DIFFERENTIAL BEHAVIOR OF AMPULLARY SUBUNITS IN THE ELECTROSENSORY SYSTEM OF THE SCALLOPED HAMMERHEAD SHARK

(SPHYRNA LEWINI)

A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAI`I AT MĀNOA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

IN

ZOOLOGY (MARINE BIOLOGY)

DECEMBER 2012

By

Christine M. Ambrosino

Thesis Committee:

Timothy C. Tricas, Chairperson Kathleen S. Cole Kim N. Holland

Keywords: electroreception, ampullae of Lorenzini, orientation, behavior

ii

To Sal and Maura,

who always encouraged my questions

and supported my pursuit of answers.

iii

ACKNOWLEDGMENTS

This project was made possible due to the help and support of many individuals. I

graciously thank my committee members, Kassi Cole and Kim Holland for their insight

and advice on the conception and development of this project. I especially thank my

committee chairperson Tim Tricas for his patience and support during my years in the

lab. My labmates and the members of the Holland lab have proven invaluable. I thank

Adam Dewan, Kelly Boyle, James Anderson, Leigh Ann Boswell, Kelly Williams,

Melanie Hutchinson, Mark Royer, Jon Dale, Yannis Papastamatiou, Toby Daly-Engel,

and Nick Whitney for advice, friendship, discussion and much needed emotional and

mental support. I thank Steve Kajiura whose work inspired my own, and whose advice

and insight helped me navigate the trials of graduate school. I also thank Kara Yopak for

her shared enthusiasm for neuroscience.

There were also many friends and eager volunteers whose enthusiasm helped to

encourage me even when fishing did not always turn into catching. I thank Joe Sanchez,

Nicole Escudero, Heather Ylitalo-Ward, Emi Yamaguchi, Julio Rivera, Mike Burns,

Kelvin Gorospe, Thomas Krueger, Matt Potenski, Dave Harrington, Julia Sullivan and

Andrew Starr for help on the water or just lending an ear. I enthusiastically thank Sal,

Maura and Ali Ambrosino, who made the completion of this project possible. I thank

Shawn Carrier and Liz Ross for their support and help on the water, and the Dive and

Boating Safety Officers of HIMB, Derek Smith and Jason Jones, who helped with

specimen collection and making sure rusty equipment still worked.

iv

Financial support was provided by grants from the University of Hawaii at Manoa

Graduate Student Organization, PADI Project AWARE, the American Elasmobranch

Society Student Travel funds, the American Elasmobranch Society Carrier Award, and

the Albert L. Tester Memorial fund.

v

ABSTRACT

The electrosensory system of elasmobranchs consists of discrete networks of gel-

filled canals that connect to a specific subgroup of a subcutaneous structure called an

ampulla. This project tested the functional subgroup hypothesis that predicts functional

differences among the ampullary subgroups using the scalloped hammerhead shark,

Sphyrna lewini. To examine electroorientation behavior, the reaction of sharks to

electrical dipoles was digitally recorded on video. After control trials, sharks then had

certain ampullary pore fields blocked with non-conductive petroleum jelly and again

exposed to the same dipoles to observe potential changes in orientation behavior. The

entire cephalofoil and either the right or left half of the cephalofoil were blocked in trials

to determine the efficacy of the treatment protocol. The Buccal (BUC) and Superficial

Ophthalmic anterior (SOa) ampullary pores were then inactivated. The BUC and SOa

pore fields were chosen for study due to their location on the cephalofoil and pore

number (the SOa includes more than half the pore field). The majority of sharks oriented

to the dipole less than 12cm away and at an angle of less than 40 degrees from the dipole

axis. Sharks with blocked BUC pores demonstrated fewer orientations to the simulated

prey field (p = 0.008), although they still fed and swam normally. The manipulated

sharks also showed decrease spiral behavior in relation to the other orientation types, and

increased their overshoot behavior with blocked SOa pores. Thus, the functional subunit

hypothesis is supported by this study and the entire ampullary pore field was

demonstrated to be necessary for proper orientation within a dipole field.

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TABLE OF CONTENTS

ACKNOWLEDGMENTS ................................................................................................. iii

ABSTRACT....................................................................................................................... iv

LIST OF FIGURES ........................................................................................................... vi

LIST OF ABBREVIATIONS AND SYMBOLS ............................................................. vii

INTRODUCTION ...............................................................................................................1

MATERIALS AND METHODS.........................................................................................7

RESULTS ..........................................................................................................................17

DISCUSSION....................................................................................................................29

REFERENCES ..................................................................................................................37

vii

LIST OF FIGURES

Figure Page

1a. Tank schematic.............................................................................................................13

1b. Dipole plate.................................................................................................................14

2. Electric field measurements...........................................................................................15

3. Orientation behavior types.............................................................................................16

4. Orientation distance from dipole ..................................................................................22

5. Dipole field strength for all approaches.........................................................................23

6. Orientation types with cephalofoil half-blocked ...........................................................24

7. Approach frequency ......................................................................................................25

8. Orientation frequency ...................................................................................................26

9. Dipole field strength for orientations ............................................................................27

10. Orientation types with BUC and SOa pores blocked...................................................28

viii

LIST OF ABBREVIATIONS AND SYMBOLS

ALLN Anterior Lateral Line Nerve

BUC Buccal Ampullary Cluster

MAN Mandibular Ampullary Cluster

SOa Superficial Opthalmic Anterior Ampullary Subcluster

SOp Superficial Opthalmic Posterior Ampullary Subcluster

V = Volts

ρ = resistivity of seawater (Ωcm)

I = applied current (A)

d = dipole separation distance (cm)

θ = angle with respect to dipole axis

r = radius distance (cm)

1

SECTION 1

INTRODUCTION

Electroreception in elasmobranchs

The orientation behavior of sharks to weak electric fields such as those produced

by prey or potential mates is well studied. Elasmobranchs are able to locate hidden prey

using electrical stimuli when visual and olfactory stimuli are absent (Kalmijn, 1966).

