The Role of Vision in Sexual Signaling in the Blue Crab

by

Jamie Baldwin Fergus

Department of Biology Duke University

Date:______Approved:

______Sönke Johnsen, Supervisor

______Manuel Leal

______Daniel Rittschof

______William Kier

______Kenneth Lohmann

Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biology in the Graduate School of Duke University

2012

ABSTRACT

The Role of Vision in Sexual Signaling in the Blue Crab

by

Jamie Baldwin Fergus

Department of Biology Duke University

Date:______Approved:

______Sönke Johnsen, Supervisor

______Manuel Leal

______Daniel Rittschof

______William Kier

______Kenneth Lohmann

Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biology in the Graduate School of Duke University

2012

Copyright by Jamie Baldwin Fergus 2012

Abstract

The dissertation work discussed here focuses on the behavioral and

physiological aspects of visual sexual signaling in the blue crab, Callinectes

sapidus. The blue crab has a pair of apposition compound eyes that are relatively

acute (1.5º resolution) for an arthropod. The eyes have two photopigments

sensitive to blue (λmax = 440 nm) and green (λmax=500 nm) light, allowing for

simple color vision. Visual cues and signals are used during antagonistic and

sexual communication and primarily involve claw-waving motions. A primary feature of the blue crab morphology is its sexually dimorphic claw coloration; males have blue and white claws and females have red claws. However, despite the potential for interesting color signaling, visual cues have typically been considered non-important, particularly in sexual communication where chemical cues have dominated blue crab signaling studies.

In a series of experiments designed to simultaneously test the role of visual cues in mating behavior and blue crab color vision, I tested males’ responses to photographs of females with differently colored claws. I found that photographs of females elicited male courting behaviors. I also found that males preferred females with red claws over those with white or isoluminant (i.e.

iv

matched brightness) gray claws. The discrimination of red from isoluminant grey

showed the use of color vision in male mate choice.

In natural populations, the claws of sexually mature females vary from

light orange to deep red. To determine males’ abilities discriminate between

similar colors, I tested male color preferences for red against several shades of

orange varying in brightness. Overall, males showed an innate preference for

red-clawed females over those with variations of orange claws. However, in tests

between red and orange shades similar in both brightness and hue, male blue

crabs did not show a distinct preference, suggesting that males are either not able

or not motivated to discriminate between these shades. Further, my results

suggest that male blue crabs may use a mixture of chromatic and achromatic

cues to discriminate between long-wavelength colors.

After confirming the use of color in mate choice, I focused on the role of claw color in intraspecific communication. To quantify claw coloration, I measured spectral reflectance of claws of a blue crab population in North

Carolina. In both sexes, the color of the claw varied with reproductive maturity and may act as a cue of reproductive readiness. Additionally, there was individual variation in claw color which could indicate individual quality. I have

v

modeled the appearance of claw coloration to the blue crab eye and found that

these color differences are visible to the blue crab eye and potentially signal

gender, reproductive readiness, and/or individual quality.

After investigating male mate choice, I began investigating visual aspects

of female mating behavior. In the blue crab, like many crustaceans, courtship

occurs during the female molting cycle and copulation takes place after the

female has shed her exoskeleton. In crustaceans and other arthropods with

compound eyes, the corneal lens of each facet is part of the exoskeleton and thus

shed during molting. I used optomotor assays to evaluate the impact of molting

on visual acuity (as measured by the minimum resolvable angle αmin) in the female blue crab. I found that visual acuity decreases substantially in the days prior to molting and is gradually recovered after molting. Prior to molting, αmin

was 1.8°, a value approximating the best possible acuity in this species. In the 24

hours before molting, αmin increased to a median of 15.0° (N=12), an eight-fold

drop in visual acuity. Six days after molting, αmin returned to the pre-molting

value. Micrographs of C. sapidus eyes showed that a gap between the corneal lens

and the crystalline cone appeared approximately five days prior to shedding and

increased in width the process progressed. This separation was likely responsible

vi

for the loss of visual acuity observed in behavioral tests. Since mating is limited to the female’s pubertal molt, a reduction in acuity during this time may have an effect on the sensory cues used in female mate choice. These results may be broadly applicable to all arthropods that molt and have particular importance for crustaceans that molt multiple times in their lifetime or have mating cycles paired with molting.

vii

Dedication

This work is dedicated to my family and my husband, Josh.

viii

Contents

Abstract…….………………………………………………………………………….iv

List of Tables………………………………………………………………………….xii

List of Figures………………………………………………………………………..xiii

Acknowledgements………………………………………………………………….xv

1. Introduction………………………………………………………………………..1

2. The Importance of Color in Mate Choice in the Blue Crab Callinectes sapidus……...…………………………………………………………………………8

2.1 Methods………………………………………………………………………14

2.1.1. Specimen collection and care:………………………………………14

2.1.2. Behavioral experiments………………………………………………15

2.2. Results………………………………………………………………………..20

2.3. Discussion……………………………………………………………………24

3. Male Blue Crabs Callinectes sapidus Use Both Chromatic and Achromatic Cues in Mate Choice………………………………………………………………...32

3.1. Methods………………………………………………………………………36

3.1.1. Study species:…………………………………………………………36

3.1.2. Measuring spectral reflectance of claw color:………………………36

3.1.3. Calculation of relative luminance and opponency to the blue crab eye:……………………………………………………………………………..38

3.1.4. Behavioral experiments evaluating male color preferences:……..40

3.2. Results………………………………………………………………………..45

3.2.1. Spectral reflectance of the claws of Callinectes sapidus:………...45

ix

3.2.2. Relative luminance and opponency of natural claw colors………..45

3.2.3. Behavioral Experiments:………………………………………………47

3.3. Discussion…………………………………………………………………….50

3.3.1. Role of claw color………………………………………………………53

4. The effects of molting on the visual acuity of the blue crab Callinectes sapidus………………………………………………………………………….58

4.1. Methods………………………………………………………………………..60

4.1.2. Study Species…………………………………………………………..60

4.1.2. Optomotor Response………………………………………………….61

4.1.3. Analysis of optomotor data……………………………………………64

4.1.4. Eye Morphology………………………………………………………..65

4.2 Results…………………………………………………………………………66

4.2.1. Optomotor Response………………………………………………….66

4.2.2. Morphology of eyes during molting…………………………………..69

4.3. Discussion…………………………………………………………………….72

5. Conclusions…………………………………………………………………………81

5.1. Color Vision in the Blue Crab………………………………………………..81

5.2. Role of color in male mate choice…………………………………………..82

5.3 The Role of Vision in Female Mate Choice………………………………...83

6. Future Directions in Callinectes Research……………………………………….84

6.1. The Role of Claw Color in Species Identification………………………….84

6.2. Investigating the Effects of Turbidity on the Perception of Visual Cues. 85

6.3. Significance of the Research………………………………………………..86

x

Appendix A……………………………………………………………………………..88

References……………………………………………………………………………..93

Biography……………………………………………………………………………..106

xi

List of Tables

Table 1. Estimation of perceived brightness of images of claw dactyls used in this study… ……………………………………………………………………...... 19

Table 2. Individual results of Red-White Binary Choice Experiment…………….23

Table 3. Individual results of Red-Black Binary Choice Experiments……………24

Table 5. Individual results of optomotor response tests showing the minimum resolvable angle αmin before, during and after molting…………………………….69

Table 4. The results of binary choice experiments testing male preferences for photographs of females with red or orange claw coloration………………………91

xii

List of Figures

Figure 1. Ventral views of male and female C. sapidus and exterior and interior views of the claws……………………………………………………………………..12

Figure 2. Claws of female C. sapidus……………………………………………….13

Figure 3. The three photographs of female C. sapidus used in the binary choice experiments…………………………………………………………………………….16

Figure 4. Spectral sensitivity and spectral reflectances of C. sapidus…………...18

Figure 5. Results of the choice experiments………………………………………..22

Figure 6. Mean spectral reflectances and photographs of both the exterior and interior exterior claw surfaces………………………………………………………...33

Figure 7. Mean spectral reflectances and standard error of the red and orange colors used in behavioral experiments………………………………………………42

Figure 8. One of the six photographs used in binary choice tests, pictured here with claws colored red…………………………………………………………………43

Figure 9. Achromatic and chromatic cues of natural crab claws with 95% confidence intervals…………………………………………………………………...46

Figure 10. A) One-dimensional plots of relative luminance and opponency of the experimental colors used during binary choice trials………………………………48

Figure 11. Results of Binary Choice Experiments………………………………….49

Figure 12. Coloration of species in the genus Callinectes………………………...54

Figure 13. Diagram illustrating the optomotor apparatus………………………….62

Figure 14. Box-and-whisker plot showing the minimum resolvable angle αmin before, during and after molting……………………………………………………...67

Figure 15. Multi-plot showing the time courses of change in minimum resolvable angle, αmin, during the molting process in individual Callinectes sapidus………68

Figure 16. Photos of C. sapidus eye before, during, and after molting………….71

Figure 17. The effects of molting on vision…………………………………………73

xiii

Figure 18. Diagrams of apposition compound eyes and ommatidia showing the progressive changes in the eye during molting, including the possible light path in the ommatidia (modified from Land and Nilsson 2002)………………………..75

xiv

Acknowledgements

I thank my advisor Sönke Johnsen for years of guidance during my graduate work. His help, advice, and friendship have been much appreciated. In addition, I thank

Mark Hooper for many fruitful discussions regarding blue crabs as well as his generous assistance in collecting and analyzing specimens. I also thank Manuel Leal for many critical discussions and comments on several aspects of my work, as well as for his assistance as a mentor. For the work discussed in Chapter’s One and Two, I would like to thank Drs. Tom Cronin and Almut Kelber for their input. Finally, I thank my dissertation committee Sönke Johnsen, Manuel Leal, Bill Kier, Ken Lohmann, and Dan

Rittschof, as well as all the members of the Johnsen Lab.

xv 1. Introduction

Mating is a complex process that requires, at minimum, the meeting of

compatible, fertile individuals and proper environmental conditions (Campbell

and Reece, 2002). In more complicated systems, animals may perform a series of

courtship rituals to attract a mate, arouse the breeding state in both genders, and

finally achieve copulation (Frings and Frings, 1977; Alcock, 2005). These rituals

involve varying signal modalities including tactile, chemical, visual, and mechanical channels (Johnstone, 1996; Candolin, 2003). Often, signalers will employ more than one modality, using multiple channels to enhance or alter the message of the signal (Candolin, 2003; Alcock, 2005). Whether sexual signals are simple or complex, the signals exchanged should allow each animal to identify both species and gender (Sherman et al., 1997). Further, in intensely aggressive and competitive species, like some decapod crustaceans, there is a need for sexual signals to clearly convey signaler intentions (Dingle and Caldwell, 1972;

Uetz, 2000).

Visual signals play a role in the sexual and agonistic behavior of several families of brachyuran crabs (Schöne, 1968). Most species’ visual displays include variations on claw movements and color (Schöne, 1968). Of the brachyurans, visual signals have been most studied in fiddler crabs (genus Uca). Visual signals

1 involving both color and movement play a large role in the social interactions of this genus (Detto et al., 2006). Uca mjoebergi and U. capricornis identify gender and recognize neighboring individuals based on learned color patterns, and mate choice in U. mjoebergi is influenced by claw color (Detto et al., 2006; Detto 2007).

Aside from fiddler crabs, vision and visual signals have not been well studied in crabs.

The blue crab, Callinectes sapidus, is of particular interest because of its exceedingly agonistic nature, wide geographical distribution, high economic value, and ecological importance. Blue crabs spend up to 40% of their time in agonistic behaviors (Clark et al., 1999). This is among the highest value reported in arthropods and “underlines the bellicose nature of the species” (Clark et al.,

1999). Blue crabs are endemic to western Atlantic coastal waters from Nova

Scotia to Uruguay and have been introduced in locations all over the world, including Hawaii, Europe, Japan, and Africa (Williams, 2007). It is perhaps best known for its edibility, and supports one of the top-grossing fisheries of the U.S.

