THE BEHAVIORAL DYNAMICS and TEMPORAL EVOLUTION of WALL- FOLLOWING BEHAVIOUR in BLIND and SIGHTED MORPHS of the SPECIES Astyanax Fasciatus

Home , Astyanax jordani, Electric fish

THE BEHAVIORAL DYNAMICS AND TEMPORAL EVOLUTION OF WALL- FOLLOWING BEHAVIOUR IN BLIND AND SIGHTED MORPHS OF THE SPECIES Astyanax fasciatus

Saurabh Sharma

A Thesis

Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

December 2008

Committee:

Sheryl L. Coombs, Advisor

Paul Moore

Robert Huber

ABSTRACT

Sheryl L. Coombs, Advisor

Mexican blind cavefish exhibit an unconditioned wall-following behavior in response to novel environments. Similar behaviors have been observed in a wide variety of animals, but the biological significance of this behavior and its evolutionary history are largely unknown. In this study, the behaviors of sighted river morphs and congenitally blind, cave-dwelling morphs of the same species, Astyanax fasciatus were videotaped during and after their initial introduction into a novel arena under dark (infrared) and visible light conditions. The swimming movements of fish in the experimental arena were tracked with an automatic image-tracking system to provide a post-hoc analysis of how the fish’s swimming speed and position (distance and orientation) with respect to the arena walls varied over time. In response to the novel environment in the dark, both sighted and blind morphs exhibited wall-following behaviors with subtle but significant differences. Blind morphs swam more nearly parallel to the wall, exhibited greater wall-following continuity and persistence and reached maximum and stable swimming speeds

(~1.5 BL/s) much more quickly than sighted morphs. In contrast, sighted morphs placed in the same novel, but well-lit environment exhibited dramatically different behaviors that consisted of either holding stationary positions near the wall for long periods of time or moving in and around the central region of the environment without moving along the walls. These results are consistent with the idea that both blind and sighted morphs have inherited primitive wall- following behaviors from their common sighted ancestor that serve an exploratory function under visually-deprived conditions. Under well-lit conditions, the proclivity of some sighted morphs to remain motionless near the wall of a novel environment suggests that near-wall iii preferences may also serve a protective function under some circumstances. It appears that wall- following behaviors of blind morphs rely more heavily on active sensing by the lateral line and have become more finely honed for exploratory purposes than those of sighted morphs. iv

DEDICATION

To my teachers and parents who have always been my guiding light. v

ACKNOWLEDGMENTS

I would like to thank my advisor, Dr. Sheryl L Coombs for her guidance and support in my research endeavors. I would also like to thank Dr. Paul Patton for his extensive technical support and advice on experimental design, Dr. Tim Bonner at Texas State University, San

Marcos, Texas for supplying the sighted Astyanax morphs, Dr. Theresa Burt De Perera and my committee members, Dr. Paul Moore and Dr. Robert Huber for their feedback on my thesis and for statistical advice. Finally, thanks to all my lab mates, especially Tristan Ula for taking care of the experimental animals for the major portion of my time as a graduate student. vi

TABLE OF CONTENTS

Page

REVIEW OF LITERATURE ...... 1

Introduction………………………………………………………………………… 1

Blind Mexican cavefish as models for studying wall-following behaviors ...... 6

The lateral line system and active hydrodynamic range ...... 9

Other non-visual sensory systems ...... 12

MATERIALS AND METHODS ...... 14

Overview of experimental design ...... 14

Experimental animals...... 15

Experimental setup...... 15

Behavioral procedures ...... 16

Data analysis ...... 17

Statistical analysis ...... 20

RESULTS ...... 22

Movement patterns, spatial distributions and body orientations of blind

sighted morphs in the dark ...... 22

Temporal development of wall-following behaviors of sighted and

blind morphs in the dark ...... 26

Behavioral results of sighted morphs in the light compared to sighted

and blind morphs in the dark ...... 29

The effects of swimming speed and fish length ...... 30

SUMMARY OF RESULTS ...... 32 vii

DISCUSSION ………...... 34

Functional significance and evolution of wall-following behaviors in

Astyanax fasciatus ...... 34

The role of active sensing by the lateral line in wall-following behaviors

of blind and sighted morphs ...... 37

Inter-individual variability within morphs ...... 41

Alternative explanations for wall-following behaviors and observed

population differences ...... 43

Switching from visual to non-visual modes of processing information ...... 44

SUMMARY AND CONCLUSIONS ...... 46

LITERATURE CITED ...... 48

APPENDIX A. TABLES AND FIGURES ...... 56 viii

LIST OF FIGURES

Figure Page

1 Distribution of the lateral line system on blind cavefish ...... 56

2 Mechanism of active flow sensing in fish ...... 57

3 Video frame record and digitized tracks of fish movements ...... 58

4 Conventions for measuring distance and orientation of the fish re: wall ...... 59

5 Swimming tracks of sighted and blind morphs in dark ...... 60

6 Frequency distributions of wall distances ...... 61

7 Frequency distributions of angular compass positions ...... 62

8 Frequency distributions of body orientations ...... 63

9 Wall distance, orientation, orientation vector strength and swimming speed

as a function of time ...... 64

10 Z statistic of angular compass distributions as a function of time ...... 65

11 Illustrations of operational criteria for wall-following behaviors ...... 66

12 Swimming tracks of two sighted individuals in the light ...... 67

13 The effects of time, morph type and light vs dark condition on key wall-following

parameters ...... 68

14 Summary of temporal effects for sighted morphs in the light and sighted and blind

morphs in the dark ...... 69

15 Temporal effects on the uniformity of angular compass positions for sighted morphs in

the light and sighted and blind morphs in the dark...... 70

LIST OF TABLES

Table Page

1 Significance levels (p values) for tests of population differences ...... 71

2 k-stat values for tests of difference between observed and hypothetical

uniform distributions of wall distances ...... 72

REVIEW OF LITERATURE

Introduction

Orientation is a critical and ubiquitous component of animal behavior that takes many different forms to function in many different behavioral and ecological contexts.

As a consequence, orienting behaviors have been classified in many different ways

(Fraenkel and Gunn 1961; Janders 1975; Schone 1984). Nevertheless, most orienting

behaviors can be grouped into general categories based on stimulus and response

duration, as well as the spatial and temporal stability of the stimulus source. During

positional orientation (Janders, 1975, Schone 1984) animals maintain a particular body

orientation with respect to a persistent and uniform stimulus such as sunlight (e.g. dorsal

light reaction, phototaxis), gravity (e.g. upright posture, geotaxis) and air or water

currents (e.g. rheotaxis). Positional orientations such as these often confer a variety of

benefits including energetic cost-savings. A second class of orienting behaviors identified

by Schone (1984) is goal orientation in which animals exhibit movement of body or body

parts towards a particular target. Schone (1984) differentiates positional and goal

orientation on the basis of whether body movements are rotational or translational.

However, goal orientation can also involve non-translational movements, such as the

transient orienting responses of the eyes, pinna, head or whole body towards (or away

from) animate sources such as predators, prey or other conspecifics. Orienting responses

in these cases are typically triggered by movement or some other form of activity (e.g.

sound production) and function in predator avoidance or the acquisition of needed

resources (e.g. food or mates). A second category of goal orientation that also involves 2 the acquisition of needed resources is object orientation (Janders, 1975), or the ability of animals to orient to (and navigate around) stationary features of the terrain (e.g. trees, rocks, hills) that have fairly stable locations over time. Spatial orientation of this type may additionally involve learning and remembering the relative locations or geometric configuration of several landmark features, as might occur for animals that maintain restricted territories and home ranges.

An unconditioned preference for following and staying near or maintaining contact (thigmotaxis) with vertically-oriented walls or boundaries of an environment is a form of object orientation that has been observed under laboratory conditions for a wide range of invertebrate and vertebrate species, including cockroaches, (Creed and Miller,

1990; Jeanson et al., 2003), fruit flies (Besson and Martin, 2004; Martin, 2004), crayfish

(Basel and Sandeman, 2000), rodents (Barnett, 1963; Treit and Fundytus, 1989; Simon et al., 1994, Wolfer et al., 1998)), blind humans (Kallai et al., 2005; Kallai et al., 2007) and blind cavefish (Breder and Gresser, 1941b; Gertychowa, 1970; Teyke, 1985, 1989;

Abdel-Latif et al., 1990; Burt de Perera, in press).

Wall-following behaviors are often, but not always associated with species that are active under visually deprived conditions and/or circumstances that limit or prevent the use of vision. In the absence of vision and other long-range senses (e.g. echolocation), animals cannot use distal cues from a single vantage point to direct their future movements, but rather must move about in the environment to bring their short- range senses within close range of landmark cues. For example, rodents rely on active movements of the head and large whiskers (macrovibrissae) to sense wall surfaces

(Fanselow and Nicolelis, 1999; Mitchinson et al., 2007), whereas crayfish utilize their

3 rostral antennae (Patullo and MacMillan 2006). Likewise, fish have a ‘touch-at a distance’, hydrodynamic sense known as the lateral line for sensing the wall’s presence.

For these short-range senses, knowledge of the spatial configuration of the environment can only be achieved through sequential samples of the environmental space and by temporally linking information from one sample at one moment in time to that of another sample at the next moment in time. In this context, it is easy to imagine how orienting behaviors might be preferentially guided and entrained by continuous (e.g. walls) or closely-spaced features vs. those that are discontinuous and widely spaced.

A common feature of many wall-following behaviors is that animals exhibit them when first introduced into a novel environment (Weissert and Campenhausen 1981;

Teyke 1985, 1989; Wolfer et al.1998; Basil and Sandemann 2000). As part of an overall response to novel and unknown circumstances, wall-following behaviors could serve a protective purpose for finding shelter and/or escape routes should threats arise. Consistent with a protective function are experimental findings indicating that wall-following behaviors are linked to anxiety in rodents (Treit and Fundytus, 1989; Simon et al., 1994,

Valle 1970) and humans (Kallai et al., 2007). In these studies, anti-anxiety drugs reduced the proclivity of animals to stay near the walls and increased the amount of time spent in the center of the test arena. For this reason, wall-seeking behaviors in these studies have been described as ‘centrophobic’ behaviors, but they might just as aptly have been described as protection-seeking (or anxiety-relieving) behaviors.

Wall-following behaviors might also function as an overall exploratory strategy for discovering and locating potential resources in a restricted environment and also for building spatial knowledge of their relative locations. For example, it has been suggested

by Kallai et al (2007) that blind humans use wall-following behaviors to build spatial

knowledge of the size and shape of the perimeter, which can then be used as an external

frame of reference or “home base” for mapping out the spatial relationships of objects in

the remaining interior space. Indeed, Hill et al (1993) report that blind humans link

objects in the interior space to the perimeter of the environment as a successful strategy

for finding and navigating around objects in a novel environment. Positional orientation

to global stimuli and short-range orienting responses to moving biotic sources typically

rely on an egocentric frame of reference (i.e. knowing the location or orientation of the

stimulus in relation to a set of body-centered coordinates). Object orientation is also likely to involve egocentric (internal) frames of reference when novel objects are initially encountered, but over time, spatial relationships between objects could be learned and mapped with respect to allocentric (external) frames of reference. Although it has been hypothesized that wall-following behaviors could represent a non-visual, exploratory strategy for developing allocentric representations of space over time, this hypothesis has not yet been experimentally tested.