Blue sharks demonstrate feeding behavior at activated dipoles suspended in the water

column (Kalmijn, 1982). Scalloped hammerhead sharks, sandbar sharks and bonnethead

sharks are able to orient to benthic dipoles simulating potential prey fields (Kajiura and

Fitzgerald, 2009; Kajiura and Holland, 2002). Male rays demonstrate sex-specific

behaviors to electric fields that mimic the fields produced by resting conspecific females

(Tricas et al., 1995). Each of these studies used free-swimming animals with intact

electrosensory systems, but discrete behaviors may be dependent upon information from

individual ampullary subgroups. Whether the differential spatial arrays of the ampullary

subclusters affect set orientation behaviors has yet to be examined.

Types of electric field stimuli

Electric fields are a potential source of environmental information for animals that

have the specialized systems necessary to exploit this sensory niche. An electric field is a

force field surrounding a charged particle. Simple monopole fields surround a single

charged source, whereas dipole and complex multipole fields surround two or more

charge sources (Kalmijn, 2000). The three types of electric fields of greatest biological

import are kinetic fields (caused by conductor movement through the earth’s magnetic

2

field), fields induced by movement of the animal, and bioelectric fields (generated by the

electric organs of electric fish and animate fields consisting of weak direct current (DC)

fields produced by electrochemical gradients within an organism (Bleckmann and

Hofmann, 1999). Using electrosensory systems, many animals utilize these fields to

detect potential prey, to locate receptive mates, or for communication (Kalmijn, 1982).

Dipole electric fields dissipate as a square of the distance from the dipole center, as

described in the model for an ideal dipole in half space (V=ρIdcosθ/πr2). Thus, these

fields must be utilized in close proximity to the source (Kajiura and Fitzgerald, 2009).

Although many taxa employ sensory systems to detect electrical fields, the system with

the greatest sensitivity is found in elasmobranchs (Bleckmann and Hofmann, 1999).

Anatomy and physiology of ampullae

To detect electric fields, elasmobranchs use small, alveolar organs called the

ampullae of Lorenzini. The ampullae are subdermally clustered along the head of sharks

and the head and pectoral fins of skates and rays (Lorenzini, 1678). Gel-filled canals

connect each ampulla to a pore at the skin’s surface. Distinct patterns in pore distribution

on scalloped hammerheads, bonnethead, sandbar sharks and stingrays have been

described in detail by several groups (Kajiura, 2001; Mello, 2009; Raschi, 1986; Raschi,

2005). Pore clusters are named by their relationship to underlying ampullae or arbitrarily

assigned letters and may be used in species identification (Kajiura, 2001; Mello, 2009;

Raschi, 1978; Raschi, 2005). Eight to eleven groups of pores have been described on the

head of the scalloped hammerhead shark and remain constant throughout the life of the

animal (Kajiura, 2001; Mello, 2009). The epithelium of the ampulla itself consists of

3

receptor and support cells. The receptor cells project a single kinocilium from their apical

surface into the ampullary lumen and detect the voltage difference between the pore and

the base of the ampulla (Wueringer et al., 2009).The support cells secrete the gel that fills

the canal and ampullary lumen. This high potassium, low resistivity gel within the

ampullary canals has electrolytic properties and may function as a thermoconductor

(Brown, 2010; Fields et al., 2007). The canal walls are highly resistive and allow the gel

to conduct an electric current from the pore to the sensory epithelium of the ampulla with

the same efficiency as seawater (Wueringer et al., 2009).

Previous studies show that the ampullae are capable of responding to a wide

variety of stimuli including salinity levels, temperature and mechanical distortion

(Loewenstein and Ishiko, 1962; Sand, 1938). However, behavioral and physiological

tests indicate that electroreception is the main sensory purpose for these structures

(Murray, 1965). The electroreceptors within the ampullae detect potential differences

within low-frequency electric fields across the apical and basal surfaces of the receptor

cell (Bennett and Clusin, 1978; Bodznick and Boord, 1986; Murray, 1965). Because a

longer canal allows for greater potential differences between the pore and the base of the

ampulla, receptors at the base of longer canals are more sensitive to field potentials

(Bennett and Clusin, 1978; Murray, 1974). The receptor sensitivity also depends upon

the orientation of the canal to the dipole axis. Electric fields parallel to the canal elicit the

greatest response from ampullary electroreceptors (Murray, 1965; Tricas, 2001). As the

angle of the field becomes perpendicular with the canal, the sensitivity decreases

(Kalmijn, 1974).

4

Functional Subunit Hypothesis

The ampullae of marine elasmobranchs are divided into distinct groups innervated

by specific branches of the anterior lateral line nerve (ALLN) (Bodznick and Schmidt,

1984; Daniels, 1967; Norris, 1929; Rivera-Vicente et al., 2011; Rivera-Vicente et al., In

prep). The number of subclusters is species dependent, ranging from three to seven.

