Polarization sensitivity in the red swamp crayfish Procambarus clarkii enhances the detection of a moving transparent object John Tuthill Abstract We tested the hypothesis that polarization sensitivity enhances the detection of a moving, transparent object by examining the escape response of the red swamp crayfish ( Procambarus clarkii ) from a visual threat. A transparent, birefringent target trans-illuminated by either partially linear polarized or unpolarized light was advanced toward individual crayfish. The optical axis of the target was aligned such that it would be conspicuous to a viewer with polarization sensitivity when trans-illuminated by polarized light. Under polarized light, significantly more crayfish retreated from the target than under unpolarized light of identical intensity (p<0.0001, Chi-square). While the potential for polarization sensitivity has been shown in neurophysiological and structural studies of the visual system of P. clarkii and the signal crayfish Pasifastacus leniusculus , our results provide the first behavioral evidence for polarization sensitivity among crayfish. The ecological function of this ability is unclear, but may enhance the detection of fish with mirror-like scales, transparent zooplankton, or macroinvertebrates. Because escape responses are generally more reliably induced than other behaviors, the method employed in this study may prove useful for examining sensory capabilities in other species. 2 Introduction Polarized light in nature In addition to wavelength and intensity, light possesses a third fundamental property that depends on the plane of polarization, often represented by the angle of the electric vector. The polarization of an electromagnetic wave is defined as the distribution of the planes of vibration of the electrical fields within the wave. A light beam oscillating in more than one plane is considered unpolarized, or depolarized. Linearly polarized light contains only a single e-vector direction, meaning that its oscillation occurs within a single plane. Natural light is partially polarized, consisting of a mixture of fully linearly polarized light with a common e-vector angle, and fully depolarized light with indiscriminate e-vector orientation. The percentage of the total field that is comprised by the fully polarized component is defined as the percent polarization. A partially polarized light field is therefore characterized by three properties: intensity, percent polarization, and dominant e-vector. Despite the fact that the sun produces fully depolarized light, partially linearly polarized light is abundant in nature. Rayleigh scattering of sunlight in the atmosphere and water column produces a robust polarization pattern that varies with solar position and direction of view. Underwater, the illumination pattern of the sky is limited to within 48 degrees from the vertical, a region called Snell’s window. Inside Snell’s Window, the polarization pattern is influenced by the same factors that determine the sky polarization, such as sun position, cloudiness, distance from the zenith, and depolarization due to anisotropy of air molecules (Shashar et al., 2004). Outside of Snell’s window, e-vector orientation depends primarily on Rayleigh scattering by submicroscopic particles in the water (Waterman, 2006). The polarization pattern in this region is perpendicular to depolarized light from the sun that is refracted at the air/water interface and 3 scattered in the water column. This e-vector distribution varies with the sun’s refracted zenith angle as well as the viewer’s line of sight, resulting in a complex polarized light field where the perpendicular e-vectors tilt varies with solar position and direction of view (Figure 1). For example, polarization is generally strongest in the horizontal viewing direction with an approximately horizontal e-vector (Shashar et al., 2004) when the sun is at the 0 ° zenith position (at noon). In addition to polarization by scattering, the reflection of light from shiny, dielectric surfaces produces strong polarization in some circumstances (Wehner, 2001). Figure 1. Effect of the solar zenith angle on sky and underwater e-vectors, as the sun moves from sunrise to ideal noon. As the sun continued across the sky, the figure would reflect the right mirror image of the diagram. The tilt of the sky and underwater polarization planes are shown for three different solar zenith angles (adapted from Waterman, 2006). Sensitivity to polarized light in animals Many animals have specific membrane structures that differentially analyze the polarization of incoming light. The ability to distinguish the e-vector of light, or polarization sensitivity, occurs in an eclectic variety of both terrestrial and aquatic species, and