A Brief Description Of Planaria Behavior And Regeneration
By
John Gibson
University Of Massachusetts, Lowell
Feb 17, 2025
Principle Of Biology II Lab
Section: 816B, Wednesday 4:30PM
Partner: Matthew Minigell
TA: Akshay Rana
Abstract
This investigation study tested planarian specimens, manipulating them to observe their behavior. Two specimens were amputated near the head, followed by one week of close observation of their recovery in spring water with and without added sodium chloride (NaCl) salt. It was hypothesized that a slight addition of salt to pure spring water mimicked the stagnant pond where water evaporates, leaving a slight elevated salt concentration and should accelerate regeneration. However, the test result showed no difference in regeneration speed.
The big question this study experiment tried to answer was whether the regeneration outward appearance, specifically white color neoblast emergence (Almazan, 2021), of the wound site, matches current research understanding of neoblast migrations.
Planarians are free-living flatworms of the Platyhelminthes phylum that gained widespread use in regenerative biology research for more than 100 years (Shibata, 2010). It is widely accepted that planarian regeneration includes 2 major biological concepts: positioning information and stem cell neoblast regeneration (Reddien, 2018).
The motive of this experiment was to highlight the outward appearance and behavior of planarians to give an intuition into the neoblast migration process. Outward behavior can also infer the neurological structure so that researchers can distinguish adequate regeneration from knock-out and knock-down mutation experiments in the gene identification processes.
The purpose, the goal of this report, was to give a concise and brief description of planarian behavior and regeneration progress as an integrated whole.
Key final result of this study experiment was that, regardless of the salt concentration difference, planarians completely regenerated a new head and tail at the same speed within 6.5 days. There was no difference in regeneration speed.
In summary, the experiment’s final results were successful. The planarian's healthy behavior was recorded, and the amputated fragment regenerated an entire animal, more robust than expected (insensitive to slight salt concentration variations).
Introduction
Planarians are free-living flatworms of the Platyhelminthes phylum, which are often used in regenerative biology research. They are related to parasitic flatworms such as tapeworms. They are naturally found in fresh water bodies such as ponds and household aquariums. Their nervous system is composed of 2 parallel nerve cords running parallel from the head region to the tail. At the head regions, the 2 parallel nerve chords form nodules, called ganglia. The eye spots sense light. The auricles sense mechanical vibrations and have chemosensors that contribute to reactions to food stimuli (Almazan, 2021). The auricles are particularly pointy in North American Girardia doroto/cephala (G. dorotocephala) species. Figure 1 points out the 3 regions of the planarian anatomy. Notice that Dugesia genus planarians do not have pointy auricles with an acute angle like in North American Girardia species (Almazan, 2021).
Planarians can regenerate severed body parts from most body fragments to produce the entire animal except the part anterior to the eye spots and the pharynx (Shibata, 2010). It is widely accepted that planarian regeneration includes 2 major biological concepts: positioning information and stem cell neoblast regeneration (Reddien, 2018). The more dense the neoblasts, the higher the regeneration capability at that body region. Positioning information signals the newly generating cells at the wound site what tissues to form, and the neoblast cells migrate to regenerate the correct cell types and propagate the signaling.
Planarians are robust, low-cost, easy-to-care-for animals that reproduce enough offspring, hence they are model organisms. In addition, planarians are particularly suitable for studying animal regeneration due to the well-known robustness of their regenerations. Researchers study the positional signaling molecules that are cross-species conserved in evolutionary background, such as Wnt interaction with β-CATENIN (Sureda-Gómez, 2016). Human health applications using regenerative knowledge can be reached when enough cross-species conserved knowledge is accumulated.
The longitudinal positional information for regeneration is encoded in the β-catenin-1 protein concentration. As shown in Figure 2, the normal distribution of β-catenin-1 concentration is high at the tail, and low near the head (Reddien, 2018).
During regeneration after amputation near the head, the β-catenin-1 gradient near the wound site of the posterior piece, as shown in Figure 3, is characteristic of a missing head, so neoblasts can be guided to generate a new head. Neoblasts tissues, at least at the auricle, are white in color (Almazan, 2011).
In this study experiment, the basal behavior of the planarian was briefly observed. Animal movements can be either kinesis (non-intentional, nondirected) or taxis (intentional). Why study the behavior of planarians? The nervous system is part of the regeneration, and only a healthy behavior can verify the complete regeneration of a newly generated animal from a fragment.
This study uses simple manipulations for 1) handedness, 2) thigmotaxis, 3) phototaxis, 4) rheotaxis, and 5) chemotaxis to establish an intuition of a healthy individual. Handedness is tested by flipping the planarian, thigmotaxis is tested by touching the 3 regions of the planarian, phototaxis is tested with a white flashlight with red and blue light filters, rheotaxis is tested with a pipet blowing water, and chemotaxis is tested with glucose and NaCl liquid. 2 planarians are subjected to head amputation to establish basal regeneration speed and tissue recovery progression.
