Comparison of Planarian (Dugesia tigrina) Regeneration Rate in Caffeine and Aloe vera Solutions
Diane Kwon and Joel Tatum
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
Dugesia tigrina, commonly known as planarians, are triploblastic flatworms that have unique regenerative abilities, which make them an ideal specimen for investigating regeneration and stem cell biology. Planarians can regenerate into new and complete organisms after a staggering 279 cuts. Caffeine is a stimulant derived from plants, which, as some studies reveal, may inhibit cell growth or proliferation. Aloe vera is a succulent plant that has fleshy leaves containing mucilage tissue (or Aloe vera gel), which contains polysaccharides that stimulate skin growth and healing. For this study, planarians were transversely cut and regeneration rates were observed for 23 days. Planarians (n=30) were divided into three groups: spring water (control group), 0.00001 M caffeine solution, and Aloe vera solution. By observing changes in the transected tail portion of the planarians exposed to nonlethal concentrations of caffeine and Aloe vera, it was predicted the planarian regeneration rate would decrease compared to that of the control group in spring water. There is no statistical difference in planarian regeneration rate from initial cut to eyespot differentiation at stage 4 of regeneration, based upon solution medium as determined by single-factor ANOVA (F(2,25)=2.919, p=0.0726, Bonferroni Correction).
Introduction
Dugesia tigrina, commonly known as planarians, are part of the phylum Platyhelminthes, which includes flatworms such as planarians but also parasitic flukes and tapeworms. Platyhelminthes are triploblastic (composed of three germ layers: ectoderm, mesoderm, and endoderm), acoelomate (no true body cavity), and bilaterally symmetrical. Platyhelminthes in the class Turbellaria are free-living freshwater animals that exhibit anteriorly located sense organs and a well-developed muscular system.
Planarians have a unique ability to regenerate into a complete organism after multiple cuts. These organisms possess the ability to regenerate an entire organism from small body fragments from a population of stem cells called neoblasts (Baguñà, 1989). When a cut is inflicted upon a planarian, neoblasts at and around the wound site begin to divide rapidly, and the generation of new tissue at the wound site via cell proliferation is called blastema formation. The blastema is characterized as a colorless area at the tip of the growing, regenerating tissue. Cells in the blastema differentiate over a period of several days to replace missing body structures (Reddien and Sánchez Alvarado, 2004).
Planarians are studied for their regenerative abilities after being cut. A transverse cut anterior to the pharynx results in a transected tail portion with a mouth, which is located posterior to the pharynx. The mouth also serves as the anus and during regeneration allows planarians to excrete waste and regenerate normally.
Caffeine, an alkaloid part of a group of compounds known as methylxanthines occurring naturally in many plants, can be found in many popular beverages, including cocoa, tea and coffee. Although caffeine is typically used to combat sleepiness, some studies suggest that caffeine may inhibit the growth and proliferation of cells. One study found that caffeine has an antiproliferative effect on human liver cancer cells. Caffeine inhibits the growth of a variety of cancer cells through cell cycle arrest at G0/G1 phase and the induction of apoptosis (Okana, et al., 2008).
Aloe vera is a succulent plant that grows readily in hot and arid regions. Aloe vera contains important compounds, such as several sugars (glucose, mannose, and cellulose) and various enzymes (oxidase, amylase, and catalase). It also contains vitamins B1, B2, B6, C, E, and folic acid and minerals like calcium, sodium, magnesium, zinc, copper, and chrome (Hashemi, et al., 2015). The center of the thick, fleshy leaves are lined with mucilage tissue, commonly known as Aloe vera gel. Aloe vera gel is used for cosmetic and medicinal purposes. The mucilage tissue contains glucomannan enriched in polysaccharides that stimulate skin growth and healing.
By observing changes in the transected tail portion of the planarians exposed to nonlethal concentrations of 0.00001 M caffeine and Aloe vera, it is predicted the planarian regeneration rate will decrease compared to that of the control group in spring water. A study performed by a student at East Tennessee State University researched the effects of caffeine and ethanol on the regeneration rates in planarians. It was discovered that all concentrations of caffeine greater than 0.00001 M were toxic to all or nearly all the specimens (Collins, 2007). To prevent a terminal study, a 0.00001 M concentration caffeine solution was used and the same mass of Aloe was utilized to allow comparability between the two solutions.
