ELECTRORECEPTION IN THE , DASYATIS SABINA

by

David W. McGowan

A Thesis Submitted to the Faculty of

The Charles E. Schmidt College of Science

In Partial Fulfillment of the Requirements for the Degree of

Master of Science

Florida Atlantic University

Boca Raton,

May 2008 IN THE EURYHALINE STINGRAY, DASYATIS SABINA

by David W . McGowan

This thesis was prepared under the direction of the candidate's thesis advisor, Dr. Stephen M. Kajiura, Department of Biological Sciences, and has been approved by the members of his supervisory committee. It was submitted to the faculty of The Charles E. Schmidt College of Science and was accepted in partial fulfillment of the requirements for the degree of Master of Science.

SUPERVISORY COMMITTEE

Thes1s A v1sor ~~

ii. ACKNOWLEDGEMENTS

I would like to thank my committee members Dr. Mike Salmon of Florida Atlantic

University and Dr. Joseph Sisneros of the University of Washington for their time and invaluable input throughout the length of this project. This study would not have been possible without the support of my colleagues at the Florida Fish & Wildlife Research

Institute's Tequesta and Deleon Springs Field labs in collecting and transporting the stingrays. My fellow lab mates in the FAU Elasmobiology Lab, Chris Bedore, Laura

Macesic, Mikki McComb, Tricia Meredith, and Anthony Cornett, were of such great support throughout this endeavor, as well the numerous undergraduate volunteers.

They constantly assisted me with husbandry, transportation of and supplies, and in the trials and tribulations of graduate school. Special thanks to my amazing wife,

Veronica, for her unconditional love and support, and limitless patience over these past four years. Finally, I would like to thank my advisor and mentor, Dr. Stephen Kajiura, for accepting such an untraditional student and tolerating all of my distractions (as important as they were). He has opened my to another side of science that I had taken for granted, and for that I am forever grateful.

lll ABSTRACT

Author: David W. McGowan

Title: Electroreception in the euryhaline elasmobranch,

Oasyatis sabina

Institution: Florida Atlantic University

Thesis Advisor: Dr. Stephen M. Kajiura

Degree: Master of Science

Year: 2008

This study determined the electrosensitivity of a euryhaline elasmobranch,

the Atlantic stingray, Dasyatis sabina, throughout the range of that it

would naturally encounter. It quantified the behavioral response of the stingrays . to prey-simulating electric stimuli in freshwater, brackish, and full strength

. The electroreceptive capability of stingrays from a permanent

freshwater population in the St. Johns River system was also compared with

stingrays that inhabit the tidally-dynamic Indian River Lagoon in east Florida.

This study demonstrated that D. sabina can detect prey-simulating electric fields

in freshwater, but the function of its electrosensory system is significantly

reduced. The SJR stingrays did not demonstrate an enhanced electrosensitivity

in freshwater, nor did they have reduced sensitivity when introduced to higher

salinities. The reduction in electrosensitivity and detection range in freshwater is

attributed to both an environmental factor (electrical resistivity of the water) and

the physiological limitations of the ampullary canals.

IV TABLE OF CONTENTS

LIST OF TABLES ...... vi

LIST OF FIGURES ...... vii

ELECTRORECEPTION IN THE EURYHALINE STINGRAY, DASYATIS SABINA

INTRODUCTION ...... 1

MATERIALS AND METHODS ...... 4

Study area ...... 4

Stingray collection and maintenance ...... 4

Experimental apparatus ...... 5

Experimental protocol...... 6

Data analysis ...... 7

RESULTS ...... 13

DISCUSSION ...... 19

LITERATURE CITED ...... 27

v LIST OF TABLES

TABLE 1. Stingray disc width and observed electric field intensities that resulted in

initiation of a feeding response by treatment...... 15

Vl LIST OF FIGURES

FIGURE 1. Map of the two sample areas: Harney in the St. Johns River system

and the southern half of the Indian River Lagoon system ...... 10

FIGURE 2. The experimental apparatus used to measure the electrosensitivity of the

stingrays ...... 12

FIGURE 3. Electric field intensities for each stingray's best response to the prey

simulating dipole ...... 16

FIGURE 4. Empirical orientation distances and derived detection ranges for brackish-

saltwater and freshwater groups ...... 17

FIGURE 5. The distribution of voltage equipotentials produced by a prey-simulating

electric dipole in an electrically conductive marine environment and a

resistive freshwater environment...... 25

Vll ELECTRORECEPTION IN THE EURYHALINE STINGRAY, DASYATIS SABINA

INTRODUCTION

All elasmobranchs (, skates, and rays) possess an extremely sensitive

electrosensory system that enables them to detect weak extrinsic electric fields in their

environment. This sensory capability has been demonstrated to function in the detection and localization of weak bio-electric fields of less than 0.5 nV generated by prey

(Kalmijn, 1971; Tricas, 1982; Haine et al., 2001; Kajiura and Holland, 2002) and

con specifics (Tricas et al., 1995). It has also been shown to detect the relatively low frequency electric fields produced by predators, triggering a defensive response

(Sisneros et al., 1998), and theorized to aid in geomagnetic orientation and navigation

(Kalmijn, 1974; Paulin, 1995).

