are coral reef fishes able to learn to avoid...

ARE CORAL REEF FISHES ABLE TO LEARN TO AVOID UNPALATABLE PREY BASED ON VISUAL CUES?

Andrew M. Miller

A Thesis Submitted to the University of North Carolina Wilmington in Partial Fulfillment

of the Requirements for the Degree of Master of Science

Department of Biology and Marine Biology

University of North Carolina Wilmington

2011

Approved by

Advisory Committee

Christopher Finelli J. Wilson White Joseph Pawlik

Chair

Accepted by

Dean, Graduate School

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TABLE OF CONTENTS

ABSTRACT……………………………………………………………………………………...iii

LIST OF TABLES……………………………………………………………………………….iv

LIST OF FIGURES……………………………………………………………………………….v

INTRODUCTION………………………………………………………………………………...1

METHODS………………………………………………………………………………..……....5

Experiment 1…………………………………………………………………….………...7

Experiment 2……………………………………………………………………….….…..8

Experiment 3……………………………………………………………………….……...9

Experiment 4……………………………………………………………………….…….10

Statistical Analysis……………………………………………………………….………10

RESULTS………………………………………………………………………………………..11

Experiment 1……………………………………………………………………………..11

Experiment 2………………………………………………………………………..……11

Experiment 3…………………………………………………………………………..…12

Experiment 4…………………………………………………………………………..…14

Discussion………………………………………………………………………………….……14

LITERATURE CITED…………………………………………………………………..………18

FIGURE LEGENDS……………………………………………………………………………..26

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ABSTRACT

Previous attempts have been made to understand the ability of marine fish to learn to

avoid unpalatable prey based on visual cues. However, none of these studies have separated

visual cues from distastefulness to assure that fish were learning because of visual recognition

and not an olfactory or gustatory response. In the present study, we tested whether the relative

position of artificial foods affected the ability of bluehead wrasse, Thalassoma bifasciatum, a

common Caribbean coral reef fish, to learn to avoid unpalatable prey. Next, we tested the ability

of T. bifasciatum to learn to avoid unpalatable prey using only visual cues by presenting them

with artificial foods of 7 different colors. These experiments also tested the ability of T.

bifasciatum to learn to avoid unpalatable prey against a color bias. Finally, we investigated

whether T. bifasciatum learns to avoid unpalatable prey more effectively using color or pattern

signals. Fish learned to avoid unpalatable prey using visual cues independent of prey position

and were able to learn to avoid some colors of unpalatable prey based on visual cues alone,

including red, blue and orange. Fish were unable to learn to avoid all other colors due to strong

preexperimental biases for or against experimental colors. Fish were able to learn to overcome a

bias against red prey, a color that these fish had a bias against. There was no difference in the

ability of these fish to learn to avoid prey using color or pattern. We conclude that T. bifasciatum

is able to learn to avoid unpalatable prey exclusively using visual signals and that these fish are

able to quickly adapt to novel prey.

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LIST OF TABLES

Table Page

1. List of experimental colors used, food coloring name and product #, concentration of food coloring used and approximate Munsell color achieved………………………………………………………………………………….24

2. Summary of results from experiment 2……………………………………………….....25

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LIST OF FIGURES

Figure Page

1. Diagram of experiment 1………………………………………………………………...35 2. Diagram of experiment 2………………………………………………………………...35

3. Diagram of experiment 3………………………………………………………………...36

4. Diagram of experiment 4………………………………………………………………...36

5. Mean percent of prey consumed from control cells while testing

yellow during experiment 1…………………………………………………………...…37

6. Mean percent of prey consumed from experimental cells while testing yellow during experiment 1……………………………………………………………...37

7. Proportion of gray prey consumed while testing yellow during experiment 1……………………………………………………………………...38

8. Mean percent of prey consumed from control cells while testing

gray during experiment 1…………………………………...……………………………38

9. Mean percent of prey consumed from experimental cells while testing gray during experiment 1……………………………………………………..………….39 10. Proportion of control prey consumed while testing gray

during experiment 1……………………………………………………………..……….39

11. Mean percent of prey consumed and proportion of gray prey consumed while testing red during experiment 2……………………………………………...……40 12. Percent of red and gray prey consumed during each session of experiment 2…………..41 13. Mean percent of prey consumed and proportion of gray prey consumed while testing blue during experiment 2…………………………………………….……42 14. Percent of blue and gray prey consumed during each session of experiment 2………....43 15. Mean percent of prey consumed and proportion of gray prey consumed while testing yellow during experiment 2……………………………………….………44 16. Percent of yellow and gray prey consumed during each session of experiment 2……....45