Because feeding disturbs planarian regeneration, planarians were not fed throughout the duration of the experiment. As a head grows back, cells in the tail end will self-destruct and provide the regenerating animal with the energy needed to survive. Over time, the tail portion will shrink to a proportion that exactly matches that of the regenerating head. Once the planarian is whole again, it will begin to feed and grow back to a normal size. Researchers are still examining the proportion-readjustment regeneration of planarians (Exploratorium 2015).
Materials and Methods
Brown planarians (Dugesia tigrina) from Carolina Biological Supplies were used for this experiment (n=30). The planarians were maintained at ambient room temperatures of approximately 20°C at Saddleback College SM 244 in Arrowhead spring water. (It is crucial to use spring water, as tap water and deionized water may kill the planarians.) On October 23, 2015, three days before experimentation began, the planarians were fed a pea-sized amount of hard-boiled egg for 30 minutes (10:17 AM to 10:49 AM). Feeding was limited to approximately 30 minutes to prevent overfeeding.
The experiment required two solutions: 0.00001 M caffeine solution (caffeine molar mass=191.19 g/mol) and an Aloe vera solution with an equivalent mass of Aloe as that used by the caffeine, which allowed the substances to be comparable. Spring water served as the solvent for both solutions. The mass of the solid substances needed for each solution was determined using stoichiometry. Below is the calculation used to determine the amount of solid caffeine required to achieve a 0.00001 M caffeine solution (lab grade caffeine provided by Saddleback College):
On October 26, 2015, experimentation began with preparing the solutions and cutting the planarians. Thirty planarians were divided into three groups of ten planaria each: spring water (control group), caffeine solution, and Aloe vera solution. Each planarian was individually separated into Petri dishes (6015mm) labeled W1 though W10 for spring water, C1 through C10 for the caffeine solution, and A1 through A10 for the Aloe solution.
The caffeine and Aloe solutions were prepared. Using a milligram balance, 0.0019 g of solid caffeine was weighed and added to 1 L of spring water. A magnetic stir bar was added to the beaker, which was then set on a stir/hot plate at stir speed 7 and heat level 4 to dissolve the caffeine. The solution was mixed for 30 minutes until the caffeine had visibly dissolved. One Aloe leaf was cut from the plant, and the interior gel-like sap was extracted. Utilizing a pipet, the aqueous part of the gel was separated into a clean 50 mL beaker. From this, 0.0019 g of the aqueous Aloe extraction were obtained and added to 1 L of spring water. Following the same procedure as the caffeine solution preparation, a magnetic stir bar was added to the beaker, which was then placed on the stir/hot plate at stir speed 7 and heat level 4. After 30 minutes on the stir/hot plate, the Aloe appeared to dissolve completely. Both solutions were then transferred to appropriately labeled 1 L dark storage bottles with airtight lids.
The ten Petri dishes labeled W1 through W10 were half filled with spring water, the ten Petri dishes labeled C1 through C10 were half filled with the caffeine solution, and the ten Petri dishes labeled A1 through A10 were half filled with the Aloe solution. The solutions were stored at 20°C for periodic solution replacement to maintain a clean environment for the planarians.
Using a new Bard Parker carbon steel #15 surgical scalpel blade, transverse cuts were made on each specimen anterior to the pharynx, ensuring that each cut resulted in two sections to prevent fusing at the incision site. After the incision, each planarian was placed into their respective Petri dish containing the appropriate solution. Photographs were taken of each of planarian from all three groups to document the regeneration process. The planarians were stored in the dimly lit biology hallway adjacent to SM 244 at approximately 20°C for later observation.
For 23 days (October 26, 2015 to November 18, 2015), planarians were photographed and observed for four major stages of head regeneration: wound healing (stage 1), blastema development (stage 2), growth (stage 3), and differentiation (stage 4). Results of experimentation are based on the observation of the tail portion after a transverse cut, as the regeneration of the head is more distinct than the regeneration of the tail. The days taken to reach stages 2 through 4 of regeneration were noted.
Figure 1. Stage 1 (wound healing) of planarian regeneration. During this stage, cells rapidly form around the wound site. Regeneration is not visible at this stage.
Figure 2.Stage 2 (blastema development) of planarian regeneration. During this stage, a rounded blastema forms at the site of the cut. However, distinct head characteristics are not apparent, as the cells have not differentiated yet.
