Behavioral modification of visually deprived lemon sharks (Negaprion ...

Journal of Experimental Marine Biology and Ecology 453 (2014) 131–137

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Behavioral modification of visually deprived lemon sharks (Negaprion brevirostris) towards magnetic fields C.P. O'Connell a,⁎, T.L. Guttridge b,c, S.H. Gruber b, J. Brooks b, J.S. Finger b,d, P. He a a

School for Marine Science and Technology, University of Massachusetts Dartmouth, New Bedford, MA 02740, USA Bimini Biological Field Station, Bimini, Bahamas School of Biosciences, Cardiff University, Cardiff CF10 3XQ, UK d Humboldt University, Berlin, Germany b c

a r t i c l e

i n f o

Article history: Received 10 May 2013 Received in revised form 13 January 2014 Accepted 16 January 2014 Available online 5 February 2014 Keywords: Beach nets Elasmobranchs Electrosensory repellents Lemon sharks Negaprion brevirostris Permanent magnets

a b s t r a c t The ability of elasmobranchs to orient to weak electromagnetic fields is well documented. Recently, scientists have employed the use of strong electrosensory stimuli, such as permanent magnets, as a means to evaluate the repellent responses of elasmobranchs and assess the utility of these materials for bycatch repellent technologies. However, several studies have produced contrasting results both between and within species. To explain these results, we hypothesized that conditions leading to vision loss (i.e. turbid water) may be a factor affecting electrosensory repellent success. To simulate a visually deprived environment, the nictitating membranes of juvenile lemon sharks (Negaprion brevirostris) were temporarily sutured closed and the behavioral responses of sharks towards a magnetic apparatus were observed in a pen within the shallows of Bimini, Bahamas. Results demonstrate that the magnet-associated behavior of visually deprived sharks significantly differed from control sharks in regard to: (1) avoidance distance, (2) visit quantity prior to first entrance through the magnet zone, and (3) total entrances/total visits. These findings suggest context-dependent switching, where elasmobranchs may exhibit a heightened reliance on their electrosensory system when the extent of their visual range is reduced. These findings also provide insight into favorable environments (e.g. estuary or other coastal ecosystems) and applications (e.g. inshore fisheries and beach nets) that may yield more consistent and successful future implementations of electrosensory repellents for sharks. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Context-dependent switching – the capacity to flexibly tailor behavior based on the current ecological and biological state – has been extensively demonstrated in a wide variety of both marine and terrestrial organisms (Hoare et al., 2004; Leahy et al., 2011; McIntyre and McCollum, 2000; Ranåker et al., 2012; Smith and Belk, 2001; Zuberbühler, 2001). For example, in teleosts, ecological factors, such as competition or presence of predators, can impact shoal size (Hoare et al., 2004), whereas environmental factors, such as turbidity, can impact chemo-sensory system reliance (Leahy et al., 2011; Ranåker et al., 2012). Studies related to context-dependent switching have been conducted on diverse taxa, including teleosts (Hoare et al., 2004; Krause, 1993), amphibians (McIntyre and McCollum, 2000) and mammals (Zuberbühler, 2001); however, there is little information pertaining to elasmobranchs (i.e. sharks, skates, and rays). Elasmobranchs have highly developed vision and electroreception which they use for, inter alia, prey detection (Cohen, 1991; Gruber and Cohen, 1978, 1985; Kalmijn, ⁎ Corresponding author. E-mail address: [email protected] (C.P. O'Connell). 0022-0981/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jembe.2014.01.009