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17. Mean percent of prey consumed and proportion of gray prey consumed while testing purple during experiment 2………………………………………….….…46 18. Percent of purple and gray prey consumed during each session of experiment 2….…....47 19. Mean percent of prey consumed and proportion of gray prey consumed while testing green during experiment 2…………………………………………………48 20. Percent of green and gray prey consumed during each session of experiment 2………..49 21. Mean percent of prey consumed and proportion of gray prey consumed while testing orange during experiment 2…………………………………………….…50 22. Percent of orange and gray prey consumed during each session of experiment 2……....51 23. Mean percent of prey consumed and proportion of gray prey consumed while testing white during experiment 2…………………………………………...……52 24. Percent of white and gray prey consumed during each session of experiment 2…….….53 25. Mean percent of prey consumed and proportion of gray prey consumed while testing red during experiment 3…………………………………………...………54 26. Percent of white and gray prey consumed during each session of experiment 3………..55 27. Mean percent of prey consumed and proportion of gray prey consumed while testing yellow with pattern during experiment 4…………………………….……56 28. Percent of yellow with pattern and gray prey consumed during

each session of experiment 3……………………………………………………...……..57

INTRODUCTION

Throughout the animal kingdom, animal body color and colorful signals serve many

purposes (Cott 1940; Edmunds 1974; Osorio & Vorobyev 2008). The color of an animal’s body

can be used for thermoregulation, camouflage, mimicry or communication (Cott 1940; Endler

1990). Animal body color can also be the indirect result of physiological processes, not related to

the previously stated examples (Edmunds 1991; Pawlik et al. 1995). Color and colorful patterns

are frequently used by animals to signal to conspecifics as well as predators (Cott 1940;

Edmunds 1974). Examples of this include: courtship displays, intraspecific communication and

interspecific communication (Osorio & Vorobyev 2008).

Animal body color and pattern is controlled by three distinct mechanisms: pigments, thin-

layer interference and scattering. Pigments are chemical compounds that absorb specific

wavelengths of light and reflect the remaining wavelengths. A receiver would perceive the color

of the light that is not absorbed. Interference is a mechanism by which iridescent coloration is

produced. A thin layer of wax or keratin causes light to be refracted as it enters and exits. This

causes two reflections of light that are in phase for specific wavelengths at specific angles.

Scattering produces a blue color in birds, fish and lizards. This blue coloration is created by a

coating of transparent material imbedded with tiny particles or air spaces covering the body of

the animal. As visible light enters the surface of these animals violet, blue and green wavelengths

are scattered and longer wavelengths are absorbed by lower layers. This results in a blue or

greenish blue coloration. Color can also be controlled behaviorally, such as color flashing in

lizards (Bradbury & Vehrencamp 1998). Some animals such as amphibians, reptiles, fish,

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cephalopods and insects can change their body color using dermal chromatophores. This is

accomplished by either controlling the dispersion of pigment granules in chromatophores or

covering clusters of pigment granules using muscle fibers connected to motor neurons (Parker

1948; Cloney & Florey 1968; Bagnara & Hadley 1973).

In many terrestrial ecosystems, brilliant colors and disruptive body patterns are important

warning signals that increase the ability of predators to learn to avoid undesirable or unpalatable

prey (Cott 1940; Edmunds 1974; Ruxton et al. 2004). Aposematism, or the use of warning

signals to denote the unpalatable or harmful aspects of an organism, is potentially a very

important aspect of predator-prey relationships, as warning signals may be more evolutionarily

advantageous than camouflage in many terrestrial systems (Cott 1940; Edmunds 1974; Ruxton et

al. 2004). Aside from color and pattern, warning signals can also be auditory, olfactory or

behavioral (Hauglund et al. 2006). According to Edmunds (1987), four criteria need to be met for

an organism to be considered an aposeme, or having an aposematic appearance or behavior: the

organism must (1) be sufficiently unpalatable to deter predators, (2) conspicuously displays

itself, (3) be avoided by some predators because of its conspicuousness, and (4) be better

protected from predation by the foregoing than by using an alternative strategy, such as crypsis.

While aposematism and mimicry are well-studied phenomena among terrestrial animals

such as insects, snakes and frogs (Mappes et al. 2005) and freshwater fish (Kruse & Stone 1984),

much less is known about the ecological importance of the warning signals of marine organisms.

There are many brightly colored or highly contrasting marine organisms that are also chemically

defended (Edmunds 1991). These include sponges, corals, flatworms, mollusks, annelids and the

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larvae of numerous marine organisms (Edmunds 1991; Pawlik et al. 1995; Ang & Newman

1998; Meredith et al. 2007). Many of these organisms are assumed to be aposematic, however

very few examples of aposematism have actually been demonstrated (Edmunds 1991). Most of

these potentially aposematic organisms would be preyed upon by fishes, so a first step in testing

for aposematism is to determine whether the potential predators are actually able to avoid

unpalatable prey based on visual signals. Tropical marine fish have been shown to use a variety

of signals to learn to avoid unpalatable prey (Gerhart 1991; Long & Hay 2006; Ritson-Williams

& Paul 2007), but it is unknown if marine fish can use visual signals alone to do so.

Marine fish, like other vertebrates, have a complex and highly specialized eye (Marshall

2000). The eyes of over 70 species of marine fish have been analyzed and have been found to

have at least two different types of cone cells (Marshall et al. 2006). Multiple types of cone cells

are a requirement for color vision (Siebeck et al. 2008). Five distinct types of photopigments

have been isolated from the eyes of coral reef fish: ultra-violet-sensitive, violet-sensitive, blue-

sensitive, green-sensitive, and red-sensitive (Munz & McFarland 1975; Loew & Lythgoe 1978;

Levine & MacNichol 1979; Lythgoe et al. 1994; McFarland & Loew 1994). As a result of this

evidence, it has been suggested that coral reef fish are physiologically capable of color vision

(Marshall 2000). Some coral reef fish may only be able to distinguish between 2 colors, while

others may have the ability to distinguish between three or more colors (Marshall 2000).