Figure 3. Stage 3 (growth) of planarian regeneration. During this stage, the blastema has proliferated, and the distinct arrowhead shape of the head is visible.
Figure 4. Stage 4 (differentiation) of planarian regeneration. During this stage, the cells are becoming specialized, which is demonstrated by the development of eye spots.
Results
The planarians exposed to Aloe vera had the fastest overall average regeneration rate (Figures 5 and 6). Overall regeneration rate is determined by the amount of days it took each specimen to reach stage 4 after being cut. The Aloe group reached this stage at an average 18.56% faster than the control group and 2.51% faster than the planarians exposed to caffeine. The caffeine-exposed planarians were only 2.51% slower than the aloe group but were 16.47% faster than the control group. The control group containing only spring water had the slowest regeneration rate: 18.56% slower than the Aloe group and 16.47% slower than the caffeine group.
Table 1.Average days to the stages of blastema development, growth, and differentiation for each tested group.
Stage 2: Blastema Development (days) / Stage 3: Growth (days) / Stage 4: Differentiation (days)Control / 7 / 13.22 / 18.56
Caffeine / 6.7 / 10.9 / 15.5
Aloe / 7.33 / 11.22 / 15.11
Figure 6. Line chart showing visual progress between stages 1 and 4 for planarian regeneration amongst each of the three tested groups (spring water, caffeine exposed, and Aloe exposed) after being cut. There is no statistically significant difference in planarian regeneration rate from initial cut to stage 4, based on the solution as determined by single-factor ANOVA (F(2,25)=2.919, p=0.0726).
Figure 7. Column chart comparing time elapsed (days) from initial cut to stages 2-4 of planarian regeneration for the three tested groups (spring water, caffeine exposed, and Aloe exposed). There is no statistically significant difference in planarian regeneration rate from initial cut to stage 4, based on the solution as determined by single-factor ANOVA (F(2,25)=2.919, p=0.0726). Error bars are ± SEM.
Two of the specimens (W10 of the spring water group and A2 of the Aloe group) were excluded from the results. W10 and A2 were missing tail regions for observation the day after incisions and data could not be obtained for their regeneration.
Regeneration rates in Dugesia trigina varied at stages 2 through 4 for each of the groups. At stage 2 (blastema development), the average rate for the caffeine group (6.7 days) was lower than both the spring water group (7.0 days) and the Aloe group (7.33 days). At stage 3 (growth), the caffeine group again had the quickest average (10.9 days), but the Aloe group slowed (11.22 days) while the spring water group showed the slowest regeneration (13.22 days). By stage 4 (differentiation), the Aloe group had the fastest regeneration rate (15.11 days) followed closely by the caffeine group (15.5 days) with the spring water group displaying the slowest rate (18.56 days). Overall, the Aloe group showed the fastest average regeneration rate (18.56% faster than the spring water group and 2.51% faster than the caffeine group), followed by the caffeine group (2.51% slower than the Aloe group but 16.47% faster than the spring water group). The spring water group showed the slowest rate (18.56% slower than the Aloe group and 16.47% slower than the caffeine group).
The following tables summarize the data results:
Table 2.ANOVA: Single Factor
Groups / Count / Sum / Average / VarianceSpring Water (Control) / 9 / 167 / 18.55556 / 9.777778
Caffeine / 10 / 155 / 15.5 / 16.5
Aloe Vera / 9 / 136 / 15.11111 / 6.361111
Table 3.ANOVA results
Source of Variation / SS / df / MS / F / P-value / F critBetween Groups / 64.81746032 / 2 / 32.40873 / 2.918537 / 0.072583 / 3.38519
Within Groups / 277.6111111 / 25 / 11.10444
Total / 342.4285714 / 27
Table 4.Bonferroni correction post-hoc test confidence intervals
Comparison / Mean 1 - Mean 2 / 98.33% Cl of Difference1: Spring Water vs Caffeine / +3.05556 / - 1.58946 to + 7.70058
2: Spring Water vs Aloe / +3.44445 / - 1.32124 to + 8.21014
3: Caffeine vs Aloe / -0.01111 / - 4.65613 to + 4.63391
Table 5.Statistical significance
Comparison / Significant? (P <0.0167?) / t1: Spring Water vs Caffeine / No / 1.996
2: Spring Water vs Aloe / No / 2.193
3: Caffeine vs Aloe / No / 0.007
A single-factor ANOVA determined no statistical difference in overall regeneration between the three tested groups (p=0.0726). A Bonferroni post-hoc correction test was applied to determine if any statistical difference lied between the three tested groups. With the Bonferroni correction test taking number of groups into consideration (n=3), a p-value below 0.0167 signifies a statistical difference. The Bonferroni corrected post-hoc test resulted in no significant statistical difference between each of the three groups.
