Quantitative measurement of the optomotor response in free-swimming Daphnia

Quantitative measurement of the optomotor response in free-swimming Daphnia Abstract Daphnia are trophically important, common zooplankton in lakes and ponds worldwide, and hence have become a significant model system in ecology and evolutionary biology. Daphnia have conspicuous eyes that confer both fitness benefits and costs, though little is known about the functional capabilities of Daphnia vision. We sought to determine whether free-swimming Daphnia exhibit an optomotor response, a reflexive behavior present in many animals that could be used in studies of visual function. Optomotor responses are movements that allow an individual to track motion of the surrounding environment and thus stabilize its visual field. We constructed an apparatus that rotates a striped drum around Daphnia, while allowing an observer to quantify swimming behavior. Across a range of angular stripe widths and rotational speeds, we demonstrate that Daphnia have an optomotor response composed of at least two distinct behaviors. Our results suggest Daphnia could use vision for predator avoidance in some circumstances, but could not see other Daphnia individuals beyond more than a few millimeters, or food particles at all. We discuss how the optomotor response may be used to study visual function in Daphnia to better understand visually mediated ecological interactions. INTRODUCTION The optomotor response (OMR) is an innate reflex involving eye, head or body movements that stabilize an animal’s visual field when the surrounding environment is moving (Land and Nilsson, 2012). The presence of an OMR in an animal demonstrates that the animal can detect and respond to motion. Therefore, the OMR in an animal can be used to determine the range of motion that the animal can see and the conditions under which it can be seen (e.g. Srinivasan et al., 1999; Lunau, 2014; Sharkey et al., 2015). Prior work has shown that many aquatic animals have an OMR, including fish (Anstis et al., 1998; Neuhauss, 2003; Rinner et al., 2005; Lerner et al., 2017) and invertebrates (Buskey, 2000; Baldwin and Johnsen, 2011; Sharkey et al., 2015; Tomsic, 2016). Daphnia are small freshwater zooplankters (1–2 mm long as adults) that live in lakes and ponds. Daphnia are often the keystone herbivore in freshwater ecosystems and are amenable to both field and laboratory experiments, leading them to become one of the main model organisms in ecology (reviewed in Benzie, 2005; Stollewerk, 2010; Lampert, 2011; Seda and Petrusek, 2011). They are also an important model in evolutionary biology, in part because they are cyclic parthenogens, which simplifies ascertainment of genetic and environmental effects in genetically diverse populations (e.g. Lynch, 1983; Weider and Hebert, 1987; Boersma et al., 1998; Dudycha, 2004; Walsh and Post, 2011; Dudycha et al., 2012). Their physiological characteristics are less well known, and few studies address the functional capabilities of organs or organ systems, particularly in the context of ecological consequences. Daphnia possess a conspicuous apposition compound eye (Ringelberg, 1987), and many species have an additional inconspicuous simple eye called an ocellus (Fig. 1). The dark, pigmented compound eye contains 22 ommatidia, arranged in a 360-degree manner with each ommatidium sampling different spatial regions (Frost, 1975; Young and Downing, 1976). The compound eye forms as two separate eyes that fuse during embryonic development, and is known to be developmentally plastic in response to resource abundance, but not light intensity (Brandon and Dudycha, 2014). Because the compound eye is the darkest part of largely transparent zooplankton, it has been suggested that predation pressure from visual hunters in lakes would select for smaller eyes (Zaret, 1972; Zaret and Kerfoot, 1975; Lenz et al., 1996). However, this has not been tested directly in Daphnia, and an analysis in a fishless pond found evidence for selection favoring larger eyes in D. obtusa Kurz (Brandon et al., 2016). Fig. 1. View largeDownload slide Daphnia pulex. The compound eye is the large black structure, with ommatidia lenses appearing as clear circles on the surface of the eye. The simple eye (ocellus) is the small black circle below the eye. Inset: a whole female, showing the size of the eye relative to the body. Fig. 1. View largeDownload slide Daphnia pulex. The compound eye is the large black structure, with ommatidia lenses appearing as clear circles on the surface of the eye. The simple eye (ocellus) is the small black circle below the eye. Inset: a whole female, showing the size of the eye relative to the body. Prior work has shown that Daphnia vision is capable of detecting the orientation of light polarization (Schwind, 1999; Flamarique and Browman, 2000), which may be used in horizontal positioning within lakes. Several studies have shown phototaxis in both the lab and the field (e.g. De Meester, 1991, 1993; Ringelberg and Flik, 1994), and phototaxis has been linked to the diel vertical migration of lake zooplankton (reviewed in Ringelberg, 1999). Other work has indicated that Daphnia can detect, track and fixate on a moving light source (Frost, 1975; Consi et al., 1990). Daphnia can move the compound eye within the head, changing its orientation and position. Frost (1975) made a detailed study of these movements in anchored Daphnia, and described parameters of eye tremors and positional movement in response to several stimuli. This work showed that the compound eye is capable of responding to variation in light and motion. An electrophysiological study of the compound eye has shown that Daphnia vision is at least tetrachromatic (Smith and Macagno, 1990), and their genome contains an unusually large family of opsins that dates back to the Mesozoic (Brandon, et al., 2017). Due to the potential for contrasting selection pressures on compound eye size and the likely high energetic costs of visual tissue, we sought to better understand the functional capability of Daphnia vision. In particular, we wanted to determine whether free-swimming Daphnia exhibit behavioral responses to motion. Ecologically, such a capability might be used in predator avoidance or evasion, mating or foraging. We were further interested in estimating their visual acuity, the ability of an eye to precisely sample the surrounding environment, involving both spatial and temporal resolution. To accomplish these goals, we constructed an apparatus to test for an OMR in free-swimming Daphnia pulex Leydig. We predicted that D. pulex would have an OMR, and that it would be evident when environmental structure exceeded the inter-ommatidial angle of 38° (Young and Downing, 1976). We then manipulated the spatial and temporal scales of motion around experimental individuals in a randomized two-factor experiment to evaluate the functional boundaries of Daphnia vision. METHODS OMR apparatus We designed an experimental apparatus based on similar work addressing OMR in guppies (Anstis et al. 1998) and blue crabs (Baldwin and Johnsen 2011). The machine consisted of a cylindrical drum (inner diameter: 24 cm, height 30 cm) attached to a DC motor, a 20 cm diameter water dish, and a second, smaller glass dish at the center of the water dish (Fig. 2). The water dish served as the experimental arena for individual Daphnia during testing, and was filled with water to a depth of 4 cm. The smaller central dish blocked swimming in the middle of the arena, and functioned to balance the competing objectives of keeping individual Daphnia at approximately the same distance from the walls of the rotating drum, while still allowing them the opportunity to swim naturally in any direction. Individuals were thus constrained to swim in a 4 cm wide circular band of open water. A Daphnia swimming at the midpoint of the band would be 4.4 cm from the drum walls, but was able to range as close as 2.4 cm or as far as 6.4 cm. The walls of the cylindrical drum were lined with paper on which alternating black and white stripes were printed. The drum was then rotated around the water tank at a constant speed by the DC motor. The motor’s voltage was controlled