Polarization sensitivity in the red swamp crayfish Procambarus clarkii enhances the detection of moving transparent objects

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).

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.

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.

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).

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.

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.

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.

The ecological function of polarization sensitivity in this species is unclear. P. clarkii is an abundant opportunistic omnivore found in seasonally flooded wetlands, lakes and streams throughout North America, often concealing itself in burrows or under rocks and logs(Gherardi, 2002). At shallow depths in streams or lakes, the overhead polarization pattern is essentially a distortion of the polarization pattern of skylight(Horváth and Varjú,2004). At angles more than 48° from the vertical, this pattern is replaced by a complex pattern due to reflections from underwater objects and scattering of downwelling light(Horváth and Varjú,2004). With increasing turbidity, the polarization vanishes.

Transparent prey (e.g. zooplankton, macroinvertebrates) viewed against polarized backgrounds may be detectable because the scattering of light within their tissues can rotate or disrupt the background polarization pattern(Shashar et al., 1998). Fish,the chief predators of crayfish(Nyström, 2002), may be detected via the polarized reflections from their mirror-like scales(Shashar et al., 2000). Analysis of reflected polarized light may also allow crayfish to detect and recognize substrata, plants, and other underwater features(Novales Flamarique and Hawryshyn,1997). Levels of polarization in the upper photic zone are highest during crepuscular periods (Novales Flamarique and Hawryshyn, 1997) when crayfish are most active(Gherardi, 2002), indicating that crayfish forage when they are most visually prepared to detect prey and avoid potential predators.

Understanding the function of polarization sensitivity in P. clarkii is complicated by the fact that the polarization information may interact with color vision. An ommatidium of P. clarkii has seven photoreceptors containing a visual pigment that peaks at 530 or 567 nm(depending on chromophore) (Zeiger and Goldsmith, 1989). With the exception of those in the dorsal retina, the ommatidia also contain an eighth cell expressing a visual pigment that peaks at 440 nm (Cummins and Goldsmith, 1981). Although sample sizes are low, it appears that,in the anterior ommatidia, most of the 440 nm receptors are sensitive to horizontal polarization, and most of the 530/567 nm receptors are sensitive to vertical polarization. In the dorsal retina, which contains only the 530/567 nm receptors, most of the cells are sensitive to vertical polarization(Waterman and Fernandez,1970). These results suggest both regional and chromatic specialization of polarization sensitivity and complicate any attempt to understand its function.

One of the difficulties in research on polarization sensitivity is that the behavioral responses have generally been marginal (reviewed by Waterman, 1981). Thus, many significant results have been based on small differences and large samples sizes. This makes experiments difficult to replicate and generates concerns about potential biasing artifacts. In contrast, this study achieved a P value less than 0.00005 with a sample size of only 40 animals. We attribute the strength of these results to the stereotypical and critical nature of escape responses. Whereas feeding, orientation, and social behaviors are important to the survival and fitness of an organism, they tend to be less reliably induced than escape responses, particularly after a period of captivity. For this reason, we believe that sensory assays based on escape responses may be quite useful for other species and other sensory abilities.

We thank Rachel Merz for a critical reading of the paper. The Norman A. Meinkoth Award at Swarthmore College provided funding for J.T. S.J. was supported in part by a grant from the National Science Foundation(IOB-0444674).