Finally, it is possible that wall-following behaviors have few, if any, biologically relevant functions and are, instead, merely an artifact of artificial testing arenas, which in

many studies are small, concave and devoid of features other than the walls. Creed and

Miller (1990) used the behavior of a computer simulated agent to show that wall- following behavior in a concave environment could result from the agent following a

simple behavioral rule - move in a straight line until a barrier is encountered, then turn,

just enough to avoid the wall. They further demonstrated that animals (cockroaches P. americana), formerly believed to exhibit strong wall-following (thigmotactic) behaviors

5 actually departed from the wall surface when they reached the convex portion of an hourglass arena, as one would predict from the behavioral rule when forward movement is no longer impeded. The frequency of departures depended on the radius of curvature, suggesting that the ability of cockroaches to follow the wall around a concave surface is limited, perhaps due to mechanical or sensory constraints.

In summary, wall-following behaviors represent an unconditioned proclivity of a wide variety of animals to stay near the perimeter of a novel, bounded space. The true function or functions of this behavior are unknown for any species, but several possibilities have been suggested, including a protective function for securing shelters and/or escape routes and an exploratory strategy for finding resources and/or building spatial knowledge of restricted spaces using an allocentric frame of reference. Not yet ruled out is the possibility that this behavior is, at least in part, an incidental consequence of the concave shape and barrenness of artificial testing arenas together with a simple obstacle avoidance strategy. The sensory basis of wall-following behaviors is thought to revolve around short-range, non-visual senses, such as touch in terrestrial animals and in addition, hydrodynamic (lateral line) senses in aquatic animals. A possible role of other non-visual senses (e.g. chemosenses) has rarely, if ever, been examined. Although it is often assumed that wall-following behaviors figure prominently in the lives of animals without vision or restricted use of vision, the prevalence of this behavior in sighted animals is unknown. Likewise, the evolution of wall-following behaviors in response to the loss of vision has seldom been studied.

Before critical questions and informational gaps like these can be addressed in any satisfactory way, a careful description of the behavior under controlled conditions is

needed. Despite the wide variety of animals for which this kind of behavior has been

observed and reported, there has been surprisingly little in the way of systematic and

quantitative descriptions of the behavior that include direct measurements of e.g.

sequential distances from and orientations to the wall, direction of travel along the wall

and the degree to which the wall is continuously ‘followed’. Thus, operational criteria for defining the behavior have seldom been adopted or applied. The goals of this study are to provide a careful description of the short-term development of wall-following behaviors of the blind cavefish (an eyeless morph of the species Astyanax fasciatus) and to determine how or if the time course and nature of wall-following behaviors in blind morphs differ from those exhibited by their nearest sighted relative (sighted morphs of the same species) under both light and dark conditions.

Mexican blind cavefish as models for studying wall-following behaviors

Mexican blind cavefish are ideal animals for this study for several reasons,

including (1) their small size (<~5 cm), which makes it easy to video track their

movements in the lab over distances equivalent to tens of body lengths, (2) the fact that their spatial abilities are founded primarily or exclusively on short-range, non-visual systems, like touch and the lateral line ‘touch-at-a-distance’ system and (3) the existence of both an eyed, surface form and an eyeless, cave-dwelling morph in the same species, making direct comparisons of wall-following behaviors possible for fish that have evolved with and without vision. The following sections review what is known about the basic ecology, behavior and sensory capabilities of this species, including how the lateral line senses stationary objects like walls.

The blind cavefish was first described by Hubbs and Innes (1936) from a cave

known as La Cueva Chica, in the state of San Luis Potosi, East Central Mexico, a region

abundant in sink holes and caves (Mitchell et al., 1977). Two basic morphotypes within

Astyanax have been described: a pigmented, surface-dwelling and eyed form called the

Mexicana tetra, and an unpigmented, eyeless morph known as the Mexican blind cavefish. (Jeffrey, 2001). Morphs with intermediate characteristics also appear to be correlated with a continuum of habitats ranging from the cave interior where no light penetrates to the water inlet of the cave where light enters (Breder, 1942; 1943). Earlier studies using cytogenetic techniques to measure allozymic variation have indicated that both morphs have sufficient genetic similarity to be classified as the same species (Avise and Selander, 1972). However, the taxonomy of this genus is still debated and currently unresolved, with some taxonomists believing that sighted and blind morphs belong to a single species, Astyanax fasciatus, ranging from Texas to Central America and others claiming that there are three different species (A. fasciatus in Central America, A. mexicanus in Texas and northern Mexico and A. aeneus in southern Mexico), with two of these (A. mexicanus and A. fasciatus) independently giving rise to separate lineages of blind cavefish (Jeffery et al., 2001).

Blind and sighted Astyanax morphs show substantial differences in their morphology. Most notably, blind cavefish have a complete loss of pigmentation

(Parzefall, 1983) and degenerated eyes with only heavily pigmented choroids remaining at the base of the eye socket, which has been covered over by skin (Breder, 1943). Blind cavefish also have an increased number of external taste buds on the lower jaw

(Schemmel, 1967) and well developed lateral line specializations (see following section

8 on the lateral line). There are also differences in head and body shape – with sighted morphs having a more streamlined, torpedo shape compared to blind morphs, which have deeper bodies and wider and more rounded heads.

A number of behavioral differences have also been reported for the two morphs, including differences in feeding and social behaviors. For example, Parzefall (1993) reports different levels of agonistic behaviors in sighted and blind morphs, with sighted morphs, but not blind morphs exhibiting agonistic behaviors towards conspecifics in crowded conditions or when food availability is low. Cave populations also exhibit negative phototaxis (Breder 1943, Breder and Rasquin, 1947; Gertychowa 1970), whereas sighted morphs exhibit positive phototaxis (Breder and Rasquin, 1947).

Although blind morphs lack retinal photoreceptors, they do have extra-ocular photoreceptors in the pineal gland (Yoshizawa and Jeffery, 2008) which presumably mediate these gross, phototactic reactions.

A number of studies have demonstrated light-dependent behavioral differences between sighted and blind morphs. For example, sighted morphs shoal in the presence of light and disperse in its absence, whereas blind morphs do not shoal at all (Breder 1943;

John 1964; Parzefall, 1983; Romero and Paulson, 2001; Gregson and Burt de Perera

(2007). Likewise, sighted morphs are surface feeders in the presence of light (Parzefall,

1983), but reportedly switch to benthic feeding habits when deprived of light (Jeffrey,

2005). Blind morphs, which are benthic feeders for most of the time, appear to have both behavioral and sensory specializations for benthic feeding. Whereas sighted fish orient their bodies nearly perpendicular to the substrate when feeding in the dark, blind morphs form a much lower angle of incidence with the substrate (~450), presumably to optimize

sensory use of the lower jaw, where there are a plethora of taste buds (Schemmel, 1980).

These light-dependent differences suggest that sighted morphs, when deprived of vision,

may have many of the same behavioral capacities and tendencies as blind morphs – a

claim that Breder (1943) has made, but with little experimental evidence to support it.

A number of recent studies have shown that blind morphs respond to novel

environments by increasing their swimming speeds (Teyke 1985, Burt de Perera, 2004)

and exhibiting a tendency to preferentially swim close to the outer boundary of the

environment (Teyke 1985, Abdel-Latif et al., 1990). Both the wall-following preference

(measured as proportion of time spent near the wall vs. in the center of the tank) and

swimming speed gradually decline over approximately the same time period of up to 15

hours (Abdel-Latif et al., 1990; Teyke 1988; Hassan et al., 1992), presumably as fish

become more familiar with the environment. For animals like the Mexican blind cavefish,

confined to relatively small cave pools and streams in total darkness, the boundaries of an environment may be among its most salient spatial features. Experiments with blind cavefish have also revealed that despite the absence of vision, they can avoid stationary

obstacles (Gertychowa 1970, Johns, Teyke, 1985), discriminate between stationary

objects with different spatial characteristics (Weissert and Campenhausen, 1981), and

detect novel changes in landmark configurations to a familiar environment (Burt de

Perera, 2004).

The lateral line system and active hydrodynamic imaging

Blind cavefish are thought to rely heavily on their non-visual senses and in particular, the

lateral line system for sensing stationary objects like walls (Coombs and Montgomery,

1999). The lateral line system is a spatially distributed system of many different flow

sensors, found in all cartilaginous and, bony fish, as well as aquatic amphibians. The

system is composed of two types of flow sensors or neuromasts: superficial neuromasts

distributed along the skin surface and canal neuromasts, which reside in fluid filled canals

just below the skin surface. Canals are open to the outside environment by a series of

pores (two surrounding each neuromast) and are distributed in five locations on the head

and body: supraorbital and infraorbital canals that run above and below the eyes, the

preopercular canal down the cheek, the mandibular canal on the lower jaw, and the trunk

canal along the main body. Each neuromast consists of a patch of mechanoreceptive,

directionally-sensitive hair cells along with surrounding supporting cells that secrete a

gelatinous covering or cupula (Montgomery et al., 2001). Whereas superficial

neuromasts tend to respond best to flow velocity and low-frequency (<~20 Hz) water

motions (steady currents), canal neuromasts respond best to flow acceleration and higher-

frequency water motions (Montgomery et al., 2001). At the apical surface of each hair

cell is a ciliary bundle, which, when deflected, opens the stretch sensitive ion channels

thereby giving rise to either a hyperpolarizing or depolarizing change in the membrane

potential, depending on the direction of deflection, and either an increase or decrease in

the firing rate of primary afferent fibers carrying information from the hair cells to the

brain. The ciliary bundles from all of the hair cells on a given neuromast are embedded in

a gelatinous cupula, which couples the motions of the surrounding water to the underlying hair cells. The rate of firing of the primary afferent fibers is generally

assumed to be directly proportional to the displacement of the cupula (Montgomery et al.,

2001).

In order to understand how stationary objects like walls can be detected by the

lateral line system of fish, one needs to appreciate that the lateral line system of fish can

be used in both active and passive ways. Passive sensing involves the detection of

exogenously-generated water motions, ranging from minute currents generated by

planktonic prey to large-scale ambient water motions in e.g. a stream or river. During

active sensing, fish probe the environment with their own, self-generated hydrodynamic

signal. This active sensing strategy is analogous to echolocation by bats and dolphins,

and electrolocation by weakly electric fish in that animals use self-generated signals to

probe features in the environment (Nelson and MacIver, 2006).

In the case of active sensing by the lateral line, fish generate a flow field around

their body as they swim (Fig. 2A). The body of a swimming fish acts like a dipole to

displace the water in front of the advancing fish. As the fish swims alongside a stationary

object, the flow field along the skin surface of the fish is altered (Fig. 2B), thus providing

lateral line information about the presence of the obstacle (Hassan, 1985).

Although few differences in the number and distribution of superficial and canal

neuromasts between blind and sighted morphs have been reported (Schemmel, 1967;

1973), there are significant differences in the size of superficial neuromasts and their

overlying cupulae (Teyke, 1990). Superficial neuromasts are larger and their cupulae are longer (300 μm) in blind morphs compared to sighted morphs (50 μm). The longer cupula are thought to increase the sensitivity of the lateral line neuromasts, as they are more likely to project outside the boundary layer of the swimming fish and into the free stream region of maximum flow velocity (Teyke 1990). The boundary layer is a region of space between a fluid and solid surface, where flow velocity decreases as it approaches the

surface, in this case the skin surface of the fish. The longer cupula also provides more

surface area for viscous (frictional) forces to deflect the cupula.

Other non-visual sensory systems

Blind cavefish might also utilize other non-visual sensory systems besides the lateral line system to follow walls and explore novel environments. The use of tactile sense has been documented by Baker and Montgomery (1999) in the context of rheotactic behavior by blind cave fish. They reported that fish made tactile contacts with the substrate once every five seconds using either a fin or part of their head. John (1957) also reported frequent contact that these morphs make with the walls of an experimental tank.