Atlantic coast, with total earnings of $130-160 million per year (Bullock et al.,

2007). In addition, the blue crab is a key species in coastal benthic and pelagic ecosystems (Hines, 2007). Blue crabs prey upon and scavenge bivalves, fish, and crabs (Darnell, 1961) and are eaten by fish, reptiles, birds, and other crabs (Hines,

2007). Although a large body of literature on the biology of C. sapidus exists 2

(reviewed by Kennedy and Cronin, 2007), their sensory physiology and behavior

are poorly understood (Jivoff et. al., 2007; Hines, 2007). Specifically, the role of

vision in the mating behavior of these colorful and highly visual animals has

been overlooked.

Mating behavior in the blue crab is influenced by the sex ratio of the

population, where the less abundant gender is the pursued and the more

abundant is the pursuer (Jivoff and Hines, 1998). Therefore, both genders need to

be attractive at certain times and choosy at others. Unlike many mating systems,

where females have the higher parental investment and are therefore the

choosier sex, both male and female blue crabs make large investments. In

C.sapidus, mating is costly for both genders in terms of time spent, resources needed, and risks taken. The total time spent on mate attraction, copulation, and mate guarding can take approximately 10-12 days (Jivoff and Hines, 1998). After copulation, males need approximately nine to 20 days to fully recover ejaculate reserves, which suggests that sperm and seminal fluid production is costly

(Kendall and Wolcott, 1999). Females are sexually receptive only after the pubertal molt and may mate with multiple males during the two to three-week period following the pubertal molt (Jivoff, 1997). During the next two years, females will produce up to six broods of embryos, with potentially three million embryos produced in each brood (Hines et al., 2003). 3

Blue crabs are sexually dimorphic, exhibiting differences in abdomen shape and claw color (Jivoff et. al. 2007). Males have a narrow white abdomen that matches the ventral carapace. In contrast, mature females have a rounded abdomen with varying bands of orange, blue, and black that contrast with the white ventral carapace. Immature females, however, have triangular abdomens with bands of pale orange or blue. More apparent than the abdomen dimorphism, is the sex-related difference in claw color between males and females. Males have white and blue claws and females have orange or red claws.

Both males and females exhibit individual variations in claw color and pattern.

Females’ claws range from light orange to a deep red. The color of males’ claws ranges from pale to dark blue. However, the extent to which color varies is not documented in either gender, nor is the significance of the color variation known.

As previously mentioned, pursuing a potential mate is risky for a blue crab. Courting an unreceptive mate can result in dismemberment or cannibalism.

Female blue crabs copulate after molting and have a thin, soft cuticle, so they are particularly vulnerable to injury, predation, and cannibalism. Given the costs of and risks associated with mating, it may be predicted that blue crab sexual signals should clearly and effectively convey sexual intent.

4

These signals may have both visual and chemical components. Chemical

signaling in C. sapidus has been studied extensively, though the identity of the

chemical(s) used remains elusive (Jivoff et al., 2007). In contrast, visual signals

have been less studied and are often considered unimportant (Jivoff et al., 2007).

It is well known, however, that blue crabs wave their claws in both agonistic and

sexual interactions (Teytaud, 1971; Jachowski, 1974). During courtship, males rise

up on the tips of their walking legs, extend their claws, and sometimes raise their

swimming paddles and laterally fan them towards the potential mate (Teytaud,

1971). The higher posture and paddling direct chemical signals towards a

potential mate, but may serve a visual purpose as well (Kamio et al., 2008).

Previous work on blue crab visual behavior is limited, although the

species is known to be an excellent visual predator -- catching fish, stalking

fiddler crabs, and even burying themselves up to their eyes to ambush a prey

item (Abbott, 1967; Hines, 2007). Experiments testing visual responses to various shaped and colored objects suggest that C. sapidus is able to discern blue, yellow, and red (Bursey, 1984), although this study did not rule out intensity-dependent differences in discrimination. Behavioral studies in the 1970’s describe a number of visual signals used in agonistic and sexual behaviors of C. sapidus (Jachowski,

1974; Teytaud, 1971).

5

Blue crabs have stalked apposition compound eyes that can move independently and retract into the carapace. Using measurements of eye structure made by Eguchi and Waterman (1958), the visual acuity of the blue crab was calculated to be approximately 1.4º (Land and Nilsson, 2002). Blue crab eyes likely have two photopigments that provide them with color vision.

Previous studies using microspectrophotometry (MSP) have found a single medium-wavelength pigment in the eye of C. sapidus that absorbs light maximally at 504 nm (Cronin and Forward, 1988). The electroretinogram (ERG) results of Martin and Mote (1982) suggest the presence of a second photopigment with a peak near 440 nm in the ventral portion of the eye. Opponency between the two visual channels may allow for color discrimination.

The effect of the molting process on the blue crab’s vision is another unexplored aspect of the blue crab’s visual physiology. Prior to molting, a new cuticle forms underneath the crab’s exoskeleton, and gradually separates from it

(Smith and Chang, 2007). This is true even in the eyes, where the chitin exoskeleton functions as the lens array (Euguchi and Waterman, 1958). The separation of the old lenses and the newly forming cuticle may limit visual acuity in a molting crab by disrupting the focusing power of the lens. While the literature exists on the molting process in blue crabs is substantial (reviewed by

Smith and Chang, 2007), no information exists on the optical changes produced 6

by molting in the blue crab or any other crustacean species. The visual

constraints of molting may be particularly important in female blue crabs, whose

pubertal molt corresponds with sexual receptivity (Van Engle, 1958).

Here we explore the role of vision in sexual signaling in the blue crab, In

Chapter Two, published in the Journal of Experimental Biology, we describe the

ability of the blue crab to respond solely to visual, sexual cues (Baldwin and

Johnsen, 2009). We also established the ability of the blue crab to perceive color

separate from intensity, or luminance. In Chapter Three, also published in the

Journal of Experimental Biology, we describe the ability of the blue crab to discriminate between variations of orange and red colors, and examine the interaction of perceived luminance and color (Baldwin and Johnsen, in press). In

Chapter Four, again published in the Journal of Experimental Biology, we discuss the visual constraints of molting (Baldwin and Johnsen, 2011). Our conclusions and directions for future research are presented in Chapters Five and Six, respectively.

7

2. The Importance of Color in Mate Choice in the Blue Crab Callinectes sapidus

In many breeding systems, animals perform a series of courtship rituals to attract a mate, arouse the breeding state in both genders, and finally achieve copulation (Frings and Frings, 1977; Alcock, 2005). These rituals involve a variety of signal modalities including visual, tactile, chemical, and mechanical channels

(Johnstone, 1996; Candolin, 2003). Often, signalers will employ more than one modality, using multiple channels to enhance and/or modulate the message of the signal (Candolin, 2003; Alcock, 2005). Whether sexual signals are simple or complex, the signals exchanged should allow each animal to identify both species and gender (Sherman et al., 1997).

Further, in aggressive and competitive species, such as certain crustaceans, there is a need for sexual signals to clearly communicate signaler intentions

(Dingle and Caldwell, 1972; Uetz, 2000). Many species of crustaceans have well developed visual systems paired with colorful displays, and in these species visual cues may play a large role in communication. Moreover, visual signals may function more rapidly and over a further distance than other signal modalities, allowing individuals to communicate instantaneously from safer ranges. In many brachyuran crabs, visual cues play a large role in sexual and agonistic behaviors

(Schöne, 1968), and have been most studied in fiddler crabs (genus Uca). Visual

8

cues involving both color and movement play a large role in the social interactions

of this genus (Detto et al., 2006). Uca mjoebergi and U. capricornis identify gender

and recognize neighboring individuals based on learned color patterns, and mate

choice in U. mjoebergi is influenced by claw color (Detto et al., 2006; Detto, 2007).

Aside from fiddler crabs, visual cues have been well documented in other

malacostracan crustaceans including several species of stomatopods

(Odontodactylus scyllarus, Marshall et al., 1996; Gonodactylis sp., Haptosquilla sp., and

Oratosquilla sp, reviewed by Christy and Salmon, 1991); shrimp (Alpheus heterochaelis, Hughes, 1996; Rhynchocinetes typus, Diaz and Thiel, 2004); and in crayfish (Austropotamobius pallipes, Acquistapace et al., 2002).

The blue crab, C. sapidus, is of particular interest given the species’ exceedingly agonistic nature, wide geographical spread, economic value, and ecological importance. Blue crabs spend up to 40% of their time in agonistic behaviors (Clark et al., 1999). This is among the highest values reported in arthropods and “underlines the bellicose nature of the species” (Clark et al.,

1999). Blue crabs are endemic to western Atlantic coastal waters from Nova

Scotia to Uruguay, and have been introduced in locations all over the world including Hawaii, Europe, Japan, and Africa (Williams, 2007). They support one of the top-grossing fisheries of the U.S. coastal Atlantic, earning $130-160 million per year (Bullock et al., 2007). Additionally, the blue crab is a keystone species in 9 the coastal benthic, pelagic, and estuarine ecosystems functioning as predator, prey and scavenger (Hines, 2007). While a large body of literature on the biology of C. sapidus exists (reviewed by Kennedy and Cronin, 2007), their sensory physiology and behavior are poorly understood (Jivoff et. al., 2007; Hines, 2007).

Specifically, the role of vision in the mating behavior of these colorful and highly visual animals has been overlooked.

Previous work on blue crab visual behavior is limited, although the species is known to be an excellent visual predator -- catching fish, stalking fiddler crabs, and even burying itself up to its eyes to ambush a prey item

(Abbott, 1967; Hines, 2007). Behavioral experiments testing visual responses to variously shaped and colored objects suggested that C. sapidus was able to discern blue, yellow, and red (Bursey, 1984). However, the perceived brightness of the different hues was not controlled for during these experiments, so it is unclear whether the crabs were using color discrimination or brightness cues to differentiate between the targets. Behavioral studies in the 1970’s described a number of visual cues used in agonistic and sexual behaviors of C. sapidus

(Jachowski, 1974; Teytaud, 1971). A more recent study investigating the use of visual and chemical cues during courtship reported mixed results for both components of the experiment, with the authors reporting that visual signals were not used, but also that chemical cues were not mandatory for mating 10 interactions (Bushman, 1999). These experiments involved antennule ablation and blindfolding, which may have affected courtship and mating behaviors.

During courtship, blue crabs may use chemical cues for mate attraction.

Chemical cues in C. sapidus have been studied extensively, though the identity of the chemical(s) used remains elusive (Jivoff et al., 2007). In contrast, visual cues in blue crabs have been less studied and the use of vision during mating has often been considered unimportant (e.g. Jivoff et al., 2007). It is well known, however, that blue crabs wave their claws in both agonistic and sexual interactions (Teytaud, 1971; Jachowski, 1974). During courtship, males will rise up on the tips of their walking legs, extend their claws, and sometimes raise their swimming paddles and laterally fan them towards the potential mate (Teytaud,

1971). The higher posture and paddling help send chemical cues towards a potential mate, but may serve a visual purpose as well (Kamio et al., 2008).

11

In addition to postures and movements, male and female C. sapidus have visible sexual dimorphism of their abdomens and claws (Jivoff et. al., 2007;

Figure 1). Males have a narrow white abdomen that matches the ventral carapace. Mature females have a rounded abdomen with orange, blue, and black

Figure 1. Ventral views of male and female C. sapidus and exterior and interior views of the claws. Scale bars equal 2 cm.

bands that contrast with the ventral carapace, and immature females have triangular abdomens with pale orange or blue bands. More apparent than the abdominal dimorphism, is the striking sex-related difference in claw color. Males

12 have white and blue claws and dactyls (i.e. fingers of the claws), while females have with blue and white claws with red dactyls. Both males and females exhibit variations in claw color and pattern. The color of males’ claws ranges from pale to dark blue. Females’ dactyls range from light orange to a deep red (Figure 2).

