Hormones and Behavior 54 (2008) 549–556

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Hormones and Behavior

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Hormonal regulation of female nuptial coloration in a fish

Helen Nilsson Sköld a,⁎, Trond Amundsen b, Per Andreas Svensson c, Ian Mayer d, Jens Bjelvenmark a, Elisabet Forsgren b,e a Department of Marine Ecology, Göteborg University, 566 Kristineberg, SE-450 34 Fiskebäckskil, Sweden b Department of Biology, Norwegian University of Science and Technology, NO-7491 Trondheim, Norway c School of Biological Sciences, Monash University, VIC 3800, Australia d Department of Biology, University of Bergen, NO-5020 Bergen, Norway e Norwegian Institute for Nature Research, NO-7485 Trondheim, Norway article info abstract

Article history: Physiological color change in camouflage and mating is widespread among fishes, but little is known about Received 20 November 2007 the regulation of such temporal changes in nuptial coloration and particularly concerning female coloration. Revised 11 April 2008 To better understand regulation of nuptial coloration we investigated physiological color change in female Accepted 23 May 2008 two-spotted gobies (Gobiusculus flavescens). Females of this species develop an orange belly that acts as an Available online 10 June 2008 ornament. The orange color is caused by the color of the gonads combined with the chromathophore based – Keywords: pigmentation and transparency of the skin. Often during courtship and female female competition, a rapid Sexual selection increase in orange coloration, in combination with lighter sides and back that increases skin and body Female ornament transparency, gives the belly an intense ‘glowing’ appearance. To understand how this increased orange Chromatophores coloration can be regulated we analysed chromatic and transparency effects of neurohumoral agents on Gobiusculus flavescens abdominal skin biopsies in vitro. We found prolactin and α-melanocyte stimulating hormone (MSH) to Prolactin increase orange coloration of the skin. By contrast, melatonin and noradrenaline increased skin transparency, Melanocyte stimulating hormone but had a negative effect on orange coloration. However, mixtures of melatonin and MSH, or melatonin and Melatonin prolactin, increased both orange coloration and transparency. This effect mimics the chromatic ‘glow’ effect that commonly takes place during courtship and intra sexual aggression. Notably, not only epidermal chromatophores but also internal chromatophores lining the peritoneum responded to hormone treatments. There were no chromatic effects of the sex steroids 17β-estradiol, testosterone or 11-ketotestosterone. We hypothesize that similar modulation of nuptial coloration by multiple hormones may be widespread in nature. © 2008 Elsevier Inc. All rights reserved.

Splendid body coloration and patterns are widespread among chromatophores in the skin (morphological color changes: Sugimoto, animals and are often the result of sexual selection (Andersson, 1994). 2002), skin patterns can be modified within minutes by reflective The nuptial coloration is commonly influenced by sex hormones changes in iridophores and through aggregation or dispersion of the (Rhodes et al., 1997; Noriega and Hayes, 2000;Toft and Baatrup, 2001; pigment-containing organelles inside the chromatophores (physiolo- Larsson et al., 2002; Toft and Baatrup, 2003). Although nuptial body gical color change: Kodric-Brown, 1998; Burton, 2002; Sköld et al., coloration in teleost fishes, as well as in many other animals, is 2002; Mähtiger et al., 2003). Such temporal pigment dispersal generally more pronounced in the males, females in several fish increases body pigmentation while pigment aggregation results in species also develop nuptial color patterns as they become sexually less body pigmentation (Fujii and Oshima,1994, Svensson et al., 2005). receptive (McLennan, 1995; Beeching et al., 1998; Takahashi, 2000). Reduction in black body pigmentation due to melanophore pigment The body coloration and patterns are often caused by chromatophores, aggregation commonly also results in greater skin transparency (Fujii which are large star shaped pigment-containing cells located in the and Oshima, 1994). Long-term chromatophore pigment dispersal also skin. The chromatophores are grouped into subclasses based on their stimulates pigment transfer to surrounding cells and induces color: xanthophores (yellow), erythrophores (red/orange), irido- chromatophore production (Sugimoto, 2002). phores (reflective/iridescent), leucophores (white), melanophores Physiological color change is very widespread, perhaps ubiquitous, (black/brown) and cyanophores (blue). In addition to the seasonal among fishes and is under hormonal and/or neuronal regulation (Fujii color change, which is caused by changes in the number of and Oshima, 1994). Such color modulation may provide camouflage (DeLoach and Humann, 1999; Healey, 1999; Höglund et al., 2002)or ⁎ Corresponding author. act in social signalling (DeMartini, 1985; Kodric-Brown, 1998; Höglund E-mail address: [email protected] (H.N. Sköld). et al., 2002). Fish species showing temporal modulation of coloration