The physiological capabilities of the visual systems of labrid reef fish have been studied

(Barry & Hawrshyn 1999; Siebeck & Marshall 2000). Electrophysiological recordings were

made from the optic nerve of the Hawaiian saddle wrasse, a coral reef fish, to determine if that

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fish had the physiological capability to see the colors of conspecifics (Barry & Hawryshyn

1999). Peak spectral sensitivities of this fish correlated with the spectral reflectance of the body

colors of conspecifics, suggesting that this fish has the physiological capability to see the colors

of conspecifics (Barry & Hawryshyn 1999). A study on the transmission capabilities of ocular

media in another labrid fish, the bluehead T. bifasciatum, revealed that the eye is capable of

transmitting light throughout the majority of the visible light spectrum, indicating that there is no

physical boundary to color vision in this species (Siebeck & Marshall 2000). Until recently, no

behavioral studies had tried to test the ability of a marine fish to see color. In 2008, Siebeck et al.

sought to test whether a coral reef fish had the ability to distinguish between two colors, using

the damselfish, Pomacentrus aboinensis. The results of this study suggested that damselfish

could distinguish between at least two colors, blue and yellow, independent of brightness

(Siebeck et al. 2008).

There have been many studies that attempted to understand the ability of predators to

learn to avoid unpalatable prey have been made in terrestrial ecosystems (Sillen-Tullberg 1985;

Ham et al. 2006; Aronsson & Gamberale-Stille 2008), and considerably less in marine systems

(Gerhart 1991; Long & Hay 2006; Ritson-Williams & Paul 2007). However, no one has tested

the ability of a tropical marine fish to avoid unpalatable prey based on visual cues.

The purpose of the present study was to investigate the ability of a coral reef fish to learn

and remember to avoid unpalatable artificial prey items based on the visual signals of color and

pattern. Wild-caught bluehead wrasse, Thalassoma bifasciatum, a common Caribbean reef fish

species, were subjected to feeding assays using artificial prey of different types to assess the

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impact of various prey colors and patterns on predation. T. bifasciatum were used in this study

because they are one of the most abundant fish on Caribbean coral reefs, as well as being

generalist predators (Feddern 1965; Randall 1967). Thalassoma bifasciatum has been used in

numerous other behavioral and feeding assays (Warner and Hoffman 1980; Pawlik et al. 1995),

and responds well to laboratory conditions. Our study attempted to answer the following

questions: (1) Does relative prey position affect the ability of T. bifasciatum to learn and

remember to avoid unpalatable prey? (2) Do wild-caught T. bifasciatum have a pre-learned bias

towards specific colors of prey? (3) Are T. bifasciatum able to learn and remember to avoid

unpalatable prey using visual signals? (4) Do specific colors affect the ability of T. bifasciatum to

learn to avoid defended prey? (5) Are T. bifasciatum able to overcome a preexperimental bias?

(6) Do T. bifasciatum learn to avoid unpalatable prey more effectively using color or pattern

signals?

METHODS

Maintenance of experimental animals

Initial phase bluehead wrasse (Thalassoma bifasciatum) were collected off the coast of

Key Largo, FL and shipped to the University of North Carolina Wilmington, Center for Marine

Science in North Carolina. Approximately 100 fish were kept in five 90 x 40 x 35 cm aquaria

filled with filtered, natural seawater from Masonboro Sound, North Carolina. The five tanks

were part of a re-circulating seawater system, from which 25% of the volume was changed

weekly. Two of the aquaria were used as experimental tanks and the other 3 were used to house

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the remaining fish. All 5 aquaria were subject to a 12:12 h light:dark cycle using 175-watt metal

halide lights. Seawater temperature was maintained at 21-23°C and salinity was maintained at

30-32 psu. Fish were never food deprived; they were fed to satiety up to three times a day with

TetraMin Tropical Flake Food. The 2 aquaria used for experiments were divided into 5 cells

each using perforated plastic dividers. This resulted in 10 cells measuring 20 x 40 x 35 cm each.

Aquaria bottoms were covered in limestone gravel and provided with several sections of PVC

pipe in which fish would hide, at least 1 per cell for divided aquaria.

Artificial prey

To create the artificial prey, the protocol based on Chanas & Pawlik (1995) was used in

which 1 gram of powdered squid mantle, 0.5 g of carrageenan and 20 ml of DI water were

blended together until homogeneous. Powdered food coloring was added (Creative Cutters

Blossom Tints), and then the mixture was microwaved for 20 seconds or until it began to boil.

The molten mixture was then poured into a 1.5 x 1.7 cm mold, overlaying fiberglass window-

screen mesh that was completely wrapped around a 10 x 10 cm stone tile. This created an

artificial prey item that covered exactly 10 x 10 rectangles of window-screen, each rectangle was

approx. 1.5 x 1.7 mm.