Sphyrna lewini (Griffith & Hamilton Smith, 1834), the scalloped hammerhead shark, has

four ampullary subgroups on either side of the head: the Mandibular (MAN), the Buccal

(BUC), and the anterior and posterior Superficial Ophthalmic groups (SOa, Sop)

(Daniels, 1967; Rivera-Vicente et al., 2011; Rivera-Vicente et al., In prep). Scalloped

hammerheads have more pores distributed along the ventral surface of their cephalofoil

than on the dorsal surfaces, much like other dorso-ventrally flattened elasmobranchs such

as rays (Daniels, 1967; Tricas, 2001). These groups each have differing organizational

morphology, and as suggested by the functional subunit hypothesis, these morphologies

may allow for the behavioral subdivision of the electrosensory system (Tricas, 2001).

The MAN group has the fewest number of associated pores which are located on the

lower jaw of the shark. Thus the MAN may be responsible for regulating biting

behavior. The BUC cluster is the most distal on the cephalofoil and its’ nearly 360

degree array of canals may be key to controlling spiraling behavior as the shark hones in

on its prey. The pores of the SOa and SOp groups overlap slightly, but the ampullae are

divided by a lateral extension of cartilage. The SOa has the greatest number of associated

pores, and is located at the anterior edge of the cephalofoil. This location and pore

density may allow the shark greater sensitivity in detecting the faint edges of the

bioelectric fields emitted by prey. The canals of the SOp are the longest, on average, of

5

the subclusters and have a mostly horizontal elevation. Their length and orientation may

help the shark detect environmental uniform field lines. With an elongated cephalofoil

that exaggerates morphological differences among ampullary canals, S. lewini provides a

unique model to study this electrosensory array.

The purpose of this study is to test the functional subunit hypothesis that

functional differences among ampullary subgroups relate to specific electro-orientation

behaviors, such as the type of orientation path or frequency of response to an electric

dipole field. Understanding the relationships between ampullary subgroups and their

respective pore fields is critical to understanding the field perception of elasmobranchs if

there are differences in function among different subgroups. The technique used to

functionally block individual ampullary subgroups with non-invasive methods allows for

the release of animals after behavioral analysis. This will also be the first study to

investigate the differential roles ampullary subgroups may play in electrosensory

processing. The functional subunit hypothesis proposes that the electrosensory system of

elasmobranchs can be behaviorally and functionally subdivided (Tricas, 2001).

Although morphological data indicates differential function among the ampullary

clusters, to date no study has examined the functional or behavioral purpose of individual

subgroups (Camperi et al., 2007; Tricas, 2001). The electrosensory system is used during

final approach to potential prey, and as the shark moves rapidly through the dipole field,

the entire array is predicted to be necessary for proper dipole detection. If this is the case,

with inactivation of certain ampullary clusters the shark will have less sensory input to

calculate an accurate strike at the dipole center. In this study, behavioral trials tested the

6

BUC and SOa ampullary subgroup function and examined the neuroecological role of

these ampullary subgroups in electroorientation behaviors.

7

SECTION 2

MATERIALS AND METHODS

Specimen collection

Juvenile scalloped hammerhead sharks, S. lewini, of both sexes were collected in

the southern portion of Kane`ohe Bay on O`ahu, Hawai`i with 11/0 circle hooks set on

hand lines and baited with cuts of squid. The hook barbs were depressed to decrease

injury to the juveniles during capture. Although this increases the chances of the hook

slipping from the animal, it was necessary to prevent damage to the electrosensory pore

fields located around the mouths of the animals. Once caught, the sharks were

immediately dehooked and placed in a 1m diameter, seawater-filled, fiberglass

hemisphere and transported to the Hawaii Institute of Marine Biology (HIMB). The

sharks were placed in a holding pen (approximately 10.2 x 19.4m with max depth of

2.4m) that is part of a natural lagoon at HIMB and enclosed with mesh fencing that

allows natural tidal flushing. The fencing around the pen prevented the sharks from

escaping, but permitted smaller reef fish to swim freely in and out of the enclosure.

Overhanging mangrove trees provided shade around the enclosure and reduced UV

radiation. The fish were fed tri-weekly to satiation with squid and capelin and cared for

according to the procedures listed in the UH IACUC protocol #09-651.

Experimental Apparatus

To test the electro-orientation behavior of the sharks, a stimulus generator was

used to generate weak dipole fields, the dimensions of which simulate the sharks’ natural

prey in Kaneohe Bay (Bush and Holland, 2002; Haine et al., 2001). A 9V battery

8

controlled with a three-way high/off/low switch was used to supply current to the power

box (Figure 1). The seawater through which the current passed acted as a resistor and

additional resistors of 10kΩ (high current circuit) and 1MΩ (low current circuit) and a

1MΩ potentiometer were used to control the current flow to the electrodes. A rotary

switch channeled the current to one of four dipoles placed in a 1m2 acrylic frame placed

on the bottom of the test tank via four pairs of shielded 18AWG underwater cables and

50cm long, seawater-filled polyethylene tubing. Current passed from the cables to the

tubing via underwater connectors with gold-plated stainless steel pins. The dipole

separation distance for each dipole was 1cm, allowing a current flow of 6µA (adjustable

by the stimulus generator). Bites at the dipole and orientation behaviors were recorded

with a JVC GR-DVL 9800 digital video camera centered above the acrylic plate on the

tank floor. Digital video was recorded at 30 frames per second (fps) and saved directly

onto a Macintosh laptop hard drive via a 20ft weather-resistant firewire cable. The

start/stop record functions of the camcorder were controlled via the laptop to decrease

experimenter motion during video trials.