1974). For example, sharks are equipped with an intraocular reflecting structure known as the tapetum lucidum (Bernstein, 1961; Best and Nicol, 1967; Braekevelt, 1994), a feature that enhances visual sensitivity in low light levels, therefore giving sharks advanced nocturnal vision (Arnott et al., 1970; Ollivier et al., 2004). An elasmobranch's unique electrosensory system, known as the ampullae of Lorenzini (Kalmijn, 1966, 1971; Murray, 1960), serves a variety of functions, including the detection of bioelectric fields produced by prey (Kalmijn, 1974; Kajiura and Holland, 2002), conspecifics (Bratton and Ayers, 1987; Tricas et al., 1995) and predators (Peters and Evers, 1985; Sisneros et al., 1998). This system is also suspected to enable the detection of magnetic fields that have been hypothesized to provide geolocation information and navigational cues (Kalmijn, 1982; Klimley, 1993; Klimley et al., 2002). Recently a number of studies have exploited this acute sensitivity to weak electric and magnetic fields. These studies investigate the applicability and efficacy of much stronger electrosensory stimuli, such as magnets and electropositive metals, to overstimulate the ampullary systems of elasmobranchs, produce repellent responses and minimize elasmobranch bycatch in fisheries and beach nets (e.g. Rigg et al., 2009; Stoner and Kaimmer, 2008). Laboratory and field analyses have produced varying results, finding that repellent efficacy can be affected by a variety of factors including organismal satiation (O'Connell

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C.P. O'Connell et al. / Journal of Experimental Marine Biology and Ecology 453 (2014) 131–137

et al., 2012; Stoner and Kaimmer, 2008; Tallack and Mandelman, 2009), habituation (Brill et al., 2009; O'Connell et al., 2011), and conspecific density (Brill et al., 2009; Jordan et al., 2011; Robbins et al., 2011). However, none of these studies have revealed if the visual environment plays a role in repellent effectiveness. For the present study, we aim to examine how visual deprivation, simulating a turbid environment, may influence the repellent success of a grade C8 barium-ferrite (BaFe12O19) magnetic barrier on the lemon shark (Negaprion brevirostris). Using a similar experimental design, identical species, and magnet-type as in O'Connell et al. (2011), we aim to evaluate how turbidity, which will be simulated by nictitating membrane closure, may affect elasmobranch electroreception/magnetoreception capabilities. Similar to the studies pertaining to context-dependent switching, we hypothesize that the magnet-associated behavior of visually deprived sharks will significantly differ from control and procedural control sharks. More specifically, we predict: (1) the avoidance ratios (total avoidances/total visits) will be significantly greater and the entrance ratios (total entrances/total visits) will be significantly reduced towards the magnet zone in comparison to the control zone, (2) the avoidance ratios and avoidance distance with respect to the magnet zone will be significantly greater in visually deprived sharks in comparison to all other shark types, (3) the entrance ratio with respect to the magnet zone will be significantly lower in visually deprived sharks in comparison to all other shark types, and (4) the quantity of visits prior to first entrance through the magnet zone will be significantly greater in visually deprived sharks in comparison to all other shark types. 2. Material and methods The study was conducted at Bimini, Bahamas (25°44′N, 79°16′W), a small series of islands approximately 85 km east of Miami, Florida, USA. A total of 24 juvenile lemon sharks (mean ± standard deviation, precaudal length (PCL) = 58.6 ± 8.24 cm) were used in the experiments, with 14 being male and 10 being female. Sharks were captured using 180 m long × 2 m deep gillnets and promptly transported to a 4m diameter holding pen. Upon arrival, each shark was restrained in a trough (10 × 100 cm), sexed, measured for PCL (tip of snout to precaudal pit, see DiBattista et al., 2008), tagged intramuscularly with a passive integrated transponder (PIT) tag (see DiBattista et al., 2008), and fitted with a color code tag to permit visual identification of individual animals (see Guttridge et al., 2011). All sharks were held in semicaptive pens that exposed them to ambient environmental conditions (i.e. changes in tides, salinity, temperature, and light) (see Guttridge et al., 2009) and given one week acclimation period. All sharks were fed to satiation during non-experimental periods on a mixed diet of fresh and frozen great barracuda (Sphyraena barracuda). No sharks died during these experiments and all were released at the site of their capture. A permit (MAF/LIA/22) to conduct scientific marine animal research was supplied by the Department of Marine Resources, Bahamas.