Anecdotal evidence indicates that the red color of female claws becomes more saturated with maturity. The extent to which color varies is not documented in either gender, nor has it been explored in regards to reproductive status or individual quality.

Figure 2. Claws of female C. sapidus. The coloration of dactyls varies in both the area of the red coloration and also the hue and saturation of the color. Scale bar equals 2 cm. 13

In this study, we investigated the use of non-postural visual cues in blue

crab courtship using photographs of female crabs. We conducted binary choice

experiments to investigate whether male blue crabs respond to female visual

cues and whether they demonstrate a preference for claw dactyl coloration. Our

results show that color cues are potentially important during courtship behavior

and male blue crabs perceive and prefer red dactyls to those of other colors.

2.1 Methods

2.1.1. Specimen collection and care:

Male blue crabs were captured from Jarrett Bay near Smyrna, North

Carolina, USA (34º45’31.4”N, 76º30’44.4”W) in July and August of 2008. Crabs

were immediately placed into individual water-filled buckets and transported to

Duke University’s central campus in Durham, NC. There, crabs were kept in individual compartments within a 700-liter re-circulating artificial seawater system (salinity 31-35 ‰, temperature 25-26ºC, ambient light). Compartment walls were opaque to minimize stress and agonistic behavior. Crabs were fed pieces of fish or scallop every two days, and kept for at least 48 hours before being used in experiments.

14

2.1.2. Behavioral experiments

Binary choice experiments were conducted in a 130-liter glass aquarium

(32 cm x 91 cm x 46 cm) with a gravel bottom. During acclimation periods, water was filtered and at all times maintained at the same salinity and temperature as the holding tank. The experimental tank was kept in a separate room and visually isolated on three sides by blue cloth and on the fourth side by a light colored wall. The tank was observed via a video camera whose monitor and recorder were on the other side of the cloth barriers. The tank was lit using overhead fluorescent and incandescent lamps, resulting in a downwelling irradiance of 8 x 1014 photons/cm2/s (integrated from 400 to 700 nm).

To limit confounding variables, such as chemical or tactile cues, a photograph of a sexually mature female in a receptive sexual posture was used in place of live females. Preliminary tests showed that males preferred females with their claws out and dactyls closed. Three versions of the photograph were used: 1) an original image, 2) an image altered so that the claws were white, and

3) an image altered so that the claws were black (Fig. 3). Images were altered using the “selective color” feature of Photoshop CS (Adobe Inc., San Jose, CA,

USA). The diffuse spectral reflectances of the claws in the printed images were measured using a fiber optic-based spectroradiometer in reflectance mode

15

Figure 3. The three photographs of female C. sapidus used in the binary choice experiments. The crab was photographed with her claws out and dactyls closed, a known sexually receptive posture. A) Original image. B) Original image altered to make claws white. C) Original image altered to make claws black. Scale bar equals 2 cm.

16

(USB2000, Ocean Optics Inc., Dunedin, FL, USA; Fig. 4). The gray value of the

black claws was chosen so that its brightness (as perceived by the crabs) matched

that of the red claws as closely as possible (Table 1). Perceived brightness L was

calculated using:

700 LC= R(λ) I( λ) S( λλ) d (1) ∫400

where R(λ) is the spectral reflectance of the claw dactyl, I(λ) is the downwelling

irradiance in the tank, and S(λ) is the spectral sensitivity of the crab eye (C is a

constant that includes factors such as eye size, etc. that are independent of

wavelength and factor out when comparing different colors). The spectral

sensitivity of C. sapidus was calculated in two ways: 1) using ERG data from

Martin and Mote (1982), and 2) using MSP-determined visual pigment curves from Cronin and Forward (1988) (λmax = 504 nm) combined with measurements

of rhabdom length and absorption coefficient. Using the same methods, the

perceived brightness of the white claws was determined to be approximately 7

times that of the black and red claws (Table 1). The images were printed on

standard paper and mounted to white foam boards for stability.

17

Figure 4. A) Spectral sensitivity of C. sapidus based on ERG data from Martin and Mote (1982) plotted with the spectral reflectances of the photographs of the red, white, and black claw dactyls used in the experiments (n = 20 per image). Based on the ERG and MSP data, the red and black dactyls are nearly isoluminant. B) Spectral reflectances of actual C. sapidus females’ red and males’ white claws (n = 36 females, n = 36 males).

18

Table 1. Estimation of perceived brightness of images of claw dactyls used in this study. Measurements are normalized such that the perceived brightness of the black dactyls is one. Values given are mean ± SD. N values are in parentheses.

Relative intensity Relative intensity (based on MSP data) (based on ERG data) Black claw image (20) 1.0±0.39 1.0±0.39 Red claw image (19) 1.3±0.72 1.4±0.74 White claw image (19) 6.9±1.2 6.8±1.2

All trials were conducted between 0700 and 1900 Eastern Daylight Time

from June through September 2008. At the start of each experiment, one male

was placed in the experimental tank. After three hours of acclimation, two

photographs were presented to the crab -- one at each end of the tank.

Photograph positions were assigned randomly. Over the next hour, the male’s behavior was monitored. Most crabs made multiple stereotypic sexual displays that were unambiguously directed towards (and occurred within 5 cm of) one of the two images. During these 5-30 second displays, the crabs rose up on their walking legs, extended their claws, and waved their paddles while facing the photograph. In between displays, the crabs generally walked around the tank.

The total number of sexual displays made towards each image over one hour was counted. Choice was assigned to the image that received the greatest number of displays. Sixteen successful trials were run for the red vs. white

19

experiment and fifteen for the red vs. black experiment. Each experiment was

analyzed using the two-tailed exact binomial test.

Because these males are wild caught, it was not known how recently they

had molted or mated. Both of these factors may affect their sexual receptivity.

Therefore, if a male made no displays during the one-hour trial, he was tested

again 5 to 7 days later. Eleven of the 31 males were used in both experiments,

although no males were used more than once in a five-day period. After testing,

the males were returned to the re-circulating system and later to their original

capture area.

2.2. Results

Males responded to photographs of females with stereotypic courtship

behaviors in 65% of the first trials (26 of 40 combined white and black trials). Of

the 14 males that did not respond in their first trial, five were re-tested in the same experiment after 5 to 7 days and four of those five displayed a behavioral response and preference. Another five of the initial 14 unresponsive males were later used in the other color trial; these five males displayed courtship behaviors and preference in the subsequent experiment. Four of the initially unresponsive males were not retested or reused. Of the ten that were either re-tested or later used, nine displayed courtship behaviors giving a total response rate of 87% over

20

two rounds of trials (35 of 40 trials). Only one male, of those retested, was

unresponsive during the course of the trials.

Males chose photographs of females with red claws over those with white

claws in 14 of 16 trials (p<0.005; two tailed exact binomial test; Fig. 5a, table 2).

Males also chose females with red claws over those with black claws in 13 of 15

trials (p<0.01, Fig. 5b, table 3). In all trials except one, the first photograph to

receive a sexual display from the male was the photograph that received the

most displays. The exception, male 16 in the red vs. white experiment displayed

at the white-clawed photograph first, but ultimately (though narrowly)

displayed more often to the red-clawed photograph. If this male was assigned a

preference of white rather than red, the overall male preference for females with

red claws in this experiment is still statistically significant (P<0.025). During 4 of

the 16 red vs. white claw trials, agonistic responses, including open-dactyl claw waving and striking against the aquarium glass, were seen towards the white- clawed photograph. There was no significant bias for the left or right side of the tank in either experiment. Left was chosen 9 out of 16 times in the red vs. white claw trials and 9 out of 15 times in the red vs. black claw trials (P>0.5 in both cases).

21

Figure 5. Results of the choice experiments. A) Male C. sapidus chose the red claws over white ones in 14 of 16 trials (P <0.005, Two-Tailed Exact Binomial Test). B) Male C. sapidus also chose red claws over black ones in 13 of 15 trials (P < 0.01).

The total number of displays over the one-hour trials was quite variable, ranging from 2 to 34 in the red vs. white trials (11 ± 9.8, mean ± SD) and from 2 to

19 in the red vs. black trials (8 ± 5.5). The average percentages of displays towards the photograph with red claws were 85 ± 28 (red vs. white) and 78 ± 32

(red vs. black). Ten of the 16 crabs in the red vs. white trials and 8 of the 15 in the red vs. black trials only displayed to the photograph with the red claws.

Interestingly, the four trials showing a choice of black or white claws over red claws suggested unambiguous preferences, with the test crab performing

22 substantially more displays toward a black-clawed female (2 vs. 0 and 10 vs. 2 displays) and to a white-clawed female (4 vs. 1 and 11 vs. 4 displays).

Table 2. Individual results of Red-White Binary Choice Experiment. * Male did not respond during first trial, so data presented are from retrial.

23

Table 3. Individual results of Red-Black Binary Choice Experiments. * Male did not respond during first trial, so data presented are from retrial.

2.3. Discussion

Color vision is known to play a role in the sexual behavior of certain crabs and crustaceans, though this is the first time it has been documented in blue 24

crabs. The results given here show for the first time that visual cues alone can

stimulate courtship behavior in male C. sapidus. At no time during the trials were

males exposed to female chemical cues, which have been routinely used in

previous behavioral experiments, including those testing visual cues. Our

results also show that male C. sapidus are able to express mate preferences based

only on the color of a female’s claws.

Unexpectedly, the males’ choices suggest that blue crabs are able to

perceive red as a color, not simply a difference in brightness. While the results of

the red vs. white trials could indicate a male preference for females with red

claws or simply a preference for darker claws, the fact that the males could also

choose red claws over isoluminant black claws suggests that they were able to

discriminate the hue of the claws. It is possible that the spectral sensitivities

based on the ERG and MSP data do not accurately represent the achromatic

channel in C. sapidus, implying that the red and black claws are not isoluminant.

This will be further examined in future studies that will also test whether blue crabs can discriminate red from orange and other long-wavelength dominated hues. However, the males’ choice of red claws over claws of two quite different gray values strongly suggests that hue discrimination is taking place.

The physiological mechanism for this hue discrimination is unclear.

Previous studies using microspectrophotometry (MSP) have found only a single 25 medium wavelength pigment in the eye of C. sapidus that absorbs light maximally at 504 nm (Cronin and Forward, 1988). However, because MSP only looks at one cell at a time, regional variation in pigment expression or the presence of rare pigments can lead to false negatives. Electroretinography (ERG) by Martin and Mote (1982) suggested the presence of an additional short- wavelength channel in the ventral portion of the eye (λmax = 440 nm), and it is possible that opponency between this and the medium wavelength pigment allows for hue discrimination at longer wavelengths. The ERG data suggesting the presence of this short wavelength pigment are not robust, however. Only six samples showed evidence of this receptor, and the methods used to collect data from these six were different from the methods used during the rest of the study

(Martin and Mote, 1982). Thus, further research is necessary to determine the physiological basis for red perception and also to determine over what wavelengths hue can be discerned.

However, if one assumes that there is an opponency mechanism between a 440 nm and a 504 nm pigment in the blue crab eye, it appears that the red claws can be discriminated from the white and black claws. One simple model assumes that the two channels are balanced (i.e. have equal influence) and that the hue is the normalized difference between their perceived brightnesses. I.e.

26

Hue = , (2) 퐿504+퐿440 퐿504+퐿440 where Lλ is the perceived brightness calculated in equation (1) for a pigment with a peak wavelength of λ. Using this, the “hues” of the white and black claws are

0.10 and 0.09 respectively and that of the red claws is 0.20. While admittedly arbitrary, this suggests that the two pigments are different enough to reliably separate greys from red, at least in daylight. For example, horses (dichromats with pigments peaking at 428 nm and 539 nm) can distinguish colors with these hue values (Roth et al., 2007).