Experimental Protocol

One week before each behavioral trial, two sharks, marked with fin clips, were

moved from the holding pen to a circular, 12ft diameter flow-through tank approximately

1.5m deep which was used as the test area (Figure 1). The flow-through tank drew water

from the sharks’ natural nursery ground and was covered with shade cloth to reduce

external visual stimuli and UV radiation, and to minimize algal growth. The two sharks

were monitored daily to determine proper acclimation to the test area, during which time

9

feeding patterns and tail beat frequency were observed. If a shark did not feed within the

week, it was removed from the tank. Sharks acclimated to the arena would swim

throughout the area and not restrict their path to the peripheral boundary or touch the tank

walls.

Before each behavioral trial, the juvenile hammerheads were starved for two days

to increase the motivation to feed. For trials, the pen was divided into two sections with

mesh. An individual shark was placed within the test area (the half of the flow-through

tank with the dipole plate) and allowed to again acclimate for 2 hours before behavioral

trials. To begin a trial and induce the sharks to orient to the activated dipole, an olfactory

stimulus (squid rinse) was introduced via a syringe, into tubing connected to the center of

the dipole acrylic plate. The olfactory stimulus elicited an excited predatory behavior

measured by an increase in the tail-beat frequency of the swimming juveniles. Tail beat-

frequency provides an estimate of the shark’s motivation to feed (Kajiura and Holland,

2002). Each trial lasted for five minutes as the sharks would stop actively searching

around that time if not presented with food. Orientation within the dipole field was

recorded by the digital video camera, and at the end of each trial, the shark was fed to

satiation then released into the free half of the tank. An orientation included actively

turning towards and swimming over the dipole, ending with a bite at the dipole center.

Although four dipoles are present on the acrylic plate, during the trials only one was

activated at a time and the three inactivated dipoles served as visual controls. Current

strength (6µA) and dipole separation distance (1cm) were constant throughout the

experiments. This maintained a consistent dipole moment of 6µA cm throughout the

trials.

10

Two days after the control behavioral trials were completed for each shark,

individual ampullary clusters were inactivated and the shark’s orientation behavior was

tested again. To functionally block the electroreceptors, petroleum jelly was placed on

the dorsal and ventral surfaces of the cephalofoil over the pore fields of interest. The

petroleum jelly was dyed with food coloring to provide visual confirmation that the

treatment was intact during trials. To first determine whether this method of blocking the

pores with petroleum jelly would affect the general behavior of the sharks and their

behavior within an electric field, the entire pore field of two sharks or either the right or

left half of the cephalofoil from the medial line to the distal edge of seven sharks was

covered and the sharks were observed for 24 hours before further trials. This period

allowed the sharks to recover from any stress from handling, and the petroleum jelly

remained on the cephalofoil during this time. To inactivate either the BUC or SOa pores,

petroleum jelly was placed over the dorsal and ventral pores to inhibit current flow along

the canal, thus functionally deactivating the ampullary receptors. To control for tactile

stimulation, an electrically conductive gel was placed on the pores that would remain

active.

Pore clusters were inactivated according to the behaviors expected to be

associated with them. The dorsal and ventral pores of the BUC group were inactivated

because the BUC is the most distal group of ampullae and associated pores on the

hammerhead cephalofoil. Once a field is detected and a shark spirals towards the dipole

center, the distal ends of the cephalofoil track the curvature of the electric field lines. The

SOa was also chosen to be inactivated because this group has the greatest number of

associated pores, and is located medially along the leading edge of the shark’s rostrum.

11

Thus, the SOa pores are the first to encounter an extrinsic electrical field in front of the

shark.

After completing control and manipulation behavioral trials, the petroleum jelly

was removed with a soft, damp cloth from the head of the sharks and they were observed

for an additional three days to ensure proper swimming and feeding behavior. The sharks

were then released in Kaneohe Bay near their collection site.

Video Analysis

Video footage from the behavioral trials was analyzed with ImageJ (v1.41, NIH)

on a Macintosh laptop. To rule out observer bias, the video was also analyzed by a

researcher blind to the treatment groups. Distance was calibrated within the 960 x 540

pixel resolution frames by black, 20cm circles surrounding each dipole on the acrylic

plate. Frame-by-frame analysis determined orientation distance, orientation angle,

orientation frequency, approach frequency and bite frequency (Figure 2). Orientation

distance was measured from the center of the dipole to the nearest part of the shark’s

cephalofoil. The orientation angle was measured as the angle of the line drawn by the

orientation distance in relation to the dipole axis. The field potential was also calculated

at the orientation distance using the equation for a dipole field in half space:

V=ρIdcosθ/πr2 (Kalmijn, 1982)

The equation for half space was used since the acrylic plate lies flat on the substrate, thus

the dipoles are only exposed to the seawater on one side. The resistivity of seawater (ρ),

the current in the system (I) and dipole separation distance (d) remained constant during

the trials.

12

Orientation type was also recorded (Figure 3). Orientation and bite behavior were

defined using behavioral patterns previously described by Kajiura and Holland (Kajiura

and Holland, 2002). A straight orientation is defined as when a shark swims in a straight

line towards the dipole and bites it. A turn orientation includes a turn towards the dipole

greater than 20 degrees from the shark’s trajectory entering the camera’s field of view.

The shark spirals into the center of the dipole along the field lines was described as a

spiral orientation. Overshoot orientation was described as when a shark swims past the

dipole, then turns around and backtracks towards the dipole center. Approach distance

and rate was used to determine that sharks had equal exposure to the electric field in all

tests. An approach was defined as the head of the shark entering a 30cm circle around

the dipole center. Preliminary tests showed that 32cm was the maximum distance the

sharks reacted to the active dipole. The frequency data were arcsine transformed

(SigmaPlot 11) and analyzed with a paired t-test comparing control and treatment trials to

determine pre- and post-treatment effects of subgroup inactivation.