Fig. 1. Experimental setup. A) Perspective view, from near to far: experimental arena (4 m × 4 m) with PVC pipes (0.5 m apart) containing either barium-ferrite permanent magnets (magnet zone) or clay bricks (control zone); the transfer corridor, the recover/ acclimation pen (5 m diameter) and the holding pen (3 m diameter). B) Surrounding the PVC columns/treatment zones and placed flush against the substrate was an observation zone containing flex pipe that were spaced at 5 cm increments from 0 to 50 cm (represented by the gray parallel and horizontal lines), as a means to determine the distance of avoidance.

0.15 m (length) × 0.10 m (width) × 0.05 m (height) clay brick, or sham magnet, was inserted. The magnet zone was identical in structure and dimension to the control zone; however, sham magnets were replaced with 0.15 m (length) ×0.10 m (width) × 0.05 m, grade C8 bariumferrite (BaFe12O19) magnets (Fig. 1A). Throughout experimentation, control and magnet zone locations were randomized to avoid any side preference-based behaviors. Furthermore, to standardize the location of observable behaviors around each treatment zone and to determine the distance of avoidance in reference to each zone, an observation zone (1 × 0.5 m) was placed flush against the substrate surrounding the control and magnet zones. Within each observation zone, PVC piping (1.3 cm diameter) was placed parallel at 5 cm increments from 0 to 50 cm as a means to determine avoidance distance in reference to the treatment zones (Fig. 1B). In addition, in the center of the treatment separation region HD Go Pro 1080p cameras were positioned to permit a post-hoc identification and measurement of avoidance distance.

2.1. Experimental setup

2.2. Surgery and shark type

A pen consisting of three compartments was constructed, including: 1) recovery/acclimation pen (5 m diameter), 2) corridor (3 × 1 m), and 3) experimental arena (4 × 4 m) (Fig. 1A). Each compartment was built with diamond-shaped construction mesh (5 cm × 5 cm) with evenly spaced steel reinforcing bar. Sliding mesh doors were constructed between compartments allowing researchers to usher sharks without the stress associated with handling. The experimental arena consisted of four zones: the separation, observation, control, and magnet zones. The separation zone was a 2 m section of construction mesh placed perpendicular to the substrate that was used to separate the control and magnet zones. The control zone consisted of three 1.75 m (height) polyvinyl chloride (PVC) columns spaced by 0.5 m and placed perpendicular to the substrate. At 0.5 m intervals on each column a slot was cut and a

All sharks (N = 24) were randomly assigned to one of four types/ treatments (N = 6 per treatment): 1) ‘control’ — no manipulation, 2) procedural control ‘eyebrow’ — one suture above each eye, 3) procedural control ‘one eye’ — one suture used to temporarily close the nictitating membrane (note: for this treatment, three sharks had the left eye closed and three sharks had the right eye closed, and 4) ‘visually deprived’ — one suture for each eye to close the nictitating membrane and to severely compromise its visual acuity (Fig. 2). For treatments, sharks were lightly anesthetized in a 1:20,000 solution of tricane methanesulfonate (MS-222) in seawater to facilitate safe handling (see Newman et al., 2010). Once anesthetized, one 3–0 silk suture was used per eye (Fig. 2). After surgery, sharks were transferred to the recovery/acclimation pen, monitored until typical captive behaviors


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2.4. Data analysis 2.4.1. Pre-trial behaviors To determine if there were any associations between shark type (e.g. control, eyebrow, one-eye, and visually deprived) and behavior (e.g. chafes, accelerations, and contact with outer pen mesh), chisquare analyses were conducted.