Furthermore, Windsor (PhD thesis) recorded video images of blind cavefish periodically

contacting the wall surface with their extended pectoral fins as they swim past a wall

surface. Blind cavefish also have hearing specializations (e.g. bony connections from

the swim bladder to the ear,) that render them sensitive to the pressure rather than kinetic

component of sound. As a result, they have better sound pressure sensitivity over a wider

range of frequencies than most non-otophysan fishes (Popper 1970). However, best

sensitivities and frequency ranges of hearing of blind and sighted morphs are

indistinguishable (Popper 1970) and characteristic of an entire super order (Ostariophysi)

of fishes with very similar, if not identical specializations. Although it is conceivable that

blind cavefish could use a form of echolocation for sensing stationary objects, as reported

for marine catfish (Tavolga 1976), sound production by blind cavefish has never been documented. Nevertheless, the ability of the pressure-sensitive ears of blind cavefish to detect the ‘damming pressure’ created at the front of a fish as it advances towards a wall cannot yet be ruled out.

The possible role of chemical cues in wall-following and exploratory behaviors has not yet been studied in blind cavefish. However, exploration of novel environments has been investigated in another cave-dwelling fish, the East African cave cyprinid

Phreatichthys andruzzii (Paglianti et al., 2006). Experiments performed under low- intensity, diffuse light showed that when given a choice between unfamiliar areas with unfamiliar odors vs. an unfamiliar area with self-odors, fish always ventured into the areas without self-odor. This study suggests that odors may provide important cues about whether an environment is novel or familiar and thus, contribute to the decision of whether or not to explore a novel environment.

MATERIALS AND METHODS

Overview of Experimental Design

The behaviors of blind and sighted (Astyanax fasciatus) morphs were videotaped

during and after their initial introduction into a novel arena under dark (infrared) and

visible light conditions. To eliminate chemosensory ‘novelty’ cues and to minimize stress

factors associated with differences between water quality in home and experimental tanks

(e.g. water temperature, conductivity, pH etc), the novel test arena was filled with water

from the fish’s home tank. Since blind cavefish exhibit negative phototaxis (Breder 1943)

and may be capable of optokinetic behaviors to visual stripes on a rotating drum (Teyke,

1994), presumably using extra-retinal (pineal) photoreceptors, all testing for ‘dark’

conditions was done under IR illumination in a light-tight enclosure. Substrate borne

vibrations (e.g. those arising from some one walking across the room) were minimized by

conducting tests on a vibration-isolation table.

The swimming movements of fish in the experimental arena were tracked with an

automatic image-tracking system to provide a post-hoc analysis of how the fish’s

swimming speed and position (distance and orientation) with respect to the arena walls varied over time. Continuous wall-following bouts, defined by distance and orientation criteria, were subsequently characterized in terms of direction (clockwise or counterclockwise) and distance traveled along the wall. Various performance metrics were compared between blind and sighted morphs to determine the extent to which the

15 time-course and nature of wall-following behaviors in blind morphs resemble those in sighted morphs and by inference, their common sighted ancestor.

Experimental Animals

Naïve blind cavefish (ranging in standard length (SL) between 3-6 cm) and sighted morphs (SL = 4-6 cm) of Astyanax fasciatus were used for all experiments. Blind morphs are obtained from commercial aquarium suppliers, whereas sighted morphs were provided by Dr. Timothy Bonner at Texas State University, San Marcos, Texas. Fish were maintained in 75.8 liters aquaria at 20-25 degrees Celsius and 12:12 light/dark cycles. Protocols for the maintenance care and experimental use of fish followed the guidelines for the Care and Use of Laboratory Animals and have been approved by

Bowling Green State University Institutional Animal Care and Use Committee.

Experimental Setup

Fish were tested in a circular arena (30 cm or ~ 6 body lengths in diameter) (Fig.

3A) housed inside a larger rectangular tank resting on top of a vibration-isolation table. A video camera (Sony Handicam DCR-HC 42) mounted ~1 m above the testing arena recorded the swimming behavior of fish. The entire set-up was enclosed in a light-tight enclosure made with thick black curtains to prevent any visible light from entering the testing arena. A 115 x 77 cm matrix of infra red LED diodes (~10 Amp, 20 volts) provided an upwelling source of illumination for the dark conditions of these experiments, whereas two, 150 W (Halco PureLite) incandescent neodymium bulbs provided an upwelling source of broad spectrum visible light for the light conditions. To minimize depth of field errors due to vertical excursions of the fish, water depth in the experimental arena was kept shallow (5 cm).

Behavioral Procedures

Fish were transferred from their home tank to a transport bucket and the

experimental arena using a plastic-lined, water-filled net to prevent any mechanical

damage to superficial neuromasts of the lateral line system. The circular testing arena was

temporarily removed from the outer square tank and fish were introduced into the square

tank where they were corralled by a small holding ring (~ 15 cm in diameter). The

circular testing arena was then placed around the holding ring, and fish were given 5

minutes to acclimate and recover from the transfer process. After 5 minutes of

acclimation, the holding ring was slowly removed to minimize water motions and fish

were released into the experimental arena where their swimming behavior was videotaped for 10 - 20 minutes. In an additional set of experiments on sighted individuals, the 5 minute period of acclimation was extended to 1 hour to give individuals more time

to adapt to the dark conditions before testing began and to control for the possibility that

behavioral responses were due to an abrupt loss of vision rather than exposure to a novel

environment. One hour was based on the typical time (~ 40 – 60 minutes) that it takes for

the vertebrate retina to recover light sensitivity when background lights are abruptly

turned off (Dowling 1967).

Data Analysis

Video Capture Software (Winnow, Version 3.2.4185) was used for online viewing and capturing of digital images of the swimming behavior of the fish at the rate of 5 frames per second (Fig. 3A). The frame-to-frame position of the fish was

subsequently tracked using Image Pro Software (Version 6.0 Media Cybernetics) (Fig.

3B). The software has an automatic image detection and tracking feature that first fits an

ellipse to the outline of the fish’s main body (from ~the tip of the snout to the base of the

caudal peduncle) and then determines the centroid of the ellipse. For each video frame,

the software determines (a) the position of the centroid in Cartesian co-ordinates relative

to a fixed reference point on the video screen and (b) the major and minor axis of the ellipse. Custom MATLAB subroutines subsequently compute the ellipse’s orientation to and distance from the walls of the arena, as well as distance moved from one frame to the

next in order to derive a characteristic swimming velocity for each frame pair.

Distance to the wall was computed in three different ways as the shortest distance

from (1) the surface of the ellipse to the wall (the surface distance), (2) the centroid of the

ellipse to the wall (the centroid distance) and (3) the head end of the ellipse (defined by

the direction of motion) to the wall (the snout distance) (Fig. 4A). Orientation to the wall

was defined by the direction in which the snout was pointing with respect to the wall

(Fig. 4 B). By convention, angles of 0 and 1800 represent cases in which the long axis of the fish was orthogonal to the wall surface and the fish was heading directly towards and away from the wall, respectively. In contrast, 90 and 2700 represent cases in which the

fish’s body was parallel to the wall and the fish was moving in a clockwise (90) or

counterclockwise (270) direction around the circular arena.

In order to determine if fish exhibited preferred wall orientations, a modified version of the vector strength of a circular distribution (Batschelet 1981), which varies from 0 (fish headings uniformly distributed across 3600) to 1 (all fish headings the same), was computed. Rather than computing the vector strength from a full 360 degrees worth of data, however, clockwise (0 – 1800) and counterclockwise (180 – 3600) directions of movement along the wall were collapsed into a single 1800 distribution. This modification preserves the relative orientation of the fish’s body with respect to the wall while removing direction of travel differences due to CW vs. CCW movements around the rim.

To determine if fish exhibited regional preferences or evidence of non-uniform travel around the perimeter of the tank, the angular position of the fish with respect to the tank perimeter was measured using a compass coordinate system (i.e. with respect to an arbitrary north (00), south (1800), east (2700) or west (900) side of the tank) for cases in which the distance to the wall was <0.5 BL. The vector strength of the full 3600 circular distribution of angular positions was also computed (Batschelet 1981).

Potential sources of measurement error include pixel resolution, which was measured to be < 1 mm, and the goodness of fit between the ellipse and the fish’s body.

Because fish body shapes deviate from that of a pure ellipse, ellipse-based estimates of surface distances are likely to be the least reliable; thus, unless otherwise specified, centroid distances are the primary distances reported in this study. Errors in estimating centroid distances depend largely on the bending angle of the fish. When the fish’s body is straight, the ellipse fitting procedure gives a reliable estimate of the centroid position on the fish. If the fish is involved in a wide-angle turn and the bending angle is large, the

fit is such that the centroid of the ellipse is shifted a few mm relative to the true centroid

of the fish. However, measurement errors due to large bending angles are assumed to be infrequent, small and randomly distributed over time and between populations.

Three additional indices (the wall-preference index, the bout length index and the

long-bout index) were used to characterize the strength and nature of wall-following

behaviors. The wall-preference-index (the fraction of video frames in which fish-to-wall

distances were less than 0.5 body length) was used to determine the degree to which fish

were near the wall vs. other areas of the tank. The bout length index provided a measure

of the degree to which fish continuously maintained a given orientation and distance with

respect to the wall. For this index, a single bout of continuous wall-following behavior

was defined by three operational criteria. Fish had to maintain (1) a distance of less than

0.5 body lengths away from the wall, (2) a body orientation +/- 300 of parallel to the wall

circumference and (3) a minimum travel distance of 2 body lengths along the wall

surface. The total distance traveled during consecutive wall-following bouts was then

divided by the total path length (distance traveled near or away from the wall during a

specified time interval) to provide a bout-length index of continuous wall-following

strength for one- or 10-minute time intervals. Because high bout-length indices could

result from many, closely spaced short bouts or a few long bouts, an additional metric,

the long-bout index, was created to distinguish between these two possibilities. This

index was simply the percent contribution of long bouts (those greater in length than 10

BL) to the total distance traveled.

Statistical Analysis

One- and two-tailed, two-sample ‘t’ tests (assuming unequal variances) were

used to determine if various behavioral parameters differed between blind and sighted

morphs during any 10 minute time interval. The significance level for each of 30 pair- wise comparisons (Table 1) was adjusted to 0.0017, using the Bonferroni correction, to

yield an experiment-wide significance level of 0.05. In cases where parameters were

distributed non-normally (e.g. distance to the wall), Kolmogorov-Smirnov tests were applied to determine if distributions differed from that expected by chance (one-sample test) or between blind and sighted populations (two-sample test).

Angular data (in degrees) were converted to radians and arc tan transformed to compute mean values for any given individual and time interval. Wall-preference and bout-length indices, which varied from 0 to 1, were arc sine transformed before statistical tests were performed. Watson-Williams tests, rather than T-tests, were used on angular data for tests of significant differences between populations. A modified Rayleigh (V) test (Greenwood and Durand 1955) was used to statistically discriminate between uniform (random) and non-uniform distributions of angular metrics centered on a particular direction. For this test, a statistical criterion, the Z stat, was computed as r2 N,

where r = vector strength of the circular distribution and N is the sample size (Batschelet

1981)

Linear regression was used to determine (1) the effect of time on various

dependent variables and (2) the relationship between two dependent variables (e.g. wall-

following index and swimming speed). Regression statistics were based on 1-min means

across all individuals in a given population or treatment group for the former case and in

21 the latter case, on individual means over the first and/or second 10-minute period of the trial.

RESULTS

Individuals from both blind (N=8) and sighted (N=8) populations exhibited evidence of wall-entrainment behaviors when placed in a novel, dark environment, meaning that fish were observed moving near the wall in more video frames than elsewhere in the tank.