The elasmobranch electrosensory system consists of hundreds to thousands of bulb-like electroreceptive organs known as the . These ampullae are grouped into 3 to 5 subdermal clusters that are distributed over the head of galeoids and also the body of batoids (Chu and Wen, 1979; Zakon, 1988). A single ampulla consists of multiple alveolar sacs continuous with a narrow canal that terminates in a pore on the skin surface (Waltman, 1966). The canal is filled with a low-impedance glycoprotein gel and the cells comprising the canal wall are bound by tight junctions that form a high impedance electrical barrier (Murray and Potts, 1961; Waltman, 1966). The lumen of the ampulla is lined by a single-layer epithelium that is comprised of sensory and support cells (Waltman, 1966; Zakon, 1988; New and Tricas, 2001 ).

The elasmobranch electrosensory system evolved in a highly conductive seawater environment. However, there are several elasmobranch that have subsequently transitioned to a freshwater environment. The high impedance freshwater environment presents a challenge to their electrosensory system. The ampullary 1 electroreceptors of obligate freshwater elasmobranchs, such as the South American freshwater stingrays (), have undergone substantial morphological adaptations to compensate for their high impedance environment. The ampullae are significantly smaller "microampullae", individually distributed in the dermis rather than in subdermal clusters, and the canals are much shorter (Raschi and Mackanos, 1989; New and Tricas, 2001 ). To aid in , the skin is thicker, enabling the to maintain an internal ionic concentration that is greater than that of the surrounding freshwater (New and Tricas, 2001 ). The thicker skin increases transcutaneous electrical resistance, forming a high impedance barrier that results in the greatest voltage change occurring across the skin (Kalmijn, 1974; Raschi and Mackanos, 1989; New and Tricas,

2001 ). Thus, the microampullae are ideally located in the dermis to detect these voltage changes (Kalmijn, 197 4; Zakon, 1988; New and Tricas, 2001 ).

Whereas the electrosensory systems of freshwater elasmobranchs have evolved to function in a high impedance environment, the electroreceptors of euryhaline elasmobranchs remain morphologically similar to those of marine species {Whitehead,

2002). Nonetheless, euryhaline species retain electroreceptive capabilities in freshwater, as demonstrated in the bull (Carcharhinus leucas) {Whitehead, 2002).

How the function of the elasmobranch electric sense is affected when euryhaline species enter freshwater remains unknown.

The goal of this study was to determine the electroreceptive capabilities of a euryhaline elasmobranch throughout the range of salinities, and hence conductivities, that it would encounter in its natural environment. The organism selected for this study was the Atlantic stingray, Dasyatis sabina. This species is locally abundant throughout the primarily brackish Indian River Lagoon (IRL) system in east Florida where it is found over shallow open sand and silt bottoms, associated with sea grass beds and spoil

2 islands (Snelson and Williams, 1981; Snelson et al., 1988). The presence of a permanent freshwater population in the nearby St. Johns River (SJR) provided the opportunity to compare electrosensory capabilities of populations of the same species that inhabit electrically dissimilar environments. The SJR population completes its full life cycle in freshwater, with no significant differences in size of maturity and reproductive success compared to marine populations in the nearby IRL and the northeast Gulf of

Mexico (Johnson and Snelson, 1996). The freshwater population is believed to have been established from the coastal populations approximately 100,000 years ago, and may have undergone adaptations to its freshwater environment (Cook, 1939; Johnson and Snelson, 1996). Comparing individuals from the two populations may provide insight into possible evolutionary adaptations of the electrosensory system in this species.

The objectives of this study were to behaviorally determine the electrosensitivity of the Atlantic stingray to prey-simulating electric fields in marine (35 ppt), brackish (15 ppt), and (0 ppt) treatments, and to then compare the electrosensitivities of stingrays from the IRL population with that of the SJR population to determine if permanent exposure to freshwater has affected the electric sense in this species.

