Alvarado, Alejandro Sánchez and Peter W. Reddien. FUNDAMENTALS OF PLANARIAN REGENERATION Vol. 20: 725-757 (Volume publication date November 2004) http://arjournals.annualreviews.org/doi/abs/10.1146/annurev.cellbio.20.010403.095114

Carolina Protozoa and Invertebrates Manual, 1977. http://web.esc20.net/livsci/pdf/Handouts/LMP-13%20Planaria.pdf

The Columbia Electronic Encyclopedia, 6th ed. 2007, Columbia University Press."Planarian”. http://www.infoplease.com/ce6/sci/A0839279.html

Sparling, Brien. Ultraviolet radiation. http://www.nas.nasa.gov/About/Education/Ozone/radiation.html

Increased exposure to UV light appears to impair proper blastema formation and morphallaxis in Dugesia tigrina. The number of fully regenerated planarians increased as the UV exposure decreased, which demonstrated that UV has the ability to damage cell structures and impair the repair of cells. Amplified levels of UV caused more planarians to die, which demonstrated that they could no longer regenerate as expected. This is most likely due to cell damage at a nuclear level, which would prevent the proper coding for proteins and lipids required to rebuild the planarian at the damaged site.

Dugesia tigrina is particularly sensitive to light and in its natural habitat is generally found in dark environments, so it is possible that any exposure to light, including exposure to light during observations and rotations, could have caused environmental stress to the planarians and increased their inability to fully regenerate. The osmolarity of the distilled water that was used in the experiment may have been different from the osmolarity found in the planarian’s natural environment, and this could have also affected the way in which the planarians regenerated.

Future research on Dugesia tigrina may include larger sample sizes, which would enable more accurate results to be recorded, and increase the accuracy of any statistical tests performed. A larger sample size would better represent the population as a whole. An effort to reduce any other outside effects of regeneration, such as reducing light sources other than the UV light source, may also help to yield more accurate results.

A Chi-square test was performed for the death rates of planarians to determine whether there was a significant difference of regeneration depending on differing levels of UV radiation exposure. The calculated value was determined to be 12.22, and at a degree of freedom of 5 and a probability of 5% the critical Chi-square value was 11.07. This demonstrated that there was a significant difference in the rate of death of brown planarians based on ultraviolet exposure. The data also indicated a trend that death rates increased as UV exposure increased.

The percentages of planarians based on full regeneration, partial regeneration, and death demonstrate that as UV exposure increases the fully regenerated planarians decrease in percent and the death rates of planarians increase. Also, as UV exposure increased, the blastema failed to form correctly, and improper cell division was found more frequently at the damaged site. One day following the cutting of the planarians the blastema was beginning to form in the control and the 6-hour test subjects, as seen in Figures 2.A and 2.B, but in the 12-hour subject the blastema region had failed to properly form, as seen in Figure 2.C. Planarians that were exposed to 12 hours of exposure also demonstrated an incomplete blastema, which led to the pharynx being ejected from the mouth, as in Figures 2.F and 2.H. In Figure 2.I the failure of the blastema to form resulted in incomplete regeneration of the head of the planarian, as can be seen by the indentation in the epithelium.

The goal of the experiment was to determine what effect exposure to variable levels of ultraviolet light had on the regeneration of Dugesia tigrina, the brown planarian.

Planarians have the ability to regenerate completely into two individuals after being bilaterally cut. This is due in part by neoblasts, groups of stem cells scattered throughout the organism’s tissue, and the process of morphallaxis which remodels pre-existing tissue to restore symmetry and function. After sustaining injury, somatic cells around the neoblasts at the blastema, a structure that forms at the wound site, are consumed to complete the regeneration process. This accounts for the lost mass of the individuals while simultaneously permitting their complete return to normal morphology (Alvarado and Reddien 2004).

The species used in the study, Dugesia tigrina (brown planarian), is a freshwater inhabiting free-living flatworm native to the North American continent. Dugesia tigrina is of the Phylum: Platyhelminthes, Class: Turbellaria, and Order: Tricladida. Known for their regenerative abilities, the species has been the subject of scientific study for decades. Brown planarians are hermaphroditic and prefer to reproduce sexually, but are capable of asexual reproduction by splitting into two individuals from the middle. The length of these animals varies between 1/8th of an inch and 1 inch. Brown planarians are scavenging carnivores that digest food externally using their pharynx before withdrawing it into their gut. Two photoreceptors on the head aid planarians in avoiding direct light ("Planarian,” 2007).

What we call ultraviolet light represents a wavelength band (400-150nm) of the electromagnetic spectrum that has a frequency too high for the human eye to perceive but is weaker than X-rays (Sparling, 2001). Ultraviolet radiation can penetrate cells and directly damage the genetic material in the nucleus, raising the possibility for mutation or growth disorders (cancer) to develop in addition to cell death. DNA absorbs UV-B radiation, and the energy can dislodge nitrogen bases from their configuration or otherwise break the strands. Ultraviolet light is generated by the sun, but is also artificially produced for use in tanning beds and sterilizing laboratory equipment.

In the pilot experiments, attempts were made to introduce variable concentrations of Aloe Vera juice to the water-filled fingerbowls that housed the planarians. A concentration gradient ranging from 50% to 5% Aloe Vera juice resulted in 100% mortality of all subjects. A second attempt featured a concentration range from 5% to 1%, with majority mortality. It was at this point that the Aloe Vera juice was removed from the experiment’s design.

The UV exposure ranges were determined also within a pilot experiment, which demonstrated that after 18 or more hours of exposure, the subjects experienced 100% mortality. The upper threshold of 12 hours was later the benchmark for exposure times in the final experiment.

Distilled water was added to each of the 6 ~100mL fingerbowls, nearly filling them. The planarians were pulled out of the specimen jar via pipette and squirted onto a glass slide, where they were cut in half with a scalpel. The severed head ends were placed in one bowl, while the tails were placed in another. This process was repeated two more times, with a total of 10 planarian halves per fingerbowl. After labeling the bowls and sealing the tops with clear polyethylene plastic wrap, they were placed on a rack in an environmental chamber as in Figure 1.

The environmental chamber was prepped to hold a constant 19 degrees Celsius (Carolina, 1977). The UV lamps were placed on the top rack, with another rack roughly six inches beneath it as a holding area for the planarian bowls undergoing exposure. The chamber’s internal fluorescent lights were turned off, except for the one on the bottom rack. A black garbage bag was wrapped around the rack to shield the exposed planarians beneath from further exposure. The control specimen fingerbowl was placed on the bottom of the chamber at experiment start. The remaining five bowls were placed on the holding rack. After a time passage increment of one hour, a labeled specimen fingerbowl would be moved to the bottom of the chamber. This was to be repeated at the intervals of 3, 6, 9, and 12 hours, until all specimen bowls were on the bottom. Exposure was not repeated. On the first day (after exposure), as well as the fourth and seventh days, photographs were taken of one specimen from each bowl to observe the extent of the subject’s regeneration.

Figure 1

Figure 2.CFigure 2.BFigure 2.A

Figure 2.G

Figure 2.FFigure 2.EFigure 2.D

Figure 2.IFigure 2.H

Figure 3