However, the time-course and nature of wall-entrainment behaviors differed between the populations. In contrast, sighted individuals provided with light and thus access to long- range visual cues (N = 4), while sometimes found near the wall, exhibited little, if any evidence of wall-entrainment behaviors. Results from blind and sighted morphs under dark conditions are compared in 2 sections which focus on (1) movement patterns, spatial distributions and body orientations within the novel arena and (2) the temporal development and strength of wall-following behaviors. In each of these sections, detailed results from two individuals, one sighted (SF 5) and one blind (BC 30) are highlighted as examples that bracket the range of variability observed across the two populations in the dark. A third section presents results from sighted morphs under visible light as compared to results from sighted and blind morphs in the dark. The last section examines the effects of swimming speed and fish length on wall-following behaviors.

Movement Patterns, Spatial Distributions and Body Orientations of Blind and

Sighted Morphs in the Dark

All blind individuals showed evidence of repeated and continuous travel around

the perimeter of the tank during both the first (Fig. 5B) and second (Fig. 5D) ten minutes of the trial period, as illustrated by BC 30. By comparison, many, but not all sighted fish,

23 spent less time near the wall during the first 10 minutes (Fig. 5A), but more time during the second ten minutes (Fig. 5C), as illustrated by SF 5. As a result, the wall-preference index for SF 5 increased from 0.25 to 0.8 from the first to second ten minutes. In contrast, the wall preference index of BC 30 was similarly high (0.85 and 0.93) for both halves of the test session. At the population level, wall-preference indices were almost (one-tailed t-test), but not quite significantly greater for blind compared to sighted morphs for the first, but not second ten minutes (Table 1).

A fish with no preference for the wall or any other area of the tank should exhibit a uniform distribution of locations throughout the tank. However, the frequency distribution of distances from the wall from such a distribution (solid black line in Fig 6) will vary according to the area encompassed by any given bin width, meaning that expected frequencies should be higher around the perimeter of the tank than near the center. Distance distributions among individuals varied from those that differed very little from the expected uniform distribution (e.g. SF 5, blue line with symbols) to those that differed substantially (e.g. BC 30, red line with symbols) (Fig. 6). Measured distributions for all blind and sighted individuals for both the first and second 10 minutes showed significantly greater wall preference than expected from a uniform distribution (one- sample, KS test, p < 0.001). However, it is worth keeping in mind that for samples sizes this large (N = 3000), almost any difference will yield a statistically significant result, no matter how small the difference. The magnitude of the difference is indicated by the KS statistic (KS stat), which was generally smaller for sighted than blind individuals during the first 10 minutes (Table 2). For example, this value, which ranges from 0 (samples are

24 identical) to 1 (maximally different), was 0.04 for SF 5 compared to 0.62 for BC 30

(Table 2, first column).

To determine if blind or sighted morphs exhibited evidence of non-uniform travel around the perimeter of the tank, the distributions of angular compass positions with respect to an arbitrary north (00), south (1800), west (2700) or east (900) side of the tank perimeter were plotted for all individuals (Fig. 7). As would be expected from the pathways depicted in Fig. 5A, SF 5’s positions were distributed non-randomly (vector strength = 0.6) and were more frequently clustered in the southeast region of the tank

(mean angle ~ 1230) during the first 10 minutes (Fig. 7A, blue line with symbols). By contrast, BC 30 positions were more uniformly distributed (r = 0.06) around the perimeter (Fig. 7A, red line with symbols). Several sighted individuals (blue lines) exhibited large deviations from uniform distributions around the perimeter, whereas the majority of blind morphs (red lines) showed very small deviations from a uniform during the first ten minutes. Nevertheless, distributions were significantly different from uniform

(Rayleigh test of uniformity, p <0.001) for all but two individuals – one sighted and one blind. Distributions were likewise significantly different from uniform during the second half of the trial at p < 0.001 for all but one blind individual.

As the circular motions of SF 5 towards and away from the wall suggest (Fig.5A), the orientation of this individual’s body with respect to the wall was more variable during the first 10 minutes than the second ten minutes when a greater percentage of movement tracks were along the wall. As a consequence, the distribution of body orientations were more nearly uniform during the first 10 minutes (Fig. 8A, blue line) than the last (Fig. 8B, blue line), as confirmed by the corresponding vector strengths of the orientation

distribution (0.79 and 0.88, respectively). In contrast, the bimodal distributions of BC 30

(Fig. 8, A, B, red line) reveal that this fish was swimming nearly parallel to the wall in

either clockwise (900) or counterclockwise (2700) directions. Corresponding vector

strengths of the orientation distributions were 0.93 and 0.97, respectively, for first and

second halves of the test session.

Although the orientation vector strengths of SF 5 appears to increase with time

towards levels exhibited by the blind morph, the modes of SF5’s angular distribution

appear to be slightly displaced inwards towards the wall relative to those of BC 30 (Fig.

8B red vs. blue line). These results are consistent with observations of videotaped

behaviors of many sighted and blind individuals near the wall. That is, sighted morphs angled their snout towards the wall as they approached it, often appearing to touch the

wall with the side of their snout. Snout or near-snout contacts like these were often made

in sweeping movements over short distances, alternating repeatedly from clockwise to

counterclockwise directions. In contrast, blind morphs tended to swim more nearly

parallel to the wall, maintaining on average some minimum distance between the wall

and their body surface, while traveling for much longer distances along the wall without

changing directions. To assess these potential differences in behaviors, angular

orientations to the wall and the length of continuous travel along the wall were analyzed

further under near-wall conditions (fish <0.5 BL away from the wall).

When near-wall orientations of CW and CCW directions are combined into a

single 1800 distribution for different individuals of both blind (red) and sighted (blue) populations, a clear picture of consistent differences between the two populations emerges during the second half of the session (Fig. 8D). That is, the modes of the sighted

morph distributions are displaced by about 100 towards the wall relative to those of the

blind morph distributions. A second, slightly different measure, based on the difference

between centroid and snout distances, was used to confirm this preference for a snout-

towards-the-wall orientation. The mean difference should be near zero if fish are on

average oriented parallel to the wall, some positive value if fish are angled more

frequently towards the wall and some negative value if fish are angled more frequently

away from the wall. This metric (centroid minus snout distance) yielded nearly

significant differences in the hypothesized direction between blind and sighted morphs

for both first (one sample t-test, p=0.003) and second (one sample p = 0.0025) 10-minute

test periods.

Temporal Development of Wall-Following Behaviors of Sighted and Blind Morphs

in the Dark

To further assess the temporal development of wall-following behaviors, wall

proximity and other parameters were plotted on a minute-by-minute basis (Figs. 9 A,B,C,

D ). In terms of distance to the wall (Fig. 9A), the results of BC 30 (red line with

symbols) are similar to the majority of blind morphs (red lines without symbols) in that

the median distance to the wall stays consistently low, generally < 0.5 body lengths

(BL), during the entire 20 minutes. Similarly, mean orientation to the wall (Fig. 9B) and

corresponding vector strength of the orientation distribution (Fig. 9C), as well as

swimming speed (Fig. 9D) remained relatively constant over time with perhaps a slight

decrease in swimming speed. In contrast, SF 5’s results (blue line with symbols) are

typical of many, but not all sighted fish (blue lines without symbols), in that it took a much longer time for wall distances to drop below 0.5 BL (Fig. 9A). Following a similar

time course of change, the mean orientation angle decreased (Fig 9B), the orientation

vector strength increased (Fig. 9C) and the mean swimming speed increased (Fig. 9D) for

SF 5, as well as for many, but not all sighted individuals. Finally, Z stat values of angular

compass positions over 1 min intervals (Fig. 10) illustrate that the majority of blind

morphs (red lines), including BC 30 (red lines with symbols) are moving uniformly or nearly uniformly with respect to the perimeter of the tank (Z stat < ~ 6.9, dashed line in

Fig. 10) more frequently than sighted morphs (blue lines), including SF5 (blue line with symbols).

Fig. 11 illustrates the temporal structure of wall following behavior by co-plotting orientation (left-hand Y-axis) and distance (right-hand Y-axis) as a function of time (5 samples/sec) for the sighted (SF 5) (Fig. 11A) and blind (BC 30)(Fig. 11B) individuals used as previous examples. Consistent with pathway plots (Fig. 5A), it can be seen that the orientation and distance of SF 5 with respect to the wall varied dramatically with time during the first 10 minutes (Fig. 11A). By comparison, the blind morph, BC 30, exhibited long periods of wall-following behavior during which both wall distances and orientations remained relatively stable to meet the operational criteria illustrated by the solid (wall distance < 0.5 BL) and dashed lines (wall orientation +/- 300 of parallel) in

Fig. 11B. Each period of time during which both criteria are simultaneously met is

defined as a single, continuous wall-following bout. The bout-length index is the

cumulative length of travel during sequential wall-following bouts divided by the total

distance traveled during a specified time interval (see methods).

As the data from SF 5 illustrate, the 1-minute bout-length indices for this sighted

individual increased from < 0.2 to > 0.6 from the first to second half of the 20 minute

period (blue line with symbols, Fig. 11C), whereas the indices remained equally high

(>~0.7) for BC 30 throughout most of the 20 minute period (red line with symbols, Fig.

11 C).

It should be noted that a robust bout-length index can result from many, closely- spaced short bouts or a few long bouts. Indeed, SF 5, like all sighted individuals, had very few long bouts compared to BC 30 (Fig. 11A, B), despite the fact that bout length indices

for both individuals reached fairly high levels by the end of the session (Fig. 11C). To

determine if populations differed in this respect, two additional parameters were

measured for each individual. One was the maximum bout length and the other was the

long-bout index, defined as the fractional contribution of bouts greater in length than 10

BL to the total distance traveled during the first or second half of the session. Maximum

bout lengths ranged from 3 to 40 BL (14 – 222 cm) in sighted morphs and from 13 – 203

BL (43 – 790 cm) in blind morphs and were significantly greater for blind than sighted

morphs for the first 10-minute time period (Table 1, first column). Long-bout indices were likewise significantly greater for blind morphs during the first 10 minutes but not second ten minutes of the test session (Table 1, first column).

To determine if the temporal development of wall-following behaviors might be altered if sighted morphs were given longer periods of time to adapt to the dark, data were collected from 4 sighted individuals after pre-adapting them to the dark for 1 hour

(the time normally required for dark adaptation by the vertebrate retina). Results from these tests revealed no significant differences between long-term (1 hour) and short-term

(5 min) dark adaptation treatments in any of the reported parameters during either the first or second ten minute periods (Table 1, 2nd column).

Behavioral Results of Sighted Morphs in the Light Compared to Sighted and Blind

Morphs in the Dark

When placed into the same novel environment under well-lit conditions, naive sighted morphs exhibited movement patterns and levels of activity that differed dramatically from those observed in the dark. Behaviors in the light fell into two categories. In the first, fish were nearly motionless, maintaining a relatively stable position near the wall (N

= 2) for long periods of time (Fig. 12 A, C), resulting in high wall-preference indices

(>0.7). In the second, fish moved around the central regions of the tank (N = 2), avoiding for the most part, the perimeter of the tank (Fig. 12 B, D), resulting in much lower wall- preference indices (0.06

Despite the near-wall preferences exhibited by individuals frozen in place, wall- following behaviors (continuous movement near the wall) among sighted morphs in visible light were reduced relative to those of sighted morphs in the dark. On average, wall-following behavior accounted for ~10 % of the total distance traveled by sighted individuals in the light vs. 36 (first 10 minutes) - 60% (second 10 minutes) by those in the dark) (Fig.13A). In addition, there were almost no long bouts of wall-following behavior in sighted individuals in visible light (Fig. 13C). The long-bout index was <

0.06 for all individuals for both the first and second 10 minutes and long-bout indices were likewise significantly less than those of sighted morphs in the dark for the second ten minutes (Table 1, column 3).