The significance of claw color in blue crabs has not been investigated, and it is unknown if color simply indicates sex or if it may also reflect upon the quality of the individual. In blue crabs, color is contained in the hypodermis portion of the cuticle (Smith and Chang, 2007). The colors are pigmentary and derived from carotenoids, which are not synthesized by animals. Carotenoid- based pigments are obtained from diet; therefore, animal colors may be limited in part by an animal’s foraging ability (Bagnara and Hadley, 1973; Brush and

Power, 1976). In certain species that utilize carotenoid pigments in color displays, coloration is reflective of an individual’s foraging ability and may indicate overall fitness (e.g. Endler, 1980; Hill and Montgomerie, 1994). Typically, better foragers have brighter, more attractive coloration and may be perceived as better

27

quality mates and receive mate preference (Burley and Coopersmith, 1987;

Kodric-Brown, 1989; Hill, 1990). For example, diet is directly linked to color in

guppies (Poecilia reticulata). Male guppies fed a carotenoid-enriched diet

produced more saturated red and orange patches, which increased their

attractiveness to females (Kodric-Brown, 1989). Similar studies have linked diet, color, and mate attractiveness in three-spine sticklebacks (Gasterosteus aculeatus;

Frischknecht, 1993; Bakker and Mundwiler, 1994); firemouth cichlids (Cichlasoma meeki; Evans and Norris, 1996); and in several species of birds such as house finches (Carpoducus mexicanus; Hill and Montgomerie, 1994) and greenfinches

(Carduelis chloris; Saks et al., 2003). The results of these studies suggest that the

blue and red colors of both males and female blue crabs could be indicative of

foraging ability and thereby quality if carotenoids are a limited resource.

Additionally, the claw color in blue crabs may enhance the visibility of

sexual cues through the blue crab’s environment. Blue crabs utilize various

habitat types depending on age, molt stage, gender, salinity, and season and are

typically found in coastal and estuarine waters between 0 – 15 m deep (Hines,

2007). The drab olive coloration of C. sapidus’ carapace apparently functions as

camouflage, while the bright claw colors may be advertisements, particularly the

red claws of females. In some aquatic environments, red colored cues and the

ability to distinguish red may be particularly useful. Red is conspicuous against 28 blue or green-dominated underwater light environments, especially in shallow water where red solar illumination is still present (Lythgoe, 1979). Red is used in courtship displays in several relatively shallow water species including: three- spine sticklebacks (Gasterosteus aculeatus; McLennan and McPhail, 1990), guppies

(Poecilia reticulata; Endler, 1980), cichlids (Haplochromis nyererei; Seehausen and van Alphen, 1998), California market squid (Loligo opalescens; Zeidberg, 2008), and the fiddler crab (U. pugilator; Hyatt, 1975), among others. Red coloration may also be visible at longer distances than other hues in the blue crabs’ habitat.

Displaying red color is common in freshwater systems where short-wavelength light can be heavily attenuated and longer wavelength light pervades the underwater habitat (Kodric-Brown, 1998, Lythgoe, 1979). Similarly, dissolved organic matter in coastal and estuarine environments can shift the underwater light environment to longer wavelengths. For example, underwater irradiance measurements from the Rhode River area of the Chesapeake Bay (38.71-38.89º N latitude; 76.34-76.54º W longitude) show that the underwater light environment is predominately yellow (Tzortziou et al., 2007). The visibility of blue crab claws will depend on habitat conditions and the interaction of light and color.

The red coloration of female C. sapidus may play a role in male mate choice by indicating gender, reproductive status, and/or individual quality.

While investigations of color cues and ornaments have historically focused on 29

males, more recent studies have found that female coloration and ornaments are

used in both male mate choice and female-female competition, even in systems

where sex roles are not reversed (Amundsen and Forsgren, 2001; Jones and

Hunter, 1993). In a marine fish, the two-spotted goby (Gobiusculus flavescens),

males preferred females with bright yellow-orange colored bellies to those with drab bellies (Amundsen and Forsgren, 2001). Males of the crested auklet (Aethela

christatella), a seabird, prefer females with lengthier crest ornaments (Jones and

Hunter, 1993). Female striped plateau lizards (Sceloporus virgatus) develop orange

throat coloration during the breeding season that indicates both higher

individual quality and fecundity and may function as an ornament to attract

mates (Weiss, 2006). Only a few other studies in birds and fish have shown male

preference for female ornaments and colors, but male mate choice is likely

occurring in other groups (Amundsen and Forsgren, 2001). Investigations of

female coloration or ornaments as cues or signals during female-female

competition are fewer, but studies in dotterels (Charadrius morinellus; Owens et

al., 1994), capuchinbirds (Perissocephalus tricolor; Trail, 1990), and hummingbirds

(Amazill spp.; Wolf, 1969) indicate that showy female traits or colors may be

selected through female-female competition over sexual or non-sexual resources.

Given the use of female color and ornaments in other systems, it is plausible that

30 the red color of the female blue crab sends a message of reproductive status or mate quality.

Determining the role of claw coloration in C. sapidus will require further investigation, both behavioral and physiological. Such investigations are needed to determine the importance of vision and to understand the visual ecology of this economically and ecologically important species that often finds itself inhabiting anthropogenically turbid waters.

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3. Male Blue Crabs Callinectes sapidus Use Both Chromatic and Achromatic Cues in Mate Choice

Bright, conspicuous colors are often investigated within the context of

sexual communication and sexual selection. Animals use color to communicate

information such as gender (Butcher and Rohwer, 1989; Marquez and Verrell,

1991), reproductive readiness (McLennan and McPhail, 1990; Sköld et al., 2008),

individual or species identity (Losos, 1985; Detto et al, 2006), social status

(Rohwer, 1975; Watt, 1986), and individual quality (Endler, 1980; Montgomerie

and Hill, 1994). However, colors can only be effective signals or cues if they can

be perceived by the intended receiver (Rowland, 1979). It is necessary then, when

investigating the role of an animal’s coloration, to also consider the receiver’s

visual system, which varies broadly across animal taxa (reviewed by Briscoe and

Chittka, 2001; Osorio and Vorobyev, 2008; Kelber and Osorio, 2010).

The blue crab, Callinectes sapidus, is an excellent system for simultaneously

investigating the perception of color cues and their role in sexual signaling. The

blue crab is a colorful portunid species endemic to coastal Atlantic waters along

North, Central, and South America. Male and female blue crabs have sexually

dimorphic claw coloration; adult males have white and blue claws, and adult

females have orange or red claws (Fig. 6). Immature males’ and females’ claw

coloration range from pale blue or violet to orange (Fig. 6), and qualitative

32

Figure 6. Mean spectral reflectances and photographs of both the exterior and interior exterior claw surfaces. Included are sexually mature males (n=28), immature males (n=30), immature females (n=27), prepubertal females (n=21), and sexually mature females (n=25). The error bars denote standard error.

33

evidence suggests that claw coloration changes with sexual maturity in both

males and females. These differences suggest that claw color may act as a cue of

gender and/or sexual maturity. Individual variation within each group invites speculation regarding individual quality or fitness.

Hypotheses regarding the signaling function of claw coloration must be

evaluated within the parameters of the blue crab visual system. Blue crabs likely

have a dichromatic visual system. Electroretinogram (ERG) data suggest the

presence of photoreceptors with peak sensitivity of 508 nm (green) over the

entire eye and a second set of photoreceptors in the ventral portion of the eye

that peak at about 440 nm (blue) (Martin and Mote, 1982). Thus, the detection of

chromatic cues may occur via opponency between the green and blue

photoreceptors in the ventral portion of the eye. Achromatic cues, those that are

based on signal intensity alone, are likely detected via the 508 nm photoreceptor

given that it is the predominant receptor in the eye (Martin and Mote, 1982).

Color vision was suggested in behavioral experiments where C. sapidus

showed distinct responses to yellow, red, and blue approaching objects (Bursey,

1984). However, these experiments did not control for the intensity of the colors

used, thus responses may have been based on signal intensity rather than color.

A more recent behavioral study demonstrated the blue crab’s use of chromatic

cues when males displayed a preference for images of red-clawed females over 34

those of grey-clawed females that had the same luminance (as perceived by the

blue crab eye) (Baldwin and Johnsen, 2009). Though males preferred red-clawed females to those with isoluminant grey claws, it was undetermined how or if male blue crabs can distinguish naturally occurring variations in the red and orange claw coloration of females. The blue crab’s blue-green dichromatic visual system may have a restricted range of color discrimination due to its limited sensitivity to long-wavelength light. Thus, the nuances of orange and red colors that are evident to humans may not be perceived by the blue crab eye.

Here we have investigated natural variation in claw coloration in male and female blue crabs, documenting color differences between sexually immature and mature individuals of both sexes. We then estimated the appearance of claw color to the blue crab visual system by modeling perceived luminance (achromatic cues) and opponency (chromatic cues) using ERG data.

Then, in binary choice tests using photographs of females with claws colored red and six variations of orange, we behaviorally tested the ability of male blue crabs to discriminate between various long-wavelength-dominated colors. These behavioral assays examine both chromatic and achromatic mechanisms of male mate choice and whether males are capable of discriminating between naturally occurring claw colors.

35

3.1. Methods

3.1.1. Study species:

Males and females of the blue crab, Callinectes sapidus, were captured from

Jarrett Bay near Smyrna, North Carolina, USA (34º45’31”N, 76º30’44”W) in April

2011. Gender and sexual maturity were determined by visually examining claw color, abdomen shape, and overall size. In males, sexual maturity is associated with size and individuals over 100 mm were assumed to be sexually mature

(Milikin and Williams, 1984). In females, abdomen shape is a reliable indicator of sexual maturity. Immature females have triangular shaped abdomens while mature females have wider, rounder abdomens (Newcombe et al, 1949; Jivoff et al., 2007). Prepubertal female crabs are in a transitional stage of sexual maturity, meaning that they will become sexually receptive just prior to their next molt; these can also be identified by their abdominal shape and coloration.

3.1.2. Measuring spectral reflectance of claw color:

Blue crab claw color was measured in April 2011, during the first peak of the commercial soft crabbing season in North Carolina. During this time, juvenile and adult male and female blue crabs were available in running seawater enclosures at a commercial crab fishing facility in Smyrna, NC. Crabs were separated into five groups based on gender and sexual maturity: immature

36

males, mature males, immature females, prepubertal females, and mature

females. Spectral reflectance measurements of claws were taken in a darkened

room. Prior to measurements, crabs were anaesthetized on ice for 15 – 30 min to

facilitate handling.

Spectral reflectance was collected using methods outlined by Johnsen

(2005), using a fiber optic reflectance probe (R400-7 reflection probe, Ocean

Optics. Inc., Dunedin, FL, USA) coupled with a pulsed xenon light source (PX-2,

Ocean Optics) and a multi-channel spectrometer (USB2000, Ocean Optics). The reflectance probe contained seven 400 µm diameter optical fibers in a six-around- one arrangement. The six outer fibers were coupled to the light source and illuminated the specimen. The central fiber collected the light reflected from the specimen and was coupled to the spectrometer. The end of the reflectance probe was held next to the claw surface at a 45º angle using a rigid optical mount, which illuminated and collected the back-reflection of the claw surface. This approach reduced the collection of specularly reflected light from the shiny, claw surface, and instead collected the diffuse reflectance, which is relatively independent of the angles of illumination and measurement (Palmer, 1995). The reflectance measurements were calibrated using Spectralon™, a diffuse reflectance standard that diffusely reflects nearly 100% of light from 200 to 800 nm (WS-1 Diffuse Reflection Standard, Ocean Optics). Spectral reflectance was 37

taken at the center of the dactyls (moveable fingers) of each side of the claws,

referred to as the interior and exterior claw faces relative to the crab.

Measurements were taken from the right claws of 18-29 individuals of each

group.

3.1.3. Calculation of relative luminance and opponency to the blue crab eye: Claw reflectance spectra, the illumination spectra, and the spectral

sensitivity of the visual system were used to model the perception chromatic and

achromatic signals. The luminance (L) of reflected light perceived by the blue

crab eye is given by:

700 LC= ∑ R(λ) I( λ) S( λλ)∆ (1) λ=400

where R(λ) is the diffuse spectral reflectance of the object being viewed, I(λ) is the downwelling spectral irradiance , S(λ) is the spectral sensitivity of the crab

eye, and C is a constant that includes factors such as eye size, etc. that are

independent of wavelength and factor out when comparing different samples.