Discussion
With the obtained p-value greater than 0.05 from the ANOVA, the null hypothesis is accepted, which indicates that there is no significant difference in the means for all three tested groups. Additionally, the Bonferroni correction compares each individual group to one another and found that the means between each of the three groups was not significantly different from one to another. Thus, this study found that there is no statistical difference in planarian regeneration rates exposed to spring water, caffeine, or Aloe vera.
Aloe vera, of the pharmaceutical herbs, has been used in the treatment of many infections and dermatologic condition. The influence of Aloe vera gel on dermal wound healing is evaluated. A study documented the effects of two different dressings for wound-healing management on full-faced dermabrasion patients. After 48 hours with the Aloe vera dressing, intense vasoconstriction and reduction in edema was noted; by the fourth day, less exudate and crusting were apparent. By the fifth day, reepithelialization was complete to 90% on the Aloe side compared with 40–50% on the control side. The study found that wound healing was approximately 72 hours faster at the Aloe side (Vogler and Ernst, 1999).
Rate of wound healing is affected by various factors, such as nutrition, vitamins, steroidal hormones, oxygen, and environmental factors. Further studies might consider different solution concentrations, different species of Aloe, different species of planaria, or larger test groups to effectively determine if caffeine and/or Aloe exposure alters regeneration rates.
Acknowledgements
The investigators would like to thank Professor Teh for his guidance. Thank you to the Saddleback College Department of Biological Sciences for providing necessary resources.
Literature Cited
Baguñà J., Saló E., and Auladell, C. (1989).Regeneration and pattern formation in planarians. III. Evidence that neoblasts are totipotent stem cells and the source of blastema cells. Development, 107(1): 77–86.
Chatzifotis, S., Kokou, F., Ampatzis, K., Papadakis, I. E., Divanach , P., and Dermon, C. R. (2008). Effects of dietary caffeine on growth, body composition, somatic indexes, and cerebral distribution of acetyl-cholinesterase and nitric oxide synthase in gilthead sea bream (Sparus aurata), reared in winter temperature. Aquaculture Nutrition, 14(5), 405-415. doi:10.1111/j.1365-2095.2007.00541.x
Collins, E.L. The Effect of Caffeine and Ethanol on Flatworm Regeneration (2007).Electronic Theses and Dissertations.Paper 2028.
Exploratorium [Internet]. Planaria: A window on regeneration (2015). Available from:
Hashemi, S. A., Madani, S. A., and Abediankenari, S. (2015). The Review on Properties of Aloe Vera in Healing of
Cutaneous Wounds.BioMed Research International, 2015(Article ID 714216).
Moustakas, D., Mezzio, M., Rodriguez, B. R., Constable, M. A., Mulligan, M. E., & Voura, E. B. (2015). Guarana Provides Additional Stimulation over Caffeine Alone in the Planarian Model. PLoS ONE, 10(4), e0123310.
Okano, J., Nagahara, T., Matsumoto, K., & Murawaki, Y. (2008). Caffeine Inhibits the Proliferation of Liver Cancer Cells and Activates the MEK/ERK/EGFR Signalling Pathway. Basic & Clinical Pharmacology & Toxicology, 102(6): 543-551. doi:10.1111/j.1742-7843.2008.00231.x
Rawls, S. M., Patil, T., Yuvasheva, E., & Raffa, R. B. (2010).First evidence that drugs of abuse produce behavioral sensitization and cross-sensitization in planarians.Behavioural Pharmacology, 21(4): 301–313.
Reddien, P.W. and Sánchez Alvarado, A. (2004).Fundamentals of planarian regeneration. Ann. Rev. Cell Dev. Bio., 20: 725–757. doi: 10.1146/annurev.cellbio.20.010403.095114
Saló, E. and Baguñà, J. (1984).Regeneration and pattern formation in planarians. I. The pattern of mitosis in anterior and posterior regeneration in Dugesia (G) tigrina, and a new proposal for blastema formation. J. Embryol. Exp. Morphol, 83: 63–80.