Short-term temporal changes in key parameters are summarized in Fig.14 for

sighted morphs in the light (light blue functions with filled symbols) compared to sighted

(blue functions with filled symbols) and blind (red functions with open symbols) morphs in the dark. On average, both sighted morphs in the light and blind morphs in the dark maintained relatively stable levels of wall distance (Fig. 14A) and orientation (Fig. 14B) as well as bout length indices (Fig. 14C) and swimming speeds (Fig. 14D). The slopes of regression lines through individual means were not significantly different from zero in all cases. In contrast, sighted morphs in the dark tended to show a gradual decrease in wall distance and orientation and a gradual increase in bout length index and swimming speed with the slopes of all regression lines significantly different from zero (p < 0.0001).

When sighted individuals were given a longer period (1 hour) of dark-adaptation before testing began, all but one of the temporal effects were eliminated. However, both short and long-term adapted groups showed similar effects of time on swimming speed.

The swimming speeds increased at the same rate (slopes of regression lines = 0.02

BL/min) and the slope of the regression line were significantly different from zero for the short-term adapted group (p = 0.0001) and almost, but not significantly different from

zero for the long-term adapted group (p = 0.07). Finally, angular positions around the

perimeter of the tank were uniformly or nearly-uniformly distributed for blind morphs in

the dark, but not for sighted morphs in either dark or light conditions, as evidenced by 1-

min z-stat values averaged across individuals for each population and condition (Fig. 15).

The Effects of Swimming Speed and Fish Length

Because there was a consistent trend for sighted fish to increase their swimming

speed in the dark (for at least the first 10 minutes), a linear regression analysis was

31 performed to determine if swimming speed was correlated with key wall-following parameters. Mean swimming speed was not significantly correlated with either long-bout index or maximum bout length for sighted morphs (either short-term or long-term adapted groups), but accounted for 77 and 66% of the variance for these two parameters, respectively in blind morphs (p = 0.004 and 0.01).

A final regression analysis was performed to determine if wall-following abilities might have been constrained by fish length, as might be expected if the degree of wall curvature prevented or constrained longer fish from swimming parallel to the wall and thus, from meeting the criteria for continuous wall-following bouts. Neither maximum bout lengths nor long bout indices were correlated with fish length in either blind or sighted populations.

SUMMARY OF RESULTS

Both blind and sighted morphs exhibit wall-entrainment behaviors in response to a novel,

environment in the dark, whereas sighted morphs placed in the same novel, but well-lit

environment exhibited dramatically different behavior. Half of all sighted morphs in the

light remained nearly motionless for the entire 20-minute test period – a behavior that

was never observed in the dark from either blind or sighted morphs.

Unlike sighted morphs in visible light, both sighted and blind morphs in the dark

swam more frequently along the walls than anywhere else in the novel environment.

However, their near-wall swimming behaviors differed in subtle, but significant ways.

Behavioral differences between blind and sighted morphs in the dark can be summarized

in terms of (1) the fish’s body orientation with respect to the wall, (2) the degree of

continuity and persistence of the wall-following behavior and (3) the time that it took fish

to develop maximum swimming speeds. Blind morphs swam nearly parallel to the wall

surface for long distances (up to 8 times the circumference of the arena) in the same

direction (Fig. 7A), developing maximum and stable swimming speeds of ~ 1.5 BL/s

within 1 – 2 minutes after first being introduced into the novel arena (Fig. 15D). In

contrast, sighted morphs took much longer (~ 10 – 15 min) to attain similarly high

swimming speeds (Fig. 15D) and swam for shorter distances along the wall with the head directed more inwards towards the wall. Whereas blind morphs tended to travel at high speeds in one direction along the entire perimeter of the tank, sighted morphs in the dark traveled along the wall in a less uniform fashion (Fig. 16). Instead of covering the entire perimeter in one direction, they often switched from clockwise to counterclockwise

33 directions to repeatedly cover the same, short distance. Alternatively, they swam in short loops, first towards and then away from a small region of the wall (Fig. 5A) – a pattern of behavior that was also observed in sighted fish in the light (Fig. 15A). Behavioral differences were independent of whether sighted morphs were given short or long periods of dark-adaptation time before encountering the novel arena.

DISCUSSION

Functional significance and evolution of wall-following behaviors in Astyanax

fasciatus

The functional significance of wall-following behaviors in Astyanax fasciatus and

other species remain largely unknown and untested. At least two biologically-relevant functions – exploration and predator avoidance - are worth discussing in light of these results. The exploratory hypothesis proposes that wall-following behaviors function to help animals (a) find needed resources (e.g. food, shelter, escape routes) and/or (b) acquire a general knowledge about the spatial configuration of the environment and the relative location of needed resources within the environmental space.

Reduced levels of locomotor activity and the absence of wall-following behaviors in sighted morphs under well-lit conditions are consistent with the exploratory hypothesis. That is, when visual cues are available, sighted morphs do not require

locomotor strategies to bring their short-range senses within range of different spatial

features in the environment. Thus, it is theoretically possible for sighted individuals to

acquire spatial information about either the location of a needed resource or the general size and shape of a well-lit, bounded environment without moving, but impossible for either sighted or blind individuals to do the same in the dark, assuming that other long- range cues (e.g. chemical or sonic) are unavailable as well. This is not to say that sensorimotor strategies (e.g. oculomotor movements to scan the extent of the wall) are unimportant for long-range sensing, only that long-range sensing does not require that

animals move to distal landmarks in order to sense them or determine their spatial relationships.

The use of long-range chemical cues to acquire information about the spatial configuration of the environment is unlikely given what we know about the capricious nature of the temporal and spatial dispersion of chemical cues (Koehl, 2006). In stagnant water, any chemical cues from the wall’s surface would disperse slowly and uniformly by diffusion, but in the presence of a swimming fish, dispersion by water motion is likely to be erratic in both time and space. Furthermore, the possibility of odor cue use by fish in this study was reduced by the addition of familiar, home-tank water into the novel arena.

Long-range sonic cues are likewise unlikely, given that sounds have yet to be recorded from this species (Popper 1976, personal observation).

Wall-proximity by blind or sighted morphs could also serve a protective or predator avoidance function. That is, fish could take up positions near the wall for purposes of shelter and/or to decrease the number of directions from which a predator might attack. This explanation is plausible for sighted fish, which were sometimes observed to take up stationary positions near the wall in the light. The sighted morph has natural predators and individuals in this study were sometimes observed to exhibit

possible fright responses (rapid, erratic swimming) when first placed into the test arena.

However, this explanation does not obviously account for the wall-following behaviors observed for both sighted and blind morphs in darkness, but not by sighted morphs in the light. It is, in particular, less satisfying for blind morphs, which are believed to have few, if any natural predators in the subterranean caves that they inhabit, other than possibly conspecifics preying upon the young (Poulson, 1963). In fact, the colonization of caves

by sighted ancestors of the blind morphs is thought to have been driven, at least in part,

by the absence of predatory pressures (Mitchell et al., 1977; Jeffery 2003).

One possibility worth considering is that the attraction to walls observed in both

populations serves different primary functions under the two sensory conditions of these

experiments. For the sighted morph in the light, wall proximity may serve a protective

function. When wall proximity is coupled with the absence of movement, as was the case

for two of the sighted individuals in this study, this theoretically increases the protective

value of the behavior, since visual systems, such as those of visual predators, are strongly

geared towards detecting visual motion (Palmer, 1999). For both the sighted morph and

the blind morph in the dark, however, wall-following behaviors may serve an exploratory

function. Depending on predatory pressures and levels of perceived threat (the fear

factor), exploration could be used to build general spatial knowledge of the environment over longer periods of time and/or to find shelter or escape routes as quickly as possible.

From an evolutionary perspective, both sighted and blind morphs may have inherited a primitive wall-following behavior from their sighted ancestor. In both cases, this behavior may serve an exploratory function under visually-deprived conditions experienced sometimes by the sighted morph and always by the blind morph. In both light and darkness, wall proximity may also serve a protective function, evident most strongly for sighted morphs in the light that take up stationary positions near the wall.

Over an evolutionary timescale, the wall-following behavior of blind morphs may have become more finely honed due to its greater utility in constant darkness. In light of the exploratory hypothesis, the different characteristics of wall-following behaviors in blind and sighted morphs may be understood, at least in part, in terms of the underlying

sensorimotor mechanisms for active hydrodynamic sensing and how short range senses

are used in the acquisition of spatial information.

The role of active sensing by the lateral line in wall-following behaviors of bind and

sighted morphs

There are several reasons to believe that active sensing by the lateral line plays a major

role in the sensory guidance of wall-following behaviors by blind, if not sighted morphs

in the dark. First, the lateral line system is likely to be within detection range of the wall

for the majority of near-wall distances reported in this study (Fig. 6). Whereas the passive

sensing range of the lateral line system to current-generating sources like moving prey (or

vibrating spheres) is generally restricted to less than 1 BL (Kalmijn, 1988, 1989; Coombs and Montgomery, 1999), the active sensing range for stationary sources like walls is predicted to be much less (<~0.2 BL), as estimated from obstacle avoidance tasks

(Windsor, 2008) and computational models of fish-shaped objects moving past planar surfaces (Hassan 1985; Windsor, 2008).

Although the sensing range of the tactile sense would seem to be even shorter, blind cavefish have been observed touching walls with their pectoral fins while swimming past them (Windsor, PhD Thesis). The pectoral fin lengths of blind cavefish in this study ranged from ~0.6 – 0.75 cm (~0.15 BL) and were easily within reach of the wall for the majority of wall-distances reported in this study (Fig. 6). Although a contribution of pectoral fin touch cannot be ruled out for wall-following behaviors, recent studies on blind morphs indicate that wall-following indices are dramatically decreased when the lateral line system is pharmacologically blocked (Coombs et al, 2008). In addition, the correlations reported in this study and in Coombs et al (2008) between

swimming speed (and by inference, hydrodynamic signal strength) and various metrics

for the strength of wall-following behaviors are also consistent with active sensing by the

lateral line.

A theoretical case can easily be made for how the wall-following behaviors of

blind morphs are better suited for acquiring spatial information via the lateral line than those of sighted morphs. By swimming at relatively high, but stable speeds and maintaining nearly parallel orientations to the wall, blind morphs are likely to improve their ability to detect wall-generated distortions in their own self-generated flow field. A high swimming speed increases the probability that the signal distortions will be detected since the amplitude of the flow field is a function of swimming speed (Hassan, 1985;

Teyke, 1988; Hassan, 1989, 1992a, b). A stable swimming speed ensures that different

signal strengths arising from e.g. landmarks of different sizes or at different distances will

not be confused with different signal strengths caused by variable swimming speeds.

Body orientation is also important for both signal reception and generation.

Because the fish’s body is laterally compressed and lateral line sense organs are

distributed all over the head and body (Schemmel, 1973), the field of view will be much

narrower in front of the head than along the flanks. This is particularly important for two

reasons. One involves how distance of a stimulus source is encoded by the lateral line

(Hassan, 1989; Coombs et al., 2002) and the other, how information from sequentially

encountered landmarks may be serially linked in time for the purpose of learning spatial

relationships. As distance between the fish and any flow-distorting obstacle increases, the

pattern of activity along distributed lateral line sensors broadens and the peak activity

decreases (Hassan, 1985). Distance encoding of far sources require longer arrays than

39 does distance encoding of near sources. Lateral fields of view (i.e. fish’s body oriented parallel to the wall) will thus encode a larger range of distances than frontal views (fish pointing toward the wall).

Lateral fields of view also increase the body distance over which different spatial features might be simultaneously sensed and registered as the fish swims past them. In this respect, the propensity for blind morphs to swim continuously along the wall (or in theory, other spatially distributed landmark features) for long distances in the same direction might assist them in constructing a sequential map of features encountered at different points in time. Whether or not sequentially acquired information about spatial relationships is later converted into an allocentric map that can be used for e.g. determining the shortest route to a given goal (e.g. food site) remains to be seen.

