Planarians are perhaps better known for their extraordinary regenerative capacity, which is associated with a large population of adult stem cells neoblasts. In the last century, several laboratories around the world have used planarians as a biological model in a surprisingly broad and extensive range of applications.
Systematic research on planarians was first substantiated by Thomas Hunt Morgan and Harriet Randolph, who studied regeneration in planarians Randolph ; Morgan The fact that freshwater planarians are commonly found in the wild e. Although several hundred planarian species exist, research on freshwater planarians in the last century has been restricted to species either commonly found in the wild or commercially available mostly members of the Dugesiidae family, including the genera Dugesia , Girardia , Neppia , Romankenkius , Schmidtea , and Spathula.
In the last 10 yr, attention to planarian molecular and genetic research has been greatly enhanced and has focused on work in two species in which clonal lines have been derived: S. The advantages of using S. Although most protocols and techniques can be adapted to different planarian species, here we focus on S. The taxonomic classification for S. Benazzi et al. The primary difference between the asexual and sexual strains of S. The diploid asexual strain of S.
Studies on the geographical distribution of S. Asexual specimens of S. Some of the specimens that thrived in the new laboratory conditions were individually cut and grown separately to establish clonal lines. Clonal lines were created using a strategy similar to that used to establish CIW4.
This offered a unique opportunity to study embryonic development and functional germ-cell specification in S. Furthermore, the inbreeding of these animals performed by Peter W.
Establishing and Maintaining a Colony of Planarians Oviedo et al. Besides S. Interestingly, different procedures e. Similar to the sexual strain of S. Important advances have been made possible by using D.
However, there are some important differences between S. Recently, a D. For the last century, planarians have been a favorite model system in many research areas. Not only are planarians easy to rear under laboratory conditions and relatively inexpensive to maintain, they are amenable to pharmacological, behavioral, physiological, molecular genetic, and classical surgical techniques.
Thus, in recent years, they have become a popular model for state-of-the-art studies in regenerative biology as well as a low-cost system to produce fast experimental results for classroom science projects and other educational purposes Oviedo and Levin The planarian CNS is particularly important because it exhibits evolutionary conservation with higher organisms in many neural receptors e. Moreover, the molecular conservation of the CNS also indicates that planarians are a good model in which to test the effects and mechanisms of drugs using large pharmacological screens Oviedo and Levin Remarkably, the rapid reconstruction of multiple missing parts e.
Additionally, intact adult animals have the capacity to regulate their body sizes according to metabolic status i. Interestingly, both growth and degrowth in S. Taken together, these observations indicate that planarians possess efficient mechanisms for synchronizing metabolic status and tissue maintenance. Thus, their tissues are continuously renewed. Therefore, S. The anatomy of the reproductive system of S. Unlike other commonly studied invertebrate models, planarians do not appear to segregate their germ-cell lineage during early embryogenesis; instead, the reproductive system in sexual planarians has been proposed to be determined by epigenetic mechanisms after hatching post-embryonically Zayas et al.
In planarians, tissue regeneration, germ-cell specification, tissue remodeling, and adult tissue maintenance involve a large population of undifferentiated cells known as neoblasts planarian stem cells , located throughout the body, which constantly divide. During regeneration, these cells are the source of new tissue.
In asexual worms, neoblasts are the only known cell with mitotic activity and are therefore the sole source of new cells. Thus, neoblasts coexist in a microenvironment that is tightly regulated and allows them to respond to signals to self-renew, proliferate, and migrate, giving rise to differentiated progeny that are properly incorporated into demanding tissues.
The complexity of the neoblast population is far from being understood Rossi et al. Study of the molecular conservation of regulatory molecules e. Furthermore, the identification and characterization of stem cell regulatory molecules that are conserved between vertebrates and planarians but absent in other classical invertebrate model organisms such as Drosophila melanogaster and Caenorhabditis elegans is now possible in S.
The molecular basis of germ-cell specification in adult stages is poorly understood at this time Zayas et al. Interestingly, it has been proposed that germ cells in sexual S. The plasticity of germ-cell specification and maintenance in adult S.