Fig. 2. The stages of temporarily blinding a juvenile lemon shark (Negaprion brevirostris). A) Shark is anesthetized using MS-222 and researcher takes tweezers dipped in iodine for sterilization to grasp the nictitating membrane. B) After grasping the nictitating membrane with the tweezers, the researcher gently closes the membrane. C) The researcher locks the hemostats on the suture needle and slowly places the needle through the nictitating membrane, making sure not to make contact with the eye. D) After one stitch, the nictitating membrane is successfully closed and the shark is placed in the recovery pen and observed for 30 min.

were observed, and given a 12 hour acclimation period prior to experimentation. Once completed with experimentation, sharks were captured, placed into tonic immobility (Watsky and Gruber, 1990) for suture removal, given a week to recover in the holding pen, and then released. All procedures were conducted in accordance with the University of Massachusetts Dartmouth IACUC protocol 12-05.

2.3. Experimental trials After the 12 hour acclimation period, all sharks, regardless of surgery, fed well, indicating recovery. In addition, immediately after feeding, visual observations were conducted for a total of 30 min to determine if all sharks exhibited typical captive behaviors including: chafes, accelerations, and contact (Table 1). After this observation period, an individual shark was ushered into the corridor and given 5 min to acclimatize. On completion, a manually operated sliding door was opened and the test shark was free to enter the experimental arena. Trial time was 30 min and three main behaviors were recorded: visits, avoidances, and entrances (Table 1). At the start and end of each trial environmental factors were recorded including water temperature, salinity and turbidity (YSI-Model 2030 and YSI Ecosense 9500 Photometer).

Table 1 Ethogram of juvenile lemon shark (Negaprion brevirostris) pre-trial and experimental behaviors. Behavior

Definition of behavior

Pre-trial behaviors Chafes Dorsoventral rotation of shark body in combination with substrate contact Accelerations Shark swimming speed increases Contact Shark makes physical contact with outer pen mesh Experimental behaviors Approaches Shark swam within an observation zone Avoidances Shark abruptly changed direction, such as a 45°, 90° or 180° turn and/or acceleration away, after visiting an observation zone Entrances Shark visited an observation zone and swam through the PVC pipes

2.4.2. Experimental behaviors First, to assess if sharks were deterred from magnetic fields, the ratios of behaviors for all sharks, regardless of shark type, were compared between control and magnet zones. Secondly, to assess if behavioral differences existed between shark types, four behavioral benchmarks were used: 1) ratio of avoidance — the total number of avoidances divided by the total number of visits, 2) ratio of entrance — the total number of entrances divided by the total number of visits, 3) initial visits — the number of visits prior to first entrance, and 4) avoidance distance — the distance of avoidance in reference to the magnet zone. Ratios were used, rather than count data, to standardize for differences in visit quantity between sharks. 2.4.2.1. Entrance behavior. To determine if any significant difference existed between entrance ratios and treatment zones (e.g. control zone and magnet zone), regardless of shark type (e.g. control, oneeye, eyebrows, and visually deprived), data was first subjected to a Shapiro–Wilk's test for normality and a Levene's test for equality of variances to satisfy the assumptions for a Student's t-test. A Student's t-test was then used to determine if any significant difference existed between treatment zones. Once entrance ratios were analyzed between treatment zones, a one-way ANOVA was conducted per treatment zone to determine if any variation existed among shark types. Prior to analysis, data was subject to a Shapiro–Wilk's test for normality and a Levene's test for equality of variances to satisfy the assumptions required for an ANOVA. Following the one-way ANOVA, a post hoc Tukey's HSD (Honestly Significant Difference) test was performed. As an additional means to determine if shark behavior differed in reference to the magnet zone, the quantity of visits prior to the first entrance for each shark was recorded. First, a mean for the quantity of visits prior to first entrance (±standard deviation) was computed for each shark type. Secondly, since data was not normally distributed, a Kruskal–Wallis test was used to determine if any variation in visit quantity prior to first entrance existed between shark types. Following the Kruskal–Wallis test, post-hoc Wilcoxon rank sum tests with Bonferroni correction were performed on each possible shark type pair (e.g. control versus visually deprived). 2.4.2.2. Avoidance behavior. Since data was not normally distributed, a Wilcoxon signed-rank test was used to determine if any variation in avoidance ratios existed between control and magnet zones, regardless of shark type. For magnet zone-associated data, an avoidance ratio was created for each shark. This data was then log transformed and subjected to a Shapiro Wilk's test for normality and a Levene's test for equality of variances to satisfy the assumptions required for an ANOVA. To determine if any variation existed between shark type and avoidance ratios, a oneway ANOVA was used. The avoidance distance for each shark type was compiled by conducting a post-hoc video analysis. First, a mean avoidance distance (± standard deviation) for each shark type was computed. Secondly, to determine if any variation existed between shark type and avoidance distance, a Kruskal–Wallis test was used since the data was not normally distributed. Following the Kruskal–Wallis test, post-hoc Wilcoxon rank sum tests with Bonferroni correction were performed on each shark type pair (e.g. control versus visually deprived). All statistical analyses were conducted using R, version 2.13.0 (www.r-project.org).