3 MATERIALS AND METHODS

Study area

The Indian River Lagoon (IRL) system is a dynamic located along the east coast of Florida, U.S.A. (Fig. 1a). The estuary undergoes wide ranging fluctuations throughout the year, with seasonal changes ranging from full-strength seawater in the winter/spring dry season to freshwater in the late-summer/fall wet season. In the southern half of the IRL, daily salinity fluctuations result from tidal flushing through four inlets connected to the Atlantic Ocean, mixed with freshwater inflows from rivers, creeks, and canals draining from the interior of the Florida peninsula.

Atlantic stingrays (Oasyatis sabina Leseur 1824; Dasyatidae) in the IRL are found in all salinities and are tolerant of moderate daily fluctuations (Snelson and Williams, 1981;

Snelson et al., 1988; personal observation).

The St. Johns River (SJR) system originates along the east coast of Central

Florida as a series of interconnected freshwater that merge to form a northward flowing river, eventually emptying into the Atlantic Ocean though Jacksonville, FL. Lake

Harney (28° 45' N, 81 o 03' W, Fig. 1b) is a 3211 ha freshwater lake in this system, located approximately 300 km south of the mouth of the SJR. The freshwater population of D. sabina described by Johnson and Snelson (1996) is commonly found in Lake

Harney and surrounding lakes (J. Holder, per. comm.).

Stingray collection and maintenance

Stingrays were collected using gear that avoided any damage to the electroreceptors (i.e. hook wounds near the mouth). In the IRL, stingrays were collected between August 2005 and January 2007 using a 183.5-m center-bag seine. In Lake

Harney, stingrays were collected between June 2006 and August 2006 by electrofishing

4 from an airboat. Upon capture, each stingray was sexed, measured, and placed in a live well. Only sexually mature stingrays (> 22 em disc width) were retained for this study to account for previously described ontogenetic changes in response properties of the

Atlantic stingray's electrosense (Snelson et al., 1988; Sisneros and Tricas, 2002).

IRL stingrays caught in less than 7 ppt were assigned to the freshwater IRQ treatment, those captured at salinities between 7.1 to 24.9 ppt were assigned to the brackish-water IR15 treatment and stingrays captured in salinities greater than 25 ppt were assigned to the saltwater IR35 treatment. A major drought in 2006 resulted in higher salinities throughout the IRL which precluded collection of stingrays for the IRO freshwater treatment. As a result, 7 stingrays were acclimated to freshwater, by decreasing salinity levels by 2 ppt every 24 hours, and assigned to the

IRQ treatment. Because the SJR was always fresh, all SJR stingrays were assigned to the freshwater SJO treatment.

All stingrays were maintained in identical 122 x 244 x 50 em fiberglass tanks at either the Florida Atlantic University (FAU) Marine Science facility or the Boca Raton campus. The IR35 stingrays were held at the FAU Marine Science facility in marine flow-through aquaria at 23.0-26.8° C. The IRQ, IR15, and SJO stingrays were held at the

FAU Boca Raton campus in closed-circuit flow tanks at 21.3-22.0° C. All rays were fed to satiation once daily on a diet of thawed grass shrimp.

Experimental apparatus

A stimulator based on Kajiura and Holland's (2002) design was used to produce a prey-simulating dipole electric field. An applied current ranging from 6.9 to 8.3 mA produced a DC field similar to the weak electric fields produced by the amphipods and mysids that constitute a large portion of the stingray's diet (Cook, 1994; Tricas per.

5 comm.). Current from the stimulator was delivered via a pair of shielded 18 AWG SO underwater cables. The cables terminated to gold-plated stainless steel pins connected to water-filled polyethylene tube salt bridges. The polyethylene tubing was mounted flush to the underside of an opaque acrylic plate, forming a dipole. The gap distance between the centers of the dipole openings were 1 em to simulate a small prey item.

The voltage gradient produced by the dipole was maintained at a constant intensity by continuous adjustment of the applied current. Current was monitored with an ammeter to be within 0.1 mA of the target intensity. Current from the stimulator was delivered randomly to one of four electric dipoles, identical to the one previously described. The four dipoles were equally spaced 40 em apart from one another in four quadrants of a 1- m square at one end of the acrylic plate (Fig. 2). The 122 x 213 em acrylic plate lay on the bottom of the experimental tank. An identical plate was also placed in the bottom of the holding tank to provide conditions as similar as possible. The experimental tank was identical to the holding tank and connected to the same water flow and filtration system.

A digital video camera mounted on a tripod was positioned 1 m above the surface of the water over the center of the electrode array.

