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Chromatophore Mapping of the Jewel Cichlid ( Hemichromis bimaculatus ) and the Effects of Morphological and Physiological Controls During Development By Diana Ward A Thesis Submitted to the Division of Natural Sciences New College of Florida In partial fulfillment of the requirements for the degree Bachelor of Arts Under the sponsorship of Dr. Leo Demski Sarasota, Florida April, 2010
DEDICATION I would like to dedicate this thesis to my parents and family, due to the immense amount of support that they have given me over the years. This is for you for always being there. ii
ACKNOWLEDGEMENTS I want to thank my advisor, Dr. Demski and my committee members, Dr. Beulig and Dr. Bauer for the guidance and support through the thesis proce ss. Thank you to my family for being patient and the constant love, even when it was just pho ne calls to hear about the alligators under the beds or of braiding hair. Ravi, thank you for pushing me constantly, for believing in me incessantly when I had felt like the future was bleak. You stuck by me through the stressful times and always made me smile. Amber Patti, you ar e an amazing roommate. I would not have been able to do it without you and the Pokemon epis odes, dances, songs. Carmela, you helped me dance my way through all of the turns. Brandan Cole, you were there almost daily listening to my odd rants, my random-seeming speeches and ideas, and always put up a face that they were logical. Duff Cooper, working with you for the p ast 3 years has been an exciting blessing. Bill Tiffany, your constant positive vibes have gotten me through so much, it is my turn to return the favor. Finally, Joel Beaver, you guided me, pus hed me, teased me, and mentored me through the past 4 years. I could not have done this without you. iii
TABLE OF CONTENTS Dedication......................................................................................................................ii Acknowledgements ...iii Table of Contents ...iv List of Figures & Tables .....v Abstract......vi Introduction................................................................................................................... ..1 Synthesis of Pertinent Literature.....................................................................................3 Methods........................................................................................................................ .38 Experimental and Observational Results.......................................................................42 Discussion and Conclusion............................................................................................53 Bibliography..................................................................................................................7 1 iv
LIST OF FIGURES & TABLES Table 1..........................................................................................................4 Figure 1............................................................................................................11 Table 2.............................................................................................................12 Table 3.............................................................................................................13 Table 4.............................................................................................................19 Table 5.............................................................................................................23 Table 6.............................................................................................................56 Table 7.............................................................................................................57 Table 8.............................................................................................................58 Figure 2.......................................................................................................... .62 Figure 3............................................................................................................63 Figure 4a..........................................................................................................64 Figure 4b..........................................................................................................65 Figure 5a..........................................................................................................66 Figure 5b..........................................................................................................67 Figure 5c..........................................................................................................67 Figure 5d..........................................................................................................68 Figure 6............................................................................................................69 Figure 7............................................................................................................70 v
vi Chromatophore Mapping of the Jewel Cichlid ( Hemichromis bimaculatus ) and the Effects of Morphological and Physiological Controls During Development Diana Ward New College of Florida, 2010 ABSTRACT The jewel cichlid (Hemichromis bimaculatus ) is used regularly as a model for behavior studies, commonly researched due to its unique color, ease in breeding, hardiness and prevalence in Florida. Studies have been focused on the role of color and patterns in fishes, examining its purpose in communication. Incomplete descriptions exist for the development of chromatophores for H. bimaculatus with the most complete study to da te by Baerends and Baerends-Van Roon (1950). For the present study, observations were carried out on the distribution and type of chromatophores across various stages in developmen t. The fish were photographed to mark the location of cells, placed on different backgrounds and social colorations marked. A partial chromatophore map was created as well as a staging system based on chromatophore observations. Numerous colors and hues, and thus, many signals may be produced due to the layering and distribution of the pigment cells, as well as their motile abilities. The distribution of chromatophores undergoes changes until early adulthood, at which stage red hues from erythrophores allow mating intent a nd aggression to be displayed. Dr. Leo Demski Natural Sciences Division
1 Introduction: Communication is important in animal species, as a tool for survival and it plays a crucial role in learning information about their environment and other organisms. A main form of communication in fishes is through skin color, both through hues and patterns. The ability to rapidly change colors makes t possible to produce a variety of signals. Rapid physiological color changes occur through the motile abilities of chro matophores, the color cells (Nery and de Lauro Castrucci, 1997). Slower changes in colorati on occur through developmental and seasonal changes in the fish, altering the cell number or quantity of pigment in the cells. Extensive research on color patterns has been done on the beha viors of various fishes, specifically medaka, zebrafish and jewel cichlids (Ohta and Sugimoto, 1980; Price et al., 2008; Baerends and Baerends-van Roon, 1950; Noble and Curtis, 1939). Baerends and Baerends-van Roon (1950) studied the various chromatophore patterns at th e different developmental stages in several cichlid species, including Hemichromis bimaculatus Red coloration was found to be important in mating and social recognition. This thesis addresses the development of chromatophores in H. bimaculatus including the timing of the neural innerva tions for the cells. It aims to determine when hormonal control is gained by the fish dur ing development and the importance of the color patterns seen through development. A study was conducted to determine the various chromatophores in the skin of the jewel cichlid at multiple ages and stages of devel opment. The methodology used was chosen to minimize the reduced stress and invasiveness to wards the fish (Beeching et al., 2002). Noninvasive light microscopy on live animals was used to document the patterning of melanophores and other chromatophores across the surface of the sk in. Failure to maintain broods of fry for long periods of time altered the scope of the stud y and changed it to an examination of published research on the development of color in Hemichromis bimaculatus and a discussion of their
2 findings in comparison to the limited original ob servations obtained in this thesis. This review provides insight into developmental patterns in H. bimaculatus and in other cichlids in general, as well as the importance of coloration during ontogeny The main area focused on was the specific placement of the five types of chromatophores during various stages of developmental growth. This is detailed in the review of colors and color patterns. Hemichromis bimaculatus Gill was chosen specifically due to its use in numerous be havior studies. Hardiness, bright display colors, opportunistic feeding, ease in breeding, and prevalen ce in Florida also led to the selection of the jewel cichlid for this study. An overview on colors in mammals is incorporated since models for mammals can often be based on comparisons to systems in cold-blooded vertebrates.
3 Review & Synthesis of Pertinent Literature Communication in Animals Interspecies communication is vital to a sp ecies survival. Such communication involves signals which aid in the defense of a group, signify the need to attack, and provide social cues. Vocal communication in mammals is often discu ssed in the literature. Studies range from those that examine the alarm calls of black-tailed prairie dogs (Cynomys ludovicianus ) which function to warn genetic relatives of approaching predator s (Hoogland, 1983) to mating calls which attract mates. Mating calls are common throughout animal species, extending through various families. Examples of vertebrates that produce mating calls are: the western gray mouse lemur (Microcebus murinus ); eastern rufous mouse lemurs ( M. rufus ) (Zimmermann et al., 2000); and the oyster toadfish (Opsanus tau L.) (Fine, 1978). Communication not only exists through auditory signals, but also through visual cues. Color displays can be useful for mating rituals, warning signals, and social warnings. The colors act to communicate both with interspecifics a nd intraspecifics. Various mating strategies have evolved color as a display. Such is common in polygamous mating. For example, females attempt to choose the most attractive colored mate, one who could pass down the most beneficial genes to their offspring. Optimal mates are chosen based on various visual cues, which act as true indicators to the individuals health. This sexua l selection has resulted in sexual ornamentation displays developed by some mammals (Anderss on, 1982; Barlow, 2000; Dominey, 1984; Setchell and Dixson, 2001). Colors in mating displays have been observed for the specific purpose of attracting mates. Blue-footed boobies ( Sula nebouxii ) lift their feet into the air during their courtship, with females possessing bluer feet receiving more attention from males (Torres and Velando, 2005). Agonistic displays can also be demonstrated through color. Such displays are observed in adult male vervet monkeys ( Cercopithecus aethiops sabaeus ) with darker scrotums were able to dominate those with a paler scrotum (Gerald, 2000).
4 Visual signals can present themselves through sexual dimorphism. Sexual dimorphism is common in many taxa of insects, fish, amphibians, birds and mammals. It is demonstrated in variations between color, shapes and sizes of the sexes. Color variabilit y between the sexes can play an important role in communication (Rohwer 1975; Zahavi, 1991; Andersson, 1994; Gerald, 2000). Bright advertisements may be maladaptive to an indivi dual (Lande, 1980), making them more visible to predators, however the benefit of providing them higher chances of reproductive success usually outweighs the drawbacks. Importance of Color in Fishes Colors in fishes and other vertebrates ma y provide social cues to conspecifics and intraspecifics. Color patterns are multi-compone nt signals that are formed from pigment arrangement and structural components (such as scales). Alterations in color may signify the intentions of animals to mate, fight or defend a territory. Color changes in fishes, as in other vertebrates, can be classified as cryptic or sematic From there, the classifications of the use of colorings can be further broken down (Table 1). Table 1. Classification of integumental colour schemes of animals (from Needham, 1974) Cryptic (camouflage ) 1. To escape detection by predators 2. To avoid detection by prey and to facilitate approach Sematic (Advertising, Signalling) 1. Aposematic (warning) 2. Episematic (attracting) (a) Epigamic: directed towards the opposite sex (b) Episomatic: serving non-sexual purposes such as luring prey 3. Pseudosematic (mimicking sematic schemes) (a) Pseudaposematic (b) Pseudepisematic
5 Cryptic Coloration: Cryptic coloring is a useful strategy for both prey and predators; with changes in the body pattern and colors allowing an animal to ble nd into the background. Other terms for cryptic coloring are protective coloring and concea ling coloring (Fujii, 1993) based on what the coloration is used for. Both paling and darken ing of the skin are common cryptic colorations observed in numerous fishes (Fujii, 2000; Sugimoto, 2002). Countershading as a form of cryptic coloration is important in oceanic migratory fishes, where there is a contrast of a darkened dorsal area and a whitish ventral skin (Denton and Nicol, 1966). When viewed from above, crystals in certain cells (described in detail on page 15) reflect a faint blue coloration, allowing the fish to blend in with the surrounding water; however, wh en viewed from below, the crystals reflect the light from the sky, preventing predation (Land, 1972). Sematic Coloration: Sematic coloration causes an animal to be noticed through more visible displays, providing communication with intraspecifics and inte rspecifics. It can be broken down into three categories: aposematic, episematic, and pseudosematic (Needham, 1974), based on the communicatory needs of the fish. Aposematic color advertises a warning to other fish of the same species, other species, and at times other vertebra tes which may threaten the fish. For example, territorial displays and other male-male interac tions can be linked to the color of the dominant male in the group. Male three-spined sticklebacks ( Gasterosteus aculeatus ) attack dummies with red coloration in their ventral region (Baerends, 1976). Red is a nuptial color for males during mating, and signified mate competition for the ma le three-spined sticklebacks. Certain cichlid species show a higher rate of agonistic interacti on to mating colors of conspecific males (Korzan and Fernald, 2007). Males of the swordtail fish ( Xiphophorus cortezi) displaying a pattern of
6 vertical bars show higher aggression to males with a similar pattern (Moretz, 2005) despite losing more frequently in fights against barless males of the same species. Bright, conspicuous coloration to indicate toxicity is another form of aposematic coloration witnessed in various coral-reef fishes through bright colors. Common are patterns with contrasting colors, large markings on the bodies, etc. (Reighard, 1908). The bright colors may indicate an abnormal odor, taste, unpalatability, toxi city possessed by the fish, or sharp spines that make it difficult to consume. For example, the Scorpaenidae family contains lionfish (of the genus Pterois ), which display aposematic coloration, warning predators of the venomous dorsal, anal and pelvic spines that grace the body (Schu ltz, 1986). The venom possessed in the spines is lethal to most fish (Allen and Eschmeyer, 1973) causing the conspicuous coloration to be a true warning to predators. Another true aposematic warning color for unpalatability is in the venomous red sea blenny (also called the black fang blenny) Meiacanthus nigrolineatus (Danfi and Diamant, 1984). It possesses active buccal poison glands and a color pattern of a blue rostral region trailing to a yellow caudal region, with a black stripe across. Episematic coloration is used to attract mates (epigamic) or to attract prey (episomatic). Nuptial colors are seen in a variety of cichlids, with some species (males, females, or both sexes) producing red coloration in their ventral region. Such is the case in threespine sticklebacks (Gasterosteus aculeatus ) and jewel cichlids ( Hemichromis bimaculatus ), (Korzan and Fernald, 2007; Reimchen, 1989; Rowland, 1975; Baere nds, 1976). Common nuptial colors in cichlids range from silver, black, green, red, to yellow (von Hippel, 1999; Seehausen et al., 1999; Seehausen and Schluter, 2004; Allender et al ., 2003). While stimulating aggression between the same sex, nuptial coloration characteristically attracts the opposite sex. The red color in reproductive males and females of Hemichromis bimaculatus is important in mate recognition. A female usually selects the more red of two males (Noble and Curtis, 1939; Baerends, 1976).