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Fig. 3. Inter-zone comparison of mean avoidance (total avoidances/total visits) and entrance (total entrances/total visits) ratios for the lemon shark (Negaprion brevirostris; N = 24). For these analyses, overall shark behavior was used for analysis, regardless of shark type.

Fig. 4. Mean avoidance (total avoidances/total visits) and entrance (total entrances/total visits) ratios for all four lemon shark (Negaprion brevirostris) types in response to the magnet zone. There were a total of six sharks per shark type.

2.5. Effects of surgery

associated with visually deprived sharks differed significantly: control sharks vs. visually deprived sharks (p = 0.002), one-eye sharks vs. visually deprived sharks (p = 0.012), and eyebrow sharks vs. visually deprived sharks (p = 0.0169). All other pairings (e.g. control sharks vs. eyebrow sharks) did not significantly differ (Fig. 4). For each shark type the mean quantity of visits prior to first entrance ± standard deviation were as follows: control sharks = 3.5 ± 1.97, eyebrow sharks = 4.17 ± 1.72, one-eye sharks = 4.17 ±2.926, and visually deprived sharks = 17.5 ± 9.79. A Kruskal–Wallis test revealed that visit quantity prior to first entrance through the control region did not significantly differ (H = 1.19, d.f. = 3, p = 0.755; Table 2); however, a significant difference was detected between the magnet zone and shark type (H = 11.22, d.f. = 3, p = 0.011; Table 2). Post hoc Wilcoxon rank sum tests with Bonferroni correction indicated that visit quantity prior to first entrance significantly differed between control sharks vs. visually deprived sharks (Z = 2.76, p =0.0074), one-eye sharks vs. visually deprived sharks (Z = 2.65, p =0.01), and eyebrow sharks vs. visually deprived sharks (Z = 2.49, p = 0.016) (Fig. 5). All other pairings (e.g. control sharks vs. eyebrow sharks) did not significantly differ.

To determine the effects of suturing, the eyes of sutured sharks were observed prior to surgery (day 1), three days after suture removal (day 3) and one week after suture removal (day 7). During each shark examination day, a high resolution photograph was taken using a Canon 7D digital SLR camera and an in-depth and observational descriptive analysis examining sclera color, pupil size, and nictitating membrane function was conducted. 3. Results 3.1. Pre-trial behaviors All sharks were observed to feed and displayed typical captive swimming behaviors prior to experimentation. No associations existed between observed behaviors and shark type such as chafes (χ2 = 1.789, d.f. = 3, p = 0.6172), accelerations (χ2 = 1.60, d.f. = 3, p = 0.6594), and physical contact with outer pen mesh (χ2 = 6.229, d.f. = 3, p = 0.101). 3.2. Entrance behavior

3.3. Avoidance behavior

Entrance ratios were found to be significantly different between control and magnet zones when data were pooled across shark types (Student t-test; t = 4.9257, d.f. = 22, p = 0.0044; Fig. 3; Table 2). When focusing solely on the control zone-associated behaviors, no significant variation in entrance ratios was detected between shark types (F(3,20) = 2.3732, p = 0.1007; Table 2). In reference to the magnet zone-associated behaviors, significant variation in entrance ratios was detected between shark types (F(3,19) = 4.9158, p = 0.01018; Table 1), with a post hoc Tukey HSD test demonstrating that all pairings

