Success of Dugesia tigrina regeneration in different water temperatures Abstract: The experiment at hand aimed to determine if the species Duguesia tigrina has an optimal temperature for regeneration. The experiment used three different spring water treatments of different temperatures. The treatments included a cool 12° treatment, a room temperature 25° treatment, and a warm 30° treatment. Each of the D. tigrina were cut in half, and each half was placed into a designated section of a water treatment, and observed over a one week time period.
Three replicates of the experiment were done, each over the one week time period. At the end of three weeks, the average number of successfully regenerated D. tigrina in each treatment was observed. It could be observed that in the room temperature 25° treatment, D. tigrina had the most reproduction success. Introduction: Dugesia tigrina, most commonly known as the Brown Planarian, are simple, small yet fascinating organisms. D. tigrina are “non-parasitic flatworms that have bilateral symmetry” which means their bodies can be divided into two symmetrical halves (Lobo et al 2012).
There are two eyespots at the top of their head, and two flaps on the side of their head that serve as ears. The Planarian’s nervous system is located at the anterior end of the worm. The mouth is located on the bottom side of their body, and is the one and only opening to their digestive tract (Fox, R. 2001). Most planarians live in cool, freshwater environments such as lakes, ponds, streams or even under rocks. D. tigrina are able to reproduce both sexually and asexually. A D. tigrina reproduces asexually by pulling itself in half, resulting in one half with the head, and one half with the tail.
After some time, both of these halves regenerate into two organisms with the same genetic information (Lobo et al 2012). When a D. tigrina reproduces asexually, they can only do so in an environment with an appropriate temperature. The temperature must be around ten degrees Celsius and above for this asexual reproduction to occur successfully (Bridgette, 2000). Similar to the mechanism of asexual production, if a D. tigrina is cut in half by an unnatural outside source, such as a human in a lab setting, it can also regenerate into two separate organisms as well.
To regenerate into a new organism, only 1/279th of a planarian, or a miniscule piece of the organism with as few as 10,000 cells is able regenerate into a completely new planarian in a period of one to two weeks (Lobo et al 2012). The experiment at hand will test that when cut in half, if D. tigrina will perform this regeneration process more successfully in different temperatures such as a cool, warm, or room temperature water source.
The main question the experiment aims to answer is “Does changing the temperature of the water source affect the D. igrina’s regeneration ability? ” The main purpose of the experiment is to discover the optimal regeneration temperature for the D. tigrina. This information is useful for human beings. By observing the regeneration process of D. tigrina, it gives individuals a better insight into the regeneration process as a whole. Furthermore, Brown Planarians have a gene known as hiwi, which is fairly similar to a human gene known as piwi. Both these genes are responsible for the production of a fair amount of stem cells in D. tigrina and humans respectively (Rudman et al 2012).
If the optimal regeneration temperature of the D. tigrina stem cells is found, it could give humans an insight into what the optimal temperature for the regeneration of human stem cells might be, since the genes in both organisms are similar. By studying the regeneration capabilities and optimal conditions for D. tigrina, scientists are one step closer to discovering how to control stem cell behavior in human beings (Gentile et al 2010).
A previous study on D. tigrina regeneration was conducted by V. R. Rivera and M. J. Perich. A series of four different experiments were conducted on four planarian species, one of which was D. tigrina. One of the experiments involved the regeneration of D. tigrina in four different water temperatures. D. tigrina were placed in 15°, 22°, 25°, and 30° spring water temperatures and the mean of the D. tigrina regenerated in each were recorded. Out of 30 D. tigrina tested in each treatment, an average of 11. 33 regenerated in the 15° spring water, 14. 33 in the 22° water, 10. 67 in the 25° water and 8. 33 in the 30° water. (Rivera V. R. and M. J. Perich, 1994).
The results gathered in this previously conducted experiment served as a guideline for choosing the final spring water temperatures for this experiment. It was believed that if similar temperatures The hypothesis for this experiment is that if D. tigrina are placed in cool, warm and room temperature spring water environments, the planarians will regenerate most sucessfully in the cool temperature. Since D. tigrina typically live in streams, ponds, lakes, and springs, it is believed that they will regenerate most successfully in temperatures closest to the temperatures of their natural environments.
On the other hand, if the D. igrina are placed in cold, warm, and room temperature spring water environments, and they have the most regeneration success in either the warm water treatment, or the room temperature treatment, the hypothesis will not be supported. In addition, if the D. tigrina have equal success in each of the three treatments, or overall have no success in all of the three treatments, then the null hypothesis will be supported. Methods: The D. tigrina were prepared and cut following the procedures outlined in Rudman et al. 2012. The cut Planarian were placed in their designated section of the six well plate, one half of the Planarian in each well.
The 12° treatment, was filled with an adequate amount of spring water for each D. tigrina to survive and was put in a refrigerator to maintain the cooler temperature. Similarly, for the room temperature 25° treatment, each section of the plate designated for this treatment was filled with an adequate amount of spring water. This treatment was placed in an incubator. Finally, for the warm 30° water treatment, an adequate amount of water was added to each section of the plate. This plate was placed in an incubator in order to maintain the warmer temperature.
The D. igrina were observed once or twice during the week to observe any changes occurring. After one week, the D. tigrina from each treatment were observed to determine if successful regeneration occurred. Each planarian was transferred to a culture dish with an appropriate amount of spring water, and viewed under a dissecting microscope to observe its behavior. The same steps of this experiment were repeated each week, for three weeks in order to ensure accuracy.