0018-506X/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2008.05.018 550 H.N. Sköld et al. / Hormones and Behavior 54 (2008) 549–556 include many of the splendidly colored fishes inhabiting coral reefs, understanding male ornamentation (Cotton et al., 2006) while female which undergo major or minor changes in coloration depending on ornamentation has traditionally received less attention (but see social or ecological context (e.g. Thresher, 1984; Michael, 2001; Amundsen, 2000; Amundsen and Pärn, 2006; Kraaijeveld et al., DeLoach and Humann 1999). Examples include several angelfishes 2007; Haselton et al., 2007). (Pomacanthidae) and damselfishes (Pomacentridae) in which both To better understand physiological mechanisms of color regulation sexes attain temporary colors or patterns during courtship, and in a species for which the social and sexual context of physiological cardinalfishes (Apogonidae) in which females of several species sport color change is known, we have investigated the two-spotted goby, temporal color changes (Thresher, 1984). Physiological color change Gobiusculus flavescens. The two-spotted goby is a small, semi-pelagic may allow fishes to be normally inconspicuous but to attain bright and sexually dimorphic marine fish with paternal care and dynamic colors during brief spells of courtship, as in the bluelip parrotfish sex roles (Gordon, 1983; Skolbekken and Utne-Palm, 2001; Bjelven- (Cryptotomus roseus) that sports bright gold, pink, blue and green mark and Forsgren, 2003; Forsgren et al., 2004). The females develop colors only when swooping down to court females in the seagrass colorful orange bellies as the gonads mature (Svensson, 2006). The (DeLoach and Humann, 1999). Nuptial color patterns can in many degree of belly coloration varies among fully mature females, and fishes therefore potentially become more conspicuous during court- males show a preference for females with the most colorful bellies ship by physiological color change using chromatophores (Rowland (Amundsen and Forsgren, 2001). The orange color is carotenoid-based et al., 1991; Kodric-Brown, 1998; Berglund, 2000; Berglund and and caused by the additive effect of the pigmented abdominal skin Rosenqvist, 2001; Svensson et al., 2005). and the pigmented gonads being visible through the semi-transparent Despite that physiological color change apparently plays a major skin (Svensson et al., 2005; Svensson, 2006). Thus, in breeding G. role in the mating and thus reproductive behaviour of many fishes flavescens females, the distinctive orange belly coloration is the (and other poikilothermic animals), there are remarkably few studies consequence of three components: skin transparency, skin coloration on function and physiological mechanisms behind temporary changes and gonad coloration (Svensson et al. 2005). Both during courtship in nuptial body coloration in an ecological or evolutionary context. and in agonistic interactions between females, two-spotted goby Few studies have analysed physiological mechanisms of color females actively display their strikingly orange bellies by conspicu- regulation in species for which the social and sexual context of ously bending their body towards the courted male or the female physiological color change is known. The phenomenon has further competitor (Amundsen and Forsgren, 2001; Berger, 2002; Fig. 1). rarely been studied in the wild beyond verbal descriptions. Despite The dorsal side of a mature two-spotted goby female (down to the well-described regulation of chromatophores at the cellular and around the lateral line) is rich in melanophores, in contrast to the belly molecular levels that has revealed a number of potential regulatory (ventral side, below the lateral line) which has sparse melanophore mechanisms (Fujii and Oshima, 1994; Fujii, 2000; Sköld et al., 2002), pigmentation but instead a high density of erythrophores and there is little information about the processes that modulate xanthophores (Svensson et al., 2005; Figs. 1 and 2). Observations coloration and patterns, including hormonal regulation, in larger both in the wild and in the laboratory have revealed that a striking areas of the skin or at the level of the whole fish. In addition, ecological physiological color change takes place within minutes when female research on sexual selection and signalling has been biased towards two-spotted gobies engage in courtship or female–female