Taking advantage of these properties, the Newmark lab at the University of Illinois used clonal animals of the sexual strain of S. Thus, this database will be useful for the identification of germ-cell markers as well as signaling pathways involved in germ-cell determination and tissue regeneration. When proper procedures are followed, the data conclusively show that planarians can learn.
Thus, planarians are a unique model system in which memory and regeneration can be studied in the same animal. For an in-depth discussion of memory and learning in planarians, consult Nicolas et al. At this point in time, genetic tools for gain-of-function and permanent genomic modifications in planarians are not well established. Recently, a proposal involving guidelines for gene and protein nomenclature in the planarian species S.
Applying these nomenclature guidelines will help to standardize naming and facilitate the identification of gene homology and experimental treatments e.
In addition, if there exist paralogs for a given gene, the respective planarian gene gets a numerical suffix, not a letter, at the end e. The protein name should be uppercase and not italicized e. Note that because mutant alleles and transgenes are not yet commonly used in planarian experiments, there is no standard nomenclature for such. For additional details, see Reddien et al.
Complementary information on a large collection of ESTs from sexual and asexual S. Many published protocols from other well-established invertebrate systems such as D. Basic molecular approaches include purification of mRNA to evaluate gene expression both quantitatively and qualitatively , antibody staining to evaluate the spatial distribution of molecular markers and morphogenesis during regeneration, in situ hybridization to evaluate the spatial distribution of messenger RNA, and quantitative real-time-polymerase chain reaction RT-PCR to evaluate transcript levels in planarian tissue.
Methods used successfully for developmental studies include tissue fixation for immunostaining, in situ hybridization, and gene knockdown with RNAi. Neoblast subpopulations can be isolated by flow cytometry fluorescence-activated cell sorting [FACS] Reddien et al. The effects of drug compounds on cell behavior can be conveniently tested in planaria, because regeneration is a sensitive assay for changes in proliferation, migration, differentiation, and morphogenetic cues.
Additionally, diverse physiological processes can be analyzed in the whole organism in real time e. Also, because regeneration can reveal subtle changes in cell signaling, planarians are a good system in which to identify and molecularly characterize the effects of weak electromagnetic stimulation Novikov et al.
Protocols for running behavioral experiments have been refined over the years since planarians were first used for behavioral work McConnell Although these experiments can be performed manually, the future of this field clearly rests in automated systems for training and testing worms, both for high-throughput approaches and for establishing basic quantitative and objective results.
The construction and use of such automated systems has most recently been described by Hicks et al. We thank Dr. Beane for comments and suggestions on the manuscript. We apologize to our colleagues whose work we could not cite due to space limitations. Terms of Service. Thus, each eyecup confers a left-right directional selectivity to visual information while the rostral location confers an anterior dimension to visual information transduced by the ocelli.
The photoreceptor cells are bipolar neurons whose cell bodies are located outside of the optic cup [15]. Axons from the photoreceptor neurons project posteriorly into the brain, with some fibers forming a partial optic chiasma to integrate photosensory inputs from both sides of the animal [16] , [17] , [18]. The dendrites of the planarian photoreceptors extend inside the optic cup and form a rhabdomeric structure where opsin accumulates [12] , [19].
Opsins are a highly conserved class of G-protein coupled receptors that covalently bond to a chromophore forming the visual pigment rhodopsin [20]. Transcriptome analyses reveal that the rhodopsin signaling pathway is conserved in planarians, including two R-opsin homologs [14].
The planarian species Schmidtea mediterranea was used. Boxed region shows a close up of the eyes, with an inset diagram of the light-sensing structures of the optic cup. The eye consists of two tissue types: the light capturing pigment cells and the photoreceptor neurons that transduce photons into signals sent to the brain.
Planarians are photophobic and when exposed to light they seek cover [21] — [23]. This negative phototaxis has been used to evaluate regeneration of the visual system [23] — [26] , as well as memory storage and transference [27] , [28].