by the experimenter, which, in combination with adjustments to the position of a rubber wheel driving the drum rotation, allowed for tests to be conducted at a wide range of rotational speeds. The apparatus was open at the top, allowing behaviors to be scored directly. Fig. 2. View largeDownload slide Schematic illustration of the OMR apparatus and OMR behaviors. The dashed line (not to scale) indicates the alternating white and black stripe stimulus pattern that was affixed to the inside of a rotatable drum. The experimental arena in which individual Daphnia are placed is a clear glass water tank whose center is blocked off by a clear glass dish. In the compass reaction, the Daphnia, indicated by the letter D, rotates on its vertical axis. In the circling reaction, the Daphnia swims in concert with the moving striped wall. Fig. 2. View largeDownload slide Schematic illustration of the OMR apparatus and OMR behaviors. The dashed line (not to scale) indicates the alternating white and black stripe stimulus pattern that was affixed to the inside of a rotatable drum. The experimental arena in which individual Daphnia are placed is a clear glass water tank whose center is blocked off by a clear glass dish. In the compass reaction, the Daphnia, indicated by the letter D, rotates on its vertical axis. In the circling reaction, the Daphnia swims in concert with the moving striped wall. OMR Scoring We made preliminary observations of Daphnia swimming in the arena to determine how to record Daphnia behaviors. These observations led us to define two distinct behaviors that would potentially indicate an OMR. In the “compass reaction”, individuals rotate on their vertical axis in the same direction as the motion of the striped wall which surrounds them. In the “circling reaction,” individuals are observed to swim in the same direction as the motion of the striped wall which surrounds them. Video recordings failed to show the compass response, so we relied on direct observations during our experiment. For each experimental trial, one adult Daphnia (age 14–21 d) was placed in the arena and allowed to acclimate for 2 minutes. The apparatus was then turned on, and animals were allowed to acclimate for a further 2 minutes while the drum was rotating. This ensured we avoided mis-scoring any startle response to small vibrations from the motor as an OMR. Later experiments directly comparing the first and third minutes after the apparatus was turned on showed no difference in scores (Perez and Dudycha, unpublished data). Behavior was then scored for 1 minute as the number of seconds engaged in the compass response, the number of seconds engaged in the circling response, and the number of seconds engaged in neither response. A metronome timer was used to allow the observer to count seconds in real-time. The total OMR score was the sum of the time spent circling or in the compass response. Experimental design We conducted a randomized two-factor experiment using five levels of stripe width and five rotational speeds (1.22, 1.94, 2.45, 3.16 and 4.00 rpm), for a total of 25 different treatments. Stripes were either 17.5, 27.8, 34.5, 44.0 or 55.0 mm wide. In addition, we scored behaviors at all five speeds with the walls of the rotating drum covered with a uniform 50% gray to serve as controls in which motion could not be visually detected. Trials were conducted in a randomized order, with individuals randomly assigned to treatment. Behavior was scored on five replicate adult Daphnia pulex (clone PA42, the clone used for the Daphnia reference genome; Ye et al. 2017) for each experimental condition (125 experimental trials + 25 control trials = 150 independent experimental individuals). For the first 22 trials (20 experimental, plus 2 control trials), behavior was recorded as just the sum of time spent circling or in compass rotation. Thereafter, times for circling and compass were recorded separately. Prior to behavioral assays, Daphnia were maintained under standard laboratory conditions (low density; 12:12 L:D photoperiod; 20°C; fed daily with Ankistrodesmus falcatus. For further details, see Dudycha et al., 2013; Dudycha and Hassel, 2013). We based our range of stripe widths on a prior estimate that the inter-ommatidial angle in the Daphnia eye is 38° (Young & Downing, 1976), which should define the smallest section of their field-of-view that could be clearly resolved. In our arena, Daphnia were not located at the centerpoint of the rotating drum. We assumed Daphnia would be on average 4.4 cm from the drum, at the midpoint of the space in which they could swim. Our prediction was that Daphnia should definitely be able to see stripes that appeared ≥40°, approximately our mid-sized stripe (Table I). We based rotation speeds on preliminary observations of Daphnia swimming, and set our fastest speed to approximate the swimming capabilities of Daphnia. This ensured that we would not mistakenly interpret a failure to be able to swim fast enough to keep up with the rotation as absence of an OMR. Table I: Significance of Dunnett’s post hoc tests for differences between behavior at specific stripe widths compared to the gray controls Stripe width OMR total p = Compass p = Circling p = 17.5 mm (21.7°) 0.012 0.161 0.328 27.8 mm (32.3°) 0.000 0.001 0.287 34.5 mm (38.1°) 0.000 0.000 0.058 44.0 mm (45.0°) 0.000 0.000 0.109 55.0 mm (51.3°) 0.000 0.000 0.238 df 144 122 122 MSE 96.517 0.453 0.983 Stripe width OMR total p = Compass p = Circling p = 17.5 mm (21.7°) 0.012 0.161 0.328 27.8 mm (32.3°) 0.000 0.001 0.287 34.5 mm (38.1°) 0.000 0.000 0.058 44.0 mm (45.0°) 0.000 0.000 0.109 55.0 mm (51.3°) 0.000 0.000 0.238 df 144 122 122 MSE 96.517 0.453 0.983 Stripe widths are given in absolute distance, along with the angular field of view occupied for an individual at the midpoint of the swimming space, estimated as the arctangent of the ratio of the distance to the rotating drum and the absolute width of the stripe. Degrees of freedom (df) and the pooled mean square error (MSE) are given for each trait’s test in the bottom rows. Bold = significant at the p < 0.05 level. Table I: Significance of Dunnett’s post hoc tests for differences between behavior at specific stripe widths compared to the gray controls Stripe width OMR total p = Compass p = Circling p = 17.5 mm (21.7°) 0.012 0.161 0.328 27.8 mm (32.3°) 0.000 0.001 0.287 34.5 mm (38.1°) 0.000 0.000 0.058 44.0 mm (45.0°) 0.000 0.000 0.109 55.0 mm (51.3°) 0.000 0.000 0.238 df 144 122 122 MSE 96.517 0.453 0.983 Stripe width OMR total p = Compass p = Circling p = 17.5 mm (21.7°) 0.012 0.161 0.328 27.8 mm (32.3°) 0.000 0.001 0.287 34.5 mm (38.1°) 0.000 0.000 0.058 44.0 mm (45.0°) 0.000 0.000 0.109 55.0 mm (51.3°) 0.000 0.000 0.238 df 144 122 122 MSE 96.517 0.453 0.983 Stripe widths are given in absolute distance, along with the angular field of view occupied for an individual at the midpoint of the swimming space, estimated as the arctangent of the ratio of the distance to the rotating drum and the absolute width of the stripe. Degrees of freedom (df) and the pooled mean square error (MSE) are given for each trait’s test in the bottom rows. Bold = significant at the p < 0.05 level. All trials were scored by the same observer. Trials were conducted under white fluorescent laboratory lighting (Philips Universal/Hi-Vision bulbs) that produces 7–10 μmol quanta m−2 s−2 at the bench surface on which the apparatus was placed. Statistical analyses were conducted in Systat 13. Visual inspection of distribution plots showed that the total OMR score was approximately normally distributed, but that the separate compass and circling scores were strongly skewed. Therefore, we ln-transformed those scores (+1 to account for zeroes), which produced approximately normal distributions, before applying linear models. RESULTS Behavior under control conditions We first used ordinary least-squares regression to test whether rotation speed influenced the OMR scores obtained from the gray controls. No significant effects were found for the compass reaction (df = 1, 21; F = 0.613; p = 0.442), for the circling reaction (df = 1, 21; F = 0.421; p = 0.524) or for the combined OMR score (df = 1, 23; F = 0.541; p = 0.470). Therefore, we