serves a variety of functions. Karl von Frisch was the first to demonstrate that honeybees could sense the 4 polarization pattern of the sky, and that they used the e-vector pattern as a compass for navigation while foraging (Frisch, 1967). In honeybees, desert ants, and certain other insects, the dorsal rim of the eye functions specifically to detect the overhead e-vector pattern (Labhart and Meyer, 2002). The same consistent polarization cues are exploited by aquatic organisms for orientation and navigation. In the characteristic “shore-flight” behavior, Daphnia swim toward light with a higher degree of polarization, regardless of intensity (Schwind, 1999). Polarization sensitivity may also be used to enhance the visibility of transparent or otherwise-camouflaged animals in water (Lythgoe and Hemmings, 1967). Octopods are able to recognize contrast produced by e-vector variation within a single imaged object, indicating that they use object detection based on polarized recognition as a means of contrast enhancement (Shashar and Cronin, 1996). Cuttlefish use polarization-based contrast enhancement for detecting transparent prey (Shashar et al., 1998), as well as for breaking the countershading camouflage of light-reflecting silvery fish (Shashar et al., 2000). Some species, such as cuttlefish and stomatopods, may exploit polarization signals for intraspecific recognition and communication (Marshall et al., 1999; Shashar et al., 1996). These experiments demonstrate that animals use patterns of polarized light for image formation rather than just orienting stimuli, and that polarization sensitivity provides some selective advantage for the animals that possess it. Dichroic visual pigment molecules are inherently sensitive to the e-vector orientation of linearly polarized light due to the fact that pigment molecules are maximally excited when the e- vector is parallel to the linear absorption dipole of the chromophore (Waterman, 1981). Because the visual pigments are not fixed in a specific orientation within the flat membranes of rods and cones, most vertebrate photoreceptors are unable to discriminate the e-vector of light. The mechanisms by which some vertebrate species distinguish e-vector is poorly understood 5 (Flamarique et al., 1998). In contrast, the visual pigments in invertebrate compound eyes with rhabdomeric photoreceptors are aligned such that they preferentially absorb light of a specific e- vector. Polarization sensitivity in crayfish Among invertebrates, the structural and neural basis for polarization sensitivity is particularly well-documented in crustaceans, especially the stomatopods (Marshall, 1988) and crayfish (Glantz, 2001). The ommatidia of many crustacean compound eyes possess adjacent photoreceptors with orthogonally oriented microvilli, establishing the potential for discriminating the orientation of the plane in which the e-vector of light oscillates (Waterman, 1981). The red swamp crayfish, Procambarus clarkii , appears to have all the anatomical and neural structures required to distinguish differences in the dominant e-vector of polarized light. The responses of both the long wavelength-sensitive and short wavelength-sensitive retinular cells are affected by the polarization of the incident light (Waterman and Hernandez, 1970; Muller, 1973; Waterman, 1984). In addition, the lamina and medulla externa of the visual system demonstrate polarization sensitivity in four of the neuronal classes that make up the earliest stages of the visual pathway (Glantz, 1996a; Glantz, 1996b). Inputs from photoreceptors and visual interneurons of P. clarkii converge at the medulla externa into two pathways with orthogonal e-vector sensitivity: the sustaining and the dimming fibers (Figure 2) (Glantz and McIsaac, 1998). Vertically and horizontally polarized light activate the sustaining and dimming fibers respectively (Glantz, 1996b). In addition, activation of one pathway inhibits its complement. Antagonistic inputs from these orthogonally oriented polarization analyzers project to both the brain and the medulla terminalis, where polarization opponency between 6 photoreceptors may be integrated (Glantz, 2001). Structural investigations of connectivity patterns between receptors and interneurons have indicated that opponency between these cell groups may provide a mechanism for analyzing and enhancing the sensitivity to the polarization of the signal (Glantz, 1996b). In most of the cells of this pathway, the polarization response is substantially higher for changing e-vector orientation than for fixed orientation, suggesting that polarization discrimination may enhance sensitivity to moving stimuli, a function that is particularly well-developed in crayfish (Glantz, 2001). Figure 2. A simplified schematic