Experimental protocol

Trials were conducted with individual stingrays after they had been acclimated in their holding tank to the test salinities of either 0, 15, or 35 ppt and had been feeding for a minimum of 7 days. Prior to commencement of experimental trials, stingrays were fasted for a minimum of 48 hours. One stingray was moved from the holding tank to the experimental tank and allowed to acclimate for a minimum of 10 minutes. The stingray was aroused to feed by placing one piece of thawed shrimp in the experimental tank to elicit a prey-searching behavior, determined by increased swimming velocity and turning

6 frequency. Once the stingray ingested the shrimp and began to search for more food, video recording commenced and the stimulator was switched on to deliver current to one of the four dipoles. The response of the stingray to the electric field was recorded, with a successful feeding response defined by the stingray biting at the active dipole. After a response was observed, the stimulator was turned off. Once the stingray swam away from the four dipoles, the stimulator was turned back on, delivering current to another dipole. This procedure was repeated until I observed a positive response at each dipole or until the stingray no longer showed interest, ending the trial. The stingray was then fed to satiation and returned to its holding tank.

The IRL stingrays were tested only at the same salinity in which they were captured (i.e. 0, 15, 35 ppt). The SJR stingrays were tested first in freshwater (0 ppt) then slowly acclimated to 15 ppt, increasing the tank salinity by 2 ppt every 24 hours.

Once acclimated to brackish water and feeding normally for a minimum of one week, trials were conducted and the results constituted the SJ15 treatment. The stingrays were then acclimated to 35 ppt following the same protocol, and tested again in saltwater

(SJ35 treatment).

Data Analysis

Video clips of successful responses were extracted from the source tapes and edited using the software Final Cut Pro. All video clips were renumbered prior to analysis to conceal the treatment and test salinity, thereby enabling the analysis to be conducted in a single-blind fashion to prevent bias. From the resultant video clips the frame in which the stingray initiated an orientation to the dipole was extracted, then contrast adjusted, and deinterlaced using Adobe Photoshop. From this frame, the stingray's spatial location with respect to the center of the dipole was quantified using

7 the software lmageJ. To quantify the spatial position of each orientation, two parameters were measured: the stingray's distance from the center of the dipole to the point at which the orientation was initiated (orientation distance) and the angle of the orientation point with respect to the dipole axis (orientation angle). Orientation distance was measured from the center of the dipole to the posterior edge of the closest spiracle, which closely approximates the position of the hyoid ampullary cluster. The responses for each stingray were reviewed by a second analyst in a single-blind fashion to verify the initiation point for the orientation to the dipole. The results of both analysts were in overwhelming agreement.

The orientation distance and angle were incorporated into Kalmijn's ( 1982) 'ideal dipole field' equation to determine the electric field intensity at the point where the stingray first oriented towards the simulated prey. The ideal dipole equation states:

p Id cos e = 1-l V em -l

where p is the resistivity of the water (the experimental factor), I the applied current (6 .9

- 8.3 f,LA), d the gap distance between the centers of the dipole openings (1 em), r the measured orientation distance, and e the measured orientation angle with respect to the dipole axis. The orientation angle is included in the equation to account for the cosine dependency of the voltage gradient (Kalmijn, 1982).

The weakest electric field intensity that elicited a positive response for each stingray was defined as its best response and used for statistical analysis. A natural log transformation was applied to the derived electric field intensity values to meet the assumptions of normality and homoscedasticity (Ramsey and Schafer, 2002). A one- way analysis of covariance (ANCOVA) was employed to test for differences in the

8 minimum detected electric field intensity among the treatments, with disc width included

as the covariate (Dowdy and Wearden, 1991 ). All statistical analyses were conducted

using SAS version 8.02.

Both a median and a maximum detection range were derived from the orientation

distance data. The derived detection range was defined as the area around the center

of the dipole in which stingrays were observed to have initiated an orientation to the

dipole prior to a successful feeding response. The best responses (i.e. lowest electric field intensity) for each stingray in their respective treatment were used to calculate the

median electric field intensity for each treatment. Each median electric field intensity was used in Kalmijn's 'ideal dipole field' equation to back-calculate the median orientation distance from the dipole (r) along a goo arc for its respective treatment, graphically depicting a detection area around the dipole that incorporates the cosine dependency of the electric field intensity. To calculate the maximum detection range, the single weakest electric field intensity for each treatment was used to back calculate the maximum orientation distance from the dipole.

9 St. Joh ns River System

FIGURE 1. Map of the two sample areas: Lake Harney in the St. Johns River system, located approximately 300 km upstream from its saltwater outlet in Jacksonville, FL and

10 the southern half of the Indian River Lagoon system, ranging from Vera Beach, FL

(2r38'N, 80°22'W) as the northern boundary to Jupiter, FL (26°58'N, 80°05'W) as the southern boundary, and including the St. Lucie River estuary to the west (North fork

2r14.50'N, 80°19.00'W and south fork 2r10.00'N, 80°15.25'W). The C-44 canal in the southwest corner of the St. Lucie River connects Lake Okeechobee to the IRL and is a major source of freshwater inflow into the study area.

