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Dugesia dorotocephala Regeneration Proficiency in Acidic and Neutral Solutions

1. INTRODUCTION

Dugesia dorotocephala are members of the phylum Platyhelminthes and are commonly referred to as planaria or flatworms. Planaria have variations of the ectoderm, mesoderm, and endoderm as well as the absence of organ containing cavities, called coelom, giving them a solid body (Hyman 1951). The presence of three germ layers, also known as triploblastic, leads to the production of organs and tissues within the planaria. Photoreceptors assist in helping the planaria move away from light, as well as sensory organs allowing for taxis to specific detections. Sexual reproduction is limited to cross-fertilization through hermaphroditism by switching sexes, and asexually through transverse binary and fission (Hyman 1951), the latter being the most important element of planaria. Planaria regeneration is highly important in understanding the process of cell proliferation and using the data for future advances and a greater understanding of cell regeneration.

D. dorotocephala are very unique by their ability to regenerate body parts by cell regrowth to restore their original conditions. The planaria can be split bilaterally, decapitated, or bisected, and each half will regenerate into two individual organisms (Figure 1). Upon trauma in the planaria, mitosis creates cell proliferation at the wound and leads towards the construction of  epithelial and mesenchymal, or blastema, and creates epimorphic regeneration. (Sánchez, Alvarado, & Newmark 1998). The blastema formation provides the basis of regeneration while relying on morphallaxis to restore the symmetry of the planaria.

In this experiment, the regeneration of D. dorotocephala in varying pH solutions will be observed in order to determine if regeneration is more successful in higher or lower pH solutions. By performing this experiment on planaria, it allows for a more advanced understanding in the process of cell proliferation and regeneration in general and for future discoveries. The higher pH solution of 7.5 will have the most successful rate of full regeneration in the planaria by giving the planaria a fairly neutral solution to perform their living systems. Partial head individuals will show the strongest and complete forms of regeneration of their tail counterpart, with tail individuals having long regeneration times and less likely chance of completion due to the complexity of the regenerating head.

Figure 1. A) Depicts a whole planarian with lines presenting each type of cut that can be performed. B) Planaria bisected, giving two individual organisms for regrowth. C) Planaria head with partial regeneration of a tail. D) Planaria tail with partial regeneration of a head.

2. MATERIALS AND METHODS

Three agar plates contain the samples of 4 planaria with one per divided section (Figure 2). Three planaria were bisected in half (Figure 1B) and the fourth was left intact as our control, leaving a total of 6 regeneration samples and a total of 4 replicates for each treatment overall. After the planaria had been cut and all water was removed from the agar plate via pipette,  each section received 3-4mL of three pH solutions. Agar plates 1-3 received pH solutions of 5.5, 6.5, and 7.5 respectively.  The samples were then incubated at 20°C for 7 days and removed for observation. During observation, the pH solutions were removed from each sample and replaced with identical pH solutions of 5.5, 6.5, and 7.5.  Planaria were placed back into their sections if they moved and results were recorded as well as any errors or deaths. After incubating at 20°C for 7 more days, the plates were removed and observed to determine the effectiveness each pH solutions had on regeneration. Each pH sample was looked at under the dissecting microscope in order to observe and record partial or full regeneration along with any deaths and errors.

Figure 2. Agar plate sample preparation with four planaria in each section. Three will be cut an observed, with one as the control group all in the same pH solution.

3. RESULTS

After examining the results on the D. dorotocephala regeneration samples, it was discovered that each pH solution had varying effectiveness in assisting in the regeneration of Planaria. In Group 1, the sample with a pH of 5.5 contained 6 partially regenerated Planaria, pH 6.5 had 5 partial planaria, and pH 7.5 sample had 6 partial planaria left over. The sample of pH 6.5 had one death thus leading to only 5 out of 6 planaria being partially regenerated. The combined average results of all samples within the lab saw 21/24 partial and 3/24 full regenerations in the pH 5.5 solution; 19/24 partial and 3/24 full regenerations in the pH 6.5 solution; and 20/24 partial and 4/24 full regenerations in the pH 7.5 solution (Figure 3). Figure 2 and Figure 4 depict the black planaria before and after regeneration. Overall, the pH 5.5 solution saw the largest partial regeneration at 87% and the pH 7.5 solution saw the greatest full regeneration at 17% (Figure 3).