Avoidance ratios were found to be significantly different between control and magnet zones when data were pooled across shark types (Z = 16, p = 0.029; Fig. 3; Table 2). For the magnet zone and between shark types, no significant variation existed between avoidance ratios (F(3,20) = 0.6987, p = 0.5638; Table 2); however, statistical analyses solely for the control zone were not conducted due to the lack of avoidances. The mean avoidance distance for each shark type was as follows: control sharks = 20.87 ± 12.08 cm, eyebrow sharks = 18.2 ± 10.88 cm,

Table 2 A summary of the results from all trials, excluding post-hoc analyses. P-values in bold denote significance. Behaviors analyzed

Test conducted

Test statistic

d.f.

P-value

Avoidance behaviors Avoidance ratio and treatment zone (excluding shark type) Avoidance ratio within control zone and between shark types Avoidance ratio within magnet zone and between shark types Avoidance distance within control zone and between shark types Avoidance distance within magnet zone and between shark types

Wilcoxon-signed rank test – One-way ANOVA – Kruskal–Wallis test

16 – 0.699 – 57.397

– – 3,20 – 3

0.029 – 0.564 – b0.001

Entrance behaviors Entrance ratio and treatment zone (excluding shark type) Entrance ratio within control zone and between shark types Entrance ratio within magnet zone and between shark types Visit quantity prior to first entrance through control zone and shark type Visit quantity prior to first entrance through magnet zone and shark type

Student t-test One-way ANOVA One-way ANOVA Kruskal–Wallis test Kruskal–Wallis test

4.926 2.373 4.916 1.19 11.215

22 3,20 3,19 3 3

0.004 0.101 0.002 0.755 0.011


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sustained wounds above one eye which occurred during suture removal; however, there were indications of healing and a full recovery. 4. Discussion

Fig. 5. Box and whisker plot showing the median number, 25th percentile and 75th percentile, and the range. The plot represents visit quantity prior to the first entrance for each lemon shark (Negaprion brevirostris) type towards the magnet zone.

This study examines how the behavior of visually deprived elasmobranchs is modified towards electrosensory stimuli. It remains uncertain if the behaviors elicited by the present approach serve as an accurate representation of what would typically be observed in the wild. However, these results suggest that juvenile N. brevirostris exhibit a heightened reliance on their electrosensory system in a visually deprived world and therefore may be of critical importance for elasmobranchs to both navigate and forage in high turbidity environments. 4.1. Control zone versus magnet zone

one-eye sharks = 18.95 ± 12.64 cm, and visually deprived sharks = 7.26 ± 4.11 cm (Fig. 6). In addition, a Kruskal–Wallis test revealed that avoidance distance significantly differed between shark types (H = 57.397, d.f. = 3, p b 0.0001; Table 2). Post hoc Wilcoxon rank sum tests with Bonferroni correction indicated that avoidance distance significantly differed between control sharks vs. visually deprived sharks (Z = − 6.62, p b 0.001), one-eye sharks vs. visually deprived sharks (Z = − 5.93, p b 0.001), and eyebrow sharks vs. visually deprived sharks (Z = − 5.27, p b 0.001). All other pairings (e.g. control shark vs. eyebrow sharks) were not significantly different.