13

Figure 1a. Schematic of the 12-foot diameter, saltwater flow-through tank used in experimental trials. A power box was used to supply current to the electrodes. A 9V battery was used to supply power to the circuit, and 10kΩ and 1MΩ resistors and a 1MΩ potentiometer were used to control the current flow to the electrodes. A rotary switch channeled the current to one of four dipoles placed in a 1m2 acrylic frame. The acrylic frame sat on the substrate where the sharks naturally fed. A JVC digital camcorder was used to record the sharks’ behavior during trials.

14

Figure 1b. Schematic of the acrylic frame and dipoles on the floor of testing area. The dipole separation distance was 1cm allowing a current flow of 6µA (adjustable by the power box). Current was carried via four pairs of shielded 18AWG underwater cables and seawater-filled polyethylene tubing. Current passed from the cables to the tubing via underwater connectors with gold-plated stainless steel pins. Each length of tubing from the cable to the acrylic plate was 50cm. The olfactory stimulant (squid rinse) was introduced through tubing connected to a hole in the center of the plexiglass plate.

15

Figure 2. Example of video image and measurements taken to quantify orientation behaviors observed. One of four dipoles was activated at a time on the acrylic plate as the sharks searched for food. The distance of the shark from the center of the dipole field (r) and the approach angle in relation to the dipole axis (θ) were used to calculate the field strength at the point where the shark initiated an orientation.

16

Figure 3: Orientation behaviors demonstrated by intact hammerheads. A straight approach occurred when the shark swam directly over the center of the dipole, without turning. A turn was defined as a motion towards the dipole greater than 20 degrees from the initial swim path. An overshoot occurred when the shark swam over and past the dipole, then turned back towards the center. During a spiral, the shark’s cephalofoil would follow the dipole field lines towards the center of the field. (Adapted from Kajiura and Holland, 2002)

17

SECTION 3

RESULTS

Seventeen first-year juvenile scalloped hammerhead sharks, S. lewini, captured

from Kaneohe Bay successfully acclimated to the holding pen environment, fed normally

and were used in experimental trials. Additional sharks (N=4) that did not initiate

feeding behavior within one week after capture were released back into Kaneohe Bay.

The retained juveniles demonstrated orientation and biting behavior at the active dipole,

but not at the inactive, control dipoles. The four orientation pathways as described above

were observed in control animals. Spiral orientations were the most frequent, followed

by turning, overshoot and straight behaviors. The olfactory stimulant (squid rinse) was

necessary to elicit hunting and feeding behavior during trials as without the olfactory

stimulus the sharks would not respond to the electric fields, even while swimming

directly over an active dipole.

Unmanipulated trials

All sharks oriented to and bit the active dipole center, and the majority of

orientation behaviors occurred less than 12cm from the center of the field (Figure 4).

Almost all orientations occurred within 30cm of the dipole center. The sharks

demonstrated sensitivity to very low field strength, as calculated from the orientation

distance. As described in the equation for an ideal dipole in half space, the strength of

the electric field decreases rapidly with increasing distance from the dipole center. Thus,

electroreception tends to be used as a short-range detection method. However, sharks

18

demonstrated high sensitivity to the field and responded to intensities below 100nV

(Figure 5).

Whole and half-head manipulations

After control tests with intact electrosensory systems, non-conductive petroleum

jelly, dyed with food coloring, was applied to the complete dorsal and ventral surfaces of

the cephalofoil in two sharks to determine the effects of the experimental manipulation

on general behavior. After treatment, the animals continued swimming normally as

demonstrated by tail beat frequency of about 1 Hz. The sharks also were able to feed

normally and continued interacting with other sharks in their vicinity. The sharks did not,

however, respond to the electric fields while the dipoles were active, even when excited

by the olfactory stimulus. Forty-eight hours after the petroleum jelly was initially

applied, it was removed and two days later, the sharks were exposed to the electric fields

again. With the petroleum jelly treatment removed, the sharks again demonstrated active

orientation within the electric fields with the olfactory stimulant. Following the trial, the

sharks were fed to satiation and released into Kaneohe Bay.

To examine further the effects of the petroleum jelly treatment on the behavior of

the scalloped hammerhead sharks, the right half or left half of the cephalofoil of six

randomly selected sharks was covered with non-conductive jelly on the dorsal and ventral

surfaces. To control for the effects of physical manipulation over the cephalofoil, an

electrically conductive gel was spread over the pore fields to remain active. Again the

animals were able to swim and feed normally. Although the sharks initially had

difficulty orienting properly to the center of the active dipole field, the sharks all oriented

within the field during the trials with either the right or left half of the cephalofoil

19

covered. A behavior was noted in the post-treatment animals described as a “miss.” This

behavior occurred as the animal neared the active dipole. During some approaches

within approximately 30cm of the active dipole, the post-treatment sharks began to

search for the dipole as indicated by rapid turning of the head. Although the sharks were

actively searching for the dipole, they did not locate it and properly orient. The number

of orientations in both pre- and post-treatment groups was highly variable, but animals

that were more active during control trials, remained more active during experimental

trials. The results of a matched pairs t-test between the blocked and unblocked

orientations did not differ for orientation frequency (t = 2.306, p = 0.15) or bite frequency

(t = 1.83, p = 0.13).