Prey color was carefully controlled in the formulation of all artificial prey. Table 1 lists

concentrations of food colorant used to create each color prey, as well as the resulting color on

the Munsell scale and gray scale. Food colorings were palatable to assay fish at 4x experimental

concentration in pellet feeding assays (Pawlik et al. 1995), as well as at 2x experimental

concentration in masked feeding assays. In masked feeding assays, black food coloring

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(vegetable charcoal) was used to mask the color of the powder colorant. Prey palatability was

controlled by adding denatonium benzoate to unpalatable prey items at a concentration of 2

mg/ml of gel-based food prior to microwaving. Denatonium benzoate is an extremely bitter, non-

toxic compound marketed under the names Bitrex™ and Aversion™, and used to prevent nail

biting in humans, chewing in dogs, and to make denatured alcohol unpalatable.

Pre-experimental training

Prior to testing, fish were presented with a brown-colored prey (Table 1), a color not used

during any experiments. Fish were trained to eat prey from the screen wrapped tile. Training

lasted 3 hours and was conducted each day with fresh prey items until fish consumed 50% of the

prey item from each tile within that time period. Once this was achieved, the testing would begin

the following day.

Experiment 1: Does relative prey position affect the ability of T. bifasciatum to learn to avoid

unpalatable prey?

This experiment tested the hypothesis that T. bifasciatum would be able to learn to avoid

unpalatable prey regardless of relative position. This experiment consisted of two 3-hour

sessions, a learning phase and a memory test. Each session was separated by a 1-hour delay. The

10 cells in the 2 fish aquaria were divided into 5 control cells and 5 experimental cells, with

control and experimental cells randomly assorted within each tank. In this experiment, 3 fish

were placed in each of these 10 cells. During the learning phase, both the experimental and

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control cells received identical tiles each with two prey items, a yellow unpalatable prey and a

gray palatable prey (yellow was chosen due to the apparent lack of bias against this color).

Following the 1-hour delay, both the treatment and control cells received yellow and gray

palatable prey, although the position of the yellow and gray prey in the experimental cells were

switched and the position of the prey in the control cells remained identical to the learning phase.

A diagram of this experiment is provided (Figure 1). This experiment was repeated using two

similarly colored gray prey; this was done to determine whether fish could learn to avoid an

unpalatable prey item based on relative position when prey color was the same. The amount of

prey consumed for each treatment was quantified by counting the number of rectangles of

window screen mesh exposed under the prey. If fewer than 10 squares of prey were removed

from any tile in total, that tile was scored as having no response and not used in the analysis. If

greater than five of the ten tiles in any experimental run had no response, the experimental run

was discarded.

Experiment 2: Do wild-caught T. bifasciatum have a pre-learned bias towards specific colors of

prey?

Is T. bifasciatum able to learn to avoid unpalatable prey using visual signals?

Do specific colors affect the ability of T. bifasciatum to learn to avoid defended prey?

Following pre-experimental training, groups of three fish were placed into each of the 10

cells within the experimental aquaria. This experiment consisted of four sessions over two days,

with two sessions tested each day. Each of the four sessions lasted 3 hours and on each day the

two sessions were separated by a 1-hour delay. On the first day a color bias test and a bias

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durability test were conducted, followed by a learning phase and memory test on the second day.

This experiment was repeated using seven different colors, to test the response of the fish to a

wide range of specific colors. Each experiment tested a treatment color prey against a gray

control color prey (see Table 1 for a list of colors used). For the color bias test, one treatment

color and one control prey, both palatable, were presented to each group of fish. After the delay,

for the bias durability test, fish were presented with freshly prepared, identical prey to test if any

color bias persisted. On the second day, during the learning phase, fish were presented with

freshly prepared prey of the same color as the previous day, but the treatment color was

unpalatable After the delay, during the memory test, freshly prepared prey of the same color

were again presented, but both were palatable. A diagram of this experiment is provided (Figure

2). Individual fish were used for multiple experiments, but never used more than once for the

same color treatment.

Experiment 3: Is T. bifasciatum able to overcome a preexperimental bias?

The results of Experiment 2 showed that T. bifasciatum have a pre-learned or innate

negative bias against the color red. Experiment 3 tested the ability of T. bifasciatum to overcome

this color bias. All methodological aspects of this experiment were identical to those in 2, but

this experiment consisted of only 3 sessions: a color bias test on the first day and a learning

phase and memory test on the second day. During the color bias test and the memory test, both

red (treatment) and gray (control) prey were palatable. During the learning phase the gray prey

was made unpalatable while the red control prey remained palatable. A diagram of this

experiment is provided (Figure 3).

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Experiment 4: Does T. bifasciatum learn to avoid unpalatable prey more effectively using color

or pattern signals?

Experiment 4 tested whether T. bifasciatum would learn to avoid unpalatable prey more

effectively using color or pattern signals. The subjects, housing, artificial prey and pre-

experimental training were all identical to those in previous experiments. All methodological

aspects of this experiment were identical to those in experiment 2, except for the memory test. In

sessions 1 through 3, all of the same protocol was followed, except the treatment prey was

yellow and contained 4 dots of black prey mixture imbedded into each prey (in a pattern similar

to the 4-face of a die), and the control prey was made a light gray. During session 4 the 10

groups of fish (1 in each cell) were divided into three sub-groups. All sub-groups received a

treatment prey and a light gray control prey. Sub-group 1 received a treatment prey with the

same color and pattern that they received during sessions 1-3. Sub-group 2 received a treatment

prey that was yellow with no pattern. Sub-group 3 received a treatment prey that had pattern but

its color matched the control prey (light gray). A diagram of this experiment is provided (Figure

4).