11 water level

ammeter stimulus

acryUc plate

FIGURE 2. The experimental apparatus used to measure the electrosensitivity of the stingrays. A 122 x 213 em acrylic plate was placed at the bottom a 122 x 244 x 50 em tank. The four dipoles were equally spaced 40 em apart within a 1 m2 near one end of the acrylic plate. The gap distance between each pair of dipole openings was 1 em. A stimulus generator produced a DC field with an applied current of 6.9 to 8.3 mA to simulate a weak electric field of the stingray's natural prey. The response of the stingray was recorded by a digital video camera positioned directly over the center of the four dipoles, 1 m above the surface of the water.

12 RESULTS

Fifty-one Atlantic stingrays were collected and tested for this study. Ten were collected in freshwater from Lake Harney in the St. Johns River (SJR) system. Within the Indian River Lagoon (IRL) system, 4 were collected in freshwater, 18 in brackish water, and 11 in saltwater. One male stingray (IR0-04) was retained even though its disc width was less than 22 em (OW=21.8 em) because its claspers were elongated and fully calcified. This indicated that the stingray was sexually mature, therefore meeting the study requirements of only testing sexually mature adults (Snelson et al., 1988).

The SJR stingrays were successfully acclimated from 0 ppt to 15 ppt, but experienced high mortality (60%) immediately after the second acclimation period from

15 to 35 ppt. Logistical constraints precluded collecting additional stingrays from Lake

Harney, resulting in the exclusion of the SJ35 treatment from the analysis.

Electric field intensity detection threshold

Of the 51 stingrays tested, 7 were excluded from the analysis due to lack of motivation to feed during trials. The size and electrosensitivity of the remaining experimental rays is summarized for each treatment in Table 1. Each stingray's best response (i.e. lowest field intensity) is shown in Fig. 3. The electric field intensities

differed significantly among the five treatments (ANCOVA; F5, 39=70. 79, P<0.0001, r=0.90), and the disc width covariate did not significantly affect the model (P=0.088). A

Tukey-Kramer post hoc test revealed that neither the two freshwater treatments (IRQ vs.

SJO, P=0.99), nor the saltwater versus the brackish treatments (IR15 vs. IR35, P=0.60;

IR15 vs. SJ15, P=0.19; IR35 vs. SJ15, P=0.84) differed statistically. However, the electric field intensities in both freshwater treatments were significantly greater than each of the brackish-saltwater treatments (P=<0.0001 ). The median electric field intensities in

1 1 the two freshwater treatments (IR0=1 .186 1-JV cm· , SJ0=1.532 1-JV cm- ) were 2.39

13 orders of magnitude greater than for the saltwater and two brackish water treatments

(IR35=0.00471.N em-\ SJ15=0.0031 IJV em-\ IR15=0.01121JV em-\ The weakest electric field intensity detected was 0.00057 IJV cm-1 by an IR35 stingray, whereas the weakest electric field intensity in either freshwater treatment was 0.217 IJV cm-1 by an

IRQ stingray. These results justified pooling the treatments into two groups; a freshwater

(FW) and a brackish-saltwater (8SW) treatment group.

Prey detection range

Detection ranges were derived for the two pooled groups, rather than for all five treatments. The point at which the stingray initiated its orientation to the dipole was plotted using polar coordinates for each successful response (Fig. 4A). The median

1 1 electric field intensities for the 8SW (0.0055 IJV cm- ) and FW (1.4 IJV cm- ) groups were derived from the best responses for each stingray in their respective treatment. These median electric field intensities were used to calculate the median detection range for each group. At oo to the dipole axis, the derived median detection distance was 25.03 em in brackish-saltwater and 15.33 em in freshwater (Fig. 48). Using the minimum

1 electric field intensity for each group (8SW=0.57 nV em-\ FW=0.217 IJV cm- ) the derived maximum detection distance was 44.03 em in brackish-saltwater and 28 .52 em in freshwater (Fig. 48), while the maximum observed detection distance was 38.8 em in brackish-saltwater and 24.47 em in freshwater.

14 TABLE 1. Stingray disc width and observed electric field intensities that resulted in initiation of a feeding response by treatment.