3.4. Effects of surgery Prior to surgery, the eyes of all sharks were characterized by having a fully white sclera with a vertical pupil that was approximately one third the width of the eyeball (Fig. 7). Tactile stimulation resulted in the immediate closing of the nictitating membrane (i.e. “normal”) suggesting a healthy eye. Three days after surgery, sharks were characterized by having a discolored sclera and a substantially smaller pupil (Fig. 7). During this time period, some sharks maintained “normal” nictitating membrane function, whereas other sharks had delayed or lost nictitating membrane function. Seven days after surgery, the eyes of all sharks were nearly identical to those of pre-surgery sharks, with the sclera being only slightly discolored, the pupil being properly dilated, and with “normal” nictitating membrane function (Fig. 7). Only two sharks

Previous studies suggest that certain elasmobranch species utilize the weak geomagnetic fields, ranging from 0.25 to 0.65 G, for navigation and geolocation purposes (Carey and Scharold, 1990; Klimley, 1993; Klimley et al., 2002). Using this concept, a variety of studies explored the potential to overwhelm an elasmobranch's acute electrosensory system, known as the ampullae of Lorenzini, utilizing a strong magnetic stimulus (~ 1000 G), such as a barium-ferrite permanent magnet (BaFe12O19) (e.g. O'Connell et al., 2010, 2011; Rigg et al. 2009). More specifically O'Connell et al. (2011) examined the effects of a magnetic fence-like apparatus on juvenile N. brevirostris and demonstrated that these magnets were capable of manipulating the swimming behavior of these sharks. Similarly, the results from the present study are consistent with the previously conducted magnetic repellent studies and demonstrate that strong permanent magnets can evoke repellent responses in N. brevirostris, which was evidenced by significant differences in swimming behavior (e.g. avoidance ratios and entrance ratios) occurring between control and magnet zones (Fig. 3). 4.2. Magnet zone 4.2.1. Avoidance behaviors When examining the effects of the magnet zone on avoidance ratios, no significant variation existed between shark types and therefore this finding is inconsistent with the original hypothesis. Although variation did not exist among the avoidance ratios and shark type, significant

Fig. 6. Histograms of the percentage of avoidance behaviors of the lemon shark (Negaprion brevirostris) throughout the 0–50 cm magnetic treatment zone. On the secondary y-axis, the associated magnetic field strength in gauss (G) is graphed in relation to distance from the magnet. A) Control sharks, B) eyebrow sharks, C) one-eye sharks, and D) visually deprived sharks.

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Fig. 7. The effects of surgery on a lemon shark (Negaprion brevirostris) eye. A) Represents the eye of N. brevirostris prior to surgery, or Day 1. These eyes were characterized by having a fully white sclera, a vertical pupil that was approximately one third the width of the eyeball, and a quick nictitating membrane response to tactile stimulation. B) Represents the of N. brevirostris three days after surgery. These eyes were characterized by having a discolored sclera, a substantially smaller pupil and a delayed or nonexistent nictitating membrane response to tactile stimulation. C) Represents the eye of N. brevirostris seven days after surgery. These eyes were nearly identical to day 1 sharks, with a slightly discolored sclera, a properly dilated pupil and the nictitating membrane was fully functional.