7 Attack, escape, and courtship beha viors were evoked in males of H. bimaculatus as a result of the color dummy being presented (Rowland, 1975). Episomatic coloration is used primarily to attract prey, although there are cases where certain colors are attractive to offspring of fishes (Baerends, 1976). A predatory cichlid, Haplochromis livingstoni resembles a decaying fish, and adopts death feigning (McKaye, 1981) as a hunting strategy. After dropping to the floor, th e cichlid waits for smaller fishes to approach, then attacks. Alternatively, episomatic colo ration can be useful for non-aggressive functions. Hemichromis bimaculatus fry, when removed from a school, have a preference for the color red displayed on a dummy (Noble and Curtis, 1939). Fry stayed close to the red colored dummy as if it were a parent. The preference for longer wa velengths by schooling fry increased as they developed (Baerends and Baerends-v an Roon, 1950). This was most specifically for the color red, which is considered to be the dominant nuptial color of the adults. Similar results were found for Chiclasoma biocellatum Aequidens portalegrensis and Aequidens latifrons (Baerends, 1976). Various forms of pseudosematic displays where an organism mimics another for personal gain, are possible. The levels of mimicry can range from similar appearances to complete imitations of another vertebra te or object. Nonterritorial males of Astatotilaopia burtoni from Lake Tanganyika, do not display the bright coloration of a blue or yellow background color as observed in the territorial males. Instead, they possess a similar coloration and size as the females, a brown/green background coloration (Korzan and Fernald, 2007). This coloration allows them to exist in the territory of a territo rial male without being attacked while they have side matings with the female. Pseudoaposematic or warning mimicry colors, more commonly called Batesian mimicry, are seen in fishes and other vertebrates. A harmless vertebrate displays the same coloration and pattern as an unpalatable vertebrate in order to ga in similar advantages. An example is the red sea
8 mimic blenny ( Ecsenius gravieri Pelegrin) which adopts the coloration of Meiacanthus nigrolineatus (Dafni and Diamant, 1984) to prevent predation. Other fishes mimic sematic colorations for th e purpose of attraction. This is termed pseudoepisematic or aggressive mimicry. Some pr edatory fishes adopt the coloration of harmless species to gain access to potential pr ey; such as bluestriped fangblennies ( Plagiotremus rhinorhynchos ) which mimic juvenile bluestreaked cleaner wrasse ( Labroides dimidiatus ) (Ct and Cheney, 2004). Cleaner wrasses work to rem ove ectoparasites from large reef fish, while bluestriped fangblennies use their similarities to them to get close to large reef fish, then attempt to remove tissue and scales. Nonaggressive forms of pseudoepisematic colorations do exist. A good example is found in the territorial male Haplochromis burtoni a mouth-brooding cichlid. The male has a few round, yellow patches on the an al fin, which resemble eggs. A female, who has already released her eggs and is holding them in her mouth sees the patches, she attempts to suck the patches into her mouth, allowing the male enough time to ejaculate and fertilize her recently released eggs (Fujii 1993a). Overall, colors in fishes have been shown to be crucial in communication specifically in social recognition. Through various colorations from conspicuous to inconspicuous, fishes are able to adapt to various environments and niches. The colorations themselves are created by the placement and layering of various types of chromatophores; as well as the layer of scales that overlap them. Color in Vertebrates: Color is perceived due to the stimulation of the retina by light of various wavelengths. Fishes seem capable of seeing color with spectra l ranges that are similar to humans, although some species (such as deep-sea fishes) are colo r-blind (Lythgoe, 1988). Each species capacity to see various colors is limited by their photic e nvironments and survival strategies. Colors
9 discernible in the skin of fishes are visible in a manner similar to those in humans (i.e. they are generated by light of specific wavelengths bei ng absorbed, scattered or reflected). The color presented, and thus, the light absorbed, is dependent on the type of chromatophore that is being hit by the incident light. As visible light of certain wavelengths is absorbed more than the light of other wavelengths (Bohren, 1988); the light reflected is interpreted visually as a color. Absorption of the light transforms white light while scattering redirects white light (Bohren, 1988). Chromatophores, the cells that contain colore d pigments, provide coloration and patterns in vertebrates and invertebrates. These cells are pr esent throughout the body, and can be found in the epidermis, neurons, sensory organs, muscl e, liver and other organs (Needham, 1974). Chromatophores are derived from the neural crest (Fujii, 1993a; Fujii and Oshima, 1986). Chromatoblasts (pigment cell precursors), of neural crest origin, migrate, proliferate and differentiate into chromatophores. The origin of some of the chromatophores from the neural crest has been shown in amphibians and in zebrafish, Danio rerio, (a model organism) (Kelsh, 2004). Cells specifically derive from the dorsal neural tube, and migrate to their respective locations in the body. In zebrafish, melanophores disperse along the lateral (below the developing epidermis) and medial (between somites and the neural tube) neural crest pathways (Raible and Eisen, 1994). Some chromatophore types, such as xanthophores (as discussed in detail on page 15), only travel along the lateral pathway, while iridophores travel solely along the medial pathway (Kelsh, 2004). Pigment cells destinati ons are designated before they begin their migration. Melanoblasts, xanthoblasts and iridoblasts are the terms for the pigment cells that are in the state of migrating, being colorless or partially pigmented at the time (Kelsh, 2004). In poikilothermic vertebrates there are five different classes of chromatophores that are found dermally: melanophores, erythrophores, xa nthophores, leucophores and iridophores (Fujii, 1993a; Fujii and Oshima, 1986; Fujii, 2000). In these five classes of chromatophores, there are four types of pigments present: melanins, carotenoids, pteridines, and purines. Cyanophores, a
10 fifth type of color cell perceived as a bright blue, have been found in relatively few fishes (specifically in callionymid fishes), and their pigment composition is unknown (Goda and Fujii, 1995). They will not be discussed in this paper. Chromatophores are innervated by post-ganglio nic sympathetic nerve fibers that have multiple synaptic endings in contact with the cell body. This allows the processes of that nerve fiber some control over the pigment granule move ment (Demski, 1992; Fujii and Novales, 1969; Fujii, 2000). For teleosts, most melanophores are inne rvated by nerve fibers in the skin. Higher firing rates of the postganglionic sympathetic fibers cause melanosomes to appear more centralized in the cell, while lower frequenci es cause the melanosomes to disperse (Fujii and Novales, 1969; Fujii, 2000). Movement of pigment granules can exist in th ree states: condensed to the center of the cell (aggregated); expanded throughout the cell (dis persed); and partially dispersed followed by partial/complete aggregation or vice versa (Dierksen et al., 2004). The motile activities are dependent on motor-proteins, specifically tubulin, dynein and kinesin (Fujii, 1993a; Obika, 1986). Pigments: Melanins are the most widely distributed chromatophores in fishes. In melanocytes, melanins provide black to brown tones throughout the skin. They are highly polymerized and are derived from the amino acid tyrosine (through th e Raper-Mason scheme, Figure 1) (Raper 1927; Mason, 1959; 1967). Melanin granules are pos itioned in the organelles of the melanophores, called melanosomes. Melanin exists as different polymers; such as pheomelanins which are polymers with a sulfur containing organelle that is derived from cysteine. However, fish have been found to contain only eumelanins, a brown to black pigment.
Figure 1: Melanin Biosynthetic Pathway (from Hearing, 2006) Carotenoids are polyene pigm ents that are insoluble in water, and are present in xanthophores and erythrophores. They are not produc ed by the fishes themselves and instead are derived from their food sources. Various carotenoi ds present in fishes permit conspicuous color displays: tunaxanthin is found in fishes and is a factor in yellow color; astaxanthin is common in red fishes; lutein absorbs blue light, appear ing yellow to orange-red in both freshwater and marine fishes; -carotene is found in freshwater fishes providing a red-orange coloration; taraxanthin produced an orange color; as does and doradexanthin and zeaxanthin (Fujii, 1993a). Pteridines contain a pteridine nucleus (pyrimido[4,5b ]pyridine) as the soluble basic skeleton and play an active role in creating yellow, red and browns. They are similar to carotenoids in their purpose of production of bri ght colors. The containing organelles are called pterinosomes, which are commonly found in xanthophores and erythrophores. 11
12 Purines are found in a crystalline structure in iridophores and leucophores where they actively refract light. Guanine is a common purine, and large amounts are present in the silvery ventral surface of many fishes (Fujii, 1993a). U nder the microscope, the crystal appears colorless; however the light it bends can take on a variety of hues. Table 2: Flourescence of Zoochromes (modified from Needham, 1974) Zoochrome Color and wavelength of fluorescence Solubility in a neutral aqueous environment Carotenes Orange-red Carotenoproteins and carotenologycosides are soluble Xanthophylls Yellow Carotenoid acids Blue Fuscins Green, yellow, orange Insoluble Phaeomelanin Yellow (weak) Fairly Soluble Erythromelanin Yellow (weak) Fairly Soluble Eumelanin None Insoluble Pterin Red, yellow, green or blue Low Solubility Leucopterin Violet Low Solubility Ichthyopterin Blue (450-70 nm) Low Solubility Xanthopterin Blue-green, yellow (acid) Low Solubility Guanine Reflects: pearl luster, white, metallics Insoluble
13 Chromatophores: Table 3: Types of Chromatophores Type of Chromatophore Pigment Contained Light Absorbing/Reflecting Dendritic? Color Projected Melanophore Melanin Absorbing Dendritic Black, brown Xanthophore Pteridine and Carotenoids Absorbing Dendritic Yellow Erythrophore Pteridine and Carotenoids Absorbing Dendritic Red Iridophore Purine Reflecting Not Dendritic Silver Hues Leucophore Purine Reflecting Dendritic White and Metallic Hues In the skin tissue of fishes, dermal chro matophores are the predominant pigment cells. The presence of chromatophores enables fishes to rapidly change their colors, and sometimes patterns, as needed to survive. Four of the five are dendritic cells, with several dendritic processes being delivered from the perikaryon (cell body). The processes from chromatophores develop parallel to the skin surface (Fujii, 1993a), allowing fast expansion and contraction of pigment granules. The pigment granules move centripetally when aggregating to the center of the chromatophores, or centrifugally when disper sing (Fujii, 1993a). Melanophores, xanthophores, erythrophores and leucophores are all dendritic chromatophores. Iridophores usually do not have dendrites, and instead appear as round or oval. In dendritic chromatophores, microtubules leave the perikaryon and extenf to the dendrites. They function as a cytoskeletal element that helps maintain the stellate shape of the cells, and are believed to actively aid the move ment of melanosomes in melanophores (Fujii, 1993a). Dermal chromatophores are found beneath a layer of collagen fibrils, and are not in direct contact with the bottom of the epidermis. Parallel collagen fibrils form a thin sheet, with several
14 sheets arranged as lamellae, each sheet lying in a perpendicular direction to the previous one (Fujii, 1993a). The strong covering that is provided by the collagen fibrils protects the chromatophores from damage. Melanophores are the most common dendritic chromatophores present in fish (Bagnara, 1972; Bagnara and Ferris, 1971). They consist of a nucleus, mitochondria, melanosomes, and the pigment melanin. Melanosomes are round with a diameter of roughly 0.5 m. In melanophores, melanosomes are embedded in a 3-D system of mi crotrabecular strings that connects them with microtubules and the plasmalemma. This aids in the control of pigment granule movement (Schliwa and Euteneuer, 1983; Demski, 1992). Wh en neural and hormonal stimuli are present, melanin rapidly aggregates or disperses throug hout the melanophore, creating the effect of darkening or lightening of the skin. Melanosomes are capable of traveling to the most distal extension of a melanophore (Bagnara et al., 1968). These organelles develop as multivescular bodies, with melanization occurring around the sm all melanin granules inside (Turner et al., 1975). Colorless melanophores, called amelanotic melanophores, found in mutants of medaka Oryzias latipes (Sugimoto et al., 1985), still show aggregation and dispersion. However, they present no black or brown hue. Mature melanosom es are maintained in dermal melanophores for extended lengths of time, where they darken or lighten skins. The majority of the epidermal chromatophores are melanophores, although xanthop hores have been discovered in the antarctic blenny (the emerald rockcod, Trematomus bernacchii ) (Obika and Meyer-Rochow, 1990). Melanosomes in the epidermal melanophores are move d to epidermal cells (keratinocytes) (Fujii, 1993a) darkening the hue of the skin. Melanophores are usually present at higher densities on the dorsal surface of fishes than on the ventral surfaces, as the ventral surface us ually contains highly reflective chromatophores (Novales and Novales, 1966) for camouflage in water. Melanosomes effectively absorb white light rays in the entire range of the visual spectrum (Fujii, 2000); however the other