Mean Disc Median Voltage Width Gradient 1 1 Treatment N ± S.E.M. {mm} Range {mm} ± S.E.M. {gVcm- } Range {gVcm- } IRQ 10 252.1 ± 8.44 218-308 1.1856 ± 0.4837 0.2174-5.4777

IR15 9 247.6 ± 3.67 230-265 0.0112 ± 0.0022 0.0017- 0.0205

IR35 8 252.9 ± 4.17 232-265 0.0047 ± 0.0069 0.0006 - 0.0586

SJO 10 283.8 ± 8.19 244-339 1.5323 ± 0.4535 0.2801 - 5.2725

SJ15 7 282.9 ± 5.60 270-312 0.0031 ± 0.0007 0.0015 - 0.0066

15 10 • • I I I '";- E • • (.) I > • • .3 • • .?:- ·u; 0.1 c • 2c -o Qi 0.01 I • u:: I • I (.) ·.:::: • t5 • • Q) • 0.001 • • UJ •

0.0001 IRQ IR15 IR35 SJO SJ15

FIGURE 3. Electric field intensities for each stingray's best response to the prey simulating dipole. Treatment classification: IR = stingrays captured in the Indian River

Lagoon; SJ =stingrays captured in the St. Johns River; 0 =tested in freshwater (0 ppt);

15= tested in brackish water ( 15 ppt); 35 = tested in full-strength seawater (35 ppt).

16 A. Empirical Orientation Distance go•

Brackish & Saltwater 40 Freshwater

30 30°

~------~~~~~~~~------~~ a · 40 30 20 10 0 10 20 30 40 50 Distance from the center of the dipole (em)

B. Derived Detection Range go•

60° 60° Brackish & Saltwater 40 Freshwater

...... -·; ...... · . 30 30° .. ' . . 20 •••• ~ •••• . ~ ...... '\ .. / .... _...... : .- . . . ,. .. / . . ~ ...... I ·. , .. ,\ o· . 50 40 30 20 10 0 10 Distance from the center of the dipole (em)

FIGURE 4. Empirical orientation distances and derived detection ranges for brackish- saltwater (left side) and freshwater (right side) groups, The center of the dipole that produced the prey-simulating electric field is located at the intersection of the horizontal and vertical axes. Both axes represent the distance from the dipole origin in em

(horizontal axis = distance at oo to the dipole axis; vertical axis = distance at goo to the dipole axis). The upper polar plot (A) displays the spatial location of each stingray at which an orientation was initiated prior to a successful feeding response. The responses 17 are depicted within a 90° quadrant for both groups (11, o = all response points; •, • = best response points for each stingrays). The lower polar plot (B) illustrates the derived detection range as the median detection distance (dashed line) and the maximum detection distance (dotted line). See text for further details.

18 DISCUSSION

This study is the first to quantify the differences in electrosensitivity of a euryhaline elasmobranch that is regularly exposed to an electrically dynamic environment; one that ranges from a relatively high conductivity marine estuary to a relatively low conductivity (highly resistive) freshwater system. The effect of decreasing salinity on the electrosensory system is of particular interest because many euryhaline elasmobranchs are widely distributed across a range of salinities. These include several species of stingrays commonly found in freshwater tributaries and upstream of coastal areas, including Dasyatis guttata, D. garouaensis, D. bennetti, and D. sephen (Thorson et al., 1983; Thorson and Watson, 1975; Taniuchi, 1979). Larger elasmobranchs, such as the (Carcharhinus leucas) and the largetooth sawfish (Pristis perottett) also frequent freshwater systems (Thorson et al., 1966; Basset al., 1973; Taniuchi, 1979;

Thorson, 1974). Of these species, some spend extended periods of time and will undergo critical life stages in freshwater. Gravid bull sharks have been reported to enter freshwater for parturition, with the neonates often remaining in brackish waters for extended periods after the adult female returns to the sea (Springer, 1963; Snelson et al., 1984; Jensen, 1976). However, their life cycle is still tied to the marine environment since mating is not believed to occur in freshwater (Jensen, 1976). For the largetooth sawfish, parturition was reported in Lake Nicaragua, along with occasional reports of mating (Thorson, 1976). The adults would stay in the lake for long time periods but the juveniles were believed to move out to coastal marine waters. Unlike these other species, the freshwater population of Atlantic stingrays in the St. Johns River (SJR) completes their entire life cycle in freshwater (Johnson and Snelson, 1996). This represents the strongest evidence that the electrosensory system has to function in some capacity for this particular marine species to permanently inhabit a freshwater

19 environment. Prior to this study, the degree to which the electrosensory system was affected by the high resistivity of freshwater was unknown.