Fig. 1. Sexually mature female two-spotted gobies (Gobiusculus flavescens). The fish were kept in aquaria with males separated from the females by transparent plastic screens (A–D). During sigmoid display the orange coloration of the belly is intensified while the back becomes more transparent and pale (C, D) compared to non-courting females (A, B). From higher magnifications of the belly, erythrophore and xanthophore pigments appear dispersed during sigmoid display (insert in C). H.N. Sköld et al. / Hormones and Behavior 54 (2008) 549–556 551

Fig. 2. Quantitative effects of various hormones and hormone blends on transparency and coloration of abdominal skin biopsies from female two-spotted gobies. The biopsies from each individual were cut in two, and one piece incubated in Ringer's solution as a control (zero value) while the other was incubated in noradrenaline (NA), melatonin (MEL), melanocyte stimulating hormone (MSH), prolactin (PRL), testosterone (T), 11 keto testosterone (11K-T) or oestrogen (E2), tested individually or in combination. We analyzed two areas of the abdominal skin biopsies (A). First, transparency and coloration (chroma) was quantified in the belly area lateral of the gonads (A: red; B, C). Second, transparency was also quantified in the dorsal area immediately above the lateral line (A: white; D). Dorsal (d) and ventral (v) directions are indicated on the skin biopsy (A). Effects on skin transparency and chroma are visualised as the difference between the treatment and the control from the same individual (mean±SE, n=5). The P values (two tailed pair wise t-tests) are given above each treatment. 552 H.N. Sköld et al. / Hormones and Behavior 54 (2008) 549–556 competition. In these circumstances, females often become much may breed several times during the May-August breeding season. The more intensely orange than they usually are. The regulatory mechan- two-spotted goby is a substrate brooder, and clutches are typically ism for this temporal color change has not been investigated. As deposited in empty blue mussels (Mytilus edulis) or on kelp fronds (e.g. judged visually, however, the body becomes pale and nearly Laminaria spp.). Males often interact agonistically over nests or transparent, and the orange belly becomes strikingly conspicuous, females, and females may also perform intrasexual agonistic acts in not only from the sides and below but even as seen from above. This the presence of males (Forsgren et al., 2004). phenomenon has been termed ‘glow’ (Berger, 2002), as it gives the Fish for use in the study were collected by snorkellers in June to impression that the female belly actually glows (Fig. 1). Shortly after July 2006 in the Gullmar fjord, Sweden (N 58°15V, E 11°27V) using small termination of the social interaction, the body may again attain its nets. Following capture, the fish were quickly transferred to the baseline colors with a darker dorsal side and no “glow”. While the egg aquaria facility at Kristineberg Marine Research Station, where they color cannot be modified, the transparency and coloration of the skin were kept in glass aquaria for ≤1 week before use. The aquaria were can be temporarily modulated through physiological color change. As supplied with flow-through natural seawater and fish were kept melanin quenches light, the suggestive increase in transparency and under a natural photoperiod. Ethical permission for all fish experi- lightness of the skin during this “glow” phase is likely to be caused by ments was obtained from the Swedish animal welfare agency. melanophore pigment aggregation, facilitating light transmission through the body and thus increasing the light reaching the colorful Chemicals gonads. It is likely that the “glow” also involves dispersion of ery- throphores and/or xanthophores of the belly skin (Fig. 1c), resulting in Stock solutions of 1 mM noradrenaline (Sigma Aldrich, St. Louis, enhanced orange skin coloration. MO, USA) were stored at −20 °C and diluted to the experimental Previous work on chromatophore regulation in two-spotted goby concentration of 10 μM in calcium-free Atlantic cod Ringer's solution females has demonstrated that epidermal melanophores, erythrophores at pH7.5 (150 mM NaCl, 5.2 mM KCl, 1.8 mM MgSO4, 7.0 mM NaHCO, and xanthophores in the abdominal skin respond to noradrenalin by 1.9 mM NaH2PO4 and 1 mg/ml glucose), modified from Karila et al. pigment aggregation, leading to less colorful, and more pale and trans- (1993), just before use. Similarly, melatonin and