Interestingly, the sharks with half of their pore fields blocked demonstrated the

same types of orientation behaviors as unmanipulated sharks (Figure 6). The juvenile

sharks, even with half of their electrosensory pore fields functionally inactivated, were

still able to locate the center of an electric field with accuracy. After the trials, the

petroleum jelly was removed, and the sharks again demonstrated proper orientation to a

dipole target.

Single cluster manipulations

To examine the role of individual ampullary clusters in orientation behaviors

within an electric field, the dorsal and ventral pore fields of the BUC group were

inactivated in seven sharks and the SOa pore field was inactivated in another in seven

sharks. In the control trials, the sharks were able to respond to the electric field as the

other sharks in the preliminary trials. To confirm that the sharks were still exposed to

20

similar electrical stimuli in both trials, approaches within 30cm of the dipole center was

counted (Figure 7). There were no differences between control and experimental group

approaches (BUC, p = 0.38; SOa, p = 0.27).

Among experimental sharks, with the BUC ampullary subgroup inactivated the

orientation frequency was significantly reduced (Figure 8; p = 0.033). With the SOa

group inactivated, the orientation frequency approached similar levels to those observed

in the BUC treatment group, and the orientation frequencies were significantly different

from the control (Figure 8; p = 0.029). As with the sharks that had half of their pore

fields blocked, although they did not respond to the electric field with the same frequency

as during the control trials, the sharks retained their feeding behavior and actively

searched for food with the introduction of squid rinse. Also, when presented with squid

after the trials, the sharks were able to locate and eat their food to satiation.

The sharks in both the control and BUC and SOa treatment groups were again

able to behaviorally respond to an electric field at very low intensities (Figure 9). With

the BUC and SOa ampullary subgroups blocked, though, the sharks did not bite as

frequently at the dipole center. The sharks would still actively hunt for the source of the

dipole field, but more passes over the dipole would be required for the sharks to localize

the center of the field.

Even when the experimental sharks passed over the center of the dipole field, they

rarely bit at the dipole center. The sharks also displayed “incorrect” orientations to the

center of the dipole, in that as they entered the field, they often turned away instead of

towards the center. These searching behaviors were noted in both experimental treatment

groups, and often resulted in the sharks not being able to locate the dipole within the

21

testing area. When the non-conductive jelly was removed from the sharks, they were

able to correctly locate the center of the field.

The sharks all displayed the typical orientation pathways as described above in

the preliminary trials. As with the previous group of control sharks, spiral behavior was

again the most frequently observed, followed by turns and straight approaches (Figure

10). The sharks did not initially display the overshoot orientation pathway during the

control runs, but as the behavior’s frequency in previous trials was very low, the sharks

still demonstrated the correct assay of behaviors. When the individual ampullary

subclusters were blocked, the array of orientation behaviors shifted.

With the SOa pores blocked, the sharks were still able to turn fairly accurately

towards the dipole, but the spiral behavior was rarely displayed. Also, the sharks began

to exhibit the overshoot behavior more frequently. There were no straight approaches

noted with the SOa pores blocked. With the BUC pores blocked, the sharks displayed a

decrease in general orientation behaviors, but were able to rarely complete a spiral or turn

orientation properly. Straight and overshoot orientations were not seen in the BUC

treatment group. The sharks in both treatment groups continued to swim and feed

normally, even with the decrease in response to the dipole field.

22

Figure 4. Graph showing the orientation distances from the center of the active dipole during control and experimental trials. Although the majority of orientations occurred within 13cm of the dipole center, sharks were able to orient up to 32cm away.

23

Figure 5. The field strength experienced by the sharks (N = 17), calculated at the point of the shark closest to the dipole center for all approaches within 35cm. The distance of the shark from the dipole center (r) and its approach angle in relation to the dipole axis (θ) were entered into the ideal equation for a dipole in half-space (V=ρIdcosθ/πr2). The average field strength the sharks responded to within 35cm of the dipole center was 0.714 µV/cm.

24

Figure 6. Graph showing the types of orientation behaviors as percent of total orientations for sharks during control trials and trials with half of the cephalofoil pore field blocked. Although the total orientation frequency differed between the test groups, the behavioral profile of orientation types did not change (N = 6).

25

Figure 7. Approaches of sharks within 30cm of the active dipole center during control and experimental trials with dorsal and ventral BUC and SOa pores blocked. There were no differences between control and blocked groups for either BUC or SOa ampullae.

26

Figure 8. Orientations of sharks during control and experimental trials with dorsal and ventral BUC and SOa pores blocked shown as a proportion of the total approaches in each group through the dipole field (~30cm). Blocking the ampullary pores with non-electrically conductive jelly significantly decreased the orientation behavior of sharks to the active dipole in the BUC group (BUC (N=7), t = 2.758, p = 0.033), and the SOa group (SOa (N=7), t = 2.855, p = 0.029).

* *

27

Figure 9. Diagram representing the orientation distance and orientation angle in relation to the dipole angle for control (left side) and BUC and SOa blocked sharks (right side). The field potential drops dramatically from the dipole center. Although electroreception tends to be used as a short range detection method, sharks demonstrated sensitivity to field intensities below 100nV.

28

Figure 10. Behavioral assay of orientations of sharks to the dipole field center. During control trials, sharks primarily demonstrated spiral behavior. Sharks with BUC and SOa pores blocked did not demonstrate spiral behavior as frequently, and demonstrated turn and spiral misses where they were unable to locate the dipole center.