The results of this study demonstrate that D. sabina is able to detect and orient towards a prey-simulating weak electric field in a wide range of salinities, including freshwater. The sensitivity of the electrosensory system appears to be reduced in freshwater, as the detection range and electrosensitivity of the stingrays in both freshwater (FW) treatments were significantly less than those tested in the brackish and saltwater (SSW) treatments. The derived median and maximum orientation distances of both FW treatments were reduced by 38.8% and 35.2% respectively, compared to the combined SSW treatments. Differences in electrosensitivity were even more pronounced, as the FW stingrays responded to a median electric field intensity of 1.4 ~V

1 1 cm· , with a minimum response threshold of 0.217 ~V cm· , whereas the SSW stingrays'

1 1 median response was 5.5 nV cm· , with a minimum response threshold of 0.57 nV cm· .

This represents a difference of 2.39 orders of magnitude for the median electrosensitivity, and 2.26 orders of magnitude for the minimum response threshold electrosensitivity.

When compared to the IRL stingrays, the SJR stingrays did not demonstrate a significantly enhanced electrosensitivity in freshwater, nor did they show a significantly reduced electrosensitivity in brackish water. In fact, the electrosensitivities did not differ between the two populations in either freshwater or brackish water. Furthermore, the results of this study demonstrate the plasticity of the electrosensory system, as its sensitivity is determined by the resistivity of the water in which the stingray was tested, not by the environment in which it was collected. This included the freshwater SJR stingrays that were acclimated to brackish water, as well as the 7 IRL stingrays caught in brackish water that were acclimated to freshwater. The response of these stingrays was

20 similar to the responses of other stingrays from both populations that were tested at those salinities without acclimation. To prevent osmotic shock and maintain normal feeding behavior, the stingrays were slowly acclimated to the test salinity(+/- 2 pptlday), and then held at the test salinity for a minimum of one week. Individuals collected from the SJR have been previously subjected to faster rates of acclimation (5.33-8 pptlday) to brackish water (15-16 ppt) and seawater (32 ppt), but the effects of the rapid acclimation on feeding behavior were unknown because the stingrays were food-deprived throughout the one-month trial (Piermarini and Evans, 1998). The high mortality of the

SJR stingrays during their acclimation from brackish to full-strength seawater during this study may have been a result of increasing salinity too quickly (28 ppt to 35 ppt) during the last two days of the acclimation process due to logistical constraints.

The electrosensitivities determined for the BSW stingrays' are consistent with previous studies that tested other elasmobranch species in marine environments with similar experimental apparatus. Small smooth dogfish (Muste/us canis) initiated responses to prey-simulating electrical stimuli of <2 nV cm- 1 from >35.6 em (Kalmijn,

1982). Juvenile scalloped hammerhead sharks (Sphyrna lewim) and sandbar sharks

(Carcharhinus plumbeus) were reported to demonstrate minimum electrosensitivities of

0.4 nV cm-1 and 0.5 nV cm-1 and maximum detection distances of 30.6 em and 31.6 em, respectively (Kajiura and Holland, 2002). And neonatal bonnethead sharks (Sphyrna tiburo) responded to <1 nV cm-1 from a maximum detection distance of 22 em (Kajiura,

2003). Direct comparison of the FW stingrays' results to previous studies is limited due to a lack of quantified physiological or behavioral sensitivities of the electrosensory system in other freshwater elasmobranchs. Szabo et al. (1972) reported that the obligate freshwater stingray Potamotrygon exhibited a minimum behavioral threshold of

120 J1V cm- 1 while Szamier and Bennett (1980) reported the threshold sensitivity of

21 Potamotrygon to be 1 mV. Whitehead (2002) reported bull sharks initiated all responses from > 15 em, but did not report a maximum detection distance or behavioral threshold sensitivity. Although the sensitivity of the FW stingrays in this study is greater than these other species, it is still several orders of magnitude different from any other marine elasmobranch.

The differences in sensitivity between the BSW and the FW stingrays can be partially explained by the electrical properties of the water in these very distinct environments. Figure 5 illustrate the voltage equipotentials of a dipole modeled in both seawater and freshwater using the same parameters as in this study (i.e. 8 11A applied current, 1 em dipole opening separation, seawater resistivity of 19 ohm-em and freshwater resistivity of 1900 ohm-em). Each equipotential represents an order of magnitude difference in both seawater and freshwater treatments. The model clearly illustrates that the equivalent equipotentials in seawater are much closer to the origin of the dipole than in freshwater. This indicates that the voltage gradient decreases more rapidly in the conductive marine environment because the electric field is in essence being grounded, whereas the voltage gradient maintains its strength over a greater linear distance in the resistive freshwater environment. This property is what makes electrofishing an effective sampling technique in freshwater systems by delivering an electric current of sufficient strength to stun fish, but ineffective in a more conductive environment unless a greater source of power is available to drive the current through the water (Reynolds, 1983).