pooled control data across the different speeds to estimate the average OMR scores under conditions where there was no visually detectable motion. For the controls, the average compass reaction score was 7.609 ± 1.290 SE, the average circling reaction score was 5.261 ± 1.262 SE and the average total OMR score was 12.760 ± 1.257 SE. These values indicate the duration of time (seconds) in which an observed individual was, just by chance, moving in concert with environmental rotation. Behavior under experimental conditions Because speed may influence the stripe width that individuals can see, we tested for their effects in a two-factor analysis of variance (ANOVA) including an interaction term. Levene’s test was used to confirm homogeneity of variances for the OMR total score and the ln-transformed compass and circling reactions (total: df = 5, 144, F = 1.383, p = 0.234; compass: df = 4, 123, F = 0.120, p = 0.297; circling: df = 4, 123, F = 1.242, p = 0.297). The overall model was significant for the total OMR score (df = 3, 146; F = 10.739; p < 0.001), the compass reaction (df = 3, 124; F = 11.490; p < 0.001) and the circling reaction (df = 3, 124; F = 2.592; p = 0.050). None of the behavioral measures showed a significant interaction between speed and stripe width (total OMR: p = 0.531; compass: p = 0.088; circling: p = 0.505). Furthermore, the effect of speed was insignificant for all behavioral measures (total OMR: p = 0.782; compass: p = 0.310; circling: p = 0.177). Stripe width, however, had a significant effect on the compass reaction (p = 0.001) and total OMR score (p = 0.016) but not the circling reaction (p = 0.872). Because speed had no significant effect on OMR behaviors over the range we assayed, we pooled data across speeds to analyze stripe width further. At all stripe widths, OMR scores were higher than for gray controls (Fig. 3), indicating that free-swimming Daphnia have an OMR. We tested whether stripe width affected behaviors with a one-way ANOVA followed by Dunnett’s post hoc tests to determine whether each individual stripe width was significantly different from the gray controls. Stripe width had a significant effect on the total OMR response behavior (df = 5, 144; F = 11.008, p < 0.001), and each individual stripe width induced a detectable response (Table I). We analyzed each behavior separately to determine which were contributing to the overall pattern. Stripe width significantly influenced the compass reaction (df = 5, 122; F = 6.675; p = 0.001), but influence on the circling reaction was not significant (df = 5, 122; F = 1.573; p = 0.173), confirming results from the two-factor analyses. All but the narrowest stripe widths had a significant effect on the compass reaction, but only the 34.5 mm width had a marginally significant effect on the circling reaction (Table I). Fig. 3. View largeDownload slide OMRs of Daphnia pulex at different stripe widths. Points show means and standard errors. “Gray” on the horizontal axis refers to the scores from gray controls (no stripes). Horizontal dashed lines are drawn at the control scores. (A) Compass reaction, or rotation on the animal’s vertical axis in concert with the motion of the rotating drum. (B) Circling reaction, or swimming laterally in concert with the motion of the rotating drum. (C) OMR total, the sum of the compass and circling reactions. Fig. 3. View largeDownload slide OMRs of Daphnia pulex at different stripe widths. Points show means and standard errors. “Gray” on the horizontal axis refers to the scores from gray controls (no stripes). Horizontal dashed lines are drawn at the control scores. (A) Compass reaction, or rotation on the animal’s vertical axis in concert with the motion of the rotating drum. (B) Circling reaction, or swimming laterally in concert with the motion of the rotating drum. (C) OMR total, the sum of the compass and circling reactions. Because the total OMR response had smaller variances than either component behavior (Fig. 3) and had greater statistical confidence in the effects of stripes, we tested for a negative association between the two behaviors (excluding data from the gray controls). If there were instead a positive relationship, this would indicate that individuals mainly differed in magnitude of the OMR, rather than the type of OMR. The behaviors were negatively correlated (Fig. 4), with R2 = 0.238. The sum of these two measures must be ≤60, constraining the state-space in which observations could occur. However, the relationship was shallower than required, and there was no evidence of a positive relationship. Fig. 4. View largeDownload slide Association between time spent circling and time spent in the compass reaction. Each point shows a unique individual. Observations from the gray controls are excluded. Fig. 4. View largeDownload slide Association between time spent circling and time spent in the compass reaction. Each point shows a unique individual. Observations from the gray controls are excluded. Reports on OMR in most species measure responses categorically (i.e. yes–no) rather than quantitatively. To investigate how this approach may have influenced our interpretation, we re-coded our data as if they were categorical. In the basic evaluation, we considered any observation where the total OMR score exceeded the mean for the gray controls to be a “YES” and lower scores to be a “NO.” Using one-way chi-square tests, we found that all stripe widths produced a significant OMR under this criterion, with the lowest confidence for the 17.5 mm stripes and the highest at 38.5 mm with 100% of 25 observations scored “YES” (Table II). If we used a more stringent criterion to score an observation as “YES” (>gray control mean + 1 standard deviation), chi-square tests showed significant effects of stripes only at 27.8 (marginally), 34.5, and 44.0 mm (Table II). Table II: Comparison of categorical analyses made with different levels of stringency for determining whether there is an OMR Stripe width Basic χ2 = Basic p = Stringent χ2 = Stringent p = 17.5 mm (21.7°) 9.000 0.003 0.040 0.841 27.8 mm (32.3°) 21.160 0.000 3.240 0.072 34.5 mm (38.1°) Undefined 0.000 17.640 0.000 44.0 mm (45.0°) 21.160 0.000 17.640 0.000 55.0 mm (51.3°) 14.440 0.000 0.360 0.549 Stripe width Basic χ2 = Basic p = Stringent χ2 = Stringent p = 17.5 mm (21.7°) 9.000 0.003 0.040 0.841 27.8 mm (32.3°) 21.160 0.000 3.240 0.072 34.5 mm (38.1°) Undefined 0.000 17.640 0.000 44.0 mm (45.0°) 21.160 0.000 17.640 0.000 55.0 mm (51.3°) 14.440 0.000 0.360 0.549 For the “basic” analysis, we treated any total OMR score greater than the mean of the gray controls as a positive OMR. For the “stringent” analysis, only scores greater than the mean + 1 SD were considered to show an OMR. All tests are one-way chi-square tests with one degree of freedom. Bold = significant at the p < 0.05 level. Table II: Comparison of categorical analyses made with different levels of stringency for determining whether there is an OMR Stripe width Basic χ2 = Basic p = Stringent χ2 = Stringent p = 17.5 mm (21.7°) 9.000 0.003 0.040 0.841 27.8 mm (32.3°) 21.160 0.000 3.240 0.072 34.5 mm (38.1°) Undefined 0.000 17.640 0.000 44.0 mm (45.0°) 21.160 0.000 17.640 0.000 55.0 mm (51.3°) 14.440 0.000 0.360 0.549 Stripe width Basic χ2 = Basic p = Stringent χ2 = Stringent p = 17.5 mm (21.7°) 9.000 0.003 0.040 0.841 27.8 mm (32.3°) 21.160 0.000 3.240 0.072 34.5 mm (38.1°) Undefined 0.000 17.640 0.000 44.0 mm (45.0°) 21.160 0.000 17.640 0.000 55.0 mm (51.3°) 14.440 0.000 0.360 0.549 For the “basic” analysis, we treated any total OMR score greater than the mean of the gray controls as a positive OMR. For the “stringent” analysis, only scores greater than the mean + 1 SD were considered to show an OMR. All tests are one-way chi-square tests with one degree of freedom. Bold = significant at the p < 0.05 level. DISCUSSION Free-swimming Daphnia have an OMR, and broadly are able to see and respond to high-contrast elements in their environment. Prior work has shown that Daphnia can see overall fields of light, but this is the first clear demonstration that they can behaviorally respond to visual structure in their environment. Two distinct behaviors are involved, rotation around an