The irradiance was chosen to be that of the test tank used in the behavioral

experiments described later in this paper. The spectral sensitivity of C. sapidus

was based on ERG data (Martin and Mote, 1982), and perceived luminance was

found for both the blue photoreceptor (λmax = 440 nm) and the green

photoreceptor (λmax = 508 nm). In many species, the medium wavelength

38

photopigment functions as the achromatic channel, and in the blue crab the

green photoreceptor is the predominant one found throughout the eye (Martin and Mote, 1982; Cronin and Forward, 1988). Therefore, the achromatic cues we report on here are based on the quantum catch of the green photoreceptor. In addition, for easier comparison we normalized all crab-perceived luminances by

the perceived luminance of the red test color used in the behavioral experiments

described below, referring to this ratio as “relative luminance”.

To determine color as perceived by the blue crab eye, we assumed that

color vision occurred via an opponency mechanism between the putative 440 nm

and a 508 nm pigment in the blue crab eye (Martin and Mote, 1982; Baldwin and

Johnsen, 2009). We used a simple model that assumes that the influence of the

two pigments is balanced over the wavelength range of 400 to 700 nm. I.e.

– Opponency = (2)

퐿508 퐿440 퐿508+ 퐿440 where Lλ is the perceived luminance calculated in equation (1) for a pigment with

a peak wavelength of λ. The relative gain of the two channels was adjusted to

produce an opponency value of zero for an achromatic object.

While the blue crab may be sensitive to ultraviolet light, we report here

only on the visible spectrum of light due to limitations of the known spectral

sensitivity of the blue crab. In addition, preliminary analyses that extrapolated

39

the visual sensitivity of the crab eye into the ultraviolet did not give significantly

different results.

3.1.4. Behavioral experiments evaluating male color preferences:

Male blue crabs were collected from June through November 2009 and

April through August 2010. Crabs were immediately placed into individual

buckets with a shallow layer of water and transported to Duke University’s

central campus in Durham, NC, USA. There, crabs were kept in individual

compartments within a 700-liter recirculating artificial seawater system (salinity

29-31 ‰, temperature 25-26ºC, natural light cycle). Compartment walls were

opaque to minimize stress and agonistic behavior. Crabs were fed pieces of fish,

shrimp, or scallop every two days, and kept for at least 48 hours before being

used in experiments.

Binary choice experiments were conducted in three 100-liter glass aquariums (32 cm x 91 cm x 46 cm) with gravel bottoms. During acclimation periods, water was filtered and at all times maintained at the same salinity and temperature as the holding tank. Experimental tanks were kept in a separate room and visually isolated on all sides by blue cloth. The tanks were observed via a video camera and lit using overhead fluorescent and incandescent lamps,

40 resulting in a downwelling irradiance of ~ 8 x 1014 photons/cm2/s (integrated from

400 to 700 nm).

Photographs of a sexually mature female in a receptive posture on a solid grey background were used in place of live females to limit confounding variables, such as chemical, tactile, or motion cues. Photos were printed on

Staples brand recycled copy paper and mounted to white foam boards for stability. A red-clawed photograph was tested against photographs with orange claws of varied relative luminances. The red color tested was selected from a photograph of a sexually mature female blue crab’s claws using the Color Picker tool of Photoshop CS (Adobe Inc., San Jose, CA, USA). After selecting the red test color, we chose an orange color similar in relative luminance under the given illumination spectrum of the test arena. This color is described as Orange 0.85; with the number following corresponding to relative luminance. The spectral reflectances of the red and orange colors were measured using a reflecting probe coupled with a light source and spectrometer. From Orange 0.85, five other oranges were produced by increasing and decreasing the relative luminance of the color using the Brightness function in the Color Picker tool: one darker

(Orange 0.48) and four lighter variations (Orange 2.6, 5.1, 7.7, and 12). These orange variations had similarly shaped reflectance curves, but varied in average reflectance (Fig. 7). Thus, a total of six versions of the photograph were used: one 41

image with red-colored claws (Fig. 8) and five images with orange-colored claws.

The grey background used in the photographs had a relatively constant spectral reflectance (not shown), with a relative luminance of 3.8, and an opponency of

0.007.

Figure 7. Mean spectral reflectances and standard error of the red and orange colors used in behavioral experiments. n = 20 for each color.

42

Figure 8. One of the six photographs used in binary choice tests, pictured here with claws colored red. The crab is in a known sexually receptive posture.

All experimental trials were conducted between 0700 and 1900 local time from May through November 2009 and April through August 2010. Prior to the start of each experiment, one male was placed in an experimental tank and allowed to adjust to the surroundings for three hours. Then, two photographs were presented to the crab -- one at each end of the tank. Photograph positions were assigned randomly. Over the next hour, the male’s behavior was recorded on video. Videos were later watched and scored blind to the experimental conditions. Most crabs made multiple stereotypic sexual displays that were unambiguously directed towards (and occurred within 5 cm of) one of the two images. During these 5-30 second displays, the crabs rose up on their walking legs, extended their claws, and waved their paddles while facing the photograph

43

(see Baldwin and Johnsen, 2009 for further details). The total number of sexual displays made towards each image over one hour was counted. Choice was assigned to the image that received the greater number of displays. Full results of each binary choice test, including first display and number of displays, are given in Table 4 located in Appendix A. We evaluated the statistical significance of choices for each variation of orange tested using two-tailed exact binomial tests.

The statistical issues inherent in multiple testing were regulated using the

Benjamini and Hochberg (1995) procedure, which controlled for false discovery rate.

We used 126 male crabs during these binary choice tests. Because these males are wild-caught, it was not known how recently they had molted or mated. Both of these factors may have affected their sexual receptivity. If a male did not display mate preference during the one-hour trial, the trial was discarded and the male was tested again five to seven days later. No males were used more than once in a five-day period and males were not reused for the same test colors. After testing, the males were returned to the re-circulating seawater system.

44

3.2. Results

3.2.1. Spectral reflectance of the claws of Callinectes sapidus:

Measurements of claw coloration revealed clear differences in the spectral reflectance of claws between male and female blue crabs and also between sexually immature and mature crabs of each gender (Fig. 6). In most individuals, the average reflectance of the exterior of the claw was greater than that of the interior of the claw. Spectral reflectance varied between claws of sexually mature males and females, with males reflecting more light at shorter (bluer) wavelengths and females reflecting more light at longer (redder) wavelengths.

The spectral reflectance of both male and female blue crabs changed with sexual maturity (Fig 6).

3.2.2. Relative luminance and opponency of natural claw colors:

Based on our model of blue crab visual perception, the relative luminance and opponency values of claws differed between sexually immature males, mature males, immature females and mature females (Fig. 9). The perceived claw coloration appeared to be distinct between groups, although there was considerable individual variation. Claw coloration of prepubertal females overlapped with both immature and mature females, which is not surprising

45 given that this is an intermediate molt stage that transitions females from sexually immature to mature.

Figure 9. Achromatic and chromatic cues of natural crab claws with 95% confidence intervals. Achromatic cues are shown as mean relative luminance, the perceived luminance of each color relative to Red. Chromatic cues are reported as oppenecy values based on an opponent interaction between the blue (λmax = 440nm) and green (λmax = 508nm) channels. Data is reported for each side of the claw relative 46

[Figure 9 Continued] to the crab, referred to as interior and exterior claw surface. A) Interior claw surfaces. Points labeled as: MM-I, mature male interior claws (n=28); IM-I immature male interior claws (n=30); IF-I immature female interior claws (n=27); PF-I, prepubertal female interior claws (n=21); and MF-I mature female interior claws (n=25). B) Exterior claw surfaces: MM-E, mature male exterior claws (n=28); IM-E, immature male exterior claws (n=30); IF-E immature female exterior claws (n=27); PF- E, prepubertal female exterior claws (n=21); and MF-E sexually mature female exterior claws (n=25).

3.2.3. Behavioral Experiments:

Male color preferences did not appear to be mediated solely by chromatic or achromatic cues, but rather appeared to have a specific range of preferred color and intensity. Males significantly preferred red-clawed females (with an opponency value of 0.33) to those with claws colored with orange 0.48

(opponency value 0.38; p≤0.004), orange 5.1 (0.43; p ≤ 0.013), orange 7.7 (0.44; p ≤

0.001) and orange 12 (0.16; p≤0.041) (Figs.10, 11). Males significantly preferred red-clawed females over females with orange claws with both lower opponency values (orange 12, grey 5.7, and grey 0.85) and also those with higher opponency values (oranges 0.48, 5.1, and 7.7; Figs. 10, 11). However, males were either not able or not motivated to choose between red and orange claws similar in both opponency and relative luminance as perceived by the blue crab visual system.

In the trials testing for preference between Red and Orange 0.85 and Orange 2.6,

47 males chose red slightly more often, but these preferences were not statistically significant.

Figure 10. A) One-dimensional plots of relative luminance and opponency of the experimental colors used during binary choice trials. B) Relative luminance and opponency plotted together to represent how the blue crab may perceive both achromatic and chromatic cues. In both, the red cross shows the value of the red test color. Orange colors that were chosen significantly less often than red are represented

48

[Figure 10 Continued] by minus signs. Circles represent orange colors that were not chosen significantly less often than red. Data in (B) are offset for clarity. Data labels are given as R, red; G, Grey; or O, orange; followed by their luminance relative to the red test color.

Figure 11. (A) Results of the binary choice experiments testing male preference between red and orange female claws. (B) Previous results of binary choice tests showing male preference for females with red claws over those with white claws and dark grey claws that were isoluminant to the red claws (from Baldwin and Johnsen, 2009). Asterisks denote significance, * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001.

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3.3. Discussion

In the present study, models of visual perception and behavioral tests of color vision were used to evaluate the ability of the blue crab to perceive differences in natural claw colors. Previous behavioral experiments have indicated that male blue crabs use chromatic cues when choosing a female mate by demonstrating male preference for red clawed females over those with grey claws matched in relative luminance (Fig. 11B; Baldwin and Johnsen, 2009). It remained unclear, however, if males would be capable of discriminating between female claw colors found in nature (Fig 11). The behavioral trials presented here were intended to probe the ability of the blue crab to choose between similar long-wavelength colors during mate choice. Unexpectedly, our results suggest that male blue crabs use a mixture of chromatic and achromatic cues to discriminate between long-wavelength colors. Additionally, because males prefer red clawed females and can distinguish red from orange, claw color could function as a sexual signal or cue.

We found that male blue crabs could distinguish between red and orange coloration, except when the test shades were similar in both relative luminance and opponency. The results suggest that both chromatic and achromatic cues function in the discrimination of colors dominated by long-wavelength light.

Further, there may be a particular range of relative luminance (0.85-2.6) and 50 opponency (0.3 – 0.45) values that stimulates male courtship behavior (Fig. 11).

Additional studies using multiple shades of gray and other colors may help in understanding the limits of color vision in this species. Alternative models of relative luminance suggest that if the blue crab possessed a visual pigment with a

λmax between 540 nm and 600 nm, the choices we observed could be based solely on achromatic cues. However, no such photoreceptor has been detected through electroretinography (ERG) or microspectrophotometry (MSP) (Martin and Mote,

1982; Cronin and Forward, 1988).