In Kalmijn's (1982) 'ideal dipole equation', resistivity is directly proportional to electric field intensity. Consequently, the difference in resistivity between the salt and fresh water environments is approximately 2.0 orders of magnitude. The difference in

1 1 median electrosensitivity between the BSW (5.5 nV cm- ) and FW (1.4 1.N cm- ) groups

22 is approximately 2.39 orders of magnitude, resulting in an environmental differential in

resistivity that accounts for 84% of the significant difference in electric field intensity. In

spite of the environmental differential, the 16% reduction in electrosensitivity and >35%

reduction in detection range demonstrate that the sensitivity of the electrosensory

system is reduced in freshwater.

One possible explanation for this reduction in sensitivity could be inferred from

the morphological differences between other marine and obligate freshwater elasmobranch species. The function of the long ampullary canals in marine species has recently become more apparent. The varied geometry of the canals, both in length and direction of canal projection, greatly enhance the electrosensory system's ability to sense differences in the voltage gradient (Camperi et al., 2007). Central processing of the hundreds of voltage potentials instantaneously detected by the network of electroreceptors enable the animal to contrast these signals with each other. The greater the distance between the canal pores, the greater the contrast becomes, resulting in a more rapid localization of the source of the electrical stimulus. Additionally, longer canals increase electrosensitivity by serving as biological conductors (Sisneros and Tricas, 2000; Brown et al., 2005). This is due to the relative low resistivity of the skin compared to the internal tissues, which are electrically more similar with the external environment. A voltage potential at the surface of the skin will be affected by the resistivity of the internal tissue and the capacitance properties of the canal hydrogel before the greatest voltage drop occurs in the lumen of the ampulla (Kalmijn, 197 4;

Brown et al., 2005). As a result, the potential difference will be greater in longer canals.

The absence of the long ampullary canals in obligate freshwater species would suggest an evolutionary loss of function. Unlike in marine elasmobranchs, the internal tissue in freshwater species is much less resistive than the external environment,

23 creating an isopotential environment throughout the body (Kalmijn, 197 4 ). Given that

the greatest voltage drop occurs at the surface of the highly resistive skin in these

species, the potential difference from the canal pore to the ampulla would be minimal

(Kalmijn, 197 4 ). The loss of this function would render the long ampullary canals

obsolete, resulting in the morphological migration of the ampullae to the dermal layer of

the skin, as seen in obligate freshwater species. In regards to D. sabina, the production

of skin mucus was noticeably greater when the stingrays were maintained in freshwater.

This was most likely to aid in osmoregulation, but perhaps concomitantly increased the

overall resistivity of the skin, somewhat akin to the obligate freshwater Potamotrygonids.

The reduction in sensitivity of the Atlantic stingray's electrosensory system in freshwater does not necessarily equate to a decrease in function. All freshwater

stingrays were able to detect, localize, and successfully bite at the prey-simulating

stimulus, some from distances in excess of 15 em. This species' ability to withstand

regular salinity fluctuations in the dynamic Indian River Lagoon and other coastal , expand their range into freshwater for extended time periods, and permanently inhabit a freshwater system demonstrates its electrosensory system continues to function sufficiently to locate prey and mates to complete its life history.

Despite their permanent exposure to freshwater over the past 100,000 years, the SJR stingrays did not demonstrate an enhanced electrosensitivity in freshwater, nor did they have reduced sensitivity when reintroduced to higher salinities. Stingrays from both populations responded similarly to the prey-simulating stimulus when tested in similar salinities, regardless of where they were collected from in the wild . The plasticity of this sensory system to function in such diverse environments demonstrates its adaptive significance.

24 go· Saltwater Freshwater 60°

30°

-30°

-60°

-90° Electric Field 1 Equipotential (JN cm- ) 10.0 1.0 0.1 0.01 0.001 0.0001

FIGURE 5. The distribution of voltage equipotentials produced by a prey-simulating electric dipole in an electrically conductive marine environment (left) and a resistive freshwater environment (right). Voltage gradients were modeled using the same parameters as in this study (i.e. 8 JiA applied current, 1 em dipole opening separation, seawater resistivity of 19 ohm-em and freshwater resistivity of 1980 ohm-em). Each equipotential represents an order of magnitude of the voltage gradient, ranging from

1 1 10.0 J1V cm- to 0.001 J1V cm- . The center of the dipole that produced the electric field is located at the intersection of the horizontal and vertical axes. Both axes represent the 25 distance from the dipole origin in em {horizontal axis = distance at oo to the dipole axis; vertical axis = distance at 90° to the dipole axis). The inset in the lower right corner illustrates the full extent of the electric field in freshwater.

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