15 chromatosomes absorb a more na rrowed range that is complement ary to the color that they exhibit. Contributions of various stimuli trigger the motile responses of melanin, making it a key component of rapid color changes. Xanthophores and erythrophores are dendritic chromatophores, which are generally smaller than melanophores, that are seen as colors ranging from yellow to red in fish skin. They are structurally similar, being motile and res ponding to stimuli by the dispersion of pigment (Obika and Meyer-Rochow, 1990). Both xanthoph ores and erythrophores contain carotenoids and pteridines (on carotenoid vesicles and pterinos omes respectively) (Matsumoto, 1965). The main color produced by xanthosomes is yellow, while erythrosomes present as red. Both xanthophores and erythrophores contain a single cell membrane (Matsumoto, 1965), and have fewer cytoskeletal components (mic rofilaments, intermed iate filaments, and microtubules) than melanophores (Obika and Meyer-Rochow, 1990). Leucophores are in the dermis of some species of fish, primarily in Osteichthyes (Fujii, 1993a). Unlike melanophores, xanthophores and erythrophores, leucophores do not absorb light but rather reflect light (as do iridophores, see below). Leucophores contain leucosomes, which scatter visible light. Each leucosome is a globular organelle enclosed by one limiting membrane (Obika, 1988). Few dendritic processes emanate from leucophores even though their size is comparable and sometimes exceeds that of other dendritic chromatophores (Obika, 1998). Leucophores are positioned frequently just below melanophores; however, the direction of the movement (expansion or contraction of the le ucosomes) is opposite that of light-absorbing chromatophores (Fujii, 1993a). Iridophores are present in areas of skin that appear white; i.e. primarily silver and white ventral surfaces of fishes. When present in some white and silver ventral skin, they are packed closely together, so that other chromatophores ar e sparse (Fujii, 1993a). Iridophores reflect light through the use of crystalline platelets that form parallel stacks in the cytoplasm (Denton and
16 Nicol, 1966; Land, 1972; Setoguti, 1967). The spacing between stacks of platelets is uniform, with each platelet being a crystal containing guanine (however some have been found with other purines) (Fujii, 1993a). Each platelet is highly re fractive, causing the cells to scatter light through specular reflection (Fujii, 1993a). When the distan ce between the stacks of platelets increases, the iridophores reflect light of longer wavelengths (called the longer-wavelength light-reflecting response). As distance decreases, shorter wavelengths are reflected (called the shorterwavelength light-reflecting response) (Fujii, 1993 b). Iridophores are capable of reflecting the maximum amount of light (Fujii, 1993a ), acting as a reflecting material. Combined Effects: Colors viewed on the skins of numerous fishes are a combination and layering of various types of chromatophores. The wild medaka (Oryzias latipes), which is seen as a combination of brown and black colors, has melanophores, xant hophores and leucophores distributed across the skin (Yamamoto, 1975). Through layering a nd placement of chromatophores, numerous shades and colors are possible. The dispersion or aggr egation of chromatophores over the surface of the skin changes the color seen, by expanding one color/shade and contracting another. Each chromatophore responds differently to the endocrine and nervous system stimuli, allowing fish to adapt rapidly to various hues (Fujii, 1993a). In addition to being placed in various patt erns across the skin, chromatophores can layer each other, overlapping so that the contracti on of one type of chromatophore displays the underlying color cell. Bright yellow-to-red coloration in fish is often created by a layer of iridophores beneath a xanthophore or erythrophores (Fujii, 1993a). A well-studied example is the dermal chromatophores unit (Bagnara and Hadley, 1973), where a xanthophore, iridophore and melanophore form an orderly unit. Present in many amphibian and reptilian species, it is not well represented in fishes. Xanthophores form the
17 outermost layer beneath the basal lamella, followed by iridophores. Under each iridophore, a melanophore exists with projections spread up a nd around the iridophore (Bagnara et al., 1968). When the skin darkens, the melanosomes move up from the melanophore to fill the processes that are emitted around the iridophore, which then is obscured. In fishes, simple layers of chromatophores are found that create colors that would not be possible by the presence of a single pigment alone (Fujii, 1993a). The brilliant bl ue coloration of the blue damselfish ( Chrysiptera cyanea ) is created by a layer of iridophores found ju st below the epidermis, followed by a deeper layer of melanophores (Kasukawa et al., 1987; Fu jii, 1993a). Alternatively, some species have reverse configuration. When light passes thr ough xanthophores and/or erythrophores, a whitish colored lining from chromatophores is present to increase the brightness of the pigment (Fujii, 1993a), Chromatophore units exist where multiple chromatophores are lined up in close association to the point where the cell centers ove rlap (Demski, 1992; Ahmad, 1970), at dermal scale boundaries (Sire and Graudie, 1984). Small superficial melanophores are evenly distributed, with the most occurring dorsally. They are involved in darkening and protective countershading. Deeper in the scale, larger melanophores, or melanoiridophores (melanophores associated with iridophores) can exist in high densities (Demski, 1992). These can be seen in vertical bands during agonistic displays. The d eepest layer is a set of melanoiridophores found in Oreochromis (Tilapia) mossambica that cause a black coloration in males (Demski, 1992).These chromatophore units are organized in respect to scale boundaries. An example of chromatophore layers in fishes is found in the coho salmon, Oncorhynchus kisutch. In the upper dermis of dark colored skin, globular iridophores are surrounded by the dendritic arms of the mo re proximal melanophores (Hawkes, 1974). This allows the melanophores to cover and block out the iridophores under the presence of certain
18 stimuli. Deep in the ventral area of the dermis, slightly underneath the stratum compactum, layers of dendritic iridophores are partially shielded by a limited number of melanophores. Baerends and Baerends-Van Roon (1950) furthe r expanded on the layers of color cells in fishes through a comprehensive study (summarized in table 4). They report that the first layer was superficial, small melanophores that were equally represented across the body, notably on scale boundaries. These cells were in a higher density on the dorsal side of the fish, compared to the ventral. These melanophores are the cause of count ershading and may expand when a fish is scared to cover the brighter colors. The second layer is the deeper melanophores which usually associate with iridophores to comprise melanoiridosom es. The cells populate the bars seen in fish, and are predominantly viewed when fish are de monstrating aggressive or mating behaviors. The expansion of the second layer occurs rapidly, and is caused by physiological effects. The third system or layer is composed of deep-set melanophores. The cells form eye spots and other special markings. They are able to expand or cont ract rapidly, and are densely concentrated, to the level that color is still present in the skin even when they are contracted. The fourth system is a layer of melanophores that serve an unk nown purpose, and have been found in Tilapia natalensis They will not be examined in the study. Guanocytes make up the fifth chromatophore system. They provide colors seen across the skin (ex.: white, yello w, green or blue). The last system consists of the erythrophores and xanthophores which occur at different depths in the skin. These cells are marked by slower responses to stimuli.
19 Table 4: Systems of Chromatophores Present in Skin (from Baerends and Baerends Van Roon, 1950) System Type of Chromatophore Location General Effect Comments 1 Small Melanophores Superficial usually on the edge of scales Countershading Contraction/Expansion allow deeper color cells to show 2 Larger Melanophores and Melanoiridosomes Beneath System 1 Vertical Bars Motile abilities occur rapidly 3 Melanophores Beneath System 2 Longitudinal Bands and Eye Spots Expand or Contract rapidly. Dense Concentrations Only contracted when animal at rest 4 Melanophores Beneath System 3 Unknown Found in Tilapia natalensis 5 Guanocytes (Iridophores and Leucophores) Varying layers Provide colors and hues Often found in dense concentrations 6 Erythrophores and Xanthophores Varying layers Red, Orange, Yellow and Brown colors Do not expand or contract rapidly Expanded in reproductive displays Patterns : The primary determinant of coloration in mo st fishes is the distribution, activity and interaction of chromatophores (Bagnara and Hadley, 1973) grouped into color patterns. Patterns present on the skins of fishes are not necessarily stationary; instead, they can change throughout ontogeny. Very slow modifications ma de to the patterns during adaptations to the environment occur through morphological interventio n, while faster changes are by physiological causes. Physiological color cha nges are needed to rapidly adapt to a background or for communication between individuals. This primarily occurs through stimuli provided by the nervous and/or endocrine sy stem (Fujii and Oshima, 1986).
20 Morphological Versus Physiological Color Changes : Changes in the patterns and colors present on the skin can occur either by the motile movement of chromatophores or by changing th e amount of pigments (or pigment cells) present in that area of skin. When the changes occur rapidly, they are physiological color changes due to the expansion or contraction of pigment in the chromatophores (Fujii, 1993a). Rapid changes are essential for adaptation to backgrounds and for communication between conspecifics. Physiological color changes are controlled by differential neural or hormonal commands to chromatophores (Fujii, 2000), or groups of nearby chromatophores (if involved in a change of pattern). Slower changes, over days to weeks that occur are morphological color changes. These responses coincide with an increase or decrease in the number of dermal chromatophores present in a region. This occurs through the pr opagation of chromoblasts or the degradation of terminally differentiated chromatophores (Par ker, 1948). Morphological changes may be nonreversible and are normally associated with progressive development for a species (Crook, 1997). The rapid changes of color seen in physiol ogical color change are due to the motility of chromatophores (Fujii, 1993a). The changes that o ccur are not permanent, and may be reversed as rapidly (although exceptions exist) (Crook, 1997) The innervation of chromatophores by nerves allows for short-term, rapid movement of pigm ent granules to occur by neural input, which overrides the long-term hormonal control (Fujii, 2000). One accepted hypothesis concerning mechanisms, is that the microtubules, which spread from the center of the cell towards the edges of the dendritic processes, aid the movement of the pigment granules (Fujii, 1993a).The sliding force between the surface of th e chromatosomes and the micr otubules causes the pigment to contract (centripetal movement) towards the center or expand (centrifugal movement) towards the edges of the processes (Obika, 1986; Fujii, 199 3a). The motor protein cytoplasmic dynein is
21 involved with centripetal movement, and kinesin is involved with centrifugal movement (Obika, 1986; Fujii, 1993a). The physiological color changes are promoted by the endocrine and nervous systems (Fujii and Oshima, 1986). Information that is percei ved by the lateral eyes of fishes is sent to the pineal gland and processed in the central nervous system. Efferent signals then travel to chromatophores by hormonal and nervous pathways (Fujii, 1993a). Light and pressure on chromatophores (Marsland, 1944) are two types of physiological stimuli that can create direct effects, with both (potentially in the case of light) causing melanophores to expand. Morphological color changes are a result of slow endocrine effects. The hormones act on the chromatophores, causing the cells to accumulate or decrease in an area. Changes can be triggered by: developmental signa ls (aging from fry to adult); long-term background adaptation (Fujii, 2000); dietary restrictions; seasonal changes (Nery and de Lauro Castrucci, 1997); and changes in temperature and other factors. Nup tial coloration is caused by morphological changes (Fujii, 1993a). The changes occur slowly as horm ones are released into the system. The hormone prepares both males and females of the species to mate and release their respective sperm and eggs. These sex-hormones trigger color changes th at signal to mates that there is a potential reproductively active mate in the area. In some fish species, the nuptial coloration is red. The color can occur in the lower ventral region, or all over the body. Although morphological changes take a longer period to appear, they can be rapidly obscured by physiological changes. The physiologi cal changes are rapid, and often serve a direct communicatory need, such as fear. Temporary obscurity is from a contraction of the chromatophores that already exist, instead of cha nging the number or density. This obscurity is short term, and the long-term morphological coloration is still present, visible once the physiological color change dissipates. For example, territorial males of Haplichromis burtoni can develop a black bar stripe (called an eyebar) th at serves to attract mates, signal aggression and
22 communicate with young. The melanophores are c onnected to iridophores, which form a single chromatophores unit in the eyebar (Muske and Fernald, 1987b). When frightened or fleeing from an area, the eyebar can quickly disappear, and th en reappear seconds later (Muske and Fernald, 1987a). The eyebar is a long-standing trait that occurred with development. The temporary disappearance of the eyebar is from neural control which causes chromatosomes to contract towards the center of the chromatophores. When the melanophores are expanded, the eyebar is present. When melanophores are contracted, the ir idophores are visible and the skin appears lighter. Table 3 shows various morphological and physiological effects on chromatophores, including how the different chromatophores can have different reactions to the same stimulus. Iridophores are more easily seen by the contraction of other chromatophores which allow for the iridophores to be more easily viewed. This can occur because the melanophores lie underneath iridophores but have projections up and around them. When dispersed melanosomes in the projections obscure the iridophore. When concentr ated, the iridophore is visible on the surface of the skin, which appears white or reflective.
