The Effects of Creatine on Lumbriculus variegatus and its Biological Characteristics

By Joshua C. Morgan

Introduction/Background

Creatine is an organic substance that is synthesized naturally in the human body in trace amount.1 It has proven to be capable of enhancing muscular performance, and has accordingly been implemented by many athletes who desire the effects it presents.2 Most take it in the form of dietary supplements, which are necessary to maximize the chemical’s effect as it is not found in substantial amounts in human muscles. As expected, creatine has been the subject of many experiments in order for its effects to be observed adequately.

I plan to contribute to the aggregate scientific research on the subject of creatine’s use as a performance enhancer through a series of experiments with California blackworms, or Lumbriculus variegatus. Due to the accessibility and inexpensiveness of this species, it will be relatively convenient to access them and carry out these studies. In recent years, it has been realized in the scientific community that the California blackworm species is appropriate for carrying out a variety of experiments in physiological and biological areas because of certain physical characteristics, including its observable pulsations and regeneration capabilities. 3

The experiments that I will be performing will test the pulsation rate and regeneration abilities of the worms as dependent variables. The independent variable will be the concentration of the creatine solutions that the worms will be exposed to. In addition, a control group will be exposed to spring water that is synonymous with that in its environment. All other factors will remain constants, or be at least as similar as possible.

Literature Review

The species Lumbriculus variegatus represents a staple in research based on the introduction of an unfamiliar substance into the environment of an organism and that based on comparing and contrasting the effects of different types of environments to make conclusions based on experimental results. For example, it has been determined that an alteration of sediment types in the species’ environment produces an observable effect on many of its physiological behaviors, including many that I plan to study.4 In addition, it has been found that the introduction of boric acid (H₃B) into the environment of the worms slows down the regeneration of the species in question.5 If such a correlation can be found in this experiment, it is likely that a correlation between this factor and the introduction of creatine, although my hypothesis declares a positive correlation in contrast to the negative one found with the boric acid, will also be observable. While this research renders my hypothesis testable and my experimental design plausible, it should be noted that my experiment will remain original as it involves a different type of chemical, one that is organic.

Through a plethora of experimental research, the effects of creatine on organisms have been established to some degree. The substance has been used frequently by athletes to enhance their performance through increasing muscle strength and promoting energy sustenance. A previous experiment, however, demonstrated that creatine did not significantly affect the threshold level in a group of young men; these results negated a hypothesis that many had previously supported.6 At the previous time, much remains unknown about how dramatically creatine can affect human performance. Its benefits are primarily short term, and it has been determined by an experiment that it had a great effect on people performing rapid exercises that involved intense workout while it had a minimal effect on a group of people performing endurance exercises. The series of experiments that I hope to perform

will test effects relating to the overall energy of the worms and their cardiac activities with the addition of creatine. All of the research based on creatine to date needs supplementation by a study of its effects on another organism, and I hope to provide it.

Hypotheses

If the California blackworms are significantly sensitive to creatine solutions, then their pulsation rates will increase in a direct relationship with the solution concentration and the exposure time.

If the California blackworms are significantly sensitive to creatine solutions, then they will grow longer appendages, when cut and left to regenerate for a period of time, than if they are left in spring water for the same amount of time.

Methods

The first experiment I will perform is designed to determine the effect of creatine on the pulsation rate of Lumbriculus variegatus. I will make two creatine solutions, one with .4 mg/100 mLand one with .6 mg/100 mL, and a control group of the spring water that the worms are normally exposed to and put them in separate Petri dishes. Upon being placed in one of the groups, each worm will be timed individually for a period of 10 or 20 minutes; by doing this, we will also be able to determine if the exposure time to the solution has any effect on the worms. Essentially, 36 worms will be divided into six groups with six worms each in this experiment: two control groups with different exposure times and two sets of worms from each of the experimental groups. After a worm has been in its solution for the appropriate amount of time, it will be placed on a piece of filter paper and observed under a microscope. I will focus on one spot of the worm and count the number of times a rush of blood passes through it in a minute to get its pulsation rate. After this process has been carried out for every worm, I will calculate necessary statistical data, including the mean and standard deviation. I will then carry out T-tests to compare all of the groups and determine whether my hypothesis has been confirmed or negated.