Results: After running three separate trials for each one of the treatments over the course of three weeks, the average number of D. igrina that survived in the 25° treatment was around 4. 3. The average number of D. tigrina that survived in the warm 30° treatment was around 2. 6, and the average number that survived in the cool 12° treatment was around 2. As shown in Table 1, week one had two successful regenerations from treatment 1, the 12° treatment, four from treatment 2, the 25° treatment and four planarians from treatment 3, the 30° treatment. For the second week, treatment 1 had four successful regenerations, treatment 2 had three successful regenerations, and treatment 3 had zero successful regenerations.
For the final week, treatment 1 had zero successful regenerations, treatment 2 had six, and treatment 3 had four. In total, over the course of three weeks, 6 D. tigrina successfully regenerated in treatment 1, 13 successfully regenerated in treatment 2, and 8 successfully regenerated in treatment three. Table 1. Dugesia tigrina regeneration per week In this experiment, there was a Chi square critical value of 3. 84. The 12° treatment was not significant (x2=0. 00, DOF=1). The 25° treatment was significant (x2=12. 00, DOF=1). Finally, the 30° treatment was not significant (x2=1. 333, DOF=1).
Figure 1. Average D. tigrina regeneration Figure 1 shows the average amount of planarians that regenerated in three replicates of each treatment done over a three week time period. As exhibited in the graph above, about 2 D. tigrina regenerated successfully in the 12° treatment. Next, about 4. 3 D. tigrina regenerated in the room temperature 25° treatment. Finally about 2. 6 D. tigrina regenerated in the 30° degree warm water treatment. Discussion: After the experiment was performed, it was concluded that Dugesia tigrina had the most reproductive success in the 25° room temperature treatment.
The results of the 25° treatment were significant when compared to the Chi squared value of 3. 84, whereas the results for the 12° and 30° treatments were not. In conclusion, the original hypothesis that D. tigrina will regenerate the best in the cold temperature, was disproved. The null hypothesis, that D. tigrina will have equal success in all three of the treatments, was disproved. An alternative hypothesis was supported that D. tigrina will regenerate best in the room temperature treatment.
When comparing the findings of both the previous experiment conducted by V. R. Rivera and M. J. Perich and the experiment at hand, the results were somewhat different. In the 15° cool treatment of the previous experiment, a larger number of successful regenerations occurred in comparison to the 25° treatment (Rivera V. R. and M. J. Perich, 1994). In this experiment, the opposite proved to be true. The cooler 12° treatment had overall less success in comparison to the 25° treatment. These results were quite surprising, as it was assumed that both similarly preformed experiments would have a similar turnout.
Upon conducting the initial research for this experiment, it was believed that D. igrina would regenerate best in temperatures closest to their natural spring water environments outside of the lab setting. Yet as clearly exhibited from the data received after performing this experiment, D. tigrina regenerated most successfully in a room temperature spring water environment. This could be explained for a number of reasons. The D. tigrina used in this experiment were being stored in a spring water environment kept in a room temperature setting. These specific planarians most likely had become accustomed to a room temperature environment, as they had been living in it for many days.
Therefore, these D. tigrina may not have been used to the cool 12° treatment which may have been the explanation for their unsuccessful regeneration or death occurring in this treatment which was originally thought to have the most success. In this experiment, there are also few possibilities of sources of error that could have had a significant effect on the achieved results. For example, cutting a D. tigrina along an improper axis could cause the organism to regenerate unsuccessfully or simply die altogether. A second source of error could be not allowing the planarian’s wound enough time to heal.
A planarian must have enough time, approximately three minutes for it’s would to heal in order for the regeneration process to successfully begin (Rudman et al 2012). A third source of error could have been due to improper water temperature. The cool 12° treatment could have been much too cool to allow regeneration among the D. tigrina. As previously stated, the temperature needs to be at least 10° in order for asexual reproduction to occur (Bridgette, 2000). The original water temperature desired for the cool treatment was 14°. Similarly, the room temperature desired was 22°.
Yet due to limitations in the lab setting, 12° was the temperature set for the cool treatment, and 25° for the room temperature. The 12° temperature could have been too close to the minimum temperature of 10° that D. tigrina needs to regenerate successfully. In addition, although it was successful, the 25° degree treatment was three degrees above the desired room temperature, which could have affected the results. Finally, the warm 30° treatment could have been too warm to allow the Planarian to properly undergo regeneration. To improve this experiment for future studies, a few changes would be recommended.
First, a larger number of D. tigrina should be used each week in order to fully ensure the results are not due to chance. If one or two organisms die off for reasons other than the different water temperatures, it will not significantly affect the final results. Furthermore, the cuts of these D. tigrina should be performed more accurately. Each cut should be performed in the same area on each organism, using the utmost precision and care. After each cut, three minutes should pass before the organism is moved into its designated water environment, to allow for wound closure.
By using more care in performing these steps, it could allow for more D. tigrina to successfully regenerate throughout the water treatments. Finally, as previously stated, spring water temperatures should be adjusted a few degrees so the cool and warm temperatures will not be too extreme for the organisms to go through the regeneration process. In conclusion, Dugesia tigrina had the best regeneration success in a room temperature 25° spring water treatment, and therefore it is probable that the optimal temperature for their regeneration is around 25° Celsius.