23 Table 5: Stimuli Effects on Chromatophores Stimulus Melanophore Er ythrophore Xanthophore Ir idophore Leucophore MSD Disperse Disperse Disperse Contraction (if motile) Contract MCH Contract Contract Contract Disperse Melatonin Contract Contract Contract Disperse Epinephrine Disperse Disperse Disperse Contract Norepinephrine Disperse Disperse Disperse Adenosine Disperse Disperse Disperse cAMP Disperse Disperse Disperse Disperse Adrenergic Dopaminergic, and serotonergic Neurotransmitters Contract Contract Contract Incident Light Contract or Disperse (varies by species) Contract or Disperse (varies by species) Contract Disperse Ca2+ions Contract Contract Contract Contract Ultraviolet Light (before damaging) Disperse Disperse Disperse K+ ions Contract Contract Contract Na+ ions Disperse Disperse Disperse Prolactin Disperse Disperse Disperse Adrenocorticotropic Hormone Little to no effect Disperse Disperse Increased Pressure Disperse Di sperse Disperse Disperse MSD = Melanophore-stimulating hormone; MCH = Melanophore-concentrating hormone; cAMP = cyclic-adenosine monophosphate
24 Nervous Control of Chromatophores: White adaptation and dark adaptation ar e two commonly studied fields when differentiating between morphological and physio logical color changes in fishes. Background adaptation has been called protec tive coloration (Sugimoto, 2002) as it enables a fish to blend in with the environment more. Slower control ove r chromatophores by the central control of the brain generally originates from the endocrine syst em, while faster responses originate from direct mechanisms (reflexive). When stemming from central mechanisms, the current idea is that both excitatory and inhibitory systems operate in the paling center (Iwata and Fukuda, 1973). This modulates effects that are viewed as paling and darkening in fishes. When a fish is placed on a dark background, the ventral retina receives more light than the dorsal retina (as a low level of light is reflected from the black background) (Sugimoto, 2002). This causes neurons in the ventral retina to become activated and leads to the s uppression of chromatic motoneurons in the medulla (part of the inhibitory system) (Fujii, 2000; Iw ata and Fukuda, 1973). The overall result is the appearance of the fishs skin darkening. When placed on a white background, both the ventral and dorsal retinas are stimulated in almost equal proportions. The dorsal retina causes the excitatory system to amplify the spontaneous discharges of the motoneurons. This causes the chromatophores to aggregate and the skin to blanch. The effect caused by the stimulation of the ventral retina is inhibited at the level of the optic tectum and partially at the level of motoneurons in the medulla (Fujii, 2000; Iwata and Fukuda, 1973). Dark background adaptation for extended peri ods of time can result in an increased density of melanophores and xanthoph ores, and the presence of more networks of varicose fibers around the chromatophores (Sugimoto and Oshima, 1995). The increased density is caused by the -melanophore stimulating hormone being secreted from the pars intermedia (Sugimoto, 2002). In addition to the higher density of melanophores and xanthophores, an increase in the amount or
25 erythrophores present in the area has been noted (Sugimoto, 2002). This response is coupled with a decrease in the number of leucophores. Fewer chromatophores and varicose fibers were seen after prolonged exposure to a white background. Decreases in melanophore density are caused by the degradation of melanophores through apoptosis, cell migration and the secretion of melanin-concentrati ng hormone (defined in detail on page 27) (Sugimoto, 2002) More iridophores appear in the skin of the fish. This causes paling, thus, better matching the white backgr ound. When a fish is taken from a white background to a dark background, the fishs skin appears to darken which over a long period of time will cause an increase in the number of melanophores and a decrease in the number of leucophores (Sugimoto, 1993a). The immediate response of the darkening of the skin is due to the expansion of the melanophores. White ad aptation causes the reverse effect. Environmental Factors that Influence Chromatophores : Normally stimuli are detected by the various sense organs (depending on the nature of the stimulus) and relevant informa tion transmitted to the central nervous system where it is then processed. Neural signals sent to chromatophores prov ide the appropriate response to the stimuli. Sensory stimulation can occur directly to th e chromatophores. This circumvents the central nervous system control. Primary color responses of the physiological color changes may occur when chromatophores directly respond to incide nce light (Fujii and Oshima, 1994). Secondary color responses occur when the chromatophores ar e controlled by the endocrine or nervous system. Primary color responses are most notab le and have the highest occurrence during embryonic and larval stages (Fujii, 2000), when nerves may have not innervated the various chromatophores. Primary responses occur in a hi gher frequency when fishes are blinded and thus the sensory organ to perceive light has been blocked (Fujii, 2000).
26 Melanophores can respond to light either by aggregating or dispersing the melanosomes. For example, in young of Xiphophorus maculates (black platyfish) (Wakamatsu, 1978), cultures of melanophores showed aggregation of pigment when introduced to light. Responses to light vary by species. Young of the rose bitterling ( Rhodeus ocellatus ) display melanosomes dispersed in the presence of light (Ohta, 1983). Other chromatophores with motile capabilities were also shown to respond directly to light. Leucophores in Oryzias latipes (medaka) dispersed their leucosomes when light was presented (Ohta and Sugimoto, 1980). Xanthophores (both innervated and denervated) of adult medaka aggregated th eir pigments within 20 seconds (Oshima et al., 1998) at the presence of light. When the xanthophores are exposed to direct illumination, the light was absorbed in a range that increased the phos phodiesterase activity. This subsequently caused a decrease in cytosolic cyclic AMP levels and the perceived change in the xanthophores (Oshima et al., 1998). Erythrophores (both innervated and denervated) in Oreochromis niloticus (the Nile Tilapia), respond in both directions to li ght (Oshima and Yokozeki, 1999; Fujii, 2000). High frequency pulses of ultraviolet light can damage pigment cells and the nerve fibers that control the cellular responses of the cells (Fujii, 2000). Ultraviolet light can also affect chromatophore aggregation or dispersion, in a manner similar to that of melanotropin. The melanin production can be increased at relativ ely low levels of UV light (Aberdam, 1993; Erickson, 1976). At higher levels, photodamage occurs in chromatophores (and other cells in the body) thus leading to phototoxicity (Donawho et al., 1994; Setlow et al., 1993; Hearing, 2006). Hormonal Effects on Chromatophores : Hormonal actions on chromatophores are classified as morphological effects since they require longer (from minutes to months) to a ppear on the skin surface and reverse than physiological effects. Hormones affect the ch romatophores in different ways. Indeed, the same hormone can have opposing effects on different chromatophores types.
27 The major hormones that control pigmentation are melanotropin/melanophorestimulating hormone (MSH) (Fujii, 1993a), melanin-concentrating hormone (MCH), and melatonin. Proopiomelanocortin, the precursor of MSH, is produced by keratinocytes and by melanocytes (Chakraborty et al., 1995). Melanin-c oncentrating hormone from the posterior lobe of the pituitary (linked to the hypothalamus of the brain) causes pigment granules to concentrate around the center of the chromatophores (Baker, 1993; Kawauchi et al., 1993; Visconti et al., 1989), effectively lightening the skin coloration. Th e effect is opposite that of MSH. The effects from the hormones are reversible, this either occurs through the disappearance of the hormone that is causing the effect, or the presence of a hormone that stimulates an opposite effect (Castrucci et al., 1987). Melatonin synthesized in the pineal gland (Bagnara and Hadley, 1973), also contributes to skin lightening through the concentration of melanosomes (Novales, 1963). Melanophore-stimulating hormone (MSH) causes rapid diffusion of the melanosomes of melanophores for physiological color changes. The ho rmone is produced by the intermediate lobe of the pituitary, and is sometimes called mel anotropin or intermedin (Fujii, 2000). Specifically, -MSH is thought to have a major effect on the regulation of chromatosome movement (Fujii and Oshima, 1986). MSH stimulates melanogenic enzyme function and causes more eumelanins to be produced (Hearing, 2006). The effect is not solely limited to melanosomes as it also causes xanthosome and erythrosome disper sion in some brightly-colored teleosts (Fujii and Oshima, 1986; Negishi and Obika, 1980). Concerning light scattering cells, motile iridophores in some species of gobiid fishes show platelet contraction in response to MSH (Iga and Matsuno, 1986). The second mess enger in iridophores is thought to be cyclic AMP (Fujii, 2000), with lower levels of cyclic AMP bei ng correlated with platelet contraction. Adrenocorticotropic hormone (ACTH) is a nother example of a naturally occurring hormone that affects melanophores by causing the pigment granules to disperse (Fujii, 1993b). Prolactin, which is produced by the anterior lobe of the pituitary (Fujii, 2000), causes pigment to
28 disperse in xanthophores in the mudsucker Gillichthys This produces the yellow hue of the skin (Fujii, 2000). Prolactin has little effect on mela nosomes. Nuptial coloration in some species of fishes is connected to prolactins eff ect on erythrophores (Goda and Fujii, 1995). Melanin-Concentrating Hormone (MCH) is synt hesized in the hypothamalus. It is then delivered via axon transport to the posterior lobe of the pituitary and hence into the blood stream (Fujii, 2000). As the name suggests, mela nophores exposed to MCH have their pigments contracted towards the center of the cell. Erythrophores in swordtails and xanthophores in medaka respond similarly to MCH by contracti ng the pigment (Fujii, 2000). Leucophores have an opposite effect in response to MCH; i.e. the light -scattering organelles move to the edges of the cell (Castrucci et al., 1988). Melatonin similarly causes a contraction of pigment to occur in melanophores. The pineal hormone acts on melanophores (Fujii, 1993b) and similar dendritic light-absorbing chromatophores. Secreted mostly at night (Owens et al., 1978), melatonin is important in circadian rhythms. Melatonin actions are opposite to those of MSH and can be used to reverse the effects caused by MSH. Paracrine Factors: Paracrine factors regulate the physiological r esponses of color effector cells (Fujii, 2000). These factors include: opioid peptides that ar e found in the brain and peripheral tissues; eicosanoids that change the regulation of hormonal and neural signaling; endothelins produced in keratinocytes (acting as a strong mitogens and me lanogens that can contract chromatosomes in light-absorbing dendritic chromatophores); and nitric oxide which modulates smaller effects of the mobility of melanosomes (Fujii, 2000). When fishes are excited, epinephrine is released from chromaffin cells of adrenal tissue (Miyashita and Fujii, 1975). This action causes chromatophores to disperse in melanophores and
29 similar dendritic chromatophores. If the adreno ceptors of the chromatophores are mainly then blanching occurs, while if they are mainly darkening of the skin occurs (Fujii, 1993a). Norepinephrine and ATP are released from the chromatophores nerve terminals (Fujii and Oshima, 1986). The ATP is converted to adenosine, which then causes diffusion of pigment throughout the cell. The -adrenoceptors and receptors for MCH and melatonin cause pigment to concentrate into the center of the cell while -adrenoceptors and receptors for MSH and adenosine cause the pigments to expand outwa rd in the cell (Fujii, 1993a). cAMP (cyclic adenosine-3, 5-monophosphate), Ca2+ ions, and IP3 (inositol 1, 4, 5-triphosphate) are the second messengers that mediate in the responses (Fujii, 1993a). Levels of cAMP have been found to decrease when chromatosomes aggregate due to the inhibition of the cyclase system by melatonin and MCH. Cyclic AMP disperses chromatophores when presented alone (Fujii, 2000). This effect has been described in melanophores, xanthophor es, erythrophores and leucophores (Fujii, 1993b; Fujii and Oshima, 1994; Fujii, 2000). Free Ca2+ ions have the opposite effect, concentrating pigment to the center of cells (Fujii, 2000). Both melanophores and xant hophores show pigment aggregation in response to potassium ions and disp ersion of the pigments when sodium ions are present (Hawkes, 1974). In some species, an increase in Ca2+ can directly cause pigments to assemble in the center of the cell (Fujii, 2000). Melanophores are also regulated by: substrate availability; cytokine and growth factors; intracellular pH; trafficking and sorting path ways; proteasomes; and ultraviolet radiation (Hearing, 2006). Melanin production can be altered by controlling tyrosinase activity (tyrosine is the precursor to melanin) through inhibitors (which regulate activity), other post-tyrosinase enzymes (that change the nature of the melani ns produced, either eumelanin or pheomelanin), proteolytic processing and other mechanisms (Hearing, 2006). Melanin is no longer produced if tyrosine use is completely inactivated.