The second experiment will determine the effect of creatine on regeneration of Lumbriculus variegatus. For this experiment, I will use the spring water control group and two creatine solutions, one with .2 mg of creatine for every 100 mL of spring water and one with .4 mg/ 100 mL. Each worm used in the experiment will be divided into an anterior and a posterior portion. Each portion will be further segmented so that it is eight bands long, a measurement that can easily be observed through a microscope. Then the segments will be placed into separate wells based on the portion of the original worm that they represent and the test group that they will be a part of. In all, 12 worms and 24 segments will be used in each of the three test groups for a total of 36 worms and 72 segments. In order to stimulate the worms’ original environment, a small piece of brown paper will be placed at the bottom of each well and the wells will be placed in a closed cabinet so that the worms will be subjected to darkness. One week later, I will take the wells out of the closet and observe the worms. Under the microscope, I will count the new segments of the worm. This will be relatively easy to do as the regenerated portion is must lighter than the original portion. After collecting all of this data, I will compute the same statistical data as I did in the first experiment and use it to conclude the validity of my original hypothesis.

Timeline

All of the following will take place in 2008, in the laboratory sessions of Dr. Linda Davis’s biology course.

Thursday, June 19Practice the pulsation counting technique that will be necessary in the performance of the pulsation experiment

Thursday, June 26Perform the pulsation rate experiment Record resultsMake conclusion

Thursday, July 3Set up the design for the regeneration experiment

Thursday, July 10Record the results of the regeneration experimentMake conclusion

Thursday, July 17Organize the results of the two experiments and put them together in a professional format

Discussion/Problems

1 [Anonymous]. Creatine [Internet]. Medline Plus web site. 2008. Available from: http://www.nlm.nih.gov/medlineplus/druginfo/natural/patient-creatine.html Accessed 2008 July 7.

2 Jenkins MA. Creatine Supplement in Athletes-Review [Internet]. Sports MedWeb web site. 1998. Available from: http://www.rice.edu/~jenky/sports/creatine.html Accessed 2008 July 9.

3 Drewes C. Lumbriculus variegatus: A Biology Profile [Internet]. Iowa State University web site. 2004. Available from: http://www.eeob.iastate.edu/faculty/DrewesC/htdocs/Lvgen4.htm Accessed 2008 July 9.

4 Sardo, Soares, Gerhardt. Behavior, Growth, and Reproduction of Lumbriculus variegatus (Oligochaetae) in Different Sediment Types. Human and Ecological Risk Assessment 2007;13:519-526, 8p. Available from: Academic Search Premier [online database] http://web.ebscohost.com/ Accessed 2008 July 1.

5 Martinez VG, Zoran MJ, Reddy PK. Asexual Reproduction and Segmental Regeneration, but not Morphallaxus, are Inhabited by Boric Acid in Lumbriculus variegatus. Hydrobiologia 2006. Available from: Academic Search Premier [online database] http://web.ebscohost.com/ Accessed 2008 July 2.

6 Walter AA, Smith AE, Herda TJ, Ryan ED, Moon JR, Cramer JT, Stout JR. Effects of Creatine Loading on Electromyographic Fatigue Threshold in Cycle Ergometry in College-Age Men. International Journal of Sport Nutrition and Exercise Metabolism 2008;18:142-151, 10p. Available from: Academic Search Premier [online database] http://web.ebscohost.com/ Accessed 2008 July 1.

Creatine represents a substance that has often been utilized by people to gain a biological advantage in the physical activities they pursue. It has been the subject of some controversy in the scientific community, as many have theorized that it may have some limitations in the areas that it is believed to be useful. By performing a study on a biological counterpart of humans, the species Lumbriculus variegatus, I hope to determine just how widespread creatine’s effects actually are.

However, some of the experimental methods I will be applying may be questionable in terms of the validity of the conclusions I will make. In the setting in which I will perform all of my experiments, some of the variables that should remain constant may be uncontrollable to some degree. For example, the temperatures of the different solutions should be relatively similar, but the addition of different amounts of creatine may misconstrue this necessary equilibrium somewhat. I was also only able to theorize the appropriate concentration of the solution because I used a ratio of daily intake recommended for humans in terms of mass in grams. Due to the different biological makeup of the organism that I will perform the experiment with, this ratio may not be as appropriate as it should be. It is therefore possible that either the worms will not survive due to overdose or the solution will so dilute that it may hinder the accuracy of my conclusions. Either situation could catalyze a discrepancy between my experimental conclusion and reality. In addition, my experiment will be carried out under a predetermined organized schedule that is defined by a limited period of time in which I must perform it in its entirety. I would probably be able to perform more tests and synthesize more informative results if I were to perform the experiments under a prolonged and more flexible schedule.

Sources Cited