Introduction

Polarization sensitivity occurs in an eclectic variety of both terrestrial and aquatic species, and serves a variety of functions including orientation (Hawryshyn, 2000; Waterman, 1988), navigation (Goddard and Forward, 1991; Rossel, 1993; Wehner, 1976), intra-specific communication (Cronin and Cronin, 2003), camouflage-breaking (Shashar et al., 1998) and contrast enhancement (Lythgoe and Hemmings, 1967; Shashar and Cronin, 1996). 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 Fernandez, 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 (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 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).

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Fig. 1.

Schematic diagram of the experimental apparatus used in the study. The PVC half-section of pipe is transparent in this figure to show the inside of the device. In the actual setup the polarizer/diffuser sheets were sandwiched together against the side of the tank.

In this study we test whether the neural evidence for polarization sensitivity in P. clarkii translates into a behavioral response. In particular, we test whether polarization information in an otherwise cryptic moving stimulus increases the ability of P. clarkii to detect it.

Materials and methods

Male and female red swamp crayfish, Procambarus clarkii (Girard) (6–9 cm length) were bought from a biological supply company (Carolina Biological Inc., NC, USA). They were maintained in two aerated and filtered 120 l aquaria on a 12 h:12 h light:dark cycle at 20°C, and fed peas weekly in separate 40 l aquaria.

The experimental apparatus consisted of a half-section of white plastic pipe (10 cm diameter, 48 cm length) that was partitioned into two chambers separated by a sheet of acrylic (Fig. 1). The 13 cm long rear section, where each crayfish was isolated, had a second wall of opaque white plastic. A longitudinal cut (33 cm length) was made down the top of the front section of the pipe, along which a transparent target could be advanced toward the crayfish chamber. The target was attached via a short rod and monofilament to a 300 g mass. When dropped, the mass pulled the target down the length of pipe at an average velocity of 0.3 m s^–1 over 0.65 s. The cut ended 2 cm before the crayfish chamber, thus stopping the target before it struck the chamber wall.

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Fig. 2.

The target was constructed of clear acrylic covered with colorless, polarization-active Mylar. (A) Unmodified photograph of the transparent target viewed under polarized light. (B) Polarization contrast image of the target (trans-illuminated by vertically polarized light) generated by taking two photographs through a polarizing filter that was rotated by 90° between exposures. Each pixel brightness is equal to 255[(I_v–I_h)/(I_v+I_h)], where I_v and I_h are the pixel values when the transilluminated target is viewed through a vertical and horizontal polarizer respectively. (C) Same as in B but with wax-paper diffuser depolarizing the light. Because the neural responses of P. clarkii to changing e-vector depend on the rate of change (Glantz, 2001), and possibly on color, it is impossible to simulate how the moving target appears under each condition. Thus B and C are probably only an approximate indication of the contrast of the target under polarizing and non-polarizing conditions, respectively. The bar on right shows the contrast scale.

The transparent target was made of a sandwich of 0.3 cm thick clear acrylic and an optically anisotropic, colorless sheet of Mylar (Dura-lar Clear Overlay Film, Medium Weight 0.003; Graffix, Cleveland, OH, USA). The Mylar was aligned so that it converted the linearly polarized light to nearly circularly polarized light. Thus, while inconspicuous under transmitted unpolarized light, the target's optical properties rendered it highly visible when viewed under transmitted polarized light by a viewer with polarization sensitivity (Fig. 2).

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Fig. 3.

Spectral irradiance inside the crayfish chamber under both polarization conditions. The irradiance probe faced the light source.

The testing apparatus rested on a sheet of acrylic within a 40 l aquarium filled with water. A 75 W flood lamp was mounted 10 cm from the front of the tank. The light passed through a sandwich of a linear polarizer (HN38S, Polaroid Co., Waltham, MA, USA) and a wax-paper diffuser/depolarizer. For the polarized light trials, the polarizer followed the depolarizer. For the unpolarized light trials, the polarizer preceded the depolarizer. Thus, the two lighting conditions differed only in polarization and not in intensity or spectral distribution (Fig. 3).

Experiments were conducted in June and July of 2005, between 10:00 and 16:00 DST. Before each day's trials, the experimental tank was rinsed and filled to approximately 20 cm depth with deionized water. Each trial then proceeded as follows. A crayfish was placed in the experimental chamber and allowed a 5-min acclimation period. If, after 5 min, the crayfish did not face toward the light source, an extra period of up to 5 min was allowed for the crayfish to orient correctly. After 10 min, whether the crayfish was oriented towards the light or not (5 out of 40 did not), the target was advanced by dropping the mass from the top of the aquarium. Each animal was tested once under either the polarized or unpolarized condition.

The responses of the crayfish to the advancing target were recorded through the glass bottom of the aquarium using a digital video camera. The presence or absence of a response was recorded by a blind (ignorant of light condition) examination of the video recordings of each experimental trial. Based on preliminary observations prior to testing, a positive response was defined as a retreat by the crayfish of >2 cm. No animal retreated before the target moved.

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Fig. 4.

Response of Procambarus clarkii to the approach of the target under polarized and unpolarized conditions (N=20 for each treatment). Crayfish retreated from the target significantly more often in the polarized condition (P<0.00005).

Results

Under partially linearly polarized light, P. clarkii was four times as likely to retreat from an advancing, transparent, polarization-active object as under unpolarized conditions (P<0.00005, Fisher exact test; Fig. 4). Out of 20 trials that took place under un-polarized light, four of the subjects retreated from the stimulus. Under polarized light, 17 out of 20 crayfish retreated. The retreat response generally consisted of the crayfish pushing off with the walking legs and chelipeds, and occasionally of a tail-flip escape response. Tail-flip escapes were observed only under polarized light conditions, in four out of the 20 trials.

Discussion

The highly significant difference in response between treatments suggests that crayfish were more aware of the advancing target under polarized light conditions. These results provide the first behavioral demonstration of polarization sensitivity in crayfish and one of the few studies that show contrast enhancement.

Whether the increased number of retreats is due to enhanced detection of the transparent target itself, or enhanced detection of its motion (or both), is uncertain. P. clarkii are sensitive to moving visual stimuli over a velocity range of at least four orders of magnitude (Glantz, 2001), indicating that motion detection is a critical component of the crayfish visual system. A significant portion of the primary visual synapse (Glantz and Bartels, 1994) and the ascending optic tract (Wiersma and Yamaguchi, 1966) are devoted to motion sensitivity. Glantz (Glantz, 2001) proposed that the polarization sensitive-neurons might also contribute to this pathway. This hypothesis is supported by the existence of tangential cells in the crayfish optic lobe that demonstrate multidimensional selectivity to contrast, motion and e-vector, and enhanced responses to changes in e-vector orientation versus responses to fixed orientations (Glantz, 1996b; Glantz, 2001). Detection of movement using polarization opponency is analogous to using color opponency to detect movement in the absence of intensity contrast (Bernard and Wehner, 1977). The results of this study are consistent with, but do not confirm, the hypothesis that crayfish use such a polarization-opponency mechanism to enhance motion detection.