30 Color in the Brain: Chromomotor neurons are situated by neurons in the sympathetic chain ganglia and the rostral spinal cord (Demski, 1992). Basic colo r pattern formation may start in the lower and intermediate medulla in teleosts (Demski, 1992). Higher levels of control may occur through the hypothalamus, tegmentum, optic tectum, torus semic ircularis, thalamus, and telencephalon, where sexual, agonistic and other types of behavior s are mediated. The thalamus is involved in controlling coloring systems (Demski, 1992). Basic control of paling originates in the medulla with pathways that exist in the spinal cord at the level of the 15th spinal nerve to join the sympathetic trunk and hence to project as peripheral nerves (Demski, 1992). Thus preganglionic motor cells in the spinal cord synapse with the postganglionic motor neurons in the sympathetic chain (Demski, 1992). Norepinephrine is the post-ganglionic neurotransmitter (Fujii and Oshima 1986). Numerous studies relate paling to a medullary center. Darkening is a presumed result fr om inhibition of this paling center. Thus, electrical stimulation causes melanin to aggregat e in melanophores, by activating concentrating axons from the sympathetic trunks (Demski, 1992). Banding was elicited in sunfish ( Lepomis sp.) by stimulating rostrocaudally in: the preoptic area; the area between the thalamus a nd hypothalamus; the torus semicircularis and medial tegmentum of the midbrain; and the rost ral and intermediate medulla basal (Bauer and Demski, 1980; Demski, 1992). In the stimulation experiment, the vertical bands of several species and specialized spots in sea bass ( Serranus sp.) were controlled as only total units, and therefore show or do not show, with no intermediaries due to common neural connections (Demski, 1992). A similar area in other cichlid species brai ns may control dominant color characteristics. Some melano-iridophore units have been found to be directly innervated by sympathetic fibers running with the fifth cranial nerve. Haplochromis burtoni is an example, where the eyebar
31 is mediated centrally by areas that control the le vel of activity in the ne rve (Muske and Fernald, 1987b). Cichlid Color Development: Color patterns in various cichlids have been studied throughout development. Hemichromics bimaculatus, Cichlasoma nigrofasiatum, and zebrafish are a few that have been well studied (Baerends and Baerends-van Roon, 1950; Beeching, 2002; Noble and Curtis, 1939; Kelsh, 2004). The various color systems in the individuals each originated separately from waves of melanophores developing in the body regions at different times, populating different age patterns (Demski, 1992). The position of pigmented cells is first established by an accumulation of the color cell precursors or chromatoblasts (eith er uncolored or semi-pigmented) (Kelsh et al., 2000). Cichlasoma nigofasiatum (the convict cichlid) hatches three days postfertilization and show horizontal stripes and diffuse cranial and ve ntral chromatophores. Throughout development, vertical bars slowly appear as midlaterally inco mplete semibars (Beeching, 2002). Newly hatched fry are primarily translucent and contain a few chromatophores around the cranial region. Te largest densities of chromatophores are over the yolk sac and around the eye. Chromatophores making up a midlateral stripe are also present, although their placement was parallel to the caudal neural tube. After six days, a second lateral stripe forms, dorsal and parallel to the first. Twenty days after hatching, midlaterally incomplete ba rs begin to form at the base of the dorsal and caudal fins. The unpaired fins begin to obtain melanophores thirty-two days after fertilization, while the first bars are seen on day forty-two (Beeching, 2002). In early larval zebrafish, Danio rerio, (five days postfertilization), four longitudinal stripes composed of melanophores are present. Three of these have associated iridophores (Kelsh, 2004). Xanthophores are distributed evenly over th e lateral surface of the skin with highest
32 concentrations more distally. The four stripes develop in order, based on their distance from the neural tube. The first stripe is located dorsal, next one ventral, the third is lateral, and the last being the yolk sac (Kelsh, 2004). The dorsal stripe of melanophore begins to form thirty hours after fertilization. By fortyeight post fertilization, iridophores begin to populate the stripe as well. Xanthophores are also distributed throughout the trunk by forty-two hours post fertilization (Kelsh, 2004). The juvenile pattern for zebrafish is differe nt from the one viewed for adults. The adult form has its main 5 longitudinal stripes, with a few associated with the flanks. The stripes that appear to be black are formed of melanophores and a few iridophores. These stripes are separated by silver stripes that are composed of iridophores and xanthophores (Kelsh, 2004). Overall, xanthophores are positioned over iridophores throughout the flank and silver stripes. In the black stripes, the iridophore layer is very thin, and positioned over a layer of melanophores, followed by another layer of iridophores (Kelsh, 2004). This layering allows the colors to appear brighter. In the silver stripes, the iridophore layer becomes thicker which causes the stripe to appear more silver as light is reflected. Three Models for the Formation of Pigment Pattern Formation: There are three suggested models for how pa tterns of pigments form in fishes and how the patterns might be regulated and controlled. All agree that the chromatophores originate, migrate and differentiate from the neural crest (Fujii, 1993a; Fujii and Oshima, 1986). The first model theorizes that pigment cell dist ribution might be patterned by signals that occur over long distances (Kelsh, 2004). One class of such influences is based on reactiondiffusion mechanisms (Turning, 1952). Activator and inhibitor molecules are created from a localized source. They disperse into what woul d appear on the surface as a stripe or spotted pattern. The specific pattern is regulated the space available and by the diffusion coefficients of
33 the molecules (Kelsh, 2004). The theory predicts that increasing or decreasing the various growth parameters would change the patterns viewed. The second model suggests that more localized cues determine the developmental pattern; i.e. specifically that control is by envi ronmental signals from local tissues. The routes of the melanoblasts from the neural crest could be altered by the environmental signals (Kelsh, 2004). Changes in the myotomes may alter the la teral pathway that melanoblasts are able to migrate along (Kelsh, 2004). Contrary to the one melanocyte type present in mammals, fish have five different chromatophores types. This presents the possibility that sites along the skin could have different attractive/repulsive properties for each cell type. The range of attractive and repulsive abilities by each local site could then alter the pattern of pigments around that specific area. The third theoretical model depends on the id ea that different pigment cells in the same area affect each other. Various chromatophore cells w ould have varying levels of affinity to other types which would cause the cells to sort themsel ves (Kelsh, 2004). Differential cell affinity has been shown experimentally (Foty et al., 1996). Cells interact both mechanically and chemically which would allow for spots or stripes to form by the separation of chromatophores. Larval salamanders have alternating stripes of mela nophores and xanthophores, although initially they were homogenous over the skin. Repulsive inter actions between the cells, in addition to prior aggregation of the premigratory xanthophores (E pperlein and Lofberg, 1990; Kelsh, 2004) were suggested as the cause by the ontogenic pattern appearance in the amphibians. Evidence for local cell-to-cell interactions th at determine the color patterns has also been found in zebrafish; i.e. with stripe forma tion depends upon the interactions between melanophores and xanthophores (Kelsh, 2004). The melanophores, rather than the iridophores, may be the main determinant for the number of stripes seen on the body. As waves of melanophores populate a location, their dark cells may influence other chromatophores to
34 disperse into the area. Environmental patterning cues may exist within the horizontal myoseptum and the posterior lateral line nerve (Kelsh, 2004). Cichlidae: The family Cichlidae contains over three-thousand living species (Nelson, 2006) of the freshwater order Perciformes. The majority of species originate from Africa (Valente et al., 2009). Cichlidae is divided into four sub-families, with Pseudocrenilabrinae being the African division (the others being Etroplinae, Ptychochrominae, and Cichlinae). African cichlids have a modal diploid chromosome number of forty-four versus the neotropical cichlid (sub-family of Cichlinae) with a diploid number of forty-eight (Valente et al., 2009). The family Cichlidae has been well studied due to not only its vast number of species but also for their ability to radiate into a variety of ecological niches. This diversity is manifested in the various shapes and specializations of the gr oups. Many are known as important food fishes, such as tilapia (Genus: Pseudocrenilabrinae, Trib e: Trilapiini) (Nelson, 2006). The various species have become widely established geographically: i.e. from Africa, to Central America, North America (Nelson, 2006), and recently in Antarctica (Obika and Meyer-Rochow, 1990). In several areas, they have established themselves as an invasive species wh ere they readily adapt and out-compete native fishes (Courtenay, 1997). The success of the family is attributed to their opportunistic feeding habits and efficient reproduction. Cichlids can be divided into two parent al care groups, mouth-brooders and substratespawners. Mouth-brooder females deposit their e ggs, and following fertilization by males, quickly suck them into the mouth where she ca rries the eggs around until they hatch (such as Haplochromis burtoni ) (in some species, the males are also the mouth brooders). Substratespawners attach their eggs to a hard, flat substrate. A few days after fertilization, the juveniles
35 hatch and are known as wrigglers, until they are able to swim freely. Both groups show extended postnatal care by protecting and aerating th e eggs and fry for a period of time. Hemichromis bimaculatus: Jewel cichlids, Hemichromis bimaculatus Gill, are an African cichlid that is common in the aquarium trade. The species has been used in many behavioral studies due to their unique color, ease in breeding and hardiness. It is an opportunistic feeder, eating what is available locally. As a substrate brooder, females release e ggs, and males fertilize them, attached to a flat surface. The female and male H. bimaculatus form a monogamous breeding pair that, together in an established territory, protects the eggs, and la ter, the fry (de Boer and Heuts, 1973). The fry and juveniles live in schools until they approach ma turity, at which age they can form territories and become aggressive (Baerends and Baerends-van Roon, 1950). Overall, the social behaviors of the jewel cichlid appear to be largely dete rmined by visual cues from their environment and other fishes (de Boer and Heuts, 1973). It should be noted that after hatching, the parents may consume the fry if they are not removed before reaching a certain size (de Boer and Heuts, 1973). This is not likely to occur under more natural conditions. Until the young are capable of swimming on th eir own, the eggs and larvae are aerated and monitored by one parent at a time. By three to five days after the egg is hatched, the yolk is absorbed and the fry are no longer wrigglers (Baerends and Baerends-van Roon, 1950). They are now capable of basic swimming maneuvers. In captivity, as the fry begin to swim around the tank, the parents gradually stop moving them in to a small pit for protection. Fry show a preference for following a parent (notably the colo r red). The red coloration of the parent serves as a signal to the fry. Indeed young fry follow and have a preference for a red display dummy (Noble and Curtis, 1939). Fry for the most part ignored silver, green, blue and grey dummies.
36 Black dummies elicited a fright-response in fry (B aerends and Baerends-van Roon, 1950; Noble and Curtis, 1939). Single males and females both are capable of establishing and defending territories. They will display defensive coloration (which is also a notable red) at an intruder (of either sex). A lateral display often ensues following such a territori al violation; the two fish circle each other. This is showing full coloration and size to the othe r fish. Mouth fighting may occur if one fish is not deterred. The fish can lock jaws until one is chased off (Baerends and Baerends-van Roon, 1950). Inferiority (a dulling of the colors and fins not erect) is commonly shown immediately when one fish enters anothers territory. If scar ed suddenly, even a dominant fish will change from the red color and become dull with cross-ba nds. The same inferiority coloration can be used to prevent an attack near a more dominant fish. When in full color, overall the jewel cichlids range from orange to brilliant red (Baerends and Baerends-van Roon, 1950) with si x to eight rows of green spots along the length of the body, with more located on the head. Two black dots are present, one on the operculum and the other on the mid-section of the body. The spot on the operculum is surrounded by a yellow-green ring. A third spot is present near the caudal peduncle, although it is not seen when nuptial coloration is present (Baerends and Baerends-van Roon, 1950). The fins are predominantly yellow or pale green, with black on the outermost tips are black. Males are usually more colorful and slightly larger than females; however sexual dimorphism is not well established. When not reproductively active, the b ackground color of the fish is predominantly brownish-red with a green hue (Baere nds and Baerends-van Roon, 1950). The reproductive behavior of the jewel cichlid ( H. bimaculatus ) has been examined several times. The pattern and review of the studies focuses on the nuptial coloration (Rowland, 1975; Korzan and Fernald, 2007; Reimchen, 19 89; Baerends, 1976; Noble and Curtis, 1939; Baerends and Baerends-van Roon, 1950). Both male s and females that are about to spawn are red.