29

SECTION 4

DISCUSSION

The purpose of this study was to examine the functional subunit hypothesis

(Tricas, 2001) in relation to the electrosensory system of the scalloped hammerhead

shark, Sphyrna lewini. The electrosensory system of elasmobranchs can be subdivided

into differentially innervated subgroups that have slightly differing morphology and

orientation in space. Sphyrna lewini has the characteristic cephalofoil of all

hammerheads , the expansion of which increases morphological differences between the

three-dimensional canal arrays associated with the ampullary subgroups. The functional

subunit hypothesis suggests these morphological differences may infer functional or

behavioral differences to these subgroups as well (Tricas, 2001).

Distinction of clusters

The innervation of the ampullae of Lorenzini has been a cause of confusion for

many neurophysiologists because the primary afferent nerves that connect the ampullary

sensory epithelium with the central nervous system are closely associated with

surrounding cranial nerves. The ampullary nerves are associated with the Buccal and

Ophthalmic Rami of the facial nerve (CN VII), the trigeminal nerve (CN V), the

vestibulocochlear nerve (CN VIII) and the vagus nerve (CN X) (Daniels, 1967; Norris,

1929). Although the anterior lateral line nerve (ALLN) is closely associated these nerves

and leaves the chondrocranium in close association with them, the ALLN has the distinct

function of innervating the ampullae and has its own separate ganglia (Bodznick and

Boord, 1986; Raschi, 1986; Smeets et al., 1983). The branches of the ALLN follow

30

similar tracts to other critical nerves in the cephalofoil. The pores on the cephalofoil

were recently mapped according to their association with ampullary subgroups (Rivera-

Vicente et al., 2011). The BUC and MAN groups showed very distinct pore subgroup

boundaries. However, the pores of the SOp and SOa groups slightly overlap, although

the associated ampullary subgroups subdermally are distinctly separated by the lateral

rostral cartilage,.

Information from the ampullary receptors is carried to the central nervous system

by afferent neurons of the ALLN. There are no efferent nerves present in the peripheral

electrosensory system. The primary afferents project from the ampullae to the dorsal

octavolateralis nuclei (DON) of the hindbrain (Bodznick and Schmidt, 1984). From the

DON, primary neurons carry the electrosensory information from the central zone to the

contralateral medulla before terminating in certain midbrain nuclei (Bodznick and Boord,

1986). Projections from the midbrain then continue to the diencephalon and

telencephalon (Bodznick and Northcutt, 1984). The tectum, an area of known sensory

integration, also processes electrosensory information. The somatotopic relationship of

the ampullae and their receptive fields is well mapped in the DON. Little work has been

done on higher electrosensory processing within the brain, but the tectum also appears to

conserve electrosensory somatotopy (Bodznick and Boord, 1986).

Electroorientation in Sphyrna lewini

The sharks in this study demonstrated normal hunting behavior when exposed to

an olfactory cue (squid rinse). Without the olfactory cue, the sharks did not respond to

the active dipole field, even when swimming directly over the dipole center. The sharks

31

all demonstrated high sensitivity to the weak dipole field. Although the calculated field

strength was low due to the high approach angle (almost 90 degrees) in relation to the

dipole axis of sharks for some orientations paths, the rest of the cephalofoil surface may

be stimulated by the surrounding higher field intensity. The orientation distance of the

sharks was measured as the point of the shark nearest the dipole, in order to minimize this

potential complication. The field strength decreases dramatically with increasing

distance from the dipole center, and if measurements had been taken from the midline of

the shark, the field strength data might not accurately reflect the strength detected by the

ampullary pores closest to the dipole center (Kajiura and Fitzgerald, 2009; Kalmijn,

1974). Although sharks are extremely sensitive to electric fields, elasmobranchs use

electroreception as a close range detection system. Prey further away are detected with

olfactory, auditory and visual systems before electroreception (Collin and Whitehead,

2004; Wilkens and Hofmann, 2005).

It was observed that most orientations for both experimental and control fish

occurred with an orientation angle below 40 degrees. Although orientation angle (angle

in respect to the dipole axis) did affect the direction of the orientation behavior, approach

angle (position of dipole center in relation to the shark’s swimming trajectory) did not

appear to have an effect. Due to the physical properties of a dipole field, increased

orientation angle decreases field strength (Brown, 2002). As the canals on the sharks’

cephalofoil project in 360 degrees, the location of the field in relation to the shark may

not affect the sensitivity of the shark to the field. However, the approach angle did affect

orientation type. If a shark entered a field with the right side of its cephalofoil, it would

turn right to orient to the dipole. If a shark entered a field straight on, the shark would

32

complete a straight approach or overshoot the dipole and have to backtrack to orient

correctly.

Manipulated pore field trials

A novel method to functionally inactivate ampullary groups was developed to test

for behavioral differences between subgroups. An electrically non-conductive jelly was

applied to the electrosensory pores to block ampullary receptors. The sharks continued to

swim, respond to olfactory cues and feed normally with this jelly applied to their

cephalofoil. With half of their cephalofoils covered, sharks in both the control and

blocked groups showed high sensitivity to electric fields. There was no difference in the

orientation distances between the groups.

It was also observed that although the overall frequency of orientation behaviors

was affected by decreasing pore field sensitivity, the behavioral profile did not change

between test groups with only half the cephalofoil covered (Fig. 4). The ratios of

orientation behaviors shown by the control group were similar to those shown by sharks

with ablated electrosensory pore fields. Both the controls and sharks with blocked pores

had spiraling as their most frequent behavior, followed by turning. If orientation

behavior is dependent upon ampullary cluster function, then when all the clusters lost half

their pore fields, all orientation behaviors were equally affected.