variation did exist between avoidance distance and shark type. However, post-hoc analyses demonstrated that the avoidance distance of visually deprived sharks was significantly lower than all other shark types which is also inconsistent with the original hypothesis. One potential explanation for this inconsistent hypothesis pertaining to avoidance distance is that visually deprived sharks may be solely responding to the magnetic fields using their electrosensory system that operates over a shorter range than vision. In comparison, control and procedural control sharks responded to the magnet zone at greater distances and therefore, this significant increase in avoidance distance may have been a result of both the visual and magnetic stimuli associated with the magnet zone. Furthermore, graphical analyses illustrate that the avoidance responses of all visually deprived sharks occur within the magnetic field region that exceeds the strength of the Earth's magnetic field (0.25–0.65 G) whereas, control and procedural control shark aversion responses occur at greater distances (~ 35 cm) from the magnet where associated magnetic fields are weaker (Fig. 6). Therefore, although no significant variation was observed for avoidance ratio, based on the results pertaining to avoidance distance, there are indications that the behavior of visually deprived sharks changes towards magnetic stimuli. Lastly, since both avoidance behavioral benchmarks are inconsistent with the original hypotheses, it is possible that avoidance behaviors are not appropriate to assess behavioral modification based on visual deprivation. 4.2.2. Entrance behaviors When focusing solely on the magnet zone results, significant variation existed between the entrance ratios and visit quantity prior to first entrance with shark type. More specifically, as hypothesized, visually deprived sharks had (1) significantly lower entrance ratios (Fig. 4) and (2) significantly higher visit quantities prior to first entrance (Fig. 5), in comparison to the control and procedural control shark types. These trends in entrance-related behavioral benchmarks suggest that visual deprivation may result in a heightened reliance on electroreception in juvenile N. brevirostris, therefore resulting in behavioral modification towards strong permanent magnetic fields. These findings are consistent with previous studies that suggest that environmental parameters which restrict vision may result in a heightened reliance on short-range senses, such as mechanoreception and electroreception (Hutchinson et al., 2012; Kajiura, 2001). Although the method of simulating turbidity is invasive, these entrance-related behavioral benchmarks (i.e. entrance ratio and visit quantity prior to first entrance) provide the first evidence of context-dependent switching in elasmobranch electroreception. Future research is required to illustrate if enhanced electrosensory perception to magnetic fields occurs during visual deprivation and may involve topographical voltage maps of a sighted sharks brain versus a visually deprived sharks brain as conducted on humans (Röder et al. 1999) or cats (Rauschecker, 1995; Rauschecker and Korte, 1993), as these voltage maps would provide a clear distinction in neuro-physiological behavior, if it exists, of sharks and would further support the present findings.

4.3. Effects of surgery Prior to experimentation, pre-trial analyses demonstrated that the effects of suturing did not lead to any significant differences in shark behaviors, such as chafes, accelerations, or physical contact with pen mesh. In addition, although only a brief visual inspection was conducted on each shark, the recovery period after surgery was determined to be one week, with nictitating membrane function and pupil size being nearly identical to sharks prior to surgery. Due to these findings, the lack of statistically significant differences in pre-trial behavior, and a prompt recovery period, it is suggested that this technique is one method which can be used in future vision deprivation studies in sharks. 5. Conclusion It is uncertain if suturing closed the nictitating membranes of sharks serves as an accurate technique of inducing behaviors that would typically occur when sharks interact with magnetic fields in turbid water conditions. However, if the behaviors observed are an accurate representation, the results suggest that magnetic repellents would be more successful at manipulating shark behavior when used in turbid water conditions. Therefore, it is essential for future electrosensory repellent (e.g. electropositive metals and permanent magnets) studies to routinely measure water turbidity, as this parameter may be one key source of repellent success and one source which may explain variations and sometimes contrasting results between previous studies (e.g. Kaimmer and Stoner, 2008; O'Connell et al., 2012; Tallack and Mandelman, 2009). In addition, if the results are a true representation of elasmobranch behavior in turbid water conditions, to maximize the effectiveness of these repellents in future experiments and applications, it may be pertinent to utilize these deterrents in areas typically characterized as being turbid, such as inshore regions that are heavily influenced by riverine input (Mulder and Syvitski, 1995), runoff (Crivelli et al., 1995), and eutrophication (Rosenberg, 1985; Smetacek et al., 1991). Acknowledgments We would first like to thank the Bimini Biological Field Station (BBFS) Foundation for providing support for this research project. In addition, we would like to thank the BBFS staff members and volunteers, such as Lindsay Biermann, Michael Timm, T.J. Ostendorf, Lauren Portner, Kelsey Evans, and Abby Nease for their assistance in specimen capture and experimentation. A special thanks to Dr. John Mandelman, Dr. Gregory Skomal, Dr. Kevin Stokesbury, and Dr. Saang-Yoon Hyun from the University of Massachusetts Dartmouth for assistance in experimental design and manuscript review, and Dr. Jens Krause for reviewing and providing input for this manuscript. Lastly, we would like to thank the University of Massachusetts IACUC and the Bahamas Government for granting the proper permissions to conduct this research. [SS]


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