37 Females appear to choose the more red-colored of two males (Noble and Curtis, 1939). Thus redcoloration seems directly correlated to mating success. Males also prefer the more red, and larger (up to a certain limit) (Rowland, 1975) of femal es presented. Red colored fish presented to males may trigger: quivering (a courtship movement when males see red females); biting (an attack when males view red males); or fluttering (a sign of fear when viewed by a more dominant male) (Rowland, 1975). The red coloration takes several days to fully develop from the non-sexually excited state; however, in mating fishes it may o ccur rapidly due to certain stimuli (Baerends and Baerends-van Roon, 1950). Melanophores appear early in developmen t (Baerends and Baerends-van Roon, 1950). Guanocytes also can be seen over the body of the embryo. At two weeks after hatching, two longitudinal bands (dorsal and lateral) are visible. The dorsal band disappears as the fish develops, while the lateral concentrates into the three defined spots of the adult. Diffuse melanophores are visible over the body (Baerends and Baerends-van Roon, 1950; Noble and Curtis, 1939).Three to four weeks after the eggs have been fertilized the melanophores are dispersed, while guanine can be seen through the centers of these cells. The process entails the overlay of the two color cells that form the melanoiridosomes. Guanocytes begin to appear in deeper layers of the skin, making the skin app ear opaque with a white-almost-yellow base color. At five to seven weeks after hatching, guanocytes begin to form the spots seen in the adult body forms. The erythrophores are established last; i.e. usually after several months of development.
38 Methods Collection and Maintenance of Fish: The jewel cichlids used in this study were collected from local aquarium stores and various students on campus. Eleven adults and juve nile cichlids total were used continuously for the study. Ten fry from a mating jewel cichlid pa ir were studied at various ages. Specimens varied in length and age, so that the vari ous developmental stages could be examined. At Pritzker Marine Biology Research Laborat ory, the fish were maintained in various tanks in the upper level of the laboratory. Housing depended on the size of the fish, aggressiveness levels, and whethe r or not they were schooling or mating. The one mating pair was placed in a 70 gallon tank, and their sub sequent broods were maintained in similar conditions. A separated adult male was maintained in a 10 gallon tank; two juveniles shared a 10 gallon tank with a divider between them; four schooling juvenile Hemichromis bimaculatus shared a 20 gallon tank; and a male and female nonmating pair shared a 29 gallon tank with a divider in between. The water conditions were the same for all tanks: 70 F, 0 ppt salinity, and a pH range of 7.0 to 7.3. Every tank was supplied with a broken flower pot piece or shell for the fish to hide under. In tanks with multiple fish more than one hiding place was provided. Tanks were well-oxygenated using air st ones and water filters. Fish we re fed Gelly Belly gel diet (obtained from Aquatic Ecosystems) and fish fl akes (Tetramin Tropical Flakes). Fry were fed brine shrimp. All water changes and the initial wate r were conducted with R.O. water, to ensure that chlorine, chloramines and ammonia was not added into the system. Fish that showed aggressive behavior were placed in separate tanks or were isolated using a divider. The two juveniles were separated to aid in distinction. The mating pair coinhabited a tank, but were monito red for aggressive tendencies.
39 Determining the locations of Chromatophores: The smaller fish in this study were observed under a microscope (Olympus SZX16 microscope, attached to an Olympus DP71 camera). The method of catching and transporting the fish varied depending on the age and relationship status of the fish being moved. Fish handling and examination periods were as brief as possible. For the two broods of fry from the mating pair, two specimens were collected via pipette every f our days and placed in a small Petri dish under the microscope. The microscope was quickly focused and a digital photo taken to analyze the color patterns. The fish were then returned to their tank, with an average handling time of one minute and thirty seconds per fish. To reduce handlin g and stress, neither the side nor the angle of the fish observed was controlled. This was repeat ed with the fry, until the parents had consumed the brood. For the four schooling juveniles, every week one was collected in a hand dip net and quickly transferred to a medium-sized Petri dish with a small amount of water from the tank. The microscope was focused and several photos were take n of the various parts of the fish (as the fish were too large to accurately frame in one shot). Th e fish was then promptly returned to its tank, with an average handling time of two and one ha lf minutes. The side and angle of the fishs position was not controlled and the amount of light on the fish was minimized to prevent chromatophores dispersion due to light. The two separated juveniles were handled similarly. The adult mating pair was not observed, in order to not interfere with the spawning or rearing of their broods. Three other adults, two males and one female, were observed. Pictures were taken of the fish in the tanks. The side and angle of the fish was not controlled. Finally, a small dip-net was used to capture one fish, and pl ace it in a glass dish partially filled with water. The water level covered the fish when it was on its side, allowing for the flanks to be photographed (using an Olympus Stylus 790SW camer a). Multiple photos were taken and the fish were returned to their home tanks. The adults we re sampled one every four days, with the same
40 fish being sampled every twelve days. The average handling time was three minutes for the adults. The digital photographs were examined fo r the pigment cell locations and colors observed on the body. These photographs were then comp ared throughout the developmental stages studied. Background Adaptations: Two five gallon tanks were set up and filled th ree-fourths full with fresh R.O. water. Both tanks were well-oxygenated using an air stone. The water was changed every session using fresh water to prevent coloration from changing due to changes in the quality of water. One tank was placed over a black background. Black mesh placed on the bottoms and on the sides in the tank, for dark adaptation. The other tank was placed on a white tray with a second white tray pressed to one side providing the background of the tank. Both background tanks had a transparent cover over the top that allowed the passage of equal am ounts of light and maintained fish safety. Fish were transferred from their respective tanks to the background tanks using dip nets. One fish was tested at a time. Fish were first placed in the dark background tank. Once placed in the tanks, a photo of the fish was taken through the glass (without flash). The fish remained alone in the tank for sixty minutes. At the end of the adaptation period, a photo of the fish was taken (without flash). Using a dip-net, the fish was quickly transferred to the white background tank and observed for twenty minutes. During the ob servation period, changes in the expansion of chromatophores and hue of the skin were noted. After the initial observation period, the fish were maintained in the white background for forty more minutes (making sixty minutes total), with observations being made every five minutes. A phot o was taken and the fish was transferred back over to the black background. They were observe d similarly as in the white background tank. At the end of the sixty minutes, the fish were tran sferred back to their normal tanks and observed on a neutral background for ten minutes. A water change was performed on the testing tank and
41 then another fish was tested. Two fish were t ested per day, with various fish being tested throughout the week. Each fish was monitored in the tanks a minimum of two times (as possible). The mating pair was not tested in order to not disturb their courtship and brood rearing. The fry were also not tested here since they were too small to observe in the various tanks. Observations were instead made of the fry usi ng Petri dishes, one with a black background and one with a white background. Transfers were ma de with using a pipette. Due to their relative fragility, testing times were shortened to twen ty minutes for the fry in each Petri dish. Social-Color Observations: Three social observations were made to obser ve the color changes th at occur when fish were able to interact with other individuals. Social observations were made: between the mating pair; one for the school of four juveniles; and the last between the male that was introduced into a females territory. For the first observations, a video camera was set up to capture social interactions between the mating pair for periods of twenty minutes. Specifically observations focused on the pairs favorite surface to hide under. The video was later analyzed for color changes and patterns. The school of juveniles was observed for twenty minutes using a video camera. An adult male was introduced into the tank/territory of the adult female in an attempt to create a breeding pair. A camera was set-up to capture social interactions. The male was transferred in a two gallon container to the female s tank after he was slowly drip adapted with water from the females tank mixed into his cont ainer over the course of an hour and a half (to partially adapt him to the new environment). The initial temperature difference between the container and her tank was only 1 F. The male was then moved to the females tank using a dip net. Behavior of the new pair was then recorded for thirty minutes.
42 Experimental and Observational Results: First Week Post-Hatching: In the first days post-emergence from the egg, the larva of H. bimaculatus already display melanophores along two longitudinal bands, with xanthophores seen along the more dorsally located band. At two days post-hatching (Figure 2), the more dorsal band (indicated in the figures as the Dorsal Melanophore Band or DMB) begins at th e anterior end of the fish, slightly after the premaxilla. The band then extends the length of th e body. The band is less densely populated with melanophores closer to the posterior end at th e caudal peduncle. A few xanthophores are seen along the DMB that lies on the anteroposterior axis. Xanthophores are also visible along the midline of the head. The presence of the xanthophores along the DMB could be due to their dispersal from the neural crest along only the la teral pathway (as seen in zebrafish studied by Kelsh in 2004) compared to melanophores which travel along both the lateral and medial pathways. Melanophores appear to overlie the xanthophores. The more ventral of the two longitudina l bands, the Ventral Melanophore Band (VMB) originates near the lower mandible. The density of the band is not uniform, as fewer melanophores exist under the eye. The band extends behind the eye over the operculum and increases the density of melanophores over the stom ach before traveling to the caudal peduncle and caudal fin. The caudal fin displays melanin. The remaining fins are translucent and barely visible. Both bands are well represented due to the high density of the melanophores. In Figure 2, the melanophores are contracted, allowing the underl ying xanthophores to be visible. When fully expanded, the melanophores mask the yellow hue. A lack of chromatophores is seen away from the DMB and VMB makes the fish appear almost translucent. Those that do occur that are not presumably associated with the two longitudinal lines, are small and on the surface of the skin. The gut appears to have a slight pink hue. This is attributed to blood and other tissues rather than the presence of a distinctive color cell.
43 Melanophores are seen around the eyeball making an orbit outside of the pupil. No pattern in the density of the melanophores is noted. Beneath the melanophores, a yellow hue is seen due to xanthophores. Deeper-laying xanthophores cover the area of the brain before following the DMB down the length of the body. The xanthophores form a dense layer that underlies the melanophores on the head. The VMB does not have a predominant band of xanthophores along its length. Xanthophores are concentrated on the belly of the fish in higher numbers equal to those seen above the brain. Seven days post-hatching (Figure 3) the number of melanophores has increased along the VMB. The VMB still originates behind and belo w the eyeball; however, fewer melanophores are visible in that area of the band. The melanophores that are anterior to the operculum appear deeper in the skin. The VMB b ecomes dense with melanophores lying superficially to the skin surface around the operculum. The band extends past the caudal peduncle. Individual melanophores are distinguishable below the spinal cord. The DMB is now less populated with melanophor es. The band occurs before the eyeballs and ends closer to the anal fin that at two days post-hatching. The melanophores of the DMB occur heavily over the area above the brain and operculum and then appear in less density along the rest of the length. The further posterior along the fish, the fewer melanophores are in the DMB. Xanthophores are not as noticeable. This is partially due to the expansion of the melanophores overlying them. The operculum has a faint yellow hue; however, the xanthophores that reflect the color lay deeper in the skin of the fish. More st ructures are visible in the body. Greater differentiation of the fins is apparent as xanthophores have increased the area of their projections and there are higher densities of cells containing xanthosomes on the caudal fin. The density of melanophores on the caudal fin has incr eased and the xanthophores have increased the
44 area of their projections. Deeper layers of mela nin-containing cells are also visible behind the more lateral dermal chromatophores in the surface of the skin. Melanophores have become more distinct around the eyeballs, with a band of xanthophores circling it. The melanophores are more homogeneous along the surface of the eyeball and few exist immediately outside on th e skin. A yellow hue is present beneath the melanophores. It forms a yellow ring orbiting the pupil. The fish is predominantly white to transparen t (with the major exceptions being the head and two longitudinal bands). Deep-set leucophores provide the white color; however, their individual platelets are hard to distinguish. Th e white color is strongest in the head and operculum. Second Week Post-Hatching: Figure 4a and 4b show fry of H. bimaculatus eleven days post-hatching. Melanophores are clearly distinguishable in the DMB which originates between the eyes. The pattern is symmetrical with one DMB occurring on each side of the fish. A dense patch of melanophores, associated to the DMB, occurs over the brain ar ea. The patch contains both small superficial melanophores and deeper layers of melanophores that overlie xanthophores. The next dense patch of melanophores along the DMB is over the heart and liver. In both dense patches, xanthophores are present over the crucial organs. From the sec ond dense patch, the band begins to extinguish as it travels posteriorally. Due to the transparency of the fish, the VMB can be partially seen in Figure 4a below the eyeball. The band then extends past the eye a nd covers the expanse of the stomach with the highest concentration of melanophores present in the VMB. Layers of melanophores are visible with larger melanophores occurring underneath the sm all superficial ones. Longitudinal bands are more noticeable across the caudal fin.