Statistical analysis

Paired t-tests were used to determine if there was a difference in the amount of prey

consumed of each type during each session. Unpaired t-tests were used to determine if there was

a difference in the proportion of control prey consumed between the color bias test and memory

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test in each experiment. This was done to determine if the fish learned to avoid the treatment

prey based on visual signals alone. For experiment 1, unpaired t-tests were used to determine if

there was a difference in the proportion of control prey consumed between experimental and

control cells during the memory test. For experiment 4, an ANOVA was used to determine if

there were differences in the proportion of control prey consumed between sub-groups during the

memory test.

RESULTS

Experiment 1

Thalassoma bifasciatum were able to learn to avoid unpalatable prey using a visual cue,

color, regardless of the relative position of the unpalatable prey item (paired t-tests, p < 0.05; Fig.

5, 6). When yellow was tested, fish consumed a significantly greater amount of gray prey than

yellow prey in both sessions of the experiment for both the control fish and experimental fish

(paired t-tests, p < 0.05; Fig. 5,6). There was no difference in the proportion of gray prey

consumed between the control and experimental groups in either of the two sessions of the

experiment (t-test, p < 0.05; Fig. 7).

When gray was tested, there was no difference in the mean percentage of prey consumed

between the control prey and treatment prey in either of the two sessions of the experiment for

both the control fish and experimental fish (paired t-tests, p > 0.05; Fig. 8,9). There was also no

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difference in the proportion of control prey consumed between the control and experimental

groups in either of the two sessions of the experiment (t-test, p > 0.05; Fig. 10).

Experiment 2

Fish exhibited a strong bias against red prey, consuming a significantly greater amount of

gray prey than red prey during both the color bias and bias durability tests (paired t-tests, p <

0.05; Fig. 11,12). Fish also demonstrated an ability to learn to avoid unpalatable red prey,

consuming a significantly greater proportion of gray prey in the memory test than in the color

bias test (t-test, p < 0.05; Fig. 11).

Fish exhibited a strong bias against blue prey, consuming a significantly greater amount

of gray prey than blue prey during both the color bias and bias durability tests (paired t-tests, p <

0.05; Fig. 13,14). Fish also demonstrated an ability to learn to avoid unpalatable blue prey,



Fish exhibited a weak bias against yellow prey, consuming a significantly greater amount

of gray prey than yellow prey during the color bias test (paired t-test, p < 0.05; Fig. 15,16), but

not the bias durability test (paired t-tests, p > 0.05; Fig. 15,16). Fish also demonstrated an

inability to learn to avoid unpalatable yellow prey, consuming no difference in the proportion of

gray prey in the memory test and color bias test (t-test, p > 0.05; Fig. 15).

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Fish exhibited a strong bias against purple prey, consuming a significantly greater

amount of gray prey than purple prey during both the color bias and bias durability tests (paired

t-tests, p < 0.05; Fig. 17,18). Fish also demonstrated an inability to learn to avoid unpalatable

purple prey, consuming no difference in the proportion of gray prey in the memory test and color

bias test (t-test, p > 0.05; Fig. 17).

Fish exhibited a strong bias against green prey, consuming a significantly greater amount

of gray prey than green prey during both the color bias and bias durability tests (paired t-tests, p

< 0.05; Fig. 19,20). Fish also demonstrated an inability to learn to avoid unpalatable green prey,

consuming no difference in the proportion of gray prey in the memory test and color bias test (t-

test, p > 0.05; Fig. 19).

Fish exhibited no bias against orange prey, consuming no difference in the amount of

gray prey and orange prey during both the color bias and bias durability tests (paired t-tests, p >

0.05; Fig. 21,22). Fish also demonstrated an ability to learn to avoid unpalatable green prey,



Fish exhibited no bias against white prey, consuming no difference in the amount of gray

prey and white prey during both the color bias and bias durability tests (paired t-tests, p > 0.05;

Fig. 23,24). Fish also demonstrated an inability to learn to avoid unpalatable white prey,

consuming no difference in the proportion of gray prey in the memory test and color bias test (t-

test, p > 0.05; Fig. 23).

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Experiment 3

When red was used to test if fish can learn to overcome a bias against a red prey, fish

exhibited a bias against red prey (similar to experiment 2), consuming a significantly greater

amount of gray prey than red prey during the color bias test (paired t-tests, p < 0.05; Fig. 25,26).

Fish also demonstrated an ability to learn to overcome a bias against red prey, consuming no

difference in the proportion of gray prey in the memory test and color bias test (t-test, p > 0.05;

Fig. 25).