individual’s vertical axis and swimming in concert with environmental motion. Genetically identical individuals can differ in which OMR they tend to use. The resolution at which Daphnia responded to rotational motion suggests that their vision could be used in predator avoidance and possibly in some aspects of mating. We estimated the angular field of view that each stripe width would occupy for an individual swimming at the midpoint of the band of open water, 4.4 cm from the rotating drum wall (Table I). Daphnia exhibited a significant OMR with our narrowest stripes tested (17.5 mm, ~ 21.7° at the midpoint of the distance Daphnia could be from the striped wall). Therefore, it is possible that the minimum threshold for Daphnia to see a high-contrast object is even narrower than 21.7°, and considerably narrower than the 38° threshold we predicted based on the inter-ommatidial angle. Daphnia eyes are anchored by a short muscular stalk, and individuals can use the stalk to move the eye rapidly within the head, both rotationally and positionally (Frost, 1975; Consi et al., 1990). Such motion could give Daphnia a greater visual resolving power than the inter-ommatidial angle would suggest, if the eye movements allow for temporal summation of light from slightly different directions within individual ommatidia (Warrant, 1999; Greiner, 2006). Indeed, our results are consistent with Frost’s (1975) detailed observations of eye movements in anchored Daphnia pulex. Frost described four types of internal eye movements: a fast tremor, which he proposed served to increase visual acuity; slow scanning, which might play a role in searching for objects; saccadic eye movements that re-oriented eyes in response to directional light sources; and optokinetic nystagmus (OKN). Frost’s observations of OKN are most relevant to our work. Testing stripe widths that ranged from 5° to 45°, he found the strongest response at 22.5°, and no response at 10° or below. Thus, his work also indicated a visual acuity below the 38° inter-ommatidial angle, and is broadly in line with our estimate. Indeed, a curve fit to our data in Fig. 3C would suggest a lower visual threshold of ~10°. How these internal eye movements may occur and function in Daphnia that can move their bodies in response to visual cues remains unknown. Nonetheless, it seems likely that Daphnia combine eye movements and swimming to maximize the useful visual information they acquire and respond to that information. We did not find a speed threshold for the OMR, but our work suggests a spatial threshold, and we can use that to make preliminary inferences about the ecological function of vision. Daphnia showed no propensity to prefer a certain distance from the test arena walls (pers. obs.), so by assuming they were on average 4.4 cm from the rotating drum wall, we can consider how large an object would need to be for them to see it. Our narrowest stripe was 17.5 mm wide. This implies that at a distance of 1 cm, Daphnia could see a high-contrast object ~3.5 mm across. Even if our experimental Daphnia had consistently remained as far from the striped wall as possible, implying that the actual threshold angle is narrower than our estimate (15.4°), they would only be able to see a 2 mm high-contrast object at a distance of ~1.5 cm. It therefore seems unlikely that Daphnia could use their vision to find other individual Daphnia (typical adult length 1.2–2.5 mm, depending on species, and usually relatively low contrast), and vision probably plays little role in locating mates. Vision still may play a role in accepting/rejecting mates, or in achieving appropriate orientations once a potential mate is at close range. Recently, Schott and von Elert (2017) reported a behavioral study in which Daphnia magna males are likely using pheromone trails to identify females. They also noted that after reaching a female, they will swim in tandem, including circling each other. Vision may play a role in coordinating swimming between the pair. The resolution of Daphnia vision clearly precludes seeing individual particles that Daphnia consume via filter-feeding, even if we have underestimated their visual acuity. However, this does not mean that vision is not used in foraging. Patchiness in the density of different food types can alter the light field and Daphnia may still be able to identify either density or quality of resources found in different micropatches by relying on diffuse visual cues. Daphnia vision may allow them to avoid or evade some predators. While a Chaoborus may not be seen before it was in striking distance because it is narrow and relatively transparent, the size and contrast of other invertebrate predators, such as notonectids, put them near the threshold of observation. Similarly, a fish that appeared 3 cm across could be seen at a distance of at least 12 cm, which is about 50% greater than the average sighting and striking distance for juvenile bluegill (Leech and Johnsen, 2006). If a Daphnia had a broadside view of a small bluegill measuring 10 cm, they should be able to see it from ~35 cm or more away, which would allow the Daphnia to sink further into the deep before the fish is in striking range. Our quantitative approach to measuring an animal’s OMR provides a more finely grained picture of the animal’s visual capabilities than a categorical approach to scoring OMR behaviors. Indeed, the OMR at our narrowest stripe width was substantially lower than at wider stripes. We suggest that this indicates that, while the Daphnia can see these stripes, they may not see them as well as they see wider stripes. Hence, broader application of quantitative measures would allow tests of whether imperfect vision improves fitness in realistic ecological contexts. We also note that the categorical analysis we conducted on our data was highly sensitive to the choice of where to put the dividing line for a “YES” response, and that when a reasonably cautious criterion was used, the results differed from the more precise quantitative analysis. This suggests that categorical analyses fail to identify real OMRs, or that they require greater experimental replication to detect the milder responses. By showing that free-swimming Daphnia have an OMR, we have demonstrated a strategy for measuring visual function in Daphnia that is ecologically relevant. This should allow for experiments that more fully characterize their visual capabilities, for example to evaluate color vision, the degree of contrast necessary to respond to an object visually, the intensity of light necessary for Daphnia to see, or the spatial and temporal boundaries of motion detection. Such information would allow us better insight into the ecological function of vision in Daphnia. CONCLUSIONS Daphnia have an OMR that is composed of two distinct behaviors, rotation and swimming. Individuals do not engage in each behavior equally, but rather tend to rotate or swim in response to environmental motion. Overall, the rotation response is more prevalent than the swimming response. Daphnia have a visual acuity that is sharper than what would be predicted from the physical structure of their compound eye. The visual acuity is sufficient that vision may play a role in limiting predation by fish and some invertebrates. The OMRs of Daphnia provide a convenient tool for further investigation of visual capabilities. ACKNOWLEDGEMENTS Arthur Illingworth, Jr of USC’s Mechanical Prototype Facility constructed our apparatus and contributed practical advice on how to design it. David Cann provided technical support in maintaining live Daphnia. Daniel I. Speiser provided conceptual advice and guidance throughout the project. D. I. Speiser, Alexandra N. Kingston and the reviewers provided helpful comments on an earlier draft of this manuscript. FUNDING Funding for this project was provided by the South Carolina Honors College and a Magellan Fellowship to C.R.H. from USC’s Office of the Vice-President for Research. AUTHOR CONTRIBUTIONS C.R.H. and J.L.D. conceived and designed the experiment, and wrote the manuscript together. 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Quantitative measurement of the optomotor response in free-swimming Daphnia