In these planarian behavioral studies, analyses have been conducted with white light, which consists of an amalgamation of multiple wavelengths. However, many animals have been shown to have different behavioral responses to different wavelengths of light. For example, zebrafish larvae will swim toward ultraviolet UV , blue, and red light but are only weakly attracted to green light [29].
Conversely, leeches detect and exhibit complex negative phototactic responses to UV and green wavelengths, with UV producing the maximal response [30] , [31].
In Drosophila larvae, exposure to blue, violet, and UV wavelengths elicits negative phototaxis, while green and red light produces no behavioral response [32]. Similarly, the movement of C. A further complication of using white light for phototactic studies is that different sources of white light e. Even within a single source, such as the commonly used halogen light, substantial differences exist in the wavelengths included [34].
Additionally, regulation of intensity by controlling current also alters the spectral composition, giving rise to yet another poorly controlled variable. Therefore, we suggest that use of white light to study planarian photophobia may mask important behaviors associated with different wavelengths of light. We hypothesize that rather than a general photophobic response, planarians have differential responses across a range of wavelengths both within and outside of the visible spectrum.
Here, we describe a novel planarian behavioral assay developed to test behavioral responses to individual wavelengths including UV and infrared IR , which to the best of our knowledge have not previously been examined in these flatworms.
Our data show that planarians display a complex, hierarchal photophobic response to specific ranges of wavelengths, in addition to a brief general response that appears to be more wavelength-independent. Furthermore, similar to leeches and C. These results serve to improve our understanding of the basic biology of planarian eye function and suggest a previously underappreciated visual richness in these animals.
Asexual Schmidtea mediterranea were maintained as previously described [35] , except worm water was comprised of 0. LED wands fixed resistor and RCA plug attached to a 9 volt battery with switch were constructed as previously described [30] , [31].
White light was obtained using a standard LED fiber optic illuminator with goosenecks from a dissecting scope setup. Approximate relative luminosity in the testing dish was assessed using a phototransistor coupled to a 2 mm diameter fiber optic [36]. As expected, intensity was greatest in quadrant 1 Q1 and steadily decreased, with quadrant 4 Q4 being the darkest. In order to obtain a spot of light that was smaller than the worm itself, a piece of tape was placed on the end of the laser and punctured to create a pinhole that produced a circle of light approximately 2.
A rectangular 7. There was also a half circle at the origin, with its apex midway through Q1, for directing light placement. LED wands were secured above the testing dish with a clamp attached to a ring stand, while a second clamp secured the battery pack to prevent unintended movement of the wand.
The end of the LED wand was positioned about 5 cm above the top of the testing dish with the light directed into the half circle in Q1. An SLR camera was positioned over the testing dish using a tripod. On each experimental day, batteries were replaced in both flashlights and the LED wand.
The testing dish was filled to a depth of 0. In a single day, one wavelength was applied to total of 60 worms 10 groups of 6 worms, or 10 trials , repeated 3 times. For each trial, all worms were placed into Q1 before the camera was turned on. Except for controls, the light was switched on time 0 at 5 seconds after recording started. Behavior was recorded for 2 minutes. Animals were allowed to rest at least overnight before the next wavelength.
Filters used were A holder was designed from stiff foam pipe insulation to position the LED wand above the filter such that all emitted light passed through the filter. White paper was placed on the microscope stage so that laser light could be seen. Individual worms were transferred to the middle of the dish and recording was started when the worm began traveling on the bottom of the dish.
The laser beam was directed in front of the animal at a distance equal to one diameter of the circle of light approximately 2. Only a single wavelength was tested each day with 30 worms repeated twice, for a total of 60 trials , and animals were allowed to rest at least overnight before the next wavelength in the following order: red, green, and UV.
Recordings from all behavioral trials were examined using Windows Media Player. For the photophobia assay, the three repeat trials for each group were first averaged to compensate for individual animal variability.