However, the wall-following behaviors exhibited by blind morphs could in principle be used for this purpose. Indeed, there is some evidence to suggest that blind cavefish can learn both the spatial configuration of a set of landmarks (Burt de Perera 2004a), as well as the spatial order of landmarks within the configuration (Burt de Perera 2004b) by swimming around the configuration.

If more lateral (= parallel orientation) fields of view are beneficial to exploratory behaviors, why is it that the orientation of blind morphs is shifted by ~ 5 - 100 away from parallel and towards the wall? One simple explanation is that this is merely a consequence of fish being unable to maintain a strictly parallel orientation to a curved surface without frequent deviations from parallel (i.e. turning) to follow the curved surface. Another possibility is that observed body orientations could be a compromise driven by the cost/benefit ratio of lateral vs. frontal views in terms hydrodynamic signal

strength, field of view, and spatial resolution. Lateral line sense organs on the head are

more densely packed than those on the rest of the body (Schemmel, 1967, 1973), so the

sensitivity and spatial resolution of information in a frontal ‘view’ is likely to be greater

than that of a lateral view. Moreover, the strength of the hydrodynamic flow field in

front of the fish for any given swimming speed will be approximately twice that of the

flow amplitude to the side, assuming that the fish behaves like a dipole (Kalmijn, 1988).

Nevertheless, for a fish that is swimming in a forward direction, there is a stagnation

point directly in front of the snout where flow velocity plummets to zero. The stagnation

point coupled with a complete absence of lateral line sensors in this region (personal

observation), results in an effective lateral line ‘blind’ spot. To summarize, a lateral

‘view’ (parallel orientation) is good for distance encoding and sequential linking of

encountered landmark features, whereas a frontal ‘view (orthogonal orientation) yields a

higher signal strength and better spatial resolution, except directly in front of the snout.

To avoid the blind spot and to maximize sensing advantages that come with both frontal

and lateral views, body orientations to the wall would likely deviate somewhat from

purely parallel or orthogonal, depending on the sensory task.

In terms of active sensing by the lateral line, it would appear that blind morphs

have enhanced capabilities relative to sighted morphs for both signal generation and

reception. In addition to the various (putative) benefits gained from the blind morph’s

wall-following behaviors, as discussed above, there are likely to be morphological

benefits as well. For example, the amplitude of the flow field in front of a blind morph is

likely to be greater than that in front of a sighted morph swimming at the same speed, because blind morphs have broader heads (Jeffrey, 2001). Superficial neuromasts on the

41 head are also likely to be more sensitive in blind morphs than sighted morphs owing to their larger circumference and corresponding increased surface area and lengths of overlying cupulae (Teyke 1990) (see also The lateral line system and active hydrodynamic imaging in the Introduction).

Inter-individual variability within morphs

Both blind and sighted individuals exhibited considerable variability in their responses to the novel environment. In general, behavioral responses of sighted morphs appeared to be greater those of blind morphs. For example, half of sighted individuals in the light moved very little during the first 10 minutes (Fig. 12A, C), whereas the other half showed substantial movements (Fig. 12B, D). Similarly, half of all sighted individuals in the dark exhibited a preference for being near the wall during the first 10 minutes, whereas the other half did not. In contrast, all but one blind individual exhibited wall preferences during the same time period. Short-term temporal plots of various parameters vs. time also reveal a greater variability among sighted than blind individuals (Fig. 9).

In this regard, it is important to point out that swimming speed accounted for >

66% of the variance in wall-following metrics in blind individuals. In fact, the one blind individual that differed from all the rest in having no wall-preference and low wall- following indices also had the lowest mean swimming speed (0.4 BL/s). This source of inter-individual variability among blind morphs can, at least in part, be understood in terms of the hydrodynamic signal strength of the wall’s presence. Reduced swimming speeds will theoretically produce weaker signals of the wall’s presence and thus, lead to poorer wall-sensing and following capabilities. The strong argument against swimming speed as a source of variability for sighted individuals in either the light or the dark is the

absence of any correlation between swimming speed and any wall-following metrics.

Ironically, the few sighted individuals to show any preference for being near the wall in

the light were those that remained relatively motionless in one position for long periods

of time. As a consequence, their swimming velocities were near zero.

If swimming speed and by interference, ability to actively sense the wall with the

lateral line system is not a source of inter-individual variability in sighted individuals,

what accounts for the wide range of observed behaviors? One plausible explanation is

that there is greater variability among sighted individuals in terms of the shy-bold

continuum of personality traits. It is now well-documented that individual differences in

these traits can have profound effects on behavior in a wide variety of vertebrate groups,

including fish (Frost et al 2007, Sneddon 2003; Bell 2005; Yoshida et al 2005). Bold

individuals tend to be more active, take more risks, learn more quickly and show higher

levels of aggression than shy individuals. Thus, in novel environments, it is expected that

bold individuals will take more risks in actively exploring the new territory.

Furthermore, risk of predation and food availability are thought to play major, if conflicting roles in shaping the frequency of bold vs. shy traits in any given population.

Given the current state of our knowledge on the ecology of blind and sighted morphs, it is reasonable to assume that both food availability and risk of predation are lower for blind than sighted individuals in their natural habitat (Breder 1943, Parzefall, 1983). As a result, these two, interactive selective pressures would theoretically favor a higher frequency of bold than shy traits in blind morphs and perhaps more evenly balanced frequencies of bold and shy traits in sighted individuals. In light of this scenario, the absence of movement and preference for the wall exhibited by half of the sighted

individuals in the light might well be interpreted as risk-aversive, predator avoidance

behaviors near the shy end of the continuum (Fig. 13A, C), whereas active movements

away from the wall by the other half might be interpreted as less risk-aversive,

exploratory behaviors nearer the bold end of the continuum (Fig. 13 B, D). Similarly, the

variability in the temporal onset and degree of wall-following behaviors among sighted

individuals in the dark could be explained by variability in personality traits along the shy-bold continuum. In contrast, the rapid development of high swimming speeds and persistently strong wall-following behaviors by the majority of blind morphs are consistent with a higher frequency of bold than shy traits in these morphs, assuming an exploratory, rather than protective function for wall-following behaviors.

Alternative explanations for wall-following behaviors and observed population

differences

It has been suggested that wall-following behaviors serve no particular biological function at all, but are merely an incidental consequence of how the fish’s movements are redirected every time the walls of a concave environment are encountered as a barrier to forward motion (Creed and Miller, 1990). If animals obey two simple rules (move in a straight line until a barrier is encountered and turn by a small amount to continue moving if a barrier is encountered), wall-following behaviors are predicted to emerge, as simulated by computer models (Creed and Miller, 1990). A critical test of this hypothesis is how animals respond to a novel arena with convex surfaces. Given the simple, 2-rule algorithm above, animals moving along a convex surface should continue in a straight

path when nearing its apex because there is no longer a barrier to impede forward motion.

Recent studies in such a goggle shaped arena with concave and convex surfaces

of equal radii have revealed that although blind morphs departed from the convex

surface, they failed to follow the straight line path predicted by the Creed and Miller

algorithm (Coombs et al, 2008). Instead, their paths veered towards the convex surface in

a velocity-dependent manner. As swimming speed increased, the directional bias towards

the convex surface increased and the degree of departure decreased. This correlation is

similar to the correlation reported in this study between swimming speed and the

persistence of wall-following behavior in blind (but not sighted) morphs, as measured by

maximum bout lengths and long-bout indices. Taken together, these results suggest that

wall-following behaviors by blind morphs cannot be explained by a simple, reactive

strategy to avoid barriers whenever they are encountered. Rather, blind morphs seem to

be actively attracted to the wall in a manner that depends on swimming speed and by inference, the amplitude of the hydrodynamic signal to the lateral line. Although similar

experiments on sighted morphs in a goggle-shaped arena have not been done, the absence

of any correlation between swimming speed and measures of continuous wall-following

in this study suggests that hydrodynamic cues to the lateral line play a less significant

role in the wall-following behaviors of sighted morphs.

Switching from visual to non-visual modes of processing information

Unlike blind morphs in the dark or sighted morphs in the light, sighted morphs

plunged into a novel dark environment after only 5 minutes of acclimation to the dark

showed systematic short-term increases or decreases in many wall-following parameters

during the 20 minute test session (Fig. 14). In some cases, these changes gave the

appearance that sighted morphs may have been converging upon similar levels of wall-

45 following capabilities as blinded morphs, but simply at a slower rate, as might happen if the transition from a visual to non-visual mode of processing information was a gradual one.

It is already well-appreciated that many animals, including fish, are not rigidly diurnal or nocturnal and can switch their locomotor activity patterns from diurnal to nocturnal and vice versa (Fraser et al, 1993; Green wood and Metcalf, 1998). Although switching can arise as a function of developmental stage or in response to many external cues (e.g. temperature, season, food availability, etc.) (Reviewed in Reebs 2002), it can also be triggered by input to the visual system (Mrovosky and Hatter, 2005). When animals are first plunged into the dark, for example, it generally takes ~ 40 – 60 minutes for the vertebrate retina to recover light sensitivity when background lights are abruptly turned off – a process known as dark adaptation (Dowling 1967). Sighted fish cannot make use of recovered light sensitivity in this study. However, the underlying process of dark adaptation and visual input to the nervous system could inform central processing areas that control the transition from visual to non-visual guidance of behaviors. Indeed, many of the temporal effects observed in the short-term adapted group were absent in the long-term adapted group, lending support to the idea that many of these temporal effects were driven by the time it took for individuals to switch from a visual to non-visual processing mode. One temporal effect that did not disappear, however, was the systematic increase in swimming speed from < 1 BL/s to a level sustained by blind morphs throughout the test session (~1.5 BL/s). The persistence of this effect in the long- term adaptation group suggests that the observed rise in swimming speed was a response to novelty, rather than the loss of visions.

SUMMARY AND CONCLUSIONS

Both sighted river morphs and congenitally blind, cave-dwelling morphs of Astyanax fasciatus exhibited wall-following behaviors with subtle but significant differences in response to a novel environment in the dark. In contrast, sighted morphs placed in the same novel, but well-lit environment exhibited dramatically different behaviors that fell into two general categories: (1) holding stationary positions near the wall for long periods of time (a behavior never observed in the dark from either blind or sighted morphs) or (2) moving in and around the central region of the environment without moving along the walls. Differences in wall-following behaviors between blind and sighted morphs in the dark include (1) a more nearly parallel orientation of the head and body with respect to the wall in blind morphs vs. a more wall-ward orientation of the head in sighted morphs,

(2) a higher degree of continuity and persistence of wall-following behaviors in blind compared to sighted morphs, (3) a more rapid development of consistently high swimming speeds (~1.5 BL/s) for blind compared to sighted morphs and (4) lower inter- individual variation in wall-following behaviors in blind vs. sighted morphs.

Wall-following behaviors of both blind and sighted morphs in the dark suggest that both morphs use short-range, non-visual (lateral line and touch) senses and locomotor strategies to compensate for the loss of vision. In contrast, reduced levels of locomotor activity and the absence of wall-following behaviors in sighted morphs under well-lit conditions suggest that locomotor strategies associated with the use of short range senses (wall-following) are not required when long-range, visual cues are available.

Differences between wall-following behaviors of blind and sighted morphs in the dark

47 may largely be understood in terms of differences in active flow sensing capabilities and strategies for acquiring short-range information through the lateral line system. Inter- individual variation in wall-following performance in blind morphs is likely to be a function of swimming speed and corresponding hydrodynamic signal strength for active flow sensing, whereas that in sighted morphs may be related to variations in behavioral dispositions (‘personality traits’) along the shy/bold continuum.