In invertebrate species, achromatic and chromatic cues are often used for different tasks. Achromatic vision may be more useful for tasks involving motion detection and shape or object recognition (Lythgoe, 1979; Kelber et al, 2003), while chromatic cues are often used when identifying and classifying objects, such as food, oviposition sites, or potential mates (Vorobyev and Osorio, 1998;

Sumner and Mollon, 2000). Most studies investigating innate visual behaviors of invertebrates show evidence of only chromatic cues or only achromatic cues being used during specific tasks, and the simultaneous use of both is yet unclear

(Kelber and Osorio, 2010). However, experiments that involve color learning have shown evidence of the use of both. For example, studies in the honeybee,

Apis mellifera, provide evidence that either chromatic or achromatic cues can be used for object identification, depending on the size of the object (Giurfa and 51

Vorobyev, 1997; 1998; Giurfa et al., 1997). The hawkmoth, Macroglossum

stellatarum, can learn to associate rewards with either the chromatic or the achromatic aspect of a color, but appears to more readily learn the chromatic aspect (Kelber, 2005).

True color vision has been infrequently documented in crustaceans.

Certainly, spectral sensitivity and the presence of different spectral classes of

photoreceptors have been widely documented across crustacean species (Cronin

and Forward, 1988; Frank and Widder, 1999; Rajkumar et al, 2010), but the

behavioral evidence needed to confirm color vision (and discount confounding

achromatic cues) is not common. Aside from tests in C. sapidus, color vision has

been demonstrated in a fiddler crab, Uca mjoebergi, where females chose males

with painted yellow claws over those with claws painted various shades of grey

(Detto, 2007). Color vision has also been shown in a stomatopod, Odontodactylus

scyllarus (Marshall et al., 1996). This mantis shrimp was capable of discriminating

red, green, and yellow from different greys. However, individuals of O. scyllarus

were not able to discriminate blue from grey, possibly due to similarities

between the blue and grey stimulation profiles (Marshall et al., 1996). The

authors suggest that there may be a threshold value between photoreceptor catch

ratios for color detection to occur (Marshall et al., 1996).

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3.3.1. Role of claw color

Some crustaceans, most notably crabs and stomatopods, have colorful

displays paired with well-developed visual systems, and visual cues can play a role in their communication and social behavior (Schöne, 1968; Marshall et al.,

2006). Fiddler crabs (Uca spp.) have been shown to use color cues in gender recognition, individual recognition, and mate choice (Detto et al., 2006; Detto,

2007). In brachyuran crabs, the claws, in particular, are commonly used in sexual and agonistic communication (reviewed by Schöne, 1968; Christy, 1987). Claw color in the semaphore crab, Heloecius cordiformis, corresponds with gender and age (Detto et al., 2004), and may indicate sexual maturity. Given these examples, it is reasonable that claw color may act as a signal or cue in the blue crab.

There are several possible roles for claw coloration in Callinectes. First, claw color may play a role in species recognition. There are as many as 16 species of Callinectes, 10 of which have overlapping habitats with C. sapidus in the

Caribbean Sea (Robles et al., 2007). Claw color and pattern vary with species (Fig.

12) and may potentially be used to identify conspecific mates. It is possible that certain claw colors tested here were not recognized as belonging to potential mates and thus did not receive male preference.

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Figure 12. Coloration of species in the genus Callinectes. Of the 16 species of Callinectes, up to 10 species may overlap in coastal regions of Caribbean Sea. Eight of the ten Caribbean species are pictured here (C. maracaiboensis and C. affinis are not shown) Photographs courtesy of the Southeastern Regional Taxonomic Center (SERTC).

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Measures of crab claw coloration and their estimated appearance to the

blue crab eye, indicate that claw color may also function in gender identification.

Color differences between males’ blue claws and females’ red claws should be

apparent to the blue crab’s dichromatic visual system (Fig 9). While we have not

conducted tests between blue and red-clawed photos, in previous tests between red and white-clawed photos, males often addressed the white-clawed photos with agonistic behavior (Baldwin and Johnsen, 2009). Since the exterior face of male claws are largely white, this may indicate that male test subjects viewed our white-clawed photos as male competitors. Also, previous behavioral tests of color vision in the blue crab showed that male and female blue crabs had significantly different reaction times to blue, yellow, and red-colored approaching objects (Bursey, 1984).

Claw color may also act as a cue of sexual maturity. Spectral reflectance measurements show that claw color changes with sexual maturity in male and female crabs (Figs.6, 9). Also, claw colors of reproductively ready female crabs

(prepubertal and sexually mature females) fall within the boundaries of observed male preferences of both relative luminance and opponency (Fig 10 B). Together, these provide evidence that males may use claw color to identify sexually mature female blue crabs. Examples of color cues indicating sexual maturity are found in many species and are most often described in males (Kodric-Brown, 1985; 55

Frischknecht, 1993; Bakker and Mundwiler, 1994), but are increasingly

documented in females (McLennan, 1995, 2000; Amundsen and Forsgren, 2001).

Additionally, our color measurements show similarities between immature

males and immature females (Figs. 6, 9). In species where juveniles and adults

have different coloration, juvenile coloration may be a non-threatening cue to

adult conspecifics, indicating a lack of competition for territory, food, or mates

(Neal, 1993; Mahon, 1994).

Finally, individual variation in claw color also invites speculation

regarding claw color and individual quality. In the blue crab, both the blue and

red colors are due to carotenoid-based pigments located in the hypodermis

(Smith and Chang, 2007). Since carotenoids cannot be synthesized de novo in

animals, they must be ingested and may reflect an individual’s foraging ability

(Bagnara and Hadley, 1973; Brush and Power, 1976). In environments where

carotenoids are limited, carotenoid-based pigmentation may serve as an

indicator of individual quality (Endler, 1980; Hill and Montgomerie, 1994). In a number of species, carotenoid-based coloration may reflect an individual’s parasite load, immunological health, and overall condition (Borgia and Collis,

1989; McGraw and Hill, 2004; Clotfelter, 2007). Connections between coloration and mate attractiveness have been documented in several species of fish

(Frischknecht, 1993; Bakker and Mundwiler, 1994; Evans and Norris, 1996) and 56 birds (Hill and Montgomerie, 1994; Saks et al., 2003). Such results imply that both male and female blue crab claw coloration could be indicative of individual quality. While a relationship between claw color and individual quality has not yet been established for either male or female blue crabs, the possibility that claw color could advertise individual quality has intriguing applications for future studies of mate choice and intraspecific competition.

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4. The effects of molting on the visual acuity of the blue crab Callinectes sapidus.

The visual systems of the nearly 50,000 extant crustacean species are highly variable. Most crustaceans have compound eyes, with minimum inter-

ommatidial angles varying from about 50º in Daphnia (Land and Nilsson, 1990) to

about 1º in certain stomatopods (Land, 1999). While some crustaceans are monochromats, color vision is common, in some cases involving as many as twelve distinct channels (reviewed by Cronin, 2006). Overall, crustaceans can be highly visual creatures, using vision to find food and shelter and avoid predation. Vision is also used in both agonistic and sexual communication

(Schöne, 1968; Molenock, 1975; Bruski and Dunham, 1987; Christy and Salmon,

1991; Detto et al., 2006; Chiou et al., 2008).

Molting is a feature common to all crustaceans, and this process may create a visual challenge that has not yet been examined. In crustaceans with compound eyes, the multiple facet lenses are composed of modified exoskeleton

(Land and Nilsson, 2002). When a crustacean molts, the entire exoskeleton is shed, including the facet lenses. Prior to molting, the exoskeleton separates from the underlying hypodermis, which then secretes a new epicuticle (Smith and

Chang, 2007). Then, the outer exoskeleton ruptures, the animal extracts itself from its old cuticle, and the body quickly expands its new cuticle via rapid water

58 intake (Smith and Chang, 2007). Calcification of the new exoskeleton begins within 12 hours of molting and continues for up to 30 days after molting

(Dendinger and Alterman, 1983; Cameron, 1989). Thus, prior to molting, the existing lenses of each ommatidium must be partially resorbed, resulting in a separation of the lenses from the underlying tissue. Following this separation, the underlying tissue secretes new lenses. This process introduces a cavity between the old and new lenses, and the secretion of the new cuticle temporarily adds an additional tissue layer in the eye. These changes increase the distance between the lenses and the photoreceptors and possibly disrupt the light paths in the eye, either of which would affect visual acuity.

The blue crab Callinectes sapidus has large, stalked apposition compound eyes with a minimum inter-ommatidial angle of about 1.5° (Eguchi and

Waterman, 1958; Land and Nilsson, 2002). Blue crabs are often thought to simply act as scavengers, but they are excellent visual predators, reportedly using vision to catch fish, stalk fiddler crabs, and even bury themselves up to their eyes to ambush a prey item (Abbott, 1967; Hines, 2007). Behavioral experiments testing visual responses to various shaped and colored objects suggest that C. sapidus is able to discern blue, yellow, and red (Bursey, 1984; Baldwin and Johnsen, 2009).

Vision also plays an important role in blue crab communication, with a number of visual postures being used in agonistic (Jachowski, 1974) and 59

courtship behaviors (Teytaud, 1971). The male blue crab can visually choose

mates and prefers females with red claws over those with claws of other hues

(Baldwin and Johnsen, 2009), and female blue crabs may use visual cues in

identifying males (Teytaud, 1971). Since females pair with mates just prior to

molting (Jivoff and Hines, 1998), possible changes in visual acuity during this

period could affect female’s use of vision in mate choice.

In the present study, we investigated changes in blue crab visual acuity

during the molting process using behavioral and morphological assays. Using

the optomotor response, an innate reflex that stabilizes the visual field, we

evaluated blue crab visual acuity during molting. In addition, eyes from individuals undergoing molting were examined for changes in structure.

4.1. Methods

4.2.1. Study Species

Blue crabs were collected in April 2010, from Jarrett Bay near Smyrna,

North Carolina, USA (34º45’31”N, 76º30’44”W). Portunid crabs are, in part,

characterized by having the fifth pair of walking legs flattened into swimming

paddles. In the blue crab, a visual examination of these paddles can reveal signs

of an impending molt, such as a separation between the existing exoskeleton and

the underlying tissue (see Figure 2 in Smith and Chang, 2007). Thus, visual

60 inspection of the paddle is used by crab fishermen to identify premolt crabs, and changes in the appearance of the blue crab paddle can be used to estimate the number of days prior to an individual’s molt. Twelve female crabs approaching their terminal, adult molt and predicted to molt within five days were collected, transported to Durham, NC and kept in individual compartments in a recirculating seawater system at a temperature of 22-24°C and a salinity 28-30‰ on a 12:12 hour light: dark cycle. Pieces of shrimp were offered to crabs daily throughout the trials, though crabs only fed after molting.

4.1.2. Optomotor Response

The average visual acuity of the eye can be determined via stereotypical behaviors such as the optomotor response, which can be elicited by placing an animal inside a rotating black and white striped drum (Fig. 13). The animal reflexively turns or moves its eyes in the same direction as the rotation of the stripes, thereby stabilizing its visual field (Reichardt, 1961; McCann and

MacGinitie, 1965). This response can be used to determine an animal’s visual acuity (assessed via the minimum resolvable angle αmin) by finding the angular width of the stripe pattern that the animal no longer responds to. It should be noted that, while visual acuity in this study is assessed and quantified by the

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minimum resolvable angle, this parameter is the inverse of visual acuity. In other

words, a decrease in αmin corresponds to an increase in visual acuity.

Figure 13. Diagram illustrating the optomotor apparatus. A test subject is placed in a cylindrical acrylic tank suspended inside a striped drum that is mounted to a computer controlled stepper motor. Overhead lighting illuminates the arena and crab behavior is observed via video camera.

Our optomotor apparatus consisted of a 56 cm diameter drum that was

rotated from below by a computer-controlled stepper motor (STP-MTRH-23079,

AutomationDirect, Atlanta, GA, USA). Stripe patterns, with angular widths of

1.4º, 1.6º, 1.8º, 2.1º, 2.3º, 2.9º, 3.6º, 4.8º, 6.2º, 12º, 14º, 32º, and 48º for each pair of one black and one white stripe (as viewed from the center of the drum), were printed with a laser printer on standard printer paper and mounted within the drum. During testing, animals were placed within a water-filled 30 cm diameter cylindrical acrylic tank suspended inside the striped drum and observed using a

62 video camera mounted overhead. The positions of the crabs were noted during experiments, and the perceived angular widths of the stripe patterns were calculated based on the shortest distance between the eyes and the pattern.