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Abstract

Abstract Daphnia are trophically important, common zooplankton in lakes and ponds worldwide, and hence have become a significant model system in ecology and evolutionary biology. Daphnia have conspicuous eyes that confer both fitness benefits and costs, though little is known about the functional capabilities of Daphnia vision. We sought to determine whether free-swimming Daphnia exhibit an optomotor response, a reflexive behavior present in many animals that could be used in studies of visual function. Optomotor responses are movements that allow an individual to track motion of the surrounding environment and thus stabilize its visual field. We constructed an apparatus that rotates a striped drum around Daphnia, while allowing an observer to quantify swimming behavior. Across a range of angular stripe widths and rotational speeds, we demonstrate that Daphnia have an optomotor response composed of at least two distinct behaviors. Our results suggest Daphnia could use vision for predator avoidance in some circumstances, but could not see other Daphnia individuals beyond more than a few millimeters, or food particles at all. We discuss how the optomotor response may be used to study visual function in Daphnia to better understand visually mediated ecological interactions. INTRODUCTION The optomotor response (OMR) is an innate reflex involving eye, head or body movements that stabilize an animal’s visual field when the surrounding environment is moving (Land and Nilsson, 2012). The presence of an OMR in an animal demonstrates that the animal can detect and respond to motion. Therefore, the OMR in an animal can be used to determine the range of motion that the animal can see and the conditions under which it can be seen (e.g. Srinivasan et al., 1999; Lunau, 2014; Sharkey et al., 2015). Prior work has shown that many aquatic animals have an OMR, including fish (Anstis et al., 1998; Neuhauss, 2003; Rinner et al., 2005; Lerner et al., 2017) and invertebrates (Buskey, 2000; Baldwin and Johnsen, 2011; Sharkey et al., 2015; Tomsic, 2016). Daphnia are small freshwater zooplankters (1–2 mm long as adults) that live in lakes and ponds. Daphnia are often the keystone herbivore in freshwater ecosystems and are amenable to both field and laboratory experiments, leading them to become one of the main model organisms in ecology (reviewed in Benzie, 2005; Stollewerk, 2010; Lampert, 2011; Seda and Petrusek, 2011). They are also an important model in evolutionary biology, in part because they are cyclic parthenogens, which simplifies ascertainment of genetic and environmental effects in genetically diverse populations (e.g. Lynch, 1983; Weider and Hebert, 1987; Boersma et al., 1998; Dudycha, 2004; Walsh and Post, 2011; Dudycha et al., 2012). Their physiological characteristics are less well known, and few studies address the functional capabilities of organs or organ systems, particularly in the context of ecological consequences. Daphnia possess a conspicuous apposition compound eye (Ringelberg, 1987), and many species have an additional inconspicuous simple eye called an ocellus (Fig. 1). The dark, pigmented compound eye contains 22 ommatidia, arranged in a 360-degree manner with each ommatidium sampling different spatial regions (Frost, 1975; Young and Downing, 1976). The compound eye forms as two separate eyes that fuse during embryonic development, and is known to be developmentally plastic in response to resource abundance, but not light intensity (Brandon and Dudycha, 2014). Because the compound eye is the darkest part of largely transparent zooplankton, it has been suggested that predation pressure from visual hunters in lakes would select for smaller eyes (Zaret, 1972; Zaret and Kerfoot, 1975; Lenz et al., 1996). However, this has not been tested directly in Daphnia, and an analysis in a fishless pond found evidence for selection favoring larger eyes in D. obtusa Kurz (Brandon et al., 2016). Fig. 1. View largeDownload slide Daphnia pulex. The compound eye is the large black structure, with ommatidia lenses appearing as clear circles on the surface of the eye. The simple eye (ocellus) is the small black circle below the eye. Inset: a whole female, showing the size of the eye relative to the body. Fig. 1. View largeDownload slide Daphnia pulex. The compound eye is the large black structure, with ommatidia lenses appearing as clear circles on the surface of the eye. The simple eye (ocellus) is the small black circle below the eye. Inset: a whole female, showing the size of the eye relative to the body. Prior work has shown that Daphnia vision is capable of detecting the orientation of light polarization (Schwind, 1999; Flamarique and Browman, 2000), which may be used in horizontal positioning within lakes. Several studies have shown phototaxis in both the lab and the field (e.g. De Meester, 1991, 1993; Ringelberg and Flik, 1994), and phototaxis has been linked to the diel vertical migration of lake zooplankton (reviewed in Ringelberg, 1999). Other work has indicated that Daphnia can detect, track and fixate on a moving light source (Frost, 1975; Consi et al., 1990). Daphnia can move the compound eye within the head, changing its orientation and position. Frost (1975) made a detailed study of these movements in anchored Daphnia, and described parameters of eye tremors and positional movement in response to several stimuli. This work showed that the compound eye is capable of responding to variation in light and motion. An electrophysiological study of the compound eye has shown that Daphnia vision is at least tetrachromatic (Smith and Macagno, 1990), and their genome contains an unusually large family of opsins that dates back to the Mesozoic (Brandon, et al., 2017). Due to the potential for contrasting selection pressures on compound eye size and the likely high energetic costs of visual tissue, we sought to better understand the functional capability of Daphnia vision. In particular, we wanted to determine whether free-swimming Daphnia exhibit behavioral responses to motion. Ecologically, such a capability might be used in predator avoidance or evasion, mating or foraging. We were further interested in estimating their visual acuity, the ability of an eye to precisely sample the surrounding environment, involving both spatial and temporal resolution. To accomplish these goals, we constructed an apparatus to test for an OMR in free-swimming Daphnia pulex Leydig. We predicted that D. pulex would have an OMR, and that it would be evident when environmental structure exceeded the inter-ommatidial angle of 38° (Young and Downing, 1976). We then manipulated the spatial and temporal scales of motion around experimental individuals in a randomized two-factor experiment to evaluate the functional boundaries of Daphnia vision. METHODS OMR apparatus We designed an experimental apparatus based on similar work addressing OMR in guppies (Anstis et al. 1998) and blue crabs (Baldwin and Johnsen 2011). The machine consisted of a cylindrical drum (inner diameter: 24 cm, height 30 cm) attached to a DC motor, a 20 cm diameter water dish, and a second, smaller glass dish at the center of the water dish (Fig. 2). The water dish served as the experimental arena for individual Daphnia during testing, and was filled with water to a depth of 4 cm. The smaller central dish blocked swimming in the middle of the arena, and functioned to balance the competing objectives of keeping individual Daphnia at approximately the same distance from the walls of the rotating drum, while still allowing them the opportunity to swim naturally in any direction. Individuals were thus constrained to swim in a 4 cm wide circular band of open water. A Daphnia swimming at the midpoint of the band would be 4.4 cm from the drum walls, but was able to range as close as 2.4 cm or as far as 6.4 cm. The walls of the cylindrical drum were lined with paper on which alternating black and white stripes were printed. The drum was then rotated around the water tank at a constant speed by the DC motor. The motor’s voltage was controlled by the experimenter, which, in combination with adjustments to the position of a rubber wheel