It is suggested that both sighted and blind morphs may have inherited a primitive wall-following behavior from their sighted ancestor that serves an exploratory function under visually-deprived conditions for both morphs. Wall-proximity (remaining near the wall without necessarily moving along it) may also serve a protective function for sighted morphs, evident most strongly for sighted morphs that take up stationary positions near the wall in the light. Over an evolutionary timescale, wall-following behaviors of blind morphs have become more finely honed for exploratory purposes than those of sighted morphs due to its greater utility in constant darkness.

LITERATURE CITED

Abdel-Latif H, Hassan ES, Campenhausen Cv (1990) Sensory performance of blind

Mexican cave fish after destruction of the canal neuromasts. Naturwissenschaften

77:237-239

Avise JC and Selander RK (1972) Evolutionary genetics of cave-dwelling fishes of the

genus Astyanax. Evolution 26:1-19

Baker CF and Montgomery JC (1999) The sensory basis of rheotaxis in the blind

Mexican cave fish, Astyanax fasciatus. J Comp Physiol A 184:519-527

Barnett SA (1963) The rat, a study in behavior. Pp.288. Aldine Publ Co, Chicago

Basel J and Sandemann D (2000) Crayfish (Cherax destructor) use tactile cues to detect

and learn topographical changes in their environment. Ethology 106:247-259

Batschelet E (1981) Circular statistics in biology. London: Academic Press

Besson M, Martin JR (2004) Centrophobism/Thigmotaxis, a new role for the

mushroom bodies in Drosophila. J Neurobiol 62(3):386-96

Bell AM (2005) Behavioral differences between individuals and two populations of

stickelbacks (Gasterosteus aculeatus). J of Evol Biol 18: 464-473

Bourne DW, Heezen BC (1965) A wandering enteropneust from the abyssal Pacific, and

the distribution of spiral tracks on the sea floor. Science 150:60-63

Breder CM, Gresser EB (1941b) Behavior of Mexican cave characins in reference to light

and cave entry. Anat Rec 81: 112 (Abstract No. 216)

Breder CM (1942) Descriptive ecology of La Cueva Chica, with especial reference to the

blind fish, Anoptichthys. Zoologica 27:7-15

Breder CM (1943) Problems in the behavior and evolution of a species of blind cave fish.

Trans N.Y.Acad Sci 5:168-176

Breder CM, Rasquin P (1947) Comparative studies in the light sensitivity of blind

characins form a series of Mexican caves. Bull. Amer. Mus Nat Hist. 89: 324-351

Burt De Perera T (2004a) Fish can encode order in their spatial map Proc R Soc Lond

271, 2131-2134

Burt De Perera (2004b) Spatial parameters encoded in the spatial map of the blind

Mexican cave fish, Astyanax fasciatus. Animal Behaviour 68:291-295

Coombs S, Montgomery JC (1999) The enigmatic lateral line system. pp. 319-362. In:

R.R. Fay and A.N.Popper (eds.) Comparative hearing: Fish and Amphibians,

Springer Handbook of Auditory Research, Springer-Verlag, New York

Coombs S, Braun CB (2002) Information processing by the lateral line system pp. 122-

138. In: S.P.Collin and N.J.Marshall (eds.) Sensory Processing of the Aquatic

Environment, Springer-Verlag, New York

Coombs S, Patton P, Windsor S, Sharma S (2008) Active hydrodynamic imaging and

adaptive motor control by Mexican blind cavefish, Astyanax fasciatus, when

following wall surfaces. Presented at the Fourth meeting of Adaptive Motions of

Animals and Machines 2008, Cleveland, USA

Creed Jr RP, Miller JR (1990) Interpreting animal wall-following behavior.

Experientia 46(7) 758-761

Dowling JE (1967) The site of visual adaptation. Science 155(3760): 273-279

Fanselow E, Nicolelis MA (1999) Behavioral modultation of tactile responses in the rat

somatosensory system. J Neurosci 19:7603-7616

Fraenkel GS, Gunn DL (1961) The orientation of animals. Clarendon Press, Oxford.

Fraser NHC, Metcalfe NB, Thorpe JE (1993) Temperature-dependent switch between

diurnal and nocturnal foraging in salmon. Proceedings: Biological Sciences

252(1334): 135-139

Frost AJ, Winrow-Giffen A, Ashley PJ, Sneddon LU (2007) Plasticity in animal

personality traits: Does prior experience alter the degree of boldness? Proceedings

of Royal Society B-Biological Sciences 274:333-339

Gertychowa R (1970) Studies on the etiology and space orientation of the blind cave fish

Anotichthys jordani Hubs et Innes 1936 (Characidae). Folia Biologica 18(1)

Greenwood JA, Durand D (1955) The distribution and components of the sum of n

random unit vectors. The Annals of Mathematical Statistics 26(2):233-246

Greenwood MFD, Metcalfe NB (1998) Minnows become nocturnal at low

temperatures. Journal of Fish Biology 53:25-32

Gregson JNS, Burt de Perera T (2007) Shoaling in eyed and blind morphs of the

characin Astyanax fasciatus under light and dark conditions. Journal of Fish

Biology 70, 1615-1619

Hassan ES (1985) Hydrodynamic imaging of the surroundings by the lateral line of the

blind cave fish Anoptichthys jordani. In: The Mechanosensory Lateral Line (Ed.

By S.Coombs, P.Gorner & H.Munz), pp 217-228. New York: Springer -Verlag

Hassan El-S, Abdel-Latif H, Beibricher R (1992) Studies on the effects of Ca2+ and

Co2+ on the swimming behavior of the blind Mexican cave fish. J Comp

Physiol A 171:413-419

Hill EW, Reiser JJ, Hill MM, Hill M, Halpin J, Halpin R (1993) How persons with visual

impairments explore novel spaces: strategies of good and poor performers.

Journal of visual impairment & blindness. 87(8): 295-301

Hubbs CL, Innes WT (1936) The first known blind fish of the family Characidae: A new

genus from Mexico. Occasional papers of the Museum of Zoology, University of

Michigan 342:1-9

Janders R (1975) Ecological aspects of spatial orientation. Annual review of ecology and

systematics 6:171-188

Jeanson R, Blanco S, Foumier R, DeneubourgJ.-L, Fourcassie V, Theraulaz G (2003) A

model of animal movements in a bounded space. J Theor Biol 225:443-451

Jeffrey WR (2001) Cavefish as a model system in Evolutionary Developmental Biology.

Developmental Biology 231(1): 1-12

Jeffrey WR (2005) Adaptive Evolution of Eye Degeneration in the Mexican Blind

Cavefish. Journal of Hereditary 96(3): 185-196

John KR (1957) Observation on the behavior of blind and blinded fishes. Copeia 2:123-

132

Kallai J, Makany T, Karadi K, Jacobs WJ (2005) Spatial orientation strategies in

Morris-type virtual water task for humans. Behavioral Brain Research 159:187-

196

Kallai J, Makany T (2007) Cognitive and affective aspects of thigmotaxis strategy in

humans. Behavioral Neuroscience: 121 (1):21-30

Kalmijn AJ (1988) Hydrodynamic and acoustic field detection. In: Atema J, Fay RR,

Popper AN, Tavolga WN (eds.) Sensory Biology of aquatic animals, pp 83-130.

Springer, Berlin Heidelberg New York

Kalmijn AJ (1989) Functional evolution of lateral line and inner ear systems. In :

Coombs S, Gorner P, Munz H (eds.) The Mechanosensory lateral line,

neurobiology and evolution, pp 187-216. Springer, Berlin Heidelberg New York

Koehl M (2006) The fluid mechanics of arthropod sniffing in turbulent odor plumes.

Chem. Senses 31: 93 - 105

Martin J-R (2004) A portrait of locomotor behaviour in Drosophila determined by a

video-tracking paradigm. Behavioural processes 67: 207-219

Mitchinson B, Martin CJ, Grant RA, Prescott TJ (2007) Feedback control in active

sensing: rat exploratory whisking is modulated by environmental contact. Proc

Biol Sci 274(1613): 1035-41

Mitchell RW, Russell WH, Elliot W (1977) Mexican eyeless characin fishes, genus

Astyanax: Environment, distribution, and evolution. Spec Publ Mus Texas Tech

Univ 12, 1-89

Montgomery, J.C., Coombs S and Baker CF (2001) The mechanosensory lateral line

system of the hypogean form of Astyanax fasciatus. Environmental Biology of

Fishes, 25, 79-86

Mrovosky N, Hattar S (2005) Diurnal mice (Mus musculus) and other examples of

temporal niche switching. J Comp Physiol A 191:1011-1024

Nelson ME, MacIver MA (2006) Sensory acquisition in active sensing systems. J Comp

Physiol A 192:573-586

Paglianti A, Messana G, Cianfanelli A, Berti R (2006) Is the perception of their own

odour effective in orienting the exploratory activity of cave fishes? Can J Zool

84:871-876

Palmer S (1999) Vision Science: photons to phenomenology. The MIT Press, Cambridge,

Massachusetts, London, England

Parzefall J (1983) Field observations in Epigean and Cave populations of Mexican

Characid Astyanax mexicanus (Pisces, Characiadae) Memoires de

BIospeleologie 10:171-176

Parzefall J (1993a) Behavioural ecology of cave-dwelling fishes. In: Pitcher(ed)

Behaviour of teleost fishes, 2nd edn. Chapman and Hall, London, pp 573-608

Patullo BW and Macmillan DL (2006) Corners and bubble wrap: the structure and

texture of surfaces influence crayfish exploratory behavior. The Journal of

Experimental Biology 209:567-575

Popper AN (1970) Auditory capacities of the Mexican blind cave fish (Astyanax jordani)

and its sighted ancestor (Astyanax mexicanus). Animal behavior 18:552-562

Poulson TL (1963) Cave adaptation in amblyopsid fishes. American midland naturalist

70(2): 257-290

Poulson TL and White WB (1969) The Cave Environment. Science 165(3859): 971-981

Reebs SG (2002) Plasticity of diel and circadian activity rhythms in fishes. Rev Fish Bio

Fisheries 12:349-371

Romero A and Paulson KM (2001) It’s a wonderful hypogean life: a guide to the

troglomorphic fishes of the world. Environemntal Biology of fishes 62:13-41

Schemmel C (1967) Vergleichende Untersuchungen an dep Hantsinnesorganen ober- und

unterirdisch lebender Astyanax – men. Z Mo rphol Okol Tiere 61:255-319

Schemmel C (1980) Studies on the genetics of feeding behavior in the cave fish

Astyanax mexicanus f. Anoptichthys. An example of apparent monofactorial

inheritance by polygenes. Z Tierpsychol 53:9-22

Schone H (1984) Spatial Orientation: The spatial control of behavior in animals and

man. Princeton University Press, Princeton, New Jersey

Simon P, Dupuis R, Costentin J (1994) Thigmotaxis as an index of anxiety in mice:

Influence of dopaminergic transmissions. Behav Brain Res 61:59-64

Sneddon LU (2003) The bold and the shy: Individual differences in rainbow trout.

Journal of Fish Biology 62:971-975

Tavolga WN (1976) Obstacle avoidance in the sea catfish (Arius felis) In: Schuijf A,

Hawkins AD (eds) Sound reception in fish. Elsevier, Amsterdam, pp 185–204

Teyke T (1990) Morphological differences in neuromasts of the blind cave fish Astyanax

hubbsi and the sighted river fish Astyanax mexicanus. Brain Beh Evol 35: 23-30

Teyke T (1985) Collision with and avoidance of obstacles by blind cave fish

Anoptichthys jordani (Characidae). J Comp Physiol A 157:837-843

Teyke T (1989) Learning and remembering the environment in the blind cave fish

Anoptichthys jordani. J Comp Physiol A164: 655-662

Teyke T (1994) Blind Mexican cave fish (Astyanax hubbsi) respond to moving visual

Stimuli. J Exp Biol 188(1): 89-101

Treit D, Fundytus M (1989) Thigmotaxis as a test for anxiolytic activity. Pharmacol

Biochem Behav 31: 959-962

Valle FP (1970) Effects of strain, sex, and illumination on open-field behavior of rats.