When determining location, at least 50 percent of the worm had to be in the quadrant. A Bonferroni post hoc multiple comparisons test was conducted to examine differences between means. However, from available data, it is unclear whether planarians have a single general photophobic response or if their behavioral responses actually vary by wavelength as has been shown in other animals [29] — [33] , [39].
To distinguish between these possibilities, we developed a novel behavioral assay Materials and Methods. Because the LED wand was exchangeable, our setup allowed not only for testing behavioral responses to different visible wavelengths, but provided a means to investigate planarian responses to ultraviolet UV and infrared IR wavelengths as well.
One objective was to establish an easily reproducible photophobia assay with standardized testing parameters in order to improve comparability. Therefore, each LED wand was clamped above the testing dish at a fixed distance of about 5 cm Figure 2A. Additionally, a sheet of white paper was placed beneath the testing dish, with four equal quadrants Q1 to Q4 demarked Figure 2B.
To verify that the amount of light gradually decreased from Q1 to Q4, the intensity of light in each quadrant was estimated with a phototransistor. Finally, the assay used easily-constructed LED wands powered by 9 volt batteries, as previously described [30] , [31] , which allowed for some control of the ranges of wavelengths tested. For our experiments, the nominal wavelengths used were Figure 2C : near IR — nm , red — nm , green — nm , blue — nm , and two distinct wavelengths of near UV light — nm and — nm.
In addition, we also tested worm responses to white light using a standard LED fiber optic illuminator with goosenecks as typically used with a dissecting scope. The use of white light, even though there are certainly different spectra involved using LED or halogen sources, allowed us to compare responses from more restricted and narrow ranges of wavelengths with the non-specific white light typically used in planarian photophobia studies.
A The imaging setup. B Close-up of testing dish. B1 The labeled guide placed underneath the dish marks the 4 quadrants Q1—Q4 and the semi-circle where the LED light will be directed. B2 Image of testing dish during a trial, showing the resulting light-to dark gradient.
C The spectral composition of the LEDs used, and their location on the electromagnetic spectrum. For the assay, the behavioral responses of 60 worms were tested in 10 groups of 6 worms for each wavelength a single trial.
Trials were repeated 3 times and the data averaged, to compensate for variability in individual worm responses. Trial parameters were as follows: camera recording was turned on, a group of 6 worms was placed in Q1, after 5 seconds the LED wand was turned on, and behavior was recorded for 2 minutes the initial time was scored as when the light was first turned on.
Because of the remote possibility that the brief exposure to very weak UV light might cause damage, UV trials were performed last. Generally, worms were tested in order from longest to shortest wavelengths.
Using the above parameters, we performed our photophobia assay with control ambient light only , IR, red, green, blue, and UV nm and nm wavelengths, as well as with white light Figure 3.
Worm location by quadrant was scored at 30 second intervals Figure 3A , with photophobia being assessed after 2 minutes Figure 3B. Statistical significance asterisks in Figure 3B was assayed for the overall pattern of worm location throughout the entire dish across all four quadrants , rather than for individual quadrants. Control groups explored the dish in an apparently random manner Figure 3A and Video S1 , such that by 1 minute animals were evenly distributed between all quadrants and remained so for the duration of the trial with an average of This random exploration is consistent with initial exploratory behavior in new environments previously noted in planarians [40] — [42].
All worms begin in quadrant 1 Q1, red circles. While control worms randomly explore the dish, in UV trials worms move rapidly away from the light white circles. Images enhanced for visualization. B Graph showing overall photophobic responses for each wavelength, as measured by worm location in each of the four quadrants Q1—Q4 after 2 minutes.
Photophobic responses are indicated by increased presence in Q4 black bars which is farthest from the light. In most of the UV trials, the worms congregated on the wall of the dish furthest from the light Figure 3A and Video S2.
As expected, worms exposed to white light also displayed strong negative phototaxis, with a striking correlation across all quadrants between white light Q1: 1. On the other hand, neither of the IR or red wavelength responses were statistically different from controls by the end of the trial Figure 3B. Overall, these results suggest that our novel planarian photophobia assay is able to recapitulate the strong photophobia previously demonstrated by other methods.