Experiment 4

When yellow with pattern was used to test if fish learn to avoid unpalatable prey more

effectively using color or pattern signals, fish exhibited no bias against yellow with pattern prey,

consuming no difference in the amount of gray prey and yellow with pattern prey prey during

both the color bias and bias durability tests (paired t-tests, p > 0.05; Fig. 27,28). Fish also

demonstrated an inability to learn to avoid unpalatable yellow with pattern prey, consuming no

difference in the proportion of gray prey in the memory test and color bias test in the yellow with

pattern sub-group (t-test, p > 0.05; Fig. 27). Fish exhibited no difference in the ability to learn to

avoid unpalatable prey using color or pattern signals, there was no difference in the proportion of

gray prey consumed between sub-groups (ANOVA, p > 0.05; Fig. 27).

DISCUSSION

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Fish were able to learn to avoid unpalatable prey using visual signals. Their ability to

learn to avoid unpalatable prey using visual signals was dependent on the specific color used.

Fish were able to learn to avoid red, blue and orange prey effectively using only visual cues. Fish

were to unable to successfully learn to avoid purple, green, yellow and white prey. This variation

in responses was likely due to factors, such as innate or pre-learned biases against specific colors

of prey. The ability to learn to avoid unpalatable prey based on visual cues is also likely affected

by the visual system sensitivity of the bluehead wrasse. These results are contrary to those found

in an experiment using wild-caught great tits, where the researchers found no difference in

response to different colors when training these birds to avoid unpalatable prey (Ham et al. 2006)

Relative prey position had no effect on the ability of fish to learn to avoid unpalatable

prey. Instead our study indicates that visual cues such as color are one of the primary means by

which these fish learn to avoid unpalatable prey. These fish had a strong pre-learned bias against

red, blue, purple and green prey when compared to the gray control prey. There was a weak pre-

learned bias against yellow prey and no pre-learned bias against orange or white prey. Fish were

unable to learn to avoid gray colored prey when compared to red prey, which fish had a strong

bias against. It was also found that fish learn to avoid unpalatable prey more effectively using

color rather than pattern.

The fish used in this study were captured from the wild and as a result were expected to

have pre-learned biases towards what they would and would not attempt to eat. This study found

that fish had a strong pre-learned bias against some colors, a weak pre-learned bias against one

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color and no pre-learned bias against others. No other studies that the author is aware of have

tested color or pattern biases in a coral reef fish. However this subject has be studied in the naïve

and wild-caught great tits, Parus major, and the authors found that naïve and wild-caught birds

avoided similar colors of prey, regardless of prey conspicuousness or gregariousness, thus

indicating an inherited avoidance response to specific colors (Lindström et al. 1999). The present

study did not compare naïve to wild-caught fish, but the variation in responses either indicates

the result of pre-capture avoidance learning or a genetic basis for avoidance responses to

different colors.

Fish were able to learn to overcome a bias against red prey prey. This demonstrates the

flexibility in learning of these fish. This ability has been demonstrated using cleaner wrasse,

Labroides dimidiatus, in a reverse reward contingency test, where these fish were trained and

learned to choose the less preferred reward to ultimately receive the more preferred reward

(Danisman et al. 2010).

There was no difference in the ability of these fish to learn to avoid unpalatable prey

using color or pattern signals. This result is contrary to a response observed in domestic chicks,

Gallus gallus, in which the chicks attended to color and not pattern during avoidance learning

(Aronsson & Gamberale-Stille 2008). During this experiment, the fish had no bias against yellow

with pattern prey, four black dots. Fish were unable to learn to avoid yellow with pattern prey

based on visual signals, however they did consume a greater proportion of gray prey during the

memory test than the color bias test, although the difference was not significant. During the

memory test there was no difference in the proportion of grey prey consumed by any of the

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groups. This indicates that fish attended to color and pattern equally when learning to avoid

unpalatable prey.

The wrasse used in this study never learned to avoid 100% of the previously unpalatable

prey, indicating that these fish continually test potential prey by taking small bites. This is

contradictory to work done on blue jays and butterflies, which found that blue jays learned to

avoid attacking monarch and other unpalatable or emetic butterflies. The blue jays would

consume a whole insect then reject or vomit it out and subsequently avoid that butterfly and all

mimics (Brower 1958a-c).

As the physiological capabilities of the visual systems of marine predators continue to be

described (Levine & MacNichol 1979; Barry & Hawryshyn 1999; Siebeck & Marshall 2000;

Marshall et al. 2006), it becomes necessary to understand the cognitive capacities and behavioral

responses of these organisms. Although, due to the variable presence of light in the ocean visual

signals, such as color and pattern, and the ability of predators to perceive such signals are not as

important in marine systems as terrestrial systems when attempting to understand the behavior of

visual predators.

18

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24

Table 1. List of experimental colors used, food coloring name and product #, concentration of

food coloring used and approximate munsell color achieved.

Color Item Name & Number

Concentration (g/mL)

Munsell Color/

Grayscale Red Red- #C443 0.002 7.5 R 5/18

Orange Tangerine-

#C446 0.002 10 R 6/16

Yellow Egg Yellow-

#C429 0.0015 10 Y 9/12

Blue Ocean Blue-

#C418 0.0015 10 B 3/6

Green Foliage Green-

#C419 0.0015 7.5 GY 4/10

Purple Aubergine-

#C449 0.0015 5 RP 2/10 Brown #C424 0.002 2.5 YR 3/8 White None X 10 Light Gray Black #C422 0.0001 8 Gray Black #C422 0.00025 5 Black Black #C422 0.001 3

25

Table 2. Summary of results from experiment 2. Preexperimental bias was strong if fish

consumed significantly less of treatment prey in both session 1 and 2 of experiment, weak if fish

only consumed significantly less of treatment prey in session 1 of experiement and none if there

was no difference in amount of prey consumed for both session 1 and 2 of experiment. Fish

learned to avoid treatment prey based on visual cues alone if they consumed a significantly

greater proportion of control prey in session 4 (memory test) than in session 1 (color bias test).

