However, comparatively little is known about the physiology of their photoreception or how their behavior is affected by various wavelengths. Most phototactic studies have examined planarian behavior using white light. Here, we describe a novel planarian behavioral assay to test responses to small ranges of visible wavelengths red, blue, green , as well as ultraviolet UV and infrared IR which have not previously been examined. Our data show that planarians display behavioral responses across a range of wavelengths.
These responses occur in a hierarchy, with the shortest wavelengths UV causing the most intense photophobic responses while longer wavelengths produce no effect red or an apparent attraction IR. In addition, our data reveals that planarian photophobia is comprised of both a general photophobic response that drives planarians to escape the light source regardless of wavelength and wavelength-specific responses that encompass specific behavioral reactions to individual wavelengths.
Our results serve to improve the understanding of planarian phototaxis and suggest that behavioral studies performed with white light mask a complex behavioral interaction with the environment. Planarians are non-parasitic flatworms that are an important model system for understanding stem cell biology [1] — [3] , regeneration [4] — [6] , toxicology [7] , [8] , and evolution [9] , [10].
Additionally, with their true central nervous system and cerebral eyes connected to the brain, planarians have been used as a model for eye research.
Several basic features found in planarian eyes are phylogenetically conserved such as photoreceptor cells containing opsin, a pigmented cup structure, and a host of eye-specific developmental genes that are essential for eye formation [11] — [14]. These common features, combined with the relative simplicity of the planarian visual system, make flatworms a valuable addition to the models used for investigating the basic features of eye biology and increasing our understanding of eye evolution and development.
Located on the dorsal side of the body, planarian eyes are composed of two cell types: pigment cells and photoreceptor neurons Figure 1. The pigmented cells form a semi-lunar optic cup and function to absorb incoming light. 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 hatchling is called a miracidium; after several metamorphoses the parasite leaves the snail and infects fresh water fish, especially minnows and carp; it encysts in their flesh. When a person eats these fish uncooked or insufficiently cooked, the live parasites enter the digestive system and migrate from the small intestine via the bile duct to the liver.
There they mature into adult liver flukes and in about 3 weeks begin to produce eggs. Eggs return via the bile duct to the digestive system and exit from the body in feces. The Cestoda are parasitic tapeworms. The life cycles are complex see the text. All tapeworms are extremely flat; the body is divided into segments, and there is no digestive system. They absorb nutrients across their body walls.
Preserved tapeworms are available in jars. Scolex no high power is the term for the head end of a tapeworm; it has a disc of hooks at the tip, which anchor it into the lining of the host's intestine, and four large suckers for holding on.
New segments or proglottids are generated behind the scolex. As you move down the worm away from the head, these segments get larger. Each is a complete reproductive machine with testes , ovaries and uterus. Mature proglottid no high power , shows a branched uterus containing hundreds of eggs in each segment.
The chance of any one egg hatching and completing the life cycle to parasitize the final host is so small that high reproductive capacity is a must. Cestode life cycle : Mature proglottids of human tapeworms, Taenia , exit from the body in feces. The head and tail regions were placed in separate, labeled petri dishes to confirm, after regeneration, from which region the regenerated planarian was derived. Measurements of the head and tail region were taken immediately taken after decapitation, 30 minutes after decapitation, and 7 days after decapitation.
Between measurements, the labeled petri dishes were incubated at room temperature, and the water in the petri dish was changed every three 3 to 4 days. After the final measurements, each of the regenerated planarians was finally returned to the stock culture. When placed under a light microscope, the planarian attempted to move away from the light source. This was repeated several times to ensure that the planarian exhibited the same response each time. Because the planarian moved away from the light source, it exhibited negative phototaxis.
Additionally, when touched on both its posterior and anterior ends with a pipette tip, the planarian attempted to move away from the tip, exhibiting sensitivity to touch. With regards to regeneration itself, the planarian length before decapitation was 14 millimeters and each individual region grew to 11 millimeters head region and 6 millimeters tail region after 7 days of incubation at room temperature in pond water.
The average rate of growth over 7 days, as extrapolated from linear regression lines, of the head region was double that of the average rate of growth of the tail region. Figure 1: Length of the planarian at various points after decapitation. Note that the original length of the planarian was 14 millimeters.
Regenerating the central nervous system: how easy for planarians! Developmental Genes and Evolution , Farnesi, R. The frontal organ of a triclad flatworm, Dugesia lugubris. Cell and Tissue Research , Gurley, K. Science , Inoue, T. Zoological Science , Newmark, P. Developmental Biology, Reddien, P. Ruppert, E. Thomson Learning. Takano, T. Regeneration-dependent conditional gene knockdown Readyknock in planarian: Demonstration of requirement for Djsnap expression in the brain for negative phototactic behavior.
Development, Growth, and Differentiation , Umesono, Y. Evolution and regeneration of the planarian central nervous system. It may be necessary to carefully dislodge some individuals with your finger.
Feed planarians once a week. Suitable foods include fresh beef liver, hard-boiled egg yolk, Lumbriculus , pieces of earthworm, crushed aquarium snails, etc. For up to 50 planarians, feed a pea-sized portion. After 30 minutes, transfer the planaria to a fresh container of spring water. Carolina provides living organisms for educational purposes only. As a general policy, we do not advocate the release of organisms into the environment.
In some states, it is illegal to release organisms, even indigenous species, without a permit. The intention of these laws is to protect native wildlife and the environment. Tap water often contains metal ions that are detrimental to planarians. During their sexual period, generally February or March, black and brown planarians are fragile.
Do not handle or feed them during this time. They may deposit cocoons on the bottom of the culture dish. If maintained in fresh spring water, the cocoons will hatch in 2 to 3 weeks, giving rise to several small planaria. The anterior end of the planarian is more sensitive to toxins; if a toxic substance is in the water, the anterior end will degenerate first. Use spring water, not tap water. If you are using spring water from a grocery store, there may be a problem with it. Locally collected spring or pond water may contain a pollutant, or your containers may have soap or detergent residue in them.
Which planarians should I use for regeneration experiments? Black and brown planaria are your best choice. It will take them about 2 weeks to regenerate at room temperatures. White planaria will regenerate, but they take longer. Black planaria are difficult to find in the spring, so we may substitute brown for black.
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