sheep prolactin, both parent belly skin, indicating that this species can indeed modulate its from Sigma Aldrich, were stored at −20 °C as 10 mM and 150 IU/ml nuptial signal (Svensson et al., 2005). To test if any single hormone, or a stock solutions, and used at 10 μM and 0.150 IU/ml respectively. MSH combination of hormones, can result in the opposing effects on black (α-melanocyte stimulating hormone) from ICN Pharmaceuticals, Inc., (melanophore pigment aggregation) and orange (erythrophore and Costa Mesa, CA, USA, was stored as 1 mM stock solutions at −20 °C and xanthophore pigment dispersion) coloration that would correspond to used at 5 μM. Testosterone, 17b-estradiol (here described as oestrogen the combined increased skin transparency and increased orangeness as or E2) and 11-ketotestosterone, all purchased from Sigma Aldrich, seen during female ‘glow’, we performed in vitro testing of selected were all made up to the experimental concentrations of 0.1 μg/ml from neurohumoral agents on excised pieces of abdominal and dorsal skin an ethanol dissolved stock solution (1 mg/ml) kept at +4 °C. from gravid females. We quantitatively examined transparency and orange chroma of the biopsies. Chromatophores lining the inside of the Chromatophore assay body have generally been little explored, but could potentially be of importance in the color-signal of two-spotted gobies and other species For each treatment, biopsies from 5 random females with mature with semi-transparent skin. For this reason, we also examined the gonads were used. The fish were quickly decapitated, and the tissue “inside” (peritoneum) of the skin biopsies by microscopy. Noradrenaline covering the abdomen and sides of the fish was excised. The biopsies and melatonin were tested as potential pigment aggregating agents were placed in saltwater for ca 60 min to allow full chromatophore (Fujii and Oshima, 1994; Fujii, 2000). We predicted that these two pigment dispersion before onset of treatment. The biopsies were hormones could potentially act on different chromatophore types as photographed on a light table with the epidermis side facing towards they bind to different receptors (Aspengren et al., 2003). We also tested the camera as previously described in detail (Svensson et al., 2005). for effects of melanocyte stimulating hormone (MSH) which is widely The imaging procedure was standardised by covering the camera and known both to increase the amount of dorsal melanophores long term, the light table with black fabric and keeping the focal distance, to disperse chromatophore pigments (Baker et al., 1984; Fujii and exposure time and aperture constant. A black object placed adjacent Oshima, 1994; Fujii, 2000; Sugimoto, 2002; Höglund et al., 2002)andto to the biopsy was used as a zero-light reference in the image analysis. stimulate female sexual activities (Pfaus et al., 2004). Prolactin was These photos were taken with a Canon D30 digital camera equipped tested since it has been described to selectively disperse erythrophore with a macro lens. Biopsies were then cut in two pieces (each ca and xanthophore pigment in some fish (Fujii and Oshima, 1994; Fujii, 1cm2) along the central ventral line so that each fish yielded two 2000) and to enhance courtship in male newts (Toyoda et al., 2005). pieces, one from each side. From each fish one piece was incubated for Effects of sex steroids were tested since these are well known for their 1 h in the dark in Ringer's solution (control) and the other at the same role in the regulation of fish coloration long term (Larsson et al., 2002; conditions but with a hormone or a combination of hormones Toft and Baatrup, 2003), and potentially also short term (Fujii and dissolved in Ringer's solution as treatment. After incubation, the Oshima, 1994). Oestrogen and MSH were also tested since they induce biopsies were photographed a second time (same procedure as prolactin production (Matsumura et al., 2003; Elango et al., 2006). We before). To asses chromatophore responses of both the outside and the finally tested some candidate blends that might lead to opposing effects inside (peritoneum) of the skin biopsies, close-up photographs were on melanophores versus erythrophores and xanthophores. in addition taken using a Canon S40 digital camera mounted on a Wild M3Z stereo dissecting microscope with light from below (Leica Microsystems, Stockholm, Sweden). The magnification and illumina- Materials and methods tion was kept constant across treatments throughout photography.