The goal of this study, what this study tries to answer, is whether there is a set of basal behavior replicable to determine if an individual planarian is healthy. This can be used to test an animal after genetic knock-out/known-down experiments in further studies. The amputation and subsequent recovery at different NaCl concentrations try to answer whether planarian regeneration is sensitive to environmental changes. It is hypothesized that a slight addition of salt to pure spring water mimics the stagnant pond water that evaporates, leaving a slight elevated salt concentration and should accelerate regeneration.
In summary, regardless of the salt concentration difference, planarians completely regenerate a new head and tail at the same speed within 6.5 days. Most taxi tests, except red and blue light tests, can be used to determine whether a planarian individual's sensory and motor system is healthy.
Materials And Methods
Basal Behavior
Three North American G. dorotocephala planarians were tested in isolation, first by measuring their length without perturbations, then their handedness was observed by flipping them from their right and left sides, alternatingly for 3 trials for each planarian. No resting time between flipping.
The 3 planarians were tested for thigmotaxis by touching them with a micropipette tip at the head, the tail, and the center region on the lateral sides. Between touching tests, each planarian was given 1 minute of resting.
The 3 planarians were tested for phototaxis in half-covered Petri dishes with a white light flashlight 2.5 cm above the planarian. The light was placed near the head and the tail alternately. No resting time between phototaxis tests. Then the same test method was repeated, except with red and blue cellophane color filters over the flashlight while the petri dish remained half-covered by aluminum foil.
The 3 planarians were tested for rheotaxis by blowing water with a transfer pipet placed in front of the head onto the head. Pipet was behind the tail when blowing water onto the tail. There was a 1-minute rest time between each test for each planarian.
The 3 planarians were tested for chemotaxis with liquid glucose pipetted 3 drops into water at 2 cm and 5 cm distances from the planarians.
The 3 planarians were tested for chemotaxis with 1% NaCl pipetted 3 drops into water at 2 cm distances from the planarians.
Regeneration
Two G. dorotocephala planarians about 10 mm long were transversely amputated with a razor blade at the torso near the head region (Figure 3) in spring water and stored at room temperature in the dark. One planarian’s water had added 1% NaCl, 3 drops in a 5-cm diameter petri dish. Photographs of the planarians were taken at 0, 4.5, 5.5, 6.5, and 7.5 days post-amputation.
Result
Basal Behavior
As shown in Table 1, the 3 planarians were measured between 9 mm and 12 mm in length. There was no perturbation of water current, light, or chemical addition for 1 minute during the measurement, however, all 3 planarians had stretching and contraction movements during the 1 minute of measurement.
Table 1. Planarian Length Record
Legend: The lengths were rough estimates as the planarian stretched and contracted in petri dishes.
As shown in Table 2, all 3 planarians were left-handed, turning to their left to right by themselves within 1 second. This result was consistent and immediate with triplicate tests.
Table 2. Handedness Test And Behavior Time
Legend: Left and right turns are from the planarian’s perspective. Time in seconds is for the planarian to flip back to normal.
As shown in Table 3, all 3 planarians moved away from the white flashlight consistently and immediately. However, after using red and blue cellophane color filters, they gave both moving-away and moving-toward movements inconsistently.
Table 3. Phototaxis test
Legend: Movements of planarians all within 1 second of shining the light from the top.
As shown in Table 4, when water current was blown onto the head, all 3 planarian planarians first made a perpendicular movement of the water current, then moved in the same direction as the water current within 1 minute. This result was consistent among all 3 planarians. Also, when water current was blown onto the head, all 3 planarians consistently moved in the same direction as the water current within 1 minute.
Table 4. Rheotaxis test
Legend: Pipet was in the front of the head when blowing water onto the head. Pipet was behind the tail when blowing water onto the tail. All movements were observed within 1 minute.
As shown in Table 5, all 3 planarians moved toward the food source, consistently within 1 minute when the food was placed within 2 cm. There was no movement of the planarians when food glucose drop was at a 5 cm distance.
Table 5. Chemotaxis With Glucose
Legend: Food glucose was pipetted 3 drops into water. All movements were observed within 1 minute.
As shown in Table 6, all 3 planarians moved toward the 1% NaCl drop, consistently within 1 minute when the NaCl drop was at a 2 cm distance.
Table 6. Chemotaxis With NaCl
Legend: 1% NaCl was dropped 2 cm distance from the planarians. All movements were observed within 1 minute.
Regeneration
As shown in Table 7, 2 planarians regenerated both a new head and a new tail within 7.5 days post-amputation. Both planarians were in spring water; planarian #2’s water had NaCl added.
