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. 2014 Dec 10;9(12):e114708.
doi: 10.1371/journal.pone.0114708. eCollection 2014.

Planarian Phototactic Assay Reveals Differential Behavioral Responses Based on Wavelength

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Free PMC article

Planarian Phototactic Assay Reveals Differential Behavioral Responses Based on Wavelength

Taylor R Paskin et al. PLoS One. .
Free PMC article

Abstract

Planarians are free-living aquatic flatworms that possess a well-documented photophobic response to light. With a true central nervous system and simple cerebral eyes (ocelli), planarians are an emerging model for regenerative eye research. 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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Planarian Eye Anatomy.
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.
Figure 2
Figure 2. Photophobia Assay.
(A) The imaging setup. CT = Camera mounted on tripod. W = LED wand. D = Testing dish. C1/C2 = Clamps. B = Battery pack. R = Ring stand. (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. UV = Ultraviolet. IR = Infrared.
Figure 3
Figure 3. Planarian Photophobic Responses Vary by Wavelength.
(A) Images of the photophobia assay showing single trials (one group, n = 6) for control (ambient light, left) and UV 360 (right) wavelengths. All worms begin in quadrant 1 (Q1, red circles). While control worms randomly explore the dish, in UV 360 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. Significance (asterisks = p<0.001 as compared to controls) was calculated by Mann-Whitney (with Dunn’s Q), which takes into account worm location across all four quadrants simultaneously. Red dashed line = average control value.
Figure 4
Figure 4. Photophobic Responses Result from Light Stimulus.
Graph showing behavioral responses over increasingly attenuated light, as measured by the number of worms in Q4 at 2 minutes. Worms were exposed to full light, 95% attenuated light, and 99% attenuated light (or optical densities of 0, 1.3 and 2.0 respectively). The trend shows that phototactic responses decreased along with diminished behavioral stimuli (light).
Figure 5
Figure 5. Escape Responses Vary by Wavelength.
Graph showing escape responses as a measure of the severity of phototactic behavior. The escape index (formula image) 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. At 30 seconds, the data indicate that UV wavelengths elicited a significantly stronger escape response, distinct from both controls (p<0.001) and all other wavelengths (p<0.01); while IR wavelengths produced an opposite, attractive response (p<0.001). All time points are significantly different from controls (p<0.001) by two-way ANOVA, except for red at 1.5 minutes (p<0.01) and 2 minutes (not significant). Note the latter data indicate by 2 minutes worms have returned to the direct red light source in Q1.
Figure 6
Figure 6. Light Avoidance Responses Vary by Wavelength.
Avoidance assay to test worm responses when approaching areas of direct light. Red (top row), green (middle row), and UV (bottom row) wavelengths of laser light were placed in the worm’s path (photos), resulting in three distinct behaviors (shaded areas): worms moved into the light (left column), went around the light (middle column), or avoided the light by making 90–180 degree turns (right column).
Figure 7
Figure 7. Planarian Photophobic Behavior is Hierarchal.
(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.

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Grant support

This work was supported by a start-up grant from Western Michigan University to WB and a Faculty Research and Creative Activities Award from the Office of the Vice President for Research at Western Michigan University (W2013-007) to JJ. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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