Study species and general procedures Image analysis and statistics

The two-spotted goby (Gobiusculus flavescens (Fabricius, 1779)) is a We analyzed two areas of the abdominal skin biopsies (Fig. 2a). common inhabitant of shallow waters along the rocky shores of First, transparency and chroma were quantified in the area directly Western Europe (Miller, 1986). In most parts of its distribution range lateral of the gonads, i.e. the area below the lateral line and above an (including the study site), they live for one year only. However, they imagined line between the pelvic girdle and the anal pore (“belly H.N. Sköld et al. / Hormones and Behavior 54 (2008) 549–556 553 skin”). Second, transparency was also quantified in the area im- In contrast to the pigment aggregating effect of melatonin and mediately above the lateral line (“side skin”). To avoid any treatment noradrenaline, neither prolactin nor MSH treatment had any effect on bias in area selection, selections were first made in the photographs transparency (melanophore pigment aggregation) as compared to taken before exposure. Using landmarks on these biopsies, the same controls. However, both prolactin and MSH significantly increased areas were carefully selected in the images taken after exposure and belly chroma, due to a pigment dispersing effect on xanthophores and used for image analyses. The pre-treatment photographs were thus erythrophores. (Fig. 2, Table 1). only used as a guide to select the areas to be used in the image analysis None of the sex steroid treatments (testosterone, 11-ketotestos- of the post treatment photographs. terone, E2) had any effect on transparency or belly chroma (Fig. 2, Image analyses were performed in Adobe Photoshop CS2 (Adobe Table 1). Systems Inc.) using the CIE (Commission International de l'Eclairage) L⁎a⁎b⁎ colour space after normalising any light intensity differences Combined hormone treatments between the images by setting the black object adjacent to the biopsy as black and the background as white, as described previously in detail Melatonin and MSH together caused opposing effects on black and (Svensson et al., 2005). CIE L⁎a⁎b⁎ consists of three parameters: the L⁎ orange pigmentation (Fig. 2, Table 1), while no individual hormone value (lightness) which gives the relative lightness ranging from total had such an effect. While belly orange chroma increased relative black to total white, the a⁎ value (“redness”) which represents the to controls, due to dispersion of erythrophore and xanthophore pig- balance between magenta and green, and the b⁎ value (“yellowness”) ments, both abdominal (belly) and dorsal (side/back) skin became which represents the balance between yellow and blue (Margulis, more transparent and pale, as a result of melanophore pigment ag- 2006). A higher a⁎-value describes more magenta, and a higher b⁎- gregation. This is the pattern of chromatophore regulation we hy- value describes more yellow. A more intense orange hue will cor- pothesised to take place during “glow” (see Fig. 1 for a comparison). A respond to an increase in both these channels (Margulis, 2006). As similar, though weaker, pattern was found for the combination of a compound measure of orange color intensity, we therefore used melatonin and prolactin (Figs. 2 and 3, Table 1). While melatonin+ chroma (“saturation”), which in our case is the combined effect of the prolactin also caused a significant increase in belly chroma, as for two color parameters: chroma=√(a⁎2 +b⁎2)(Little, 1975). Lightness melatonin+MSH, the increase in transparency for melatonin+prolac- (L⁎) was converted to skin transparency, as described earlier tin was only close to the level of significance. The combination of (transparency=100×L⁎/255, Svensson et al., 2005). prolactin and noradrenaline caused an increase in transparency, both Two-tailed paired t-tests were used to compare the control and of belly and side skin. However, this effect was accompanied by a treatment biopsies from each female. Shapiro–Wilk's test of normality significant reduction in belly skin chroma. This pattern was similar was performed to ensure that the data met the assumptions for the to that found for Single-hormone treatments with melatonin and parametric tests (all PN0.07). Transparency values were arcsine noradrenaline, respectively, but strikingly different from the single- square root transformed before analysis to improve normality hormone effect of prolactin, which increased belly chroma but had no (Crawley, 2002). Weakly significant results (P close to 0.05) should effect on transparency on its own. be interpreted with caution, considering the multiple parameters (belly skin chroma, belly skin transparency and side skin transpar- Regulation of internal chromatophores ency) tested for each biopsy pair. When observing the abdominal biopsies, it became evident that not only the epidermal chromatophores, but also chromatophores lining the Results reflective peritoneum inside the fish, responded to the added hormones. For comparison, both the epidermis and peritoneum contained more Single-hormone treatments dorsally located dark melanophores and a paler xanthophore- and erythrophore rich ventral area. (Fig. 3). The responses to the hormonal Both noradrenaline and melatonin increased transparency of both treatments between the two sides of the biopsies were similar in effect belly and side skin compared to controls, through aggregation of (Table 1, Fig. 3a) and time, perhaps with the exception for the melanophore pigment. At the same time, both these hormones combination of prolactin and noradrenaline which appeared to result resulted in less orange belly skin (lower chroma), due to aggregation in more yellow on the epidermis side (Table 1)comparedtoatthe of erythrophore and xanthophore pigments (Fig. 2, Table 1). peritoneum side of the biopsies (not shown). Importantly, also at the