Table 7. Planaria Regeneration Progress Description And Photographs
Caption: Both planarians were in spring water; planarian #2’s water had NaCl added.
Discussion
As shown in Table 2, all planarians were left-handed. This could be coincidental or statistically significant, similar to humans with right-hand dominance.
As shown in Table 3, all planarians consistently avoid light. This is expected and makes evolutionary sense for a small animal without a thick skin coating to avoid UV light from the sun to avoid DNA damage. This infers that healthy planarians with intact eye spots are sensitive to light, and that the nervous system is healthy to innervate muscles to move with determined intention.
However, the red and blue cellophane colored light tests in Table 3 were inconclusive in the taxis direction of the normal planarian behavior. The red and blue colors should not be used as a criterion to determine if a planarian is healthy.
As shown in Table 4, the general direction of encountering a current flow was to move perpendicular to the flow as the immediate reaction. This makes physiological sense because the perpendicular movement likely will relocate the animal to a location without the current so that it can rest and conserve energy. However, the persistent current flow requires the planarian to exert energy to stay in the perpendicular orientation of the current, so, the planarian subsequently moved with the flow to further conserve energy.
As expected, Table 5 shows that planarians require energy for metabolism, and moving toward glucose through concentration gradient allowed the animal to absorb the energy-rich food. As shown in Table 5, when glucose was pipetted at 5 cm distance, due to diffusion, the glucose concentration gradient likely was evenly distributed, and hence planarians did not make any movement.
As expected, Table 6 shows that planarians, consistently, preferred to move toward water with slight salinity with NaCl. This was likely because the natural pond habitat has slight salinity due to water evaporation, and planarians have evolved to be most fit for the natural habitat.
Overall, the behavior test in this study answered the question of whether a set of basal behaviors could be used to assess the health of a planarian individual. Indeed, there are at least 4 criteria to assess it, namely 1) phototaxis, avoiding white light determines the intact photoreceptor and nervous-muscle system, 2) rheotaxis, avoiding current flows determines the intact mechanical sensors, 3) chemotaxis, seeking glucose, and 4) seeking slight salinity determine the intact chemoreceptors. In regenerative medicine studies with gene knockout experiments, this set of 4 criteria can determine if a regenerated planarian is fully unaffected by the gene manipulation.
The planarian specimens of this study indeed successfully regenerated as shown in day 7.5 photographs of Table 7. This was expected as the cut site was well known to have neoblast population and with a β-catenin-1 gradient (Figure 3). Between 0 days and 4.5 days after amputation, the wound area formed a narrow white tip at both the new head and the new tail region as shown in the 4.5 days pictures of Table 7. This agreed with neoblast migration theory (Almazan, 2021), as neoblasts were recruited to the wound sites. Overall, the regeneration test achieved the goal of this study to provide an intuitive visual check of regeneration of tissues. This is related to human regenerative medicines as newly generated tissues, such as skin, tend to have lighter color than the surrounding tissue.
Human health applications using regenerative knowledge will need the discovery of tissue morphology analog to planarian tissue morphology, and exact coloration analogs. However, this will require molecular signature investigations of all molecules involved.
The errors and limitations of this study experiment were the missed observations between day-1 to day-3 post-amputation. In further studies, it should be improved to record the exact time of the disappearance of the dark black band at the wound site. Another limitation of this study was the small number (3 specimens) of data points. Table 2’s handedness data set is not large enough to determine the statistical significance. Another error of this study was the insufficient observation of the unperturbed behavior of planarians. This can be improved by resting the specimens fully during the measurement of the lengths.
In summary, this experiment successfully demonstrated that a healthy planarian has an understandable basal behavior set for use in further regenerative biology/medicine experiments. And this experiment also successfully showed that planarian regeneration is robust and insensitive to slight salinity variations in the recovery environment after an amputation, providing that the cut site (transverse, torso region near the head) has sufficient neoblast population. This study shows that neoblast migration (light colored tissue forming) can be visually verified by color during planarian regeneration. The coloration matches known literature (Almazan, 2021).
Works Cited
Almazan, Eugene Matthew P et al. “Regeneration of Planarian Auricles and Reestablishment of Chemotactic Ability.” Frontiers in cell and developmental biology vol. 9 777951. 26 Nov. 2021, doi:10.3389/fcell.2021.777951
Reddien, Peter W. "The cellular and molecular basis for planarian regeneration." Cell 175.2 (2018): 327-345.
Shibata, Norito, Labib Rouhana, and Kiyokazu Agata. "Cellular and molecular dissection of pluripotent adult somatic stem cells in planarians." Development, growth & differentiation 52.1 (2010): 27-41.
Sureda-Gómez, Miquel, José M. Martín-Durán, and Teresa Adell. "Localization of planarian β-CATENIN-1 reveals multiple roles during anterior-posterior regeneration and organogenesis." Development 143.22 (2016): 4149-4160.
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