To confirm that the observed behavioral responses resulted from visual detection of specific wavelengths and not other variables such as heat or nociception, we repeated our photophobic assay with neutral density filters. If responses to light are in fact a result of visual detection, we would expect worm responses to diminish in a predictable fashion as light attenuation increases and the behaviorally relevant stimulus decreases. The results confirmed that the number of worms displaying photophobia steadily decreased with increased light attenuation, suggesting that the behavioral responses were the result of visual responses to specific ranges of wavelengths and not uncontrolled variables.
Graph showing behavioral responses over increasingly attenuated light, as measured by the number of worms in Q4 at 2 minutes. The trend shows that phototactic responses decreased along with diminished behavioral stimuli light. Although our data revealed that green, blue and UV light all resulted in robust photophobic responses Figure 3B , we observed that worms exposed to near UV light appeared to move away from the light faster than for other wavelengths tested.
This suggested that more complex differences exist between the photophobic responses than our scoring for photophobia at 2 minutes revealed.
Thus, we next examined the rate at which worms escaped direct light in Q1 by tracking both the number of worms that left Q1, and the number that returned, throughout the trial Figure 5. To do this, we calculated an escape index , where 0 indicated all worms remained in Q1 while 1 indicated all worms had left Q1. Therefore, higher values represented stronger photophobic responses. It should be noted that an important difference exists between the analyses in Figure 3 and the analyses here in Figure 5 that represent how fast worms escape from direct light exposure.
Because of this, the escape index as used here is a measure of the initial intensity of the response rather than a measure of overall strength of the response. Graph showing escape responses as a measure of the severity of phototactic behavior. The escape index is based on the number of worms that leave Q1 direct light , where a value of 1 indicates all worms have left Q1. Thus, higher values indicate stronger photophobic responses. Note the latter data indicate by 2 minutes worms have returned to the direct red light source in Q1.
Interestingly, white light was more similar to though not statistically different from blue escape responses Figure 5 , in contrast to overall photophobic response Figure 3 where white light was more similar to green. This may be related to the spectral composition of white light LEDs that typically contain several broad peaks, including notable amounts of energy in the blue range. For IR light, the escape index Figure 5 at all time points was significantly different from controls as well as all other wavelengths.
This is in contrast to the overall photophobic response to IR light Figure 3 , which was not statistically different from controls even at the earlier 30 second time point. In particular, the escape index showed that IR wavelengths produced an opposite phototactic response, where worms were initially more likely to remain under direct light Q1 than controls. This suggests the possibility that planarian responses to IR might be slightly photopositive, a hypothesis that would first need to be investigated in much greater detail.
These data also indicate that the planarian visual system may be able to respond to IR wavelengths in some as yet unknown manner. This was particularly unexpected given that the overall photophobic response to red at 2 minutes was not different than controls Figure 5.
This reflects the observation that at 2 minutes, worms that previously left Q1 returned, despite the continued presence of the red light exposure. These data suggest that after an initial photophobic response worms subsequently stopped responding to red wavelengths.
The overall photophobic response data, combined with escape index analyses, suggested that while planarians displayed different responses to different wavelengths with UV causing the most robust responses , there may also exist a separate, wavelength-independent photophobic response to being placed under direct light such as might be expected with broadly-tuned visual pigments.
In order to test this idea, we examined avoidance responses to different wavelengths Figure 6. Whereas previously we examined whether or not planarians would move away from light exposure, our avoidance assay tested the reverse behavior: whether or not worms would choose to enter a light source.
However, the LED wands we used in our previous assay produced a field of light that was too large to record worm movement from outside the field into the light. Therefore, we switched to the use of tiny spots of laser light under high magnification under a stereomicroscope. We covered the end of a laser pointer with a piece of tape that had a single pinhole in the center, thus obtaining a much smaller coherent circle of light.