While calculations of the angular widths would have been simplified by confining subjects to a smaller test arena, or by tethering individuals, this species did not respond in preliminary tests when confined to a smaller tank. A uniform

50% gray control pattern was also prepared and used during experiments. The test arena was illuminated by an overhead incandescent lamp fitted with a diffuser, resulting in a downwelling irradiance of 8.6 x 1014 photons/cm2/s

(integrated from 400 to 700 nm), which is similar to those that have been used in previous C. sapidus behavior experiments (Baldwin and Johnsen, 2009).

The twelve premolt female blue crabs were tested daily for four to five days prior to molting and for six days after molting. One individual perished during molting, so no postmolt data was recorded for it. Prior to the beginning of the experiment, individuals were placed in the test arena and allowed to acclimate for 15 minutes. Then the drum was rotated at a constant speed of two revolutions per minute, an optimal speed determined in preliminary experiments with this species. If the animal responded by walking in the direction of the rotation, the drum was counter-rotated to ensure that the crab was moving in response to the stripes. During initial tests, individuals were first 63 tested with narrow stripes, moving to wider stripes as needed to elicit a response. In successive trials, individuals were tested beginning with the narrowest stripe pattern that stimulated a response in the previous day’s test. If the crab responded, it was tested with increasingly narrower stripes until it failed to respond. If the crab did not respond, wider stripe patterns were tested. This approach minimized the amount of time that animals were exposed to the testing conditions. In the first three days of testing, each animal was subjected to the gray control once during its testing period. In subsequent days, each individual was tested with the gray control every other day. The gray control was tested a total of 82 times and did not elicit a response in any individual.

4.1.3. Analysis of optomotor data

The individuals used in the optomotor assays were wild-caught crabs.

While we strived to collect specimens that were at approximately the same molting phase, there was unavoidable variation in the number of days each animal was from molting. Thus, the results were aligned by molting time. This decreased the N value for days four and five prior to molting, since not all crabs were four or five days away from molting at the time of capture. We described the data using median and interquartile range, rather than mean and standard error, because the presence of outliers skewed the data far from a normal

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distribution. We compared the minimum resolvable angle αmin at each day to that

found four days prior to molting, as these values are similar to estimates of αmin

during intermolt phases. The non-parametric Mann-Whitney U-test was used because the distributions were not normal. The statistical issues inherent in multiple testing were regulated using the Benjamini and Hochberg procedure, which controls for false discovery rate (Benjamini and Hochberg, 1995).

4.1.4. Eye Morphology

Eyes were surgically excised from crabs at the Hooper Family Seafood shedding operation in Smyrna, North Carolina in May of 2010. Using the same methods described above, premolt crabs were identified and grouped by molt stage. Eyes were taken from intermolt crabs, crabs molting within 3-5 days, crabs molting within 1-3 days, molting crabs, and postmolt crabs no more than two hours after molting. Eyes were fixed in 4% formaldehyde buffered with artificial seawater for 48 hours and then stored in 70% ethanol. Twenty-four hours prior to sectioning, eyes were rinsed and stored in phosphate buffered saline (PBS). Eyes were then frozen and bisected horizontally across the long axis using a cryostat.

One half of each eye was examined and photographed under a stereoscopic dissecting microscope (Lumar V12, Zeiss Inc., Jena, Germany). Dimensions of the structures in the eye and the gap between the corneal lens, crystalline cone, and

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newly secreted lens were measured using AxioVision software (version 4.6.1.0,

Zeiss Inc.), a program that controlled the microscope and was calibrated

according to the installed objectives and distance from the sample. In eyes that

had variable separation, we measured the size of the gap at a location that

appeared to be between the two extremes, with the intention of measuring the

average separation. Four eyes were measured and analyzed at each stage.

4.2. Results

4.2.1. Optomotor Response

A significant loss in visual acuity was documented in C. sapidus in the

days prior to molting (Fig. 14, 15; Table 5). Four days prior to molting, the

median αmin was 1.8º. In the 24 hours prior to molting, the median αmin rose to

15.0º. After molting, visual acuity improved daily, with the median αmin dropping

to 1.6º within six days. Recovery of normal visual acuity was usually achieved

within six days of molting, although by three days after molting the median αmin

of 2.9º was not significantly different from that measured four days prior to

molting. There was substantial variation in individual acuity and in the rate of vision loss and recovery, with a few outliers having very poor visual acuity

during molting (Fig. 14).

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Figure 14. Box-and-whisker plot showing the minimum resolvable angle αmin before, during and after molting. The red bars are bounded by the lower (25th percentile) quartile and the median. The blue bars are bounded by the median and the upper (75th percentile) quartile. The error bars encompass the minimum and maximum of the data, with six outliers (defined as points farther than 1.5 times the interquartile range from the median) plotted as individual points.

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Figure 15. Multi-plot showing the time courses of change in minimum resolvable angle, αmin, during the molting process in individual Callinectes sapidus.

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Table 4. Individual results of optomotor response tests showing the minimum resolvable angle αmin before, during and after molting. Molting is time zero. The median and interquartile ranges are reported for each time point. The probability that αmin differs significantly from that of the intermolt crab eye was calculated using the Mann-Whitney U-Test. Significance is denoted with an asterisk, *.

4.2.2. Morphology of eyes during molting

We found that molting affected the structure of blue crab eyes (Fig. 16). In the intermolt crab eye, no gap between the corneal lens and underlying tissue was apparent. Eyes from crabs molting within 3-5 days displayed a gap of approximately 21.3 ± 9.8 µm (Mean ± SE), (N=4) between corneal lens and the crystalline cone. The gap increased to 68.8±34.4 µm (N=4) in the eyes of crabs predicted to molt within 1-3 days. These eyes also showed a thin, faceted layer

69 over the crystalline cone that was likely the newly secreted lens. In molting crabs, the photographs show the eye as it was exiting the exoskeleton. The average spacing between the eye and outer lens was 369 ± 221 µm (N=4). In the postmolt eyes, the eyes were taken from soft crabs less than two hours after molting and were not yet fully expanded. So, at the time of fixation, they were malformed and wrinkled.

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Figure 16. Photos of C. sapidus eye before, during, and after molting. The left photos in each pair are of halves of entire eyes. The right photos are close-ups that show the relationship between the corneal lens and crystalline cone. A) Intermolt crab that is at least 14 days away from molting. B) Crab that will molt in three to five days. C) Crab that will molt in two to three days. D) Crab that will molt in one to two days. E) Crab that was in the process of shedding its exoskeleton. F) Soft crab immediately after molting.

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4.3. Discussion

The behavioral results of this study indicate a significant, temporary loss

of visual acuity during molting in female blue crabs. Overall, the median values

of αmin in Callinectes sapidus increased from 1.8º to 15º during the molting period,

rising as high as 48º in certain individuals. Three days prior to and after molting, the median αmin was 2.9º, similar to that of the crepuscular dung beetle, Onitis

alexis (Warrant and McIntyre, 1990). In the 24 hour period before, the median αmin

in individual crabs was 15º, with some individuals having visual acuity

comparable to that of a planarian flatworm (Land and Nilsson, 2002). While the

duration and degree of change in visual acuity varied among individuals, all

demonstrated a clear and substantial decrease (Fig. 15). In most individuals, the onset of the decline in acuity occurred three days prior to molting. Recovery of visual acuity was also variable, but most individuals recovered normal vision within three to six days after molting. The recovery of individuals strongly

suggests against any acclimation to the optomotor assay with daily testing.

Taken together, our results imply that blue crabs are faced with approximately

six to nine days of reduced visual acuity during the molting period (Fig. 17).

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Figure 17. The effects of molting on vision. Simulated appearance of a courting male blue crab as viewed by a female blue crab from a distance of 15 cm. During intermolt periods, the median αmin is approximately 1.6º. Three days before to molting it is 2.9º, two days before it is 13.6º, and in the day before to molting the median is 15º. The images were created by convolving the top image with a Gaussian point spread function that corresponded to a modulation transfer function whose contrast reproduction at the maximum resolvable spatial frequency was less than 2% (the 2% value was chosen because it approximates the minimum contrast threshold for many organisms). The end result of the convolution is an image that contains no detectable spatial information spanning angles less than the minimum resolvable angle αmin. The convolution was performed for each color channel separately using standard Fourier methods on 2048 x 2048 pixel images. See Johnsen et al. (2004) for further details.

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Our examination of the eyes of molting blue crabs showed that the morphology of a molting eye differs from that of a typical intermolt apposition compound eye, and these differences could be responsible for the loss of visual acuity observed during molting. In the typical apposition compound eye, each individual ommatidium functions independently, sampling light from its visual field (Land, 1997). Incoming light is focused through the corneal lens onto the distal tip of the rhabdom (Fig. 18A; Land and Nilsson, 2002). In molting eyes, the lens separates from the underlying crystalline cone, introducing a gap that ranges from approximately 19 µm in early premolt crabs to about 0.5 mm in crabs close to molting (Fig. 18B). The corneal lens of each ommatidium has a set focal distance, so the large separation generated during molting results in the rhabdom receiving a defocused image. In addition, the secretion of the future lens between the crystalline cone and the lens of the existing exoskeleton (Fig.

18C) may further affect the eye’s focusing ability by increasing the distance between the lens and the rhabdom and by adding a layer of material with a high refractive index. Aside from changing the focal point, the growing gap may allow light entering one facet of the eye to fall onto multiple ommatidia, disrupting the ability of each ommatidium to sample light from only its particular field of vision and leading to further loss of acuity.

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Figure 18. Diagrams of apposition compound eyes and ommatidia showing the progressive changes in the eye during molting, including the possible light path in the ommatidia (modified from Land and Nilsson (2002). A) Intermolt eye. B) Crab that [Figure 18. Continued] will molt in three to five days. C) Crab that will molt in one to three days. The growing space between the corneal lens and crystalline cone is colored in blue. The newly secreted cuticle is shown as a tan layer. The orange lines indicate the probable light path and focal position in the eye. Labels: CL, corneal lens; CC, crystalline cone; RC, retinular cells; and RB, rhabdom.

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Further, in the immediate postmolt crab, the eye is structurally weak and

misshapen. The postmolt body, and likely the eye, is inflated by the rapid uptake

of water that occurs for several hours after molting (Mangum, 1992).

Calcification of the new exoskeleton takes several days to weeks (Dendinger and

Alterman, 1983; Cameron, 1989). The eye and the corneal lenses likely do not

function correctly until inflation of the body and partial hardening of the

exoskeleton occur. Reduced visual acuity in recently molted crabs may be

attributed to functional limitations of the soft crab eye.

We acknowledge that there are certain limitations and caveats to both the

behavioral and morphological components of our study. One, there is notable

individual variation in the results of both portions. The variation observed may

simply be due to random individual differences in the size of the gap between

the lens and the crystalline cone as the molting process occurs. While variables

such as individual or environmental conditions may play a role in an

individual’s progression through the molt cycle, it is unclear as to whether these

types of factors affect the separation size between the exoskeleton and underlying tissue.

Also, it would be fascinating to pair the behavioral and morphological

assays in individual subjects. However, at this time eye morphology cannot be

examined without removing the eyes, which would terminate any behavioral 76 assay of vision. Similarly, in the morphological examination, each crab’s progress in the molting cycle was estimated prior to removing and examining the eyes, giving an inexact pairing of eye morphology and molting stage. We could have removed the eyes and kept each individual alive until it molted in order to determine an exact time frame. However, removing the eyes induces physical damage, particularly blood loss, that reduces an individual’s survival during ecdysis. Further, we were concerned with the hormonal effects of removing the neuroendocrine X organ-sinus gland in the eyestalk. Additionally, this study would benefit from a mathematical model describing the changes in visual acuity that would result from the separation seen in the eye. However, this would require data on a number of variables, including curvature of the lens, refractive indices of the lens and crystalline cone, and focal distance, that have not yet been described for this species. With these limitations in mind, it is our opinion that there is a clear pattern that emerges from our results indicating that these crabs have reduced visual acuity during the molting process which may be due to a separation between the corneal lens and the underlying photoreceptors.