The American Journal of Psychology 83(1) 103-111

Weissert R and von Campenhausen C (1981) Detection of stationary objects by the

blind cave fish Anoptichthys jordani (Characidae). J Comp Physiol 143:369-374

Wilkens H (1988) Evolution and genetics of epigean and cave Astyanax fasciatus

(Characidae, Pisces): Support for the neutral mutation theory. Evolutionary

Biology (23): 271-367

Windsor S (2008). Hydrodynamic imaging by blind Mexican cave fish. PhD thesis,

University of Auckland

Wolfer PW, Stagljar-Bozicevic M, Errington ML, and Lipp HP (1998). Spatial memory

and learning in transgenic mice. News Physiol Sci 13: 118-123

Yoshizawa, M. and Jeffery, W. R. (2008). Shadow response in the blind cavefish

Astyanax reveals conservation of a functional pineal eye. J Exp Biol 211:292 -299

(A) (B)

Figure. 1 (A) Distribution of lateral line canals and superficial neuromasts on the head and body of a blind cavefish (from Schemmel 1967) and (B) illustrations of the hair cell (HC) and the overlying cupula (Cu)(B)(from:Teyke, 1990). Thick black dots in A depict pore openings to the canals (one neuromast between every two pores), whereas the numerous small dots denote the superficial neuromasts. In B, structures at the apical surface of the hair cell include the stereovillae (S) and single, elongated kinocilium (K) of the ciliary bundle and the afferent (AN) and efferent (EN) nerve fibers at the basal surface. 57

A B

Figure 2. Mechanism of active flow sensing in fish. The iso-velocity flow lines of a self- induced flow field around a fish’s body as it moves through the water in the absence (A) and presence (B) of a stationary obstacle. (Hassan, 1985). 58

Figure 3. (A) Video frame of an experimental fish in the circular testing arena under upwelling IR illumination to enhance contrast and facilitate automatic video tracking of the fish’s swimming behavior. (B) Pathway followed by a blind cavefish for

12.2 seconds during a 10-minute trial. Arrows depict the fish’s heading at each video frame and colors denote the characteristic swimming speed from one frame to the next. 59

Snout Distance Surface Distance Centroid Distance

0 0 180 90 2700

(CW) (CCW)

Figure 4. Conventions for measuring distance (A) and orientation (B) of the fish with respect to the wall. 60

Elapsed Time (Min) SF 5 BC 30 1 2 3 4 5 6 7 8 0.25 0.85 9 A B 10

11 12 13 14 15 16 17 0.80 0.93 18 C D 19

Figure 5. Swimming tracks of one sighed (SF5) (A, C) and one blind (BC30) (B,D) morph during the first (A,B) and second (B,D) ten minutes of a test period in the dark. Wall-preference indices associated with each track are shown in the lower right-hand corner

BCF 30 SF 5 Uniform Distribution 25 20 First 10 minutes 15 10 5 Frequency (%) 0 012345 A Distance from the wall (cm)

25 20 Second 10 minutes 15 10 5 B (%) Frequency 0 012345 Distance from the wall (cm)

Figure 6. Frequency distributions of wall distances for different blind (red) and sighted (blue) individuals for the first (A) and second (B) ten minutes compared to a theoretical, spatially uniform distribution of distances (black). SF 5’s distribution is highlighted by red symbols, whereas BC 30’s is highlighted with blue symbols. Distributions are based on centroid distances (see Fig. 4) 62

10 First 10 minutes

8 BCF 30 SF 5 6 4 2 Frequency (%) Frequency 0 A 0 90 180 270 360 N E S W N Position around the wall (deg)

10 Second ten minutes 8 6 4 se 2 Frequency (%) Frequency 0 B 0 90 180 270 360 N E S W N Position around the wall (deg)

Figure 7. Frequency distributions of angular compass positions around the perimeter of the tank (distance < 0.5 BL from the wall) for sighted (blue) and blind (red) individuals during the first (A) and second (B) ten minutes. Red and blue symbols highlight BC30’s and SF5’s distributions, respectively. 63

First 10 minutes Second 10 minutes

40 BC 30 SF 5 40 BC 30 SF 5 30 30 20 20 10 10 Frequency (%) Frequency 0 (%) Frequency 0 A 0 90 180 270 360 B 0 90 180 270 360 Orientation re: wall (deg) Orientation re: wall (deg)

60 Blind Sighted 60

40 40

20 20 Frequency (%) Frequency Frequency (%) Frequency 0 0 C 0 30 60 90 120 150 180 D 0 30 60 90 120 150 180 Orientation re: wall (deg) Orientation re: wall (deg)

Figure 8. Frequency distributions of body orientations during the first (A, C) and second (B, D) ten minutes. A and B depict body orientations irrespective of wall distance for two individuals, one sighted (SF5) (blue lines with symbols) and one blind (BC30)(red line with symbols). C and D depict near-wall body orientations for fish positions that are < 0.5 BL from the wall for several blind (red) and sighted (blue) individuals, including the two individual depicted in A and B (lines with symbols).

2 n 95 90 BCF 30 SF 5 1.5 85 80 1 75 (deg) 0.5 70 65 Median Distance (BL) 0 60

A 0 5 10 15 20 Orientatio Near-Wall Mean B 0 5 10 15 20 Time (min) Time (min) 1 2

0.9 1.5 0.8 1 0.7

0.6 0.5 Vector Strength (r) (r) Strength Vector Mean Speed (BL/s) Speed Mean 0.5 0 C 0 5 10 15 20 D 0 5 10 15 20 Time (min) Time (min)

Figure 9. Wall distance (A), orientation (B), orientation vector strength (C) and swimming speed (D) as a function of time for blind (red) and sighted (blue) individuals. Each data point is the mean of samples taken over a 1-minute interval (300 samples/min). Red and blue symbols highlight the results from BC30 and SF5 respectively. 65 ) 1000

100

0.1 Z Stat (Angular Position (Angular Z Stat 0 5 10 15 20 Time (min)

Figure 10. The Z statistic of angular compass distributions as a function of time for blind (red) and sighted (blue) individuals. Z stat = r2 N, where r is the vector strength of the distribution and N is the sample size. Each distribution is based on a maximum of 300 samples taken over a 1-minute interval; maximum Z stat = 300. Z stats > ~ 7 (dashed line) indicate that the distribution is significantly different from uniform. Sample size is determined by the number of positions for which the wall distance < 0.5. 66

SF 5 BC 30 360 4 360 4 ) 330 330 ) ) 3.5 ) 3.5 300 300 270 3 270 3 240 240 2.5 2.5 210 210 180 2 180 2 150 150 1.5 1.5 120 120 90 1 90 1 Orientation re: wall re: (deg Orientation 60 wall re: (deg Orientation 60 Distance from the wall the (BL Distance from

0.5 wall the (BL Distance from 0.5 30 30 0 0 0 0 0 5 10 15 20 0 5 10 15 20 B A Time (mins) Time (mins)

1 x 0.8

0.6

0.4

0.2 Bout-Length Inde Bout-Length 0 0 5 10 15 20 Time (min) C

Figure 11. Illustrations of operational criteria for wall-following behaviors. Orientation (right-hand y-axis) and distance (left-hand y-axis) to the wall are co-plotted as a function of time for one sighted (SF5) (A) and one blind (BC30) (B) individual to determine when both distance (< 0.5 BL) and orientation (+/- 300 from parallel) criteria for wall-following behavior are met. The resulting 1-minute bout-length index (the percent contribution of distance traveled when both criteria are met to the total distance traveled irrespective of orientation or distance) is plotted as a function of time for all blind (red) and sighted (blue) morphs with SF5’s and BC30’s results identified with symbols (C). 67

Elapsed Time (Min)

SF 20 SF 21 1 2 3 4 5 6 7 8 9 A 1.0 B 0.12 10

11 12 13 14 15 16 17 1.0 0.06 18 C D 19 20

Figure 12. Swimming tracks of two sighted individuals during the first (A, B) and second (B, D) ten minutes of a test period in the light. Wall-preference indices associated with each track are shown in the lower right-hand corner

First 10 min Second 10 min First 10 min Second 10 min

1 * 200

x 0.8 * * ) 150 0.6 * * 100 0.4 * Max Bout (BL 50

Bout Length Inde Length Bout 0.2

0 0 SL SD BD SL SD BD SL SD BD SL SD BD A B

0.9 First 10 min Second 10 min 0.8 0.7 ** 0.6 0.5 0.4

0.3 ***

Long- Bout Index Bout Long- 0.2 0.1

0 SL SD BD SL SD BD C

Figure 13. The effects of time (first vs second ten minute period), morph type (sighted (S) vs blind (B)) and condition (light (L) vs dark (D)) on key wall-following parameter: (A) bout length index, (B) maximum bout length and (C) long-bout index.

100 ) 1.4 BCF SF SF Visible 1.2 95 1 90 0.8 0.6 85 0.4 80 0.2 Mean Near-Wall Near-Wall Mean Orientation (deg) Orientation Mean Distance (BL Distance Mean 0 75 A 0 5 10 15 20 B 0 5 10 15 20 Time (min) Time (min) x 1 2 0.8 1.5 0.6 1 0.4 0.2 0.5

0 Speed (BL/s) Mean 0 Mean Bout-Length Inde Bout-Length Mean C 0 5 10 15 20 D 0 5 10 15 20 Time (mins) Time (mins)

Figure 14. Summary of temporal effects for sighted morphs in the light (light-blue) and sighted (dark-blue) and blind (red) morphs in the dark in terms of wall distance (A), orientation (B), bout length index (C) and swimming speed (D). Each data point represents the mean of individual medians (distance) or means (orientation, bout length index and speed) for each 1-minute interval

Z stat criterion BC SF SF visible ) 300

200

100

0 Z Stat (Angular position (Angular Z Stat 0 5 10 15 20 Time (min)

Figure 15. Temporal effects on the uniformity (as defined by the Z stat) of angular compass positions for sighted morphs in the light (light-blue) and sighted (dark-blue) and blind (red) morphs in the dark in terms of wall distance. 71

Table. 1. One tailed (top) and two tailed (bottom) ‘p’ values of significance levels for tests of population differences.

Parameters Blind vs sighted Dark-adapted vs non- Sighted (dark) vs Astyanax (dark) adapted sighted sighted (light) Astyanax Astyanax

1st half 2nd half 1st half 2nd half 1st half 2nd half

Wall Preference Index 0.02 NS NS NS NS NS NS NS NS NS NS NS

Orientation NS p<0.01* p<0.05 p<0.05 p<0.01* p<0.05

Bout-Length Index 0.0095* NS NS NS 0.008* 0.01* NS NS NS NS 0.016 0.022

Long-Bout Index 0.0002** NS NS NS 0.02 0.0001*** 0.0004** NS NS NS 0.04 0.0002***

Max Bout-Length 0.003* NS 0.05 NS NS 0.002* 0.007* NS NS NS 0.05 0.004* 72

Table. 2. K-stat values for tests of difference between observed and hypothetically uniform distributions of wall distances

Individual Uniformity Test Uniformity Test (First 10 min) (Second 10 min) BC 15 0.643 --

BC 16 0.76 ---

BC 17 0.174 --

BC 18 0.468 0.666

BC 19 0.542 0.531

BC 29 0.357 0.503

BC 30 0.622 0.749

BC 31 0.466 0.355

SF 2 0.106 ---

SF 3 0.104 0.485

SF 4 0.081 0.40

SF 5 0.039 0.527

SF 11 0.441 0.453

SF12 0.258 0.361

SF 13 0.559 0.665

SF 14 0.442 0.539