For illustration, compare the relative size of the light field versus a single worm in our photophobia assay UV panels in Figure 3A and in our avoidance assay Figure 6. We chose red, green and UV laser lights as representative of the range used in our photophobia assay. We expected that if wavelength-specific responses existed, worms would respond with increasing severity to avoid entering regions lit by red, green and UV wavelengths, respectively.
Avoidance assay to test worm responses when approaching areas of direct light. This ensured that worms began the assay outside the direct light source but was close enough that worms continued moving in the direction of the light.
Three distinct behaviors were observed. As worms approached the light source they either 1 did not respond and continued moving directly into the light, 2 moved around the light by making a slight directional change to one side without crossing into the light, or 3 abruptly made a 90— degree turn in the opposite direction of the light photos in Figure 6.
When confronted with the green light, the majority Furthermore, not a single worm chose to travel into the UV light, even though These results are consistent with our previous data showing that planarians exhibited differential responses to different ranges of wavelengths of light.
They also confirmed that not only did UV light produce the strongest photophobic responses and most robust initial responses, but that an intermediate and less severe photophobic response occurs with wavelengths within the visible spectrum such as green.
Furthermore, these results demonstrated that planarians lack a red wavelength-specific behavioral response, suggesting that the escape response we observed to red light reflects instead an initial wavelength-independent photophobic response Figure 7A.
A Graph showing the likely relationship between the two types of photophobic responses uncovered by our data: the general photophobic response, which occurs immediately after exposure to any wavelength, and the wavelength-specific responses. B Graph depicting the inverse relationship between photophobic responses and wavelength. Our results support the hypothesis that planarians do possess differential behavioral responses to light across a range of wavelengths.
Our data also reveal that planarian phototactic responses occurred in a behavioral hierarchy Figure 7B , where the shortest wavelengths in this case near UV light caused the most intense photophobic responses while longer wavelengths produced no effect for red or even opposite effects in the case of IR. These results highlight the importance of the spectral composition of light for planarian behavior and suggest that the current standard use of poorly characterized white light in planarian phototactic studies may mask more complex behaviors.
Unexpectedly, our data also suggested that planarian photophobic behavior may involve two different response types: a general photophobic response to luminal contrast for example a rapid phasic change in luminosity and more wavelength-specific photophobic responses Figure 7A. The general photophobic response occurred immediately after light exposure and drove planarians to escape the light source regardless of wavelength except for IR.
In contrast, the wavelength-specific responses encompass specific behavioral reactions that vary depending on the wavelengths involved. The difference between the general and wavelength-specific responses can be seen in the planarian response to red light. Although worms displayed an initial general response to escape the light source, they quickly adapted to it in order to return into the direct light Figure 5. This lack of a red-specific negative light response was confirmed in both our main photophobic assay Figure 3B and our avoidance assay Figure 6.
Together, these data illustrate that planarian photophobic behavior is complex and coordinated and not just the result of simple general light avoidance. In this hierarchy, planarian responses to near IR light were the most surprising as worms appeared to be attracted to it. The visual detection of IR has not been examined in planarians, although a few studies have shown that IR radiation causes increased stem cell proliferation [43] , [44]. Our data seem to suggest that planarians may be able to detect IR by some mechanism.
Although alternative explanations cannot be ruled out for instance, IR may create a shadow effect by reducing the activation of opsin, thus making the IR quadrant appear darker than the ambient room lighting , IR detection is found in various parts of the animal kingdom. For example, some snakes and bats possess IR receptors called pit organs that are capable of sensing thermal stimuli [45]. The spectral absorption of water depends largely on the concentration of suspended particles such as dissolved oxygen and organic material, which enhance scattering and absorption of short- and mid-wavelengths [48] — [50].
Therefore, fish living in turbid water have sensitivity to slightly longer wavelengths [51]. IR detection could also be an adaption for nocturnally active animals as both moonlight and starlight consist of longer wavelengths [46], at least in very shallow water environments where there might be some IR penetrance. Our data demonstrate that, like leeches and C. Fish that live in the ocean are typically most sensitive to blue wavelengths due to the fact that nm blue light penetrates the greatest [48] , [51].
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