The natural history of the blue crab is well documented, and evidence indicates that mating is limited to the time period surrounding female molting, with courtship behavior involving both chemical and visual cues (van Engle,

1958; Teytaud, 1971; Gleeson, 1980). Males are described as using a repertoire of 77

visual cues during courtship, including waving the claws, standing tall on the

walking legs, and rhythmically waving the swim paddles (Teytaud, 1971). The

paddling maneuver may also function in directing chemical cues towards potential mates (Kamio, 2008). While courting, males should be fully capable of receiving chemical and visual cues, while females’ reception of visual cues will depend on the molting stage of the female receiver. If a female pairs with a male

three to four days prior to molting or well after molting, visual cues may factor

into her mate choice. However, since females tend to resist pairing until one to

two days before molting (Jivoff and Hines, 1998), when visual acuity is poor,

visual cues may not be reliable when choosing a mate and females may instead

depend upon chemical cues. The visual constraints of molting may select for

multimodal cues in this species. Multimodal communication is common

(Hughes, 1996; Hölldobler, 1999), especially in the context of mating (Papke et.

al., 2006; Uetz and Roberts, 2002), and signals displayed over two modalities may

increase the probability of reception (Cardé and Baker, 1984; Conner, 1987). In

the blue crab, chemical and visual cues may function redundantly to overcome differences in receiver ability or temporary sensory limitations.

The effects of molting on visual acuity may impact numerous crustacean groups and, more generally, arthropod groups that molt. In crustaceans with compound eyes, the corneal lenses are comprised of exoskeleton which must be 78

shed; therefore, decreased visual acuity during molting is likely a general

phenomenon. Given the diversity of crustacean species, the extent and duration

of the changes in visual acuity may be quite variable amongst species.

Additionally, the linking of mating and female molting is present throughout the

crustaceans, particularly in decapods (Hartnoll, 1967; Christy, 1987; Asakura,

2009). Mating concurrent with female molting is documented in ostracods (in

Cypridinae; Morin and Cohen, 1991), isopods (in Sphaeromatidae; Shuster, 1989),

amphipods (in Gammaridae; Borosky and Borosky, 1987), shrimp (in Caridae and

Penaeidae; Bauer, 1979; Boddeke et al., 1991), American lobster (Homerus

americanus; Atema, 1986), and crabs (Portunidae and Cancridae; Christy, 1987).

Further, in our literature research, we have found a pattern indicating that visual sexual cues are nearly non-existent (or at least not documented) in groups that have paired mating and molting. Studies on sexual communication across

Crustacea are relatively sparse, and the existing body of literature has focused on chemical and mechanical cues (Ryan, 1966; Atema and Engstrom, 1971; Hughes,

1996). The few species that have well documented visual cues, primarily stomatopods and fiddler crabs (Salmon, 1984; Christy and Salmon, 1991;

Marshall et al., 1996; Detto et al., 2006; Chiou et al., 2008), do not have concurrent mating and molting. Our results showing reduced visual acuity during molting may serve as one explanation for the lack of observed visual cues in the sexual 79

behavior of crustaceans that pair mating and molting. Additional comparative work across multiple taxa is needed to further explore this hypothesis. More generally, our findings suggest that in addition to the well-known physiological

challenges associated with molting, the visual system is also compromised. This

is an area that has been previously unexplored and should be considered when

addressing the effects of molting on the life history of a species.

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5. Conclusions

5.1. Color Vision in the Blue Crab

The behavioral experiments described here confirm color vision in the

blue crab (see Chapters 1 and 2). Color vision is based on the comparison of

inputs from at least two receptor types in the eye, which allow an animal to

evaluate spectral information about a visual scene. In previous physiology

examinations of Callinectes sapidus vision, results of microspectrophotometry

examinations describe only one photosensitivity peak at 504 nm (Cronin and

Forward, 1988), while two photoreceptors were detected by electroretinography,

with peaks at 440 nm and 508 nm (Martin and Mote, 1982). However, even in the

presence of two confirmed photopigments, the presence of color vision can only

be established via behavioral experiments (Kelber et al., 2003).

In our first series of behavioral trials, the ability of the blue crab to distinguish between red and isoluminant grey, confirms the ability of the blue crab to detect differences in color. This supports the presence of two photoreceptors and color vision in C. sapidus (Baldwin and Johnsen, 2009).

Further, in the series of trials between red and varied shades of orange, we

further established the ability of the blue crab to perceive variations in both

81

luminance and color (Baldwin and Johnsen, 2012). These results support

dichromatic color vision in the blue crab.

5.2. Role of color in male mate choice

Visual cues are known to play a role in the sexual behavior of certain crabs

and crustaceans, though this was previously not documented in the blue crab.

The results given here show for the first time that visual cues alone can stimulate

courtship behavior in male C. sapidus. Our behavioral trials show that males can

detect variations in claw luminance and color that would be naturally occurring

in the blue crab population. Further, sexually receptive males have a preference

for females with claws that fall within a specific range of relative luminance and

opponency. We suggest that these preferences assist males in identifying

appropriates mates, ideally, sexually receptive females. Our investigations of natural claw coloration have determined that claw color reliably indicates gender and sexual maturity. It is less certain, although plausible, that claw color indicates individual quality and species identity. Thus, male blue crabs may be using claw color to evaluate information regarding gender, sexual maturity, quality, and/or species identification.

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5.3 The Role of Vision in Female Mate Choice

Vision is constrained during molting due to the separation and loss of the lenses of the compound eye, which are composed of exoskeleton (see Chapter 3). Blue crabs experience reduced visual acuity several days before and after molting. It is during this time, however, that females should be choosing male mates. This reduction of visual acuity implies that visual cues may not be primary components of female choice. Blue crabs use both visual and chemical cues in mate attraction, and it is possible that females rely more on chemical cues when vision is constrained by molting.

There are large variations in the degree of vision loss, as well as the onset and recovery of vision. These variations paired with the variability in the timing of female mate choice do not entirely rule out the use of visual sexual cues. Prepubertal females tend to resist courting males until just before the terminal molt, between 1-4 days (Jivoff and Hines, 1998). Vision loss due to molting also occurs in the 1-4 days prior to molting.

Depending on the individual female and her progression in the molting cycle, she may or may not be capable of seeing her chosen suitor. Further, females may mate with multiple males up to three weeks after the terminal molt (Jivoff, 1997). Since vision is recovered 3-6 days after molting, females engaging in later copulations would likely be capable of detecting visual cues. Further examination is needed to clearly understand the role of visual cues relative to the female blue crab.

83

6. Future Directions in Callinectes Research

6.1. The Role of Claw Color in Species Identification

The blue crab is one of 15 geographically overlapping species within

Callinectes, a genus of colorful swimming crabs that inhabit the coasts of the western Atlantic and eastern Pacific Oceans (Robles et al., 2007; Rosas et al.,

2004). Species of Callinectes are similar in shape and size, but have different claw colors (Williams, 1984). There is no documentation of hybridization between members of Callinectes; however recent molecular phylogenies indicate that some previously described species are actually different morphs or subspecies of larger species groups (Robles et al., 2007). It is unknown whether these are reproductively isolated, and hybridization is common in other brachyuran crabs, including one genus of swimming crab, Scylla. Since species of Callinectes have overlapping habitats, are morphologically similar, and use visual signals in courtship behaviors, it is possible that claw color functions in species recognition.

To explore this aspect of claw color, we would document specific differences in claw color by collecting numerous males and females of each species and measuring the spectral reflectances of the claws. We would then use behavioral experiments to determine whether Callinectes species can assortatively mate using visual cues alone. Field observations of behavior would also be conducted. Understanding the role of claw color is a key component in

84

understanding not only visual communication in this group of crabs, but also for

investigating the radiation of this large number of overlapping species radiating

from Caribbean waters.

6.2. Investigating the Effects of Turbidity on the Perception of Visual Cues

The blue crab mates in estuaries that can be extremely turbid due to

nutrient pollution. Because turbidity attenuates light and contrast, it interferes

with signal transmission and therefore may disrupt visually stimulated behavior,

such as sexual signaling. The relatively few studies that have examined the

effects of turbidity on visual signals have found that turbidity disrupts

interactions relying on vision, including those involved in mating. Turbidity

decreases selectivity for attractive mates and disrupts population dynamics of

sand gobies (Pomatoschistus minutes; Järvenpää and Lindstrom, 2004), threespine

sticklebacks (Gasterosteous aculeatus; Engström-Öst and Candolin, 2006), and cichlids (Haplochromis sp.; Seehausen et al., 1997). Comparable research has not been performed on crustaceans, despite the importance of visual signaling in this group.

The Chesapeake Bay would serve as an excellent study site to investigate whether turbidity interferes with visual cues and mate choice in the blue crab.

The bay has a well-known crab population, history of turbidity, and existing data

85 of both declining blue crab populations and water quality. Underwater irradiance measurements for several areas in the upper Chesapeake Bay show that the underwater light environment is predominately yellow (Tzortziou et al.,

2007). In certain areas, underwater sighting distance is limited to only a few centimeters due to water turbidity. At best, turbidity may increase the physical risks and time costs incurred during mating. At worst, turbidity may lead to the hybridization of closely related species and a reduction in biodiversity.

Mathematical estimates of visibility and behavioral assays testing sexual responses in environmentally relevant conditions will be necessary to determine how turbidity may affect visual communication in the blue crab.

6.3. Significance of the Research

Ecologically, the blue crab plays a key role in coastal foodwebs.

Economically, the blue crab is a valuable commodity in commercial and recreational fishing. A large body of research has been dedicated to many aspects of the blue crab’s biology, however, little is known about their visual ecology.

Examining the effects of poor water clarity on visual communication may be necessary for proper management of the North American blue crab population.

Further exploring the role of color in visual communication, particularly in regards to species identification, will lend insight into the speciation of this genus. The Callinectes genus has radiated from relatively warm, clear Caribbean

86 waters, where up 15 different species live, to more temperate, and sometimes turbid, coastal waters of North and South America, where only a few species thrive. Examining color cues, visual communication, and the effects of turbidity may provide interesting results for the blue crab and its sister species.

87

Appendix A

Table 5. The results of binary choice experiments testing male preferences for photographs of females with red or orange claw coloration. The number of displays made to each color, the side of first display, and the preferred side are presented. Totals are shown with results of two-tailed exact binomial tests.

88

89

90

91

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Biography

Jamie Baldwin was born in Urbana, Illinois on May 23, 1983. Baldwin received a bachelors of science from Illinois State University in May, 2005. She began graduate school at Duke University in August 2006. Since that time, she has published a three articles including: “The importance of color in the mate choice of the blue crab

Callinectes sapidus” in the Journal of Experimental Biology (2009); “Visual Constraints of

Molting in the blue crab Callinectes sapidus” in the Journal of Experimental Biology (2011); and “Male blue crabs, Callinectes sapidus, use chromatic and achromatic cues during mate choice” in the Journal of Experimental Biology (in press). As a graduate student,

Baldwin has received a number of awards including the Anne T. and Robert M. Bass

Fellowship for Undergraduate Instruction from Duke University for 2011-2012; Best

Student Oral Paper from the Society of Integrative and Comparative Biology, Division of

Invertebrate Zoology in 2011; a One-Semester Fellowship from the Duke University

Department of Biology for 2010-2011; a Duke University Dissertation Travel Grant for

2009-2010; a National Geographic Explorer’s Club Graduate Exploration Grant in 2009; the Keever Graduate Field Research Fund from the Duke University Department of

Biology in 2008; and two Honorable Mentions from the National Science Foundation

Graduate Research Fellowship Program in 2006 and 2007. After her graduation from

Duke University, Baldwin will continue teaching and studying biology.

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