45 Xanthophores are readily noticeable along the do rsal area of the head and body following the DMB and VMB. The xanthophores exist beneath the melanophores and are noticeable mainly due to the contraction of the overlaying melanophores. A brown hue is created from the layering of melanophores over xanthophores. White areas ar e beginning to form. Most likely they represent the presence of deep iridophores. Iridophores travel along a medial pathway from the neural crest (Kelsh, 2004) causing them to first a ppear deeper in the skin The crystals are not readily distinguished. A red hue, potentially fr om erythrophores, is seen along parts of the operculum and other areas. The erythrophores could exist epidermally or dermally. The first movements of xanthophores into the fins are apparent as a yellow color at the base of the fins. Light introduced directly onto the melan ophores causes melanin granules to travel to the extensions of the cell in 0.7 seconds. Direct stimulation causes the chromatophores to respond through neural control, either by contraction or stimulation. The melanophores observed contracting were the small superficial ones along the length of the DMB and VMB. When contracted, erythrophores were harder to observe (an exception being those over the brain). The erythrophores contracted similarly to the small superficial melanophores. Observation and Baerends and Baerends-van Roon (1950): After the examinations of chromatophore de velopment in the fry described above, the fry were then returned to home tank and unfortunately consumed by their parents. The developmental gap between eleven days post-hatching and five months may be bridged through the works of Baerends and Baerends-van Roon (1950). The following is taken from their monograph. Three to four weeks after the eggs were fertilized; the melanosomes begin to disperse along the projections of the melanophores. This could indicate that stronger neural and hormonal controls are being established over the chromatophores. Seen through the dispersed melanin granules, iridosomes appear to be associated with the melanophores (the combination creating melanoiridosomes).
46 Xanthophores are present deeper than the melanoiridosomes. The combined effect provides the fry with an opaque-yellow coloration. At five to seven weeks old, more rings are present around the eyes of the fish. The dorsal band displayed previously is beginning to di sappear. Melanophores become more dispersed and homogenous across the surface of the skin. The lateral band has begun to concentrate its melanophores into dense spots: one along the operculum; one closer towards the center of the body; and a third beginning to appear close to the caudal peduncle. Erythrophores which are formed last, have begun to travel to the dor sal fin which has thus taken on a reddish hue. Leucophores have populated the more ventral side of the fish. Five Months Post-Hatching Present Study: By five months (Figures 5 a-d) the fish have three predominant spots (Spots 1, 2, and 3 in figures 5-7) formed from dense congregations of deep melanophores along the VMB. Spot 1 occurs over the opercular area, Spot 2 is found in the center of the trunk, and Spot 3 on the end of the caudal peduncle. Each spot contains laye rs of melanophores (predominantly a superficial layer and a deeper layer). Although densely po pulated, individual melanophores in the spots are distinguishable. Iridophores which reflect li ght are present in Spot 1 along with the melanophores. The DMB is still visible, although the melanophores are more dispersed than before. Instead of a complete bar traveling the length of the body, melanophores appear in patches along the dorsal surface of the fish. This causes them to appear more as vertical bars (VB). More VB are noticeable along the posterior end of the fish near the caudal peduncle. Both the VB and the DMB have a multi-layer system of chromatophores. Small melanophores appear on the surface of the skin over deep-set large melanophores. VB al so show an intermediate level and size of melanophores.
47 Melanophores in the eyes are evenly distribut ed across the surface outside of the pupil. Below the melanophores, a yellow hue from xanthophores is visible. More melanophores have accumulated around the eye with each characterized as being small and close to the surface of the skin in comparison to melanophores seen in the eyes. The melanophores along the surface contract and expand rapidly to various stimuli. Melanin aggregation in the most dermal me lanophores was seen during times of fear such as movement from their tank to the microscope. Dir ect light caused expansion; however, this was not as rapidly as in eleven days post-hatch ing (1.5 seconds compared to 0.7 seconds). Erythrophores are in the tips of the dorsal fins and along the operculum. These red color cells are deep compared to the superficial melanophores. The red hue is predominantly seen during melanophore contraction. Xanthophores, positioned underneath the me lanophores and melanoiridophores, are present throughout the body. Highly reflective spot s appear along the surface of the skin. This response is caused by iridophores which are at highest density along the operculum. The more ventral region of the fish is slightly lighter th an the more dorsal area due to a lower density of melanophores and higher density of leucophores. Melanophores have traveled into the pectoral, dorsal and caudal fins in thin lines. Individual melanophores are most visible along the edges of the fins. Erythrophores are seen along the tips of the dorsal fins and the edges of the caudal fin. Xanthophores situated closer to the body of the fish are established in all three fins. Six Months Post-Hatching: By six months (Figure 6), the DMB has almost completely disappeared. Most of the band has differentiated into the VB across the body. Clo ser to the eye, the DMB is still intact, although the melanophores are more dispersed. The VMB is cl oser to the middle of the trunk, below the
48 spinal cord, and traverse from the lower mandible to the end of the caudal peduncle. The 3 spots are more distinguished, although Spot 3 can resemble a bar when the melanophores are fully expanded. A band of iridophores, underneath the VMB, begins at the operculum and travels to the caudal peduncle. The juveniles have an overall yellow hue mediated by deep xanthophores. Erythrophores have populated the tips of the dorsal fins and opercular area. Iridophores reflect light from the operculum and ventral portion of the body (where they are evenly dispersed). Small superficial melanophores can contract rapidly. They appear as small dots along the surface of the skin while deep iridophores create a whiter appearance ventrally. The brown hue along the longitudinal band is due to melanophores lying underneath the epidermal melanophores. Melanophores of the eye are evenly distributed around the outside rims. A circle of xanthophores orbits the eyeball. Higher densities of superficial xanthophores have congregated around the eyeball, creating an overall yellow hue to the area. Adult Chromatophore Patterns: The adult chromatophore pattern was th e last stage observed for chromatophores development (Figure 7). Two very dense spots are visible, one on the posterodorsal aspect of the operculum (Spot 1, S1), and one in the center of the body (about midway between the operculum and the caudal fin, S2). The spots are formed from very dense melanophores lying underneath the epidermal melanophores. Spot 2 is relatively la rger than the other two and the eye and is surrounded by large superficial iridophores. Spot 3 is at the end of the caudal peduncle with the expansions of the melanophores causing it to appear more as a VB than a spot. The eye has a dense concentration of melanophores around the pupil. The color cells may protect the optic system from radiation damage.
49 The VMB contains more melanophores deeper in the skin than at six months posthatching. Melanophores still exist ove r the bands and bars at the surface of the skin. The layering of melanophores is dense to the extent that the spot can be seen even when melanin granules are contracted towards the center of the cell. Iridophores are scattered across the body. They are specifically featured in higher densities from: the lower jaw to the operculum; along the VMB; and more ventral to the VMB. The iridophores appear in 6 rows along the body. Below the dorsal fin towards Spot 3, the DMB is partially visible along the most dorsal portion of the VB. The contraction of the most lateral melanophores faintly shows a series of stripes along the body. This pattern is normally obscured. The VB appears along the length of the fish, running from behind the eye to the beginning of the caudal peduncle. The bars themselves are a deep brown. They appear and disappear based on the motile abilities of the melanophores controlled by the excitement levels of the fish. Melanophores aggregate along the scale boundaries (see DSB in figure 7). These melanophores ar e visible when fish are in times of stress or fear. Expansion of the epidermal melanophor es allows the xanthophores to be partially covered. This presents the brown-yellow color that is seen across the skin. Erythrophores are scattered amongst the xanthop hores. This combination presents a slight redyellow hue. The erythrophores are most concentrated along the tips of the dorsal fin and ventral fins and over the operculum and lower jaw. Xanthophores have aggregated along the fins. The VMB has melanophores that overlay the yello w areas of the skin. Leucophores populate the ventral region of the body. This provides a fain t white hue in comparison with the more dorsal regions. Overall, the hue of the skin is yellow to brown. Because of the short time frame in which the je wel cichlid fry were able to be studied and the long developmental stages from fry to adults, only partial maps were made for the various stages of development. The full development betw een fry and juvenile was not tracked; however,
50 when examined in comparison with the devel opmental drawings from B aerends and Baerendsvan Roon (1950), a better approximation can be made (see Discussion). Reactions to Background Adaptation: These experiments entailed examining the reac tions to background adaptations (i.e. from dark to light background and vice versa). After seven days of development fish were able to alter their color rapidly. Morphological cues were not well tested in part because of the limited one hour period of the background exposure. The male placed on lighter gravel showed fewer melanophores along its surface and had a higher appe arance of a white hue (attributed to the presence of more leucophores) than the single male on the darker background. More fish need to be observed for longer periods of time in order to more fully examine this effect in jewel cichlids. However, the observations are similar to those from previous studies of long-term background adaptations to white and black backgrounds in the tilapia Oreochromis moassambicus zebrafish Brachydanio rerio, and others (Sugimoto, 2002; Iwata and Fukuda, 1973; Fujii, 2000; Sugimoto, 1993a). For the fry that were two days post-hatching, the background adaptation did not occur as readily as in the more developed jewel cichlids. Instead light seemed to play a more important role. Light directly applied on the body of th e cichlid caused faster expansion of melanophores than background adaptation. Since only one expe riment was run at this stage, potential for variability is high and more trials in the future need to be conducted. In the adults the surface melanophores imme diately responded in a matter of seconds when the fish were placed in the black-background tank. The yellow hue of the body became darker, closer to a brown coloration. The red color of the erythrophores were not as noticeable. Melanophores in all layers (surface, melanoiri dophores, and dark spots) expanded. In the melanophores associated with iridophores, melanin granules migrated into the cell dendrites and
51 obscured the iridophores. Iridophores were presumably also less noticeable, as less light was reflected from the dorsal ventral surface onto the skin. It is assumed that the neural control over the system occurred through the ventral retina wh ich obtained less visual stimulation than the dorsal retina (Fujii, 1993a) (see also the Introduction on page 24). When the adults were transferred to a white background, it is presumed that the ventral retina received roughly equal stimulation to the dor sal retina. This caused signals to be sent to the chromatophores, enabling the fish to lighten its appearance. Contraction of the melanosomes was noted within a second. Iridophores and leucophores were highly visible (and presumably in higher densities), as were the erythrophores and xanthophores. Social Observations: When the mating pair was observed through the camera recording, red nuptial coloration was seen in both the female and the male. This was evident both during pre-copulation and postfertilization of the eggs. The erythrophores formed dense layers across the skin with highest cell density layers in the ventral region. Reflec ting across the operculum, iridophores were more visible than in non-mating pairs. The red hue took a few days (three days from first notice) to reach its peak brilliance. Blanching occurred when the fish were disturbed by the placement of the camera. This caused more iridophores to be noticed and the red coloration to almost disappear. This physiological change occurred in less than one second. The effects were reversed rapidly as well (four seconds to return to their nuptial coloration). The red coloration that occurred rapidly in the single female when the single male was first introduced into her territory is in strong contrast to the slow development of the nuptial coloration. Erythrophores were quickly exposed across the body via the contraction of the melanophores and xanthophores. The female quickly displayed agonistic tendencies towards the male, biting his pectoral fins and chasing him around the territory. Aggressive coloration seen in
52 the female was equally as bright as that of the mating pair and appeared to be of a similar intense red hue. However, the time it took for the color to develop was rapid instead of the slow morphological color. After the aggressive displa ys were noticed, the female and male were quickly separated and the females coloration returned to its resting condition. The four juvenile cichlids observed were pr edominantly of a pale brown/tan background. Only during the presence of a disturbance near the vicinity of the tank did the juveniles show a noticeable color change. When this occurred, melanophores along the body contracted rapidly causing the skin to appear paler and the patterns less noticeable. When one juvenile jewel cichlid blanched, the remaining three quickly did so as well and all four sought refuge. After a minimum of two and a half minutes, the skin began to sl owly darken and the res ting colors returned.