When only the BUC group was inactivated, the frequency of orientation behaviors

decreased, and the types of behavior used during orientation were different than seen in

control and half-head manipulations. With inactivated BUC pores, the sharks did not use

spiral orientations as frequently and were not as accurate in locating the dipole center.

33

The BUC group is the most distal ampullary group on the cephalofoil of the scalloped

hammerhead and the spiral orientation behavior may allow the shark to follow the

electric field lines to the dipole center (Kajiura and Holland, 2002; Rivera-Vicente et al.,

2011). With inactivated BUC pore fields, the sharks demonstrated decreased spiral

behavior. Spiraling is the most frequently used orientation type in intact sharks.

Interestingly, without the BUC group, all orientation behaviors decreased, not just

spiraling, even though the sharks experienced similar exposure to the electric field (as

demonstrated by similar approach rates). The sharks also demonstrated an increase in

orientations that were not directed towards the dipole center. As the sharks entered

within 30cm of the dipole field, they would begin an orientation path, but could not

locate the center of the dipole and the spiral or turn would land them tens of centimeters

away from the activated target.

Blocking the SOa pores showed similar results as the BUC group. With

inactivated SOa receptors, the sharks showed a relative decrease in spiral behavior, but

showed an increase in the overshoot behavior. During an overshoot, the shark makes an

initial pass over the dipole center, and then backtracks to relocate the center. The SOa

pores are located medially and rostrally on the cephalofoil, and may help in the shark’s

initial detection of the electric field directly in front of the animal (Rivera-Vicente et al.,

2011). With these pores blocked, the animal might not detect the field until it has passed

over the center and the other pore groups can detect the weak field. The sharks in this

group also demonstrated a relative increase in the proportion of turn orientations. Turns

happen when the shark approaches the dipole field on either its right or left side. As the

SOa pores are only in the medial portion of the head, the shark is still able to respond to

34

electric fields perhaps using the more distal ampullary pores such as in the BUC group.

Spiral behaviors also happen when the shark first approaches the dipole along its side, but

the spiral behaviors in the SOa-blocked group decreased. The spiral behavior may

require input from both the BUC and SOa pores for the shark to properly follow the field

lines.

Conclusions and future directions

These results provide evidence that the full electrosensory system is necessary for

proper detection and localization of simulated bioelectric fields. The BUC and SOa

ampullary pore fields are each necessary for proper orientation behavior within a

simulated bioelectric field, but other hypotheses regarding functional subunits in the

electrosensory system remain to be tested. The scalloped hammerhead shark was chosen

for this study due to the laterally elongated, dorso-ventrally flattened cephalofoil that

provides exaggerated spatial morphological differences among the ampullary canal

subgroups (Rivera-Vicente et al., 2011). A comparative study examining these properties

in more conically-shaped carcharhiniform sharks could shed light on the importance of a

three-dimensional canal array in this sensory system.

Also, since only two of the four ampullary subgroups of S. lewini have been

examined, it remains to be determined what roles the mandibular (MAN) or superficial

ophthalmic posterior (SOp) ampullary groups may play in orientation behaviors. As the

MAN cluster is located on the lower jaw, it may detect when prey is directly under the

mouth of the animal. The morphology of the scalloped hammerhead’s cephalofoil

prevents it from seeing objects directly above or below its head, so the MAN may be

35

necessary for capturing prey in the final attack. The SOp ampullary group has the

longest canals with pores on the posterior edge of the cephalofoil and may not be as

important for proper orientation to the field produced by a potential prey source. This

group may play a larger role in detecting the uniform electric fields induced by

swimming through the earth’s magnetic field. Thus the SOp may provide the shark a

means of navigating with electric fields.

It also remains to be seen what interactions may occur among these ampullary

groups in the higher processing regions of the brain. Nerve tracing studies have observed

somatotopy within the electrosensory regions of the elasmobranch hindbrain, but

functional differences among ampullary subgroups have yet to be examined at the central

processing level (Bodznick and Schmidt, 1984; Bodznick and Northcutt, 1984). The

somatotopic representation of electroreception in the elasmobranch hindbrain is relative

to the number of ampullae (and thus number of pores) in each ampullary cluster, not the

body surface area covered by the pore field. However, this study determined that

electroorientation behavior is more affected by the location of the pore field, rather than

pore number. The half-head manipulations inactivated about 1,300 pores (~50% of total

cephalofoil pore field), but the behaviors of the sharks were not altered significantly.

With the BUC (~600 pores, ~23% of cephalofoil pore field) group inactivated, the sharks

responded to the electric field less frequently. With the SOa (~1,400 pores, ~55% of

cephalofoil pore field) blocked, there was a decreasing trend in frequency, but no

significant difference. The sharks also demonstrated fewer spiral and turn behaviors,

both of which were the most frequent behaviors exhibited in unmanipulated animals.

36

Although elasmobranchs rely on senses such as sight and smell for long-range

detection of prey and the electrosensory system may play its biggest role only in close-

range detection, the ampullae are still necessary for juvenile S. lewini to accurately detect

their target. The environment of S. lewini in Hawaii requires a properly functioning

electrosensory system for survival. Each summer about 10,000 S. lewini are born in

Kaneohe Bay, but by the winter most of those pups have died from starvation (Duncan

and Holland, 2006). Being able to find prey is critical for first year hammerhead pups.

The prey the pups hunt for are often buried in small burrows in the soft bottom of the bay

and can only be detected via bioelectric fields. A properly functioning electrosensory

system can thus mean the difference between life and death for these animals. This study

examined how two pore fields are necessary for proper orientation to an electric field, but

future studies will have many questions left to answer in the elasmobranch electrosensory

system.

37

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