Color Preexperimental Bias

Learned to

Avoid Red Strong Yes Blue Strong Yes

Yellow Weak No Purple Strong No Green Strong No

Orange None Yes White None No

26

FIGURE LEGENDS

Figure 1. Diagram of experiment 1.




Figure 5. Mean percent of prey consumed from control cells while testing yellow during

experiment 1. Error bars represent standard error. Arrows indicate the amount of time between

each part of the experiment. Significant differences in amount of prey consumed for each part of

the experiment are represented by a * (paired t-test P ≤ 0.05). The sample sizes are N = 5 for

session 1 and N = 6 for session 2 of the experiment.

Figure 6. Mean percent of prey consumed from experimental cells while testing yellow during


each part of the experiment. Significant differences in amount of prey consumed for each part of

the experiment are represented by a * (paired t-test P ≤ 0.05). The sample sizes are N = 7 for

session 1 and N = 10 for session 2 of the experiment.

27

Figure 7. Proportion of gray prey consumed while testing yellow during experiment 1. Error bars

represent standard error. Arrows indicate the amount of time between each part of the

experiment. There is no difference in the proportion of control prey consumed between fish in

control cells and experimental cells in sessions 1 or 2 of the experiment (unpaired t-test P >

0.05).

Figure 8. Mean percent of prey consumed from control cells while testing gray during


each part of the experiment. There is no difference in the mean percent of prey consumed

between control and treatment prey in either session of the experiment (paired t-test P > 0.05).

The sample sizes are N = 10 for session 1 and N = 10 for session 2 of the experiment.

Figure 9. Mean percent of prey consumed from experimental cells while testing gray during


each session of the experiment. There is no difference in the mean percent of prey consumed

between control and treatment prey in either session of the experiment (paired t-test P > 0.05).

The sample sizes are N = 9 for session 1 and N = 9 for session 2 of the experiment.

Figure 10. Proportion of control prey consumed while testing gray during experiment 1. Error

bars represent standard error. There is no difference in the proportion of control prey consumed

between fish in control cells and experimental cells in sessions 1 or 2 of the experiment

(unpaired t-test P > 0.05).

28

Figure 11. Mean percent of prey consumed and proportion of gray prey consumed while testing

red during experiment 2. Each session of the experiment lasted 3 hours. Error bars represent

standard error. Arrows indicate the amount of time between each session of the experiment.

Vertical bar plot represents the mean percent of prey consumed and the scatter plot represents the

proportion of gray prey consumed. The sample sizes for each session of the experiment were N =

13 for session 1, N = 14 for session 2, N = 14 for session 3 and N = 16 for session 4. Significant

differences in the mean percent of prey consumed between red and gray prey for each session of

the experiment are marked with * (paired t-test P ≤ 0.05). There is a significant difference

between the proportion of gray prey consumed in sessions 1 and 4 of the experiment (unpaired t-

test P ≤ 0.05).

Figure 12. Percent of red and gray prey consumed during each session of experiment 2. Each

session of the experiment lasted 3 hours. Arrows represent amount of time in between each

session of the experiment.


blue during experiment 2. Each session of the experiment lasted 3 hours. Error bars represent





differences in the mean percent of prey consumed between blue and gray prey for each session of

29

the experiment are marked with * (paired t-test P ≤ 0.05). There is a significant difference


test P ≤ 0.05).

Figure 14. Percent of blue and gray prey consumed during each session of experiment 2. Each

individual replicate response is shown. Each session of the experiment lasted 3 hours. Arrows

represent amount of time in between each session of the experiment.


yellow during experiment 2. Each session of the experiment lasted 3 hours. Error bars represent





differences in the mean percent of prey consumed between yellow and gray prey for each session

of the experiment are marked with * (paired t-test P ≤ 0.05). There is not a significant difference


test P > 0.05).

Figure 16. Percent of yellow and gray prey consumed during each session of experiment 2. Each



30


purple during experiment 2. Each session of the experiment lasted 3 hours. Error bars represent





differences in the mean percent of prey consumed between purple and gray prey for each session



test P > 0.05).

Figure 18. Percent of purple and gray prey consumed during each session of experiment 2. Each




green during experiment 2. Each session of the experiment lasted 3 hours. Error bars represent





differences in the mean percent of prey consumed between green and gray prey for each session


31


test P > 0.05).

Figure 20. Percent of green and gray prey consumed during each session of experiment 2. Each




orange during experiment 2. Each session of the experiment lasted 3 hours. Error bars represent





differences in the mean percent of prey consumed between orange and gray prey for each session



test P > 0.05).