Figure 3. Average results in percentage reflecting partial and full regeneration v. pH of the solution at 25°C,

4. DISCUSSION AND CONCLUSION

After completing this experiment it was found that the hypothesis was supported and regeneration of D. dorotocephala is optimal in a solution of pH 7.5 (Figure 3). The plates containing the solutions of pH 5.5 and 6.5 showed similar results of partial and full regeneration, with differing numbers due to two deaths in the pH of 6.5 solution (Figure 3). Given that there were no deaths in the entire pH 5.5 solution and the 6.5 control, the deaths must have resulted from another cause. It can be assumed that the lower pH does not affect the lives of the planaria short-term, but may have larger effects on the planaria longer-term or with a more acidic solution. With almost 20% full regeneration and 83% partial regeneration in the 7.5 solution, it can be concluded that D. dorotocephala favors neutral solutions over the slightly more acidic solutions for regeneration.

Regeneration from an individual head or individual tail also varied. From the 6 samples in each plate, the 3 head organisms experienced the largest amount of growth and completeness of full regeneration of a tail compared to the tail regeneration of a head. A planaria head from the pH 7.5 sample and a planaria tail from the pH 6.5 sample were compared and the pH 7.5 head experienced much larger amounts of tail growth being almost to completion, where as the pH 6.5 tail had much more head growth to go (Figure 4). Several articles support these findings with similar experiments done through the use of Planaria and head vs. tail regeneration. Mechanisms that determine the anterior from posterior and the replacement of the specific body parts are unknown (Gurley, Rink, & Alvarado 2008), but the use of RNA interference enables manipulation of these genes. These three individuals investigated the effect that ß-catenin has on the effects of regeneration a head or tail. In their experiment the RNA interference of ß-catenin results in the regeneration of a tail at anterior wounds, thus specifying ß-catenin as a molecular switch during regeneration that controls the presence of either a head or tail (Gurley, Rink, & Alvarado 2008). It was concluded that the head pieces were more successful in regenerating a tail than the tail pieces could regenerate a head. These findings support our observance of regeneration in D. dorotocephala, and help us understand the action of homeotic genes and how these genes can be altered to produce and generate varying body structures in other organisms.

Figure 4. A) Planaria head regenerating a tail in pH 7.5 at 25°C B) Planaria tail regenerating a head in pH 6.5 at 25°C

Time restraints and pH range also put a limitations on this experiment. Had the samples been available for observation over a longer period than two weeks, the data would have been much more precise in accounting for the complete and partial planaria. The pH range of the solutions was also fairly limited in this experiment from only 5.5 to 7.5. By testing the planaria regeneration at higher or lower ranges of pH solutions, the data would have been much more diverse and would portray the environments that planaria could not regenerate in or even survive in. Future experiments could include a much larger data sample at higher and lower pHs and also the integration of temperature and other variables to gain a better understanding of the absolute conditions that cell regeneration is most proficient.

By performing these experiments at varying conditions, it allows for discovery and advances in cell regeneration. Studying planaria and their methods of cell proliferation through blastema and morphallaxis assists in future medical advances.  Regeneration in not only planaria but also other organisms and animals is an amazing feat that can help us understand stem cell transplants and the formation of cell regeneration to eventually be used in humans.

REFERENCES

Gurley, K., Rink, J., & Alvarado, A. (2008). ß-Catenin Defines Head Versus Tail Identity During Planaria Regeneration and Homeostasis. Science, 319(5861), 323-327.

Hyman LH. (1951). The Invertebrates: Platy- helminthes and Rhynchocoela the acoelo- mate bilateia. McGraw-Hill. 2, 22;41

Sánchez Alvarado A, Newmark PA. (1998). The use of planaria to dissect the molecular ba- sis of metazoan regeneration. Wound Rep. Regen. 6, 415.

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