driving the drum rotation, allowed for tests to be conducted at a wide range of rotational speeds. The apparatus was open at the top, allowing behaviors to be scored directly. Fig. 2. View largeDownload slide Schematic illustration of the OMR apparatus and OMR behaviors. The dashed line (not to scale) indicates the alternating white and black stripe stimulus pattern that was affixed to the inside of a rotatable drum. The experimental arena in which individual Daphnia are placed is a clear glass water tank whose center is blocked off by a clear glass dish. In the compass reaction, the Daphnia, indicated by the letter D, rotates on its vertical axis. In the circling reaction, the Daphnia swims in concert with the moving striped wall. Fig. 2. View largeDownload slide Schematic illustration of the OMR apparatus and OMR behaviors. The dashed line (not to scale) indicates the alternating white and black stripe stimulus pattern that was affixed to the inside of a rotatable drum. The experimental arena in which individual Daphnia are placed is a clear glass water tank whose center is blocked off by a clear glass dish. In the compass reaction, the Daphnia, indicated by the letter D, rotates on its vertical axis. In the circling reaction, the Daphnia swims in concert with the moving striped wall. OMR Scoring We made preliminary observations of Daphnia swimming in the arena to determine how to record Daphnia behaviors. These observations led us to define two distinct behaviors that would potentially indicate an OMR. In the “compass reaction”, individuals rotate on their vertical axis in the same direction as the motion of the striped wall which surrounds them. In the “circling reaction,” individuals are observed to swim in the same direction as the motion of the striped wall which surrounds them. Video recordings failed to show the compass response, so we relied on direct observations during our experiment. For each experimental trial, one adult Daphnia (age 14–21 d) was placed in the arena and allowed to acclimate for 2 minutes. The apparatus was then turned on, and animals were allowed to acclimate for a further 2 minutes while the drum was rotating. This ensured we avoided mis-scoring any startle response to small vibrations from the motor as an OMR. Later experiments directly comparing the first and third minutes after the apparatus was turned on showed no difference in scores (Perez and Dudycha, unpublished data). Behavior was then scored for 1 minute as the number of seconds engaged in the compass response, the number of seconds engaged in the circling response, and the number of seconds engaged in neither response. A metronome timer was used to allow the observer to count seconds in real-time. The total OMR score was the sum of the time spent circling or in the compass response. Experimental design We conducted a randomized two-factor experiment using five levels of stripe width and five rotational speeds (1.22, 1.94, 2.45, 3.16 and 4.00 rpm), for a total of 25 different treatments. Stripes were either 17.5, 27.8, 34.5, 44.0 or 55.0 mm wide. In addition, we scored behaviors at all five speeds with the walls of the rotating drum covered with a uniform 50% gray to serve as controls in which motion could not be visually detected. Trials were conducted in a randomized order, with individuals randomly assigned to treatment. Behavior was scored on five replicate adult Daphnia pulex (clone PA42, the clone used for the Daphnia reference genome; Ye et al. 2017) for each experimental condition (125 experimental trials + 25 control trials = 150 independent experimental individuals). For the first 22 trials (20 experimental, plus 2 control trials), behavior was recorded as just the sum of time spent circling or in compass rotation. Thereafter, times for circling and compass were recorded separately. Prior to behavioral assays, Daphnia were maintained under standard laboratory conditions (low density; 12:12 L:D photoperiod; 20°C; fed daily with Ankistrodesmus falcatus. For further details, see Dudycha et al., 2013; Dudycha and Hassel, 2013). We based our range of stripe widths on a prior estimate that the inter-ommatidial angle in the Daphnia eye is 38° (Young & Downing, 1976), which should define the smallest section of their field-of-view that could be clearly resolved. In our arena, Daphnia were not located at the centerpoint of the rotating drum. We assumed Daphnia would be on average 4.4 cm from the drum, at the midpoint of the space in which they could swim. Our prediction was that Daphnia should definitely be able to see stripes that appeared ≥40°, approximately our mid-sized stripe (Table I). We based rotation speeds on preliminary observations of Daphnia swimming, and set our fastest speed to approximate the swimming capabilities of Daphnia. This ensured that we would not mistakenly interpret a failure to be able to swim fast enough to keep up with the rotation as absence of an OMR. Table I: Significance of Dunnett’s post hoc tests for differences between behavior at specific stripe widths compared to the gray controls Stripe width OMR total p = Compass p = Circling p = 17.5 mm (21.7°) 0.012 0.161 0.328 27.8 mm (32.3°) 0.000 0.001 0.287 34.5 mm (38.1°) 0.000 0.000 0.058 44.0 mm (45.0°) 0.000 0.000 0.109 55.0 mm (51.3°) 0.000 0.000 0.238 df 144 122 122 MSE 96.517 0.453 0.983 Stripe width OMR total p = Compass p = Circling p = 17.5 mm (21.7°) 0.012 0.161 0.328 27.8 mm (32.3°) 0.000 0.001 0.287 34.5 mm (38.1°) 0.000 0.000 0.058 44.0 mm (45.0°) 0.000 0.000 0.109 55.0 mm (51.3°) 0.000 0.000 0.238 df 144 122 122 MSE 96.517 0.453 0.983 Stripe widths are given in absolute distance, along with the angular field of view occupied for an individual at the midpoint of the swimming space, estimated as the arctangent of the ratio of the distance to the rotating drum and the absolute width of the stripe. Degrees of freedom (df) and the pooled mean square error (MSE) are given for each trait’s test in the bottom rows. Bold = significant at the p < 0.05 level. Table I: Significance of Dunnett’s post hoc tests for differences between behavior at specific stripe widths compared to the gray controls Stripe width OMR total p = Compass p = Circling p = 17.5 mm (21.7°) 0.012 0.161 0.328 27.8 mm (32.3°) 0.000 0.001 0.287 34.5 mm (38.1°) 0.000 0.000 0.058 44.0 mm (45.0°) 0.000 0.000 0.109 55.0 mm (51.3°) 0.000 0.000 0.238 df 144 122 122 MSE 96.517 0.453 0.983 Stripe width OMR total p = Compass p = Circling p = 17.5 mm (21.7°) 0.012 0.161 0.328 27.8 mm (32.3°) 0.000 0.001 0.287 34.5 mm (38.1°) 0.000 0.000 0.058 44.0 mm (45.0°) 0.000 0.000 0.109 55.0 mm (51.3°) 0.000 0.000 0.238 df 144 122 122 MSE 96.517 0.453 0.983 Stripe widths are given in absolute distance, along with the angular field of view occupied for an individual at the midpoint of the swimming space, estimated as the arctangent of the ratio of the distance to the rotating drum and the absolute width of the stripe. Degrees of freedom (df) and the pooled mean square error (MSE) are given for each trait’s test in the bottom rows. Bold = significant at the p < 0.05 level. All trials were scored by the same observer. Trials were conducted under white fluorescent laboratory lighting (Philips Universal/Hi-Vision bulbs) that produces 7–10 μmol quanta m−2 s−2 at the bench surface on which the apparatus was placed. Statistical analyses were conducted in Systat 13. Visual inspection of distribution plots showed that the total OMR score was approximately normally distributed, but that the separate compass and circling scores were strongly skewed. Therefore, we ln-transformed those scores (+1 to account for zeroes), which produced approximately normal distributions, before applying linear models. RESULTS Behavior under control conditions We first used ordinary least-squares regression to test whether rotation speed influenced the OMR scores obtained from the gray controls. No significant effects were found for the compass reaction (df = 1, 21; F = 0.613; p = 0.442), for the circling reaction (df = 1, 21; F = 0.421; p = 0.524) or for the combined OMR score (df = 1, 23; F = 0.541; p = 0.470). Therefore, we pooled control data across the different speeds to estimate the average OMR scores under conditions