Figure 22. Percent of orange and gray prey consumed during each session of experiment 2. Each



32


white during experiment 2. Each session of the experiment lasted 3 hours. Error bars represent





differences in the mean percent of prey consumed between white and gray prey for each session

of the experiment are marked with * (paired t-test P ≤ 0.05). There is a significant difference


test P ≤ 0.05).

Figure 24. Percent of white and gray prey consumed during each session during experiment 2.

Each individual replicate response is shown. Each session of the experiment lasted 3 hours.

Arrows represent amount of time in between each session of the experiment.


red during experiment 3. Each session of the experiment lasted 3 hours. Error bars represent




18 for session 1, N = 11 for session 2 and N = 13 for session 3. Significant differences in the

mean percent of prey consumed between red and gray prey for each session of the experiment

are marked with * (paired t-test P ≤ 0.05). There is not a significant difference between the

33

proportion of gray prey consumed in sessions 1 and 3 of the experiment (unpaired t-test P >

0.05).

Figure 26. Percent of red and gray prey consumed during each session of experiment 3. Each




yellow with pattern during experiment 4. Each session of the experiment lasted 3 hours. Error

bars represent standard error. Arrows indicate the amount of time between each session of the

experiment. Vertical bar plot represents the mean percent of prey consumed and the scatter plot

represents the proportion of gray prey consumed. The sample sizes for each session of the

experiment were N = 29 for session 1, N = 28 for session 2, N = 28 for session 3, N = 10 for the

yellow only group in session 4, N = 8 for the yellow with pattern group in session 4 and N = 10

for the pattern only group in session 4. Significant differences in the mean percent of prey

consumed between yellow with pattern and gray prey for each session of the experiment are

marked with * (paired t-test P ≤ 0.05). There is a significant difference between the proportion of

control prey consumed in the yellow only group and pattern only group during session 4 of the

experiment (unpaired t-test P ≤ 0.05). There was no difference in the proportion of control prey

consumed between the yellow with pattern group and any other group (unpaired t-test P > 0.05).

34

Figure 28. Percent of control and treatment prey consumed during each session of experiment 4.

Each individual replicate response is shown. Each session of the experiment lasted 3 hours.

Arrows represent amount of time in between each session of the experiment.

35

Figure 1

Figure 2

36

Figure 3

Figure 4

37

1 hour

Experiment session

1 2

Mea

n pe

rcen

t of p

rey

cons

umed

0

20

40

60

80

100Yellow- Mean Gray- Mean

* *

Figure 5

Experiment session

1 2

Mea

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rcen

t of p

rey

cons

umed

0

20

40

60

80

100Yellow- Mean Gray- Mean

**

1 hour

Figure 6

38

Experiment session

1 2

Pro

porti

on o

f gra

y pr

ey c

onsu

med

0.0

0.2

0.4

0.6

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1.0

Control CellsExperimental Cells

Figure 7

Experiment session

1 2

Mea

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t of p

rey

cons

umed

0

20

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60

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100Treatment- Mean Control- Mean

1 hour

Figure 8

39

Experiment session

1 2

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rey

cons

umed

0

20

40

60

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100Treatment- Mean Control- Mean

1 hour

Figure 9

Experiment session

1 2

Pro

porti

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rey

cons

umed

0.0

0.2

0.4

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1.0

Control CellsExperimental Cells

Figure 10

40

Experiment session

1 2 3 4

Mea

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t of p

rey

cons

umed

0

20

40

60

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100

Pro

porti

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0.0

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1.0

Red Gray

* *

**

1 hour 1 hour17 ~hours

Figure 11

41

Figure 12

42

Experiment session

1 2 3 4

Mea

n pe

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rey

cons

umed

0

20

40

60

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Pro

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0.0

0.2

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1.0

BlueGray

**

**


Figure 13

43

Figure 14

44

Experiment session

1 2 3 4

Mea

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t of p

rey

cons

umed

0

20

40

60

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100

Pro

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0.0

0.2

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0.6

0.8

1.0

YellowGray

** *

17 ~hours 1 hour1 hour

Figure 15

45

Figure 16

46


Experiment session

1 2 3 4

Mea

n pe

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cons

umed

0

20

40

60

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100

Pro

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0.0

0.2

0.4

0.6

0.8

1.0

PurpleGray

**

**

Figure 17

47

Figure 18

48

Experiment session

1 2 3 4

Mea

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t of p

rey

cons

umed

0

20

40

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Pro

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0.0

0.2

0.4

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1.0

GreenGray

**

**


Figure 19

49

Figure 20

50

Experiment session

1 2 3 4

Mea

n pe

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t of p

rey

cons

umed

0

20

40

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Pro

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0.0

0.2

0.4

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0.8

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OrangeGray

* *


Figure 21

51

Figure 22

52

Experiment session

1 2 3 4

Mea

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0

20

40

60

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Pro

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WhiteGray

*


Figure 23

53

Figure 24

54

1 hour17 ~hours

Experiment session

1 2 3

Mea

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0

20

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RedGray

* *

Figure 25

55

Figure 26

56

*

Experiement session

1 2 3 4

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1.0Color & Pattern Gray Color & PatternColor OnlyPattern Only

*

1 hour 17 ~hours 1 hour

Figure 27

57

Figure 28

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