Plastic spoon 50ml conical Distilled water 1000mg Vitamin C powder tablets 5ml and 10ml macro-pipettes Pair of scissors Pair of rubber gloves (for handling) Gram scale Plastic weigh-boat UV culture hood Notebook and pencil (for data recording) Sharpie marker (for labeling)

General Procedure 48 paper cups were filled roughly halfway with topsoil. Concentrations of 20g/L of Vitamin C were prepared from 1000mg Vitamin C capsules. 10mL of new Vitamin C solution were pipetted into each of 24 paper cups. 16 live worms were placed in Vitamin C cups, 16 in soil-only cups; a screen was placed over all the cups to ensure the worms wouldn’t escape (Worms were stored in a garage, with temperatures ranging from 4.5 to 6.6 degrees Celsius). After 3 days, half of the worms (8-VC, 8-NVC) were cut medially, dividing the worms into “head” and “tail” segments, and then tail segments were placed in the leftover cups in their respective spots. Cut worms were all weighed using a gram scale, and data was recorded. On this same day, half of all worms (including non-cut) were transferred from soil cups to empty plastic cups and given a 30-second UV exposure, then re- transferred back to their respective soil cups and covered with the screen. Survivorship was checked after one and two week intervals for ALL worms, and cut worms were weighed at one and two week intervals, with all data recorded.

General Procedure (cont.) Earthworms Cut Heads UV VC No VC No UV Tails Not Cut *12 total groups, x4 replicates of each = 48 total data points

ANOVA Statistical Analysis Was there significant variation between the Head and Tail groups as a whole? ANOVA Source of Variation SS df MS F P-value F crit Between Groups 0.762766 1 16.28121 0.00034 4.170877 Within Groups 1.405483 30 0.046849 Total 2.168249 31  

Further Analysis Question P-value Significance Did UV have an effect w/ VC present in heads? 0.06 Not Significant Did VC have an effect w/ UV present in heads? 0.63 Did UV have an effect w/o VC present in heads? 0.88 Did VC have an effect w/o UV present in heads? 0.03 Significant Did UV have an effect w/ VC present in tails? 0.34 Did VC have an effect w/ UV present in tails? 0.77 Did UV have an effect w/o VC present in tails? Did VC have an effect w/o UV present in tails? 0.70

Conclusions Hypothesis #1: Because the p-value received from the data was <.05, the null hypothesis was rejected, and so the alternative hypothesis was accepted. There appeared to be significant variation between the head and tail groups’ changes in body mass. Hypothesis #2: Because all of the p-values (excluding set 4) received from the head and tail data sets respectively were >.05, the null hypothesis was generally accepted, and so the alternative hypothesis was generally rejected. The presence of neither UV radiation nor Vitamin C appeared to have a significant effect on the change in body mass of either the head or tail groups.

Limitations and Extensions Only one Vitamin C concentration and one UV exposure time were used. Worms may have lost large volumes of blood or have been exposed to infection when cut in half. Other methods of Vitamin C action besides its anti-oxidant properties may have been at work Other methods of UV damage besides free radical production may have been present. Extensions Allow for additional exposure times and VC concentrations. Keep worms in more controlled, nutritious environment. More accurate gram scale. Additional replicates.

References “Anti-Oxidants and Free Radicals”. Rice.edu. <http://www.rice.edu/~jenky/sports/antiox.html>. “Lumbricus Terrestris”. Lander.edu. <http://webs.lander.edu/rsfox/invertebrates/lumbric us.html>. “Oxidative Stress”. University of Valencia. <http://www.uv.es/frag/oxidative_stress.htm>. “Ultra-Violet Radiation”. Hps.org. <http://www.hps.org/hpspublications/articles/uv.htm l>. “Vitamin C”. Wikipedia.org. <http://en.wikipedia.org/wiki/Vitamin_C>.