where there was no visually detectable motion. For the controls, the average compass reaction score was 7.609 ± 1.290 SE, the average circling reaction score was 5.261 ± 1.262 SE and the average total OMR score was 12.760 ± 1.257 SE. These values indicate the duration of time (seconds) in which an observed individual was, just by chance, moving in concert with environmental rotation. Behavior under experimental conditions Because speed may influence the stripe width that individuals can see, we tested for their effects in a two-factor analysis of variance (ANOVA) including an interaction term. Levene’s test was used to confirm homogeneity of variances for the OMR total score and the ln-transformed compass and circling reactions (total: df = 5, 144, F = 1.383, p = 0.234; compass: df = 4, 123, F = 0.120, p = 0.297; circling: df = 4, 123, F = 1.242, p = 0.297). The overall model was significant for the total OMR score (df = 3, 146; F = 10.739; p < 0.001), the compass reaction (df = 3, 124; F = 11.490; p < 0.001) and the circling reaction (df = 3, 124; F = 2.592; p = 0.050). None of the behavioral measures showed a significant interaction between speed and stripe width (total OMR: p = 0.531; compass: p = 0.088; circling: p = 0.505). Furthermore, the effect of speed was insignificant for all behavioral measures (total OMR: p = 0.782; compass: p = 0.310; circling: p = 0.177). Stripe width, however, had a significant effect on the compass reaction (p = 0.001) and total OMR score (p = 0.016) but not the circling reaction (p = 0.872). Because speed had no significant effect on OMR behaviors over the range we assayed, we pooled data across speeds to analyze stripe width further. At all stripe widths, OMR scores were higher than for gray controls (Fig. 3), indicating that free-swimming Daphnia have an OMR. We tested whether stripe width affected behaviors with a one-way ANOVA followed by Dunnett’s post hoc tests to determine whether each individual stripe width was significantly different from the gray controls. Stripe width had a significant effect on the total OMR response behavior (df = 5, 144; F = 11.008, p < 0.001), and each individual stripe width induced a detectable response (Table I). We analyzed each behavior separately to determine which were contributing to the overall pattern. Stripe width significantly influenced the compass reaction (df = 5, 122; F = 6.675; p = 0.001), but influence on the circling reaction was not significant (df = 5, 122; F = 1.573; p = 0.173), confirming results from the two-factor analyses. All but the narrowest stripe widths had a significant effect on the compass reaction, but only the 34.5 mm width had a marginally significant effect on the circling reaction (Table I). Fig. 3. View largeDownload slide OMRs of Daphnia pulex at different stripe widths. Points show means and standard errors. “Gray” on the horizontal axis refers to the scores from gray controls (no stripes). Horizontal dashed lines are drawn at the control scores. (A) Compass reaction, or rotation on the animal’s vertical axis in concert with the motion of the rotating drum. (B) Circling reaction, or swimming laterally in concert with the motion of the rotating drum. (C) OMR total, the sum of the compass and circling reactions. Fig. 3. View largeDownload slide OMRs of Daphnia pulex at different stripe widths. Points show means and standard errors. “Gray” on the horizontal axis refers to the scores from gray controls (no stripes). Horizontal dashed lines are drawn at the control scores. (A) Compass reaction, or rotation on the animal’s vertical axis in concert with the motion of the rotating drum. (B) Circling reaction, or swimming laterally in concert with the motion of the rotating drum. (C) OMR total, the sum of the compass and circling reactions. Because the total OMR response had smaller variances than either component behavior (Fig. 3) and had greater statistical confidence in the effects of stripes, we tested for a negative association between the two behaviors (excluding data from the gray controls). If there were instead a positive relationship, this would indicate that individuals mainly differed in magnitude of the OMR, rather than the type of OMR. The behaviors were negatively correlated (Fig. 4), with R2 = 0.238. The sum of these two measures must be ≤60, constraining the state-space in which observations could occur. However, the relationship was shallower than required, and there was no evidence of a positive relationship. Fig. 4. View largeDownload slide Association between time spent circling and time spent in the compass reaction. Each point shows a unique individual. Observations from the gray controls are excluded. Fig. 4. View largeDownload slide Association between time spent circling and time spent in the compass reaction. Each point shows a unique individual. Observations from the gray controls are excluded. Reports on OMR in most species measure responses categorically (i.e. yes–no) rather than quantitatively. To investigate how this approach may have influenced our interpretation, we re-coded our data as if they were categorical. In the basic evaluation, we considered any observation where the total OMR score exceeded the mean for the gray controls to be a “YES” and lower scores to be a “NO.” Using one-way chi-square tests, we found that all stripe widths produced a significant OMR under this criterion, with the lowest confidence for the 17.5 mm stripes and the highest at 38.5 mm with 100% of 25 observations scored “YES” (Table II). If we used a more stringent criterion to score an observation as “YES” (>gray control mean + 1 standard deviation), chi-square tests showed significant effects of stripes only at 27.8 (marginally), 34.5, and 44.0 mm (Table II). Table II: Comparison of categorical analyses made with different levels of stringency for determining whether there is an OMR Stripe width Basic χ2 = Basic p = Stringent χ2 = Stringent p = 17.5 mm (21.7°) 9.000 0.003 0.040 0.841 27.8 mm (32.3°) 21.160 0.000 3.240 0.072 34.5 mm (38.1°) Undefined 0.000 17.640 0.000 44.0 mm (45.0°) 21.160 0.000 17.640 0.000 55.0 mm (51.3°) 14.440 0.000 0.360 0.549 Stripe width Basic χ2 = Basic p = Stringent χ2 = Stringent p = 17.5 mm (21.7°) 9.000 0.003 0.040 0.841 27.8 mm (32.3°) 21.160 0.000 3.240 0.072 34.5 mm (38.1°) Undefined 0.000 17.640 0.000 44.0 mm (45.0°) 21.160 0.000 17.640 0.000 55.0 mm (51.3°) 14.440 0.000 0.360 0.549 For the “basic” analysis, we treated any total OMR score greater than the mean of the gray controls as a positive OMR. For the “stringent” analysis, only scores greater than the mean + 1 SD were considered to show an OMR. All tests are one-way chi-square tests with one degree of freedom. Bold = significant at the p < 0.05 level. Table II: Comparison of categorical analyses made with different levels of stringency for determining whether there is an OMR Stripe width Basic χ2 = Basic p = Stringent χ2 = Stringent p = 17.5 mm (21.7°) 9.000 0.003 0.040 0.841 27.8 mm (32.3°) 21.160 0.000 3.240 0.072 34.5 mm (38.1°) Undefined 0.000 17.640 0.000 44.0 mm (45.0°) 21.160 0.000 17.640 0.000 55.0 mm (51.3°) 14.440 0.000 0.360 0.549 Stripe width Basic χ2 = Basic p = Stringent χ2 = Stringent p = 17.5 mm (21.7°) 9.000 0.003 0.040 0.841 27.8 mm (32.3°) 21.160 0.000 3.240 0.072 34.5 mm (38.1°) Undefined 0.000 17.640 0.000 44.0 mm (45.0°) 21.160 0.000 17.640 0.000 55.0 mm (51.3°) 14.440 0.000 0.360 0.549 For the “basic” analysis, we treated any total OMR score greater than the mean of the gray controls as a positive OMR. For the “stringent” analysis, only scores greater than the mean + 1 SD were considered to show an OMR. All tests are one-way chi-square tests with one degree of freedom. Bold = significant at the p < 0.05 level. DISCUSSION Free-swimming Daphnia have an OMR, and broadly are able to see and respond to high-contrast elements in their environment. Prior work has shown that Daphnia can see overall fields of light, but this is the first clear demonstration that they can behaviorally respond to visual structure in their environment. Two distinct behaviors are involved, rotation around an individual’s vertical axis and swimming in concert with environmental motion. Genetically identical