53 Discussion and Conclusion Chosen Methodology: An approach similar to Beeching et al. (2002) was used. The study was conducted using non-invasive light microscopy and observance of patterns and colors on changing background colors. Other studies utilized measurements of the layers of chromatophores and innervation occurrences during development which required the removal of the skin. Various hormones and ions could thereby be tested on the skin for their specific effects on the light scattering and light absorbing properties of cells at various stages. These methods have been used in several studies (Fujii, 2000; Wakamatsu, 1978; Ohta, 1983; Oshima et al., 1998; Oshima and Yokozeki, 1999; Fujii, 1993b; Fujii and Oshima, 1994; Kawauchi et al., 1993; Castrucii et al., 1988) and the results have been well explained. Similar results without harm to the fish were obtained and they further confirmed the hormonal and chemi cal movements seen for physiological and morphological control from other studies. The design of this experiment did not include a detailed investigation into the morphological effects of long-term background adaptation and the effects it might have on chromatophores. In a previous study by Sugimoto (2002), this color change was tested in other species of cichlids. The authors findings indicated that long term adaptation to a black background caused an increase in the number and density of melanophores and a decrease in the number of leucophores seen on the surface of the skin. A long-term adaptation to a white background showed the reverse effects with melanophores undergoing apoptosis and an increase in the density of leucophores and iridophores. Mapping of chromatophores: The lack of crucial data on fish througho ut their transition from fry to adult (from two weeks to five months; and six months to an adult) caused gaps in the observations of the study for
54 the development of color pattern. Some of the ga ps could be bridged because they were observed in a study by Baerends and Baerends-van Roon ( 1950). Erythrophores, the cause of reproductive and aggressive color in Hemichromis bimaculatus, were the last chromatophore type to develop in the skin. Aggressive tendencies, well noted in th e adults of this species, are not present in the juveniles which school for protection (de Boer a nd Heuts, 1973). Sexual maturity is also only noted in the adults (Noble and Curtis, 1939). Th erefore the strong presence of erythrophores, does not appear important for social behaviors in young fish. Adulthood is reached when the fish begin to show aggressive behaviors, take on territories, and prepare to spawn. Red color is the predominant cue for social interactions in adults. J uveniles also react to red coloration; i.e. follow the red dummies more than those of any other colors (Noble and Curtis, 1939). Red coloration occurs after the sixth month of development, when the juveniles began to show aggressive tendencies (Noble and Curtis, 1939). Baerends and Baerends-van Roon (1950) observed the color patterns throughout development for Hemichromis bimaculatus and other cichlid species. The majority of the observations in the previous study and the curre nt study overlapped; however, differences were observed. In the current study, deep melanophor es were seen around the organs, notably the brain and heart (Table 6) in two days post-hatching a nd seven days post-hatching fry. It is postulated that the deeper melanophores aid in protecting the fragile organs from harmful UV rays and are necessary for survival. Melanophores occurring deep er in the skin were not mentioned in Baerends and Baerends-van Roon (1950) study. Other differences in observation involved the stage of 1-2 weeks post-hatching (Table 7). Similar to the previous divergence, deep chromatophores over the organs were not noted in Baerends and Baerends-van Roon; specifically they did not report the xanthophores beneath the melanophores and over the brain. These xanthophores could serve similar protective purposes as the deeper melanophores described previously. The other difference in the two studi es observed for the 1-2 weeks post-hatching stage
55 was the presence of melanophores over the operc ulum. Observed herein, a dense patch of melanophores over the operculum at eleven days post-hatching. This was not reported in the previous study.
56 Table 6: Comparison of Studies for 1 week Post-Hatching Baerends and Baerends-van Roon (1950) Curre nt Study <1 week post hatching < 1 week post hatching 2 LM B Eye M YR around the eye X around the brain case DM around O Transparent Body DMB and VMB X Eye M X YR around the Eye X X around the brain case X DM around O C Transparent Body X Key: 2 LMB = 2 longitudinal bands of melanophores; YR = yellow ring; X= Xanthophores; DM = deep melanophores; O = organs; DMB = Dorsal Melanophore Band; VMB = Ventral Melanophore Band; M= melanophores X = seen in both; B seen only in Baerends a nd Baerends-van Roon; C = seen only in current study
57 Table 7: Comparison of Studies for 1-2 Weeks Post-Hatching Baerends and Baerends-van Roon (1950) Curre nt Study 1-2 week post hatching 1-2 week post hatching 2 L M B Ey e M YR AE X A B D M W T B M on Op. X along 2 LMB RH AS M on fins M I DMB and VMB X Eye M X YR AE X X AB C DM X WTB X M on Op. C X along 2 LMB X RH AS X M on fins X MI X Key: 2 LMB = 2 longitudinal bands of melanophores; YR = yellow ring; X= Xanthophores; DM = deep melanophores; O = organs; DMB = Dorsal Melanophore Band; VMB = Ventral Melanophore Band; Op. = Operculum; MI= Melanoiridosomes; AE, Around the Eye; AB, Around the Brain; WTB, White-transparent Bo dy; RH, Red Hue; AS, Around the Stomach X = seen in both; B seen only in Baerends a nd Baerends-van Roon; C = seen only in current study
58 Table 8: Comparison of Studies for Adult Color Patterns in Hemichromis bimaculatus Baerends and Baerends-van Roon (1950) Cu rre nt Stu dy Adult Color Pattern Adult Color Patter n D M B V M B V B E y e M Y R A E X A E D M M o n O p. X al on g V M B R H A S M o n Fi ns M .I. 3 S I on Ve nB D ee p E B on E ye DMB C VMB C VB X Eye M X YR AE X X AB X DM X M on Op. X X along VMB X RH AS X M on fins X M.I. X 3 S X I on VenB X Deep E X B On Eye B Key: 2 LMB = 2 longitudinal bands of melanophores; YR = yellow ring; AE = Around the Eye; X= Xanthophores; AB = Around Brain; DM = deep melanophores; O = organs; DMB = Dorsal Melanophore Band; VMB = Ventral Melanophore Band; Op. = Operculum; M.I.= Melanoiridosomes; I= Iridophores; E= Erythrophor es; VenB = Ventral Body; RH AS = Red Hue around Stomach; S=Spots; B= Band X = seen in both; B seen only in Baerends a nd Baerends-van Roon; C = seen only in current study
59 Baerends and Baerends-van Roon (1950) ob served a cross-band over the eye of adults. This was not witnessed in the current studies exam inations of the adult male jewel cichlid. This distinction between the studies could be caused by the cross-band being visible only in reproductively active adults displaying nuptial co loration. The male examined was maintained alone in a tank, and so demonstrated none of the red hues or other color changes that would have been seen in mating fish. The cross-band could form by the expansion of deep melanophores of System 3, and this might obscure the underlying chromatophores. The last difference noted was the presence of the two longitudinal bands (DMB and VMB) in the adult form. The DMB is dispersed along the dorsal portion of the VB, where it differentiated. The VMB is intersected by all 3 spots, from the eye traveling to the operc ulum. This band was not noted by Baerends and Baerends-van Roon (1950). Indivi dual variations in the darkness of the bands could cause the divergence in observation. Perhaps the band appeared lighter in the examined fish in the previous study. Using the Baerends and Baerends-v an Roon (1950) chromatophores layering methodology (Table 4 on page 18), the melanophores along the DMB and VMB are classified as being composed of System 1 (the small supe rficial melanophores) and System 3 (the deeper melanophores). When the melanophores were coupled with the xanthophores from System 6, the deep brown bands (known as the longitudinal bands) were possible across the body. In addition to the previous systems, when DMB began to differe ntiate into the VB, an intermediate layer of melanophores (System 2) were observed. The rapi d motility of both the first and second system causes the bars to disappear (by contraction of the melanophores) during nuptial coloration. Spots 1, 2, and 3 are composed of Systems 1-3, with iridophores from System 5 providing bright reflectance for Spot 1. Spot 1, on the operculum, is common in many species of fishes; as are the iridophore patches. The operculum is flared during frontal displays, which most likely causes the head to appear la rger to other fishes. The opercular spot appears similar to a
60 large eye (an ocellus). As such the fish may resemble a larger individual and thus it could intimidate the other fishes. System 4 was not seen in this study. Red coloration (from erythrophores in System 6) in this study occurs in both a reproductively active pair of jewel cichlids and in the aggressive displays of a female guarding her territory against a new male. Red color on th e body of jewel cichlids seems to be a true indicator of social activity. When a male was in troduced into a females tank, her body rapidly became red and vertical bars became less apparent. This was followed by the female performing a frontal display to the male and biting his fins. In the reproductively active pair, both fish turned red shortly before a brood of eggs were found attached to the surface. Melanophores are the first chromatophores to populate areas on the skin of fishes (Raible and Eisen, 1994). They are also the most widely distributed chromatophore in fishes, covering most of the body surface. They may be especially important in protection of the fish from harmful UV rays (Bagnara and Ferris, 1971). Their role in blanching and darkening has been previously discussed. The contraction of melanophores allows a j uvenile to change its colors to better blend in with the environment. The response provides an additional level of protection that it would otherwise not have. Protection against predators a nd the environment seems to be more important than social communication during the first days of life. Such allows melanophores to be more advantageous to their survival than erythrophores. More complete studies on the specific age at which neural and hormonal control is gained over chromatophores is needed. Such studies will likel y require raising a brood of fry completely from hatching to adulthood. In future studies, more than one breeding pair could be maintained in order to produce continuous broods of fish at di fferent levels of devel opment. This would allow for the same developmental period to be tested more than once. The separation of fry from their parents would also prevent the cannibalism seen in this study.
61 For better observations of morphological color change, adaptation to a background for longer times (e.g. overnight) should be helpful. This would provide the fish with longer periods of both rest and melanophores adjustment to optical input. Lastly, the experiment may benefit from a change in the observation frequency. Examining the chromatophore migrations daily versus every several days should provide more comple te results. Increasing the observation frequency to every day for background adaptations would allow fo r more runs to be taken. There are multitude of factors that affect color cells and much wo rk needs to be done on both hormonal and neural control. Further study on the chromatophores of jewel cichlids, and fishes in general, could provide better insight into the use of color as a component of visual communication.
Figure 2: H. bimaculatus two days post-hatching. Explanations for abbreviations in the figure are arranged from left to right: VMB, Ventral Melanophore Band; DMB, Dorsal Melanophore Band; X, Xanthophores; DM, Deep Melanophores. 62
Figure 3: H. bimaculatus Seven days post-hatching. Explanations for abbreviations in the figure are arranged from left to right: DMB, Dor sal Melanophore Band; SSM, Small Superficial Melanophores; DM, Deep Melanophores; X, Xant hophores; VMB, Ventral Melanophore Band. 63
Figure 4a: H. bimaculatus Head Eleven days post-hatching. Explanations for abbreviations in the figure are arranged from left to right : VMB, Ventral Melanophore Band; DM, Deep Melanophores; DMB, Dorsal Melanophore Band; SSM, Small Superficial Melanophores; X, Xanthophores. 64
Figure 4b: H. bimaculatus Tail Eleven days post-hatching. Explanations for abbreviations in the figure are arranged from left to right: M, Me lanophores; VMB, Ventral Melanophore Band; SSM, Small Superficial Melanophores; DM, Deep Melanophores; X, Xanthophores; DMB, Dorsal Melanophore Band. 65
Figure 5a: H. bimaculatus Head Five months post-hatching. Explanations for abbreviations in the figure are arranged from left to right: VMB, Ventral Melanophore Band; DMB, Dorsal Melanophore Band; I, Iridophore; E, Erythr ophore; MI, Melanoiridophore; SSM, Small Superficial Melanophore; DM, Deep Melanophore; X, Xanthophore. 66
Figure 5b: H. bimaculatus Trunk Five months post-hatching. Explanations for abbreviations in the figure are arranged from left to right: ; X, Xanthophore; E, Erythrophore; S2, Spot 2; VB, Vertical Bars; VMB, Ventral Melanophore Band; DMB, Dorsal Melanophore Band; I, Iridophore; S1, Spot 1. Figure 5c: H. bimaculatus Spot 2 Five months post-hatching. Explanations for abbreviations in the figure are arranged from left to right: SSM, Small Superficial Melanophore; X, Xanthophore; DM, Deep Melanophore; IM, Intermedia te Depth Melanophore; I, Iridophore. 67
Figure 5d: H. bimaculatus Tail Five months post-hatching. Explanations for abbreviations in the figure are arranged from left to right: S3, Spot 3; VMB, Ventral Melanophore Band; X, Xanthophore; E, Erythrophore; DM, Deep Mela nophore; SSM, Small Superficial Melanophore; S2, Spot 2; DMB, Dorsal Melanophore Band. 68
Figure 6: H. bimaculatus Six months post-hatching. Explanati ons for abbreviations in the figure are arranged from left to right: DMB, Dorsal Melanophore Band; S1, Spot 1; E, Erythrophore; VB, Vertical Bar; I, Iridophore; S2, Spot 2; VMB, Ventral Melanophore Band. 69
Figure 7: Adult H. bimaculatus Male. Explanations for abbreviations in the figure are arranged from left to right: S2, Spot 2; SSM, Small Superficial Melanophore; DMB, Dorsal Melanophore Band; IM, Intermediate Depth Melanophore; VB, Ve rtical Bar; E, Erythrophore; VMB, Ventral Melanophore Band; X, Xanthophore; L, Leucophore; S3 Spot 3; I, Iridophore; S1, Spot 1; DM, Deep Melanophore; DSB, Dermal Scale Boundary. 70
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