individuals can differ in which OMR they tend to use. The resolution at which Daphnia responded to rotational motion suggests that their vision could be used in predator avoidance and possibly in some aspects of mating. We estimated the angular field of view that each stripe width would occupy for an individual swimming at the midpoint of the band of open water, 4.4 cm from the rotating drum wall (Table I). Daphnia exhibited a significant OMR with our narrowest stripes tested (17.5 mm, ~ 21.7° at the midpoint of the distance Daphnia could be from the striped wall). Therefore, it is possible that the minimum threshold for Daphnia to see a high-contrast object is even narrower than 21.7°, and considerably narrower than the 38° threshold we predicted based on the inter-ommatidial angle. Daphnia eyes are anchored by a short muscular stalk, and individuals can use the stalk to move the eye rapidly within the head, both rotationally and positionally (Frost, 1975; Consi et al., 1990). Such motion could give Daphnia a greater visual resolving power than the inter-ommatidial angle would suggest, if the eye movements allow for temporal summation of light from slightly different directions within individual ommatidia (Warrant, 1999; Greiner, 2006). Indeed, our results are consistent with Frost’s (1975) detailed observations of eye movements in anchored Daphnia pulex. Frost described four types of internal eye movements: a fast tremor, which he proposed served to increase visual acuity; slow scanning, which might play a role in searching for objects; saccadic eye movements that re-oriented eyes in response to directional light sources; and optokinetic nystagmus (OKN). Frost’s observations of OKN are most relevant to our work. Testing stripe widths that ranged from 5° to 45°, he found the strongest response at 22.5°, and no response at 10° or below. Thus, his work also indicated a visual acuity below the 38° inter-ommatidial angle, and is broadly in line with our estimate. Indeed, a curve fit to our data in Fig. 3C would suggest a lower visual threshold of ~10°. How these internal eye movements may occur and function in Daphnia that can move their bodies in response to visual cues remains unknown. Nonetheless, it seems likely that Daphnia combine eye movements and swimming to maximize the useful visual information they acquire and respond to that information. We did not find a speed threshold for the OMR, but our work suggests a spatial threshold, and we can use that to make preliminary inferences about the ecological function of vision. Daphnia showed no propensity to prefer a certain distance from the test arena walls (pers. obs.), so by assuming they were on average 4.4 cm from the rotating drum wall, we can consider how large an object would need to be for them to see it. Our narrowest stripe was 17.5 mm wide. This implies that at a distance of 1 cm, Daphnia could see a high-contrast object ~3.5 mm across. Even if our experimental Daphnia had consistently remained as far from the striped wall as possible, implying that the actual threshold angle is narrower than our estimate (15.4°), they would only be able to see a 2 mm high-contrast object at a distance of ~1.5 cm. It therefore seems unlikely that Daphnia could use their vision to find other individual Daphnia (typical adult length 1.2–2.5 mm, depending on species, and usually relatively low contrast), and vision probably plays little role in locating mates. Vision still may play a role in accepting/rejecting mates, or in achieving appropriate orientations once a potential mate is at close range. Recently, Schott and von Elert (2017) reported a behavioral study in which Daphnia magna males are likely using pheromone trails to identify females. They also noted that after reaching a female, they will swim in tandem, including circling each other. Vision may play a role in coordinating swimming between the pair. The resolution of Daphnia vision clearly precludes seeing individual particles that Daphnia consume via filter-feeding, even if we have underestimated their visual acuity. However, this does not mean that vision is not used in foraging. Patchiness in the density of different food types can alter the light field and Daphnia may still be able to identify either density or quality of resources found in different micropatches by relying on diffuse visual cues. Daphnia vision may allow them to avoid or evade some predators. While a Chaoborus may not be seen before it was in striking distance because it is narrow and relatively transparent, the size and contrast of other invertebrate predators, such as notonectids, put them near the threshold of observation. Similarly, a fish that appeared 3 cm across could be seen at a distance of at least 12 cm, which is about 50% greater than the average sighting and striking distance for juvenile bluegill (Leech and Johnsen, 2006). If a Daphnia had a broadside view of a small bluegill measuring 10 cm, they should be able to see it from ~35 cm or more away, which would allow the Daphnia to sink further into the deep before the fish is in striking range. Our quantitative approach to measuring an animal’s OMR provides a more finely grained picture of the animal’s visual capabilities than a categorical approach to scoring OMR behaviors. Indeed, the OMR at our narrowest stripe width was substantially lower than at wider stripes. We suggest that this indicates that, while the Daphnia can see these stripes, they may not see them as well as they see wider stripes. Hence, broader application of quantitative measures would allow tests of whether imperfect vision improves fitness in realistic ecological contexts. We also note that the categorical analysis we conducted on our data was highly sensitive to the choice of where to put the dividing line for a “YES” response, and that when a reasonably cautious criterion was used, the results differed from the more precise quantitative analysis. This suggests that categorical analyses fail to identify real OMRs, or that they require greater experimental replication to detect the milder responses. By showing that free-swimming Daphnia have an OMR, we have demonstrated a strategy for measuring visual function in Daphnia that is ecologically relevant. This should allow for experiments that more fully characterize their visual capabilities, for example to evaluate color vision, the degree of contrast necessary to respond to an object visually, the intensity of light necessary for Daphnia to see, or the spatial and temporal boundaries of motion detection. Such information would allow us better insight into the ecological function of vision in Daphnia. CONCLUSIONS Daphnia have an OMR that is composed of two distinct behaviors, rotation and swimming. Individuals do not engage in each behavior equally, but rather tend to rotate or swim in response to environmental motion. Overall, the rotation response is more prevalent than the swimming response. Daphnia have a visual acuity that is sharper than what would be predicted from the physical structure of their compound eye. The visual acuity is sufficient that vision may play a role in limiting predation by fish and some invertebrates. The OMRs of Daphnia provide a convenient tool for further investigation of visual capabilities. ACKNOWLEDGEMENTS Arthur Illingworth, Jr of USC’s Mechanical Prototype Facility constructed our apparatus and contributed practical advice on how to design it. David Cann provided technical support in maintaining live Daphnia. Daniel I. Speiser provided conceptual advice and guidance throughout the project. D. I. Speiser, Alexandra N. Kingston and the reviewers provided helpful comments on an earlier draft of this manuscript. FUNDING Funding for this project was provided by the South Carolina Honors College and a Magellan Fellowship to C.R.H. from USC’s Office of the Vice-President for Research. AUTHOR CONTRIBUTIONS C.R.H. and J.L.D. conceived and designed the experiment, and wrote the manuscript together. 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For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Journal of Plankton ResearchOxford University Press

Published: May 7, 2018

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