Table 1 Summary of hormonal effects on chromatophores, coloration and transparency of skin biopsies from female two-spotted gobies

Treatment Erythrophore (red/orange) Xanthophore (yellow) Melanophore⁎ (black) Belly skin color Belly skin Side skin t value/p value transparency transparency t-value/p value t-value/p value Mel −−−−−3.95/0.017 + 4.27/0.013 + 7.55/0.002 NA −−−−−3.47/0.026 + 8.83/b0.001 + 12.1/b0.001 Prl + + 0 + 4.82/0.009 0 −0.13/0.90 0 −1.69/0.17 MSH + + 0 + 5.96/0.004 0 0.68/0.53 0 0.21/0.83 Prl+Mel + + − + 7.13/0.002 + 2.87/0.045 0 2.35/0.08 MSH+Mel + + − + 5.93/0.004 + 7.18/0.002 + 8.28/0.001 Prl+NA − +/0⁎⁎ −−−3.42/0.027 + 4.63/0.008 + 8.78/b0.001 T0 0 0 0−0.69/0.53 0 −0.65/0.55 0 −0.67/0.55 11kT 0 0 0 0 0.60/0.57 0 −0.84/0.43 0 −0.43/0.67 E2 0 0 0 0 −1.91/0.13 0 0.11/0.92 0 −1.51/0.21

Chromatophore responses were assessed from images of the epidermis and peritoneum of skin biopsies covering belly and body side (+ dispersion, − aggregation, 0 no change). “Belly skin color” represents effects on epidermal belly orange chroma, and “skin transparency” represents lightening effects due to melanophore pigment aggregation on skin biopsies covering belly and side (+ increase, - reduction, 0 no significant change). Mel = melatonin, NA = noradrenalin, Prl = prolactin, MSH = melanocyte stimulating hormone, T = testosterone, 11kT = 11 keto testosterone, E2 = oestrogen. The combined increase of belly coloration (dispersion of erythrophore and xanthophore pigment) and body transparency/lightness (aggregation of melanophore pigment), amplifies the orange belly coloration ”glow” and was achieved by the combinations MSH + Mel and Prl + Mel (bold). ⁎ Most control melanophores had dispersed pigment. ⁎⁎In Prl+NA, it was difficult to distinguish dispersal from no effect in xanthophores but the epidermis side appeared yellow. 554 H.N. Sköld et al. / Hormones and Behavior 54 (2008) 549–556

Fig. 3. Regulation of chromatophores and coloration of abdominal skin biopsies from female two-spotted gobies. A: Both the “inside” (peritoneum: left panel) and the “outside” (epidermis: right panel) of the skin biopsies showed overall enhancement of the orange skin coloration either by the combinations of melatonin and melanocyte stimulating hormone (MEL+MSH) or melatonin and prolactin (MEL+PRL). The biopsies were each cut in two mirror pieces where one piece was incubated in Ringer's solution as a “control” and the other in hormone “treatment”. The images of each biopsy pair is positioned with the thin and paler xanthophore-rich ventral side facing inwards (v) and the thick and dorsal melanophore-rich side facing outwards. Scale bar 1 mm. B: Presence and regulation of the inside (peritoneal) melanophores and erythrophores are exemplified by close-up images from a biopsy treated with melatonin (MEL), melatonin combined with prolactin or a control incubated in Ringer's solution only (Control). While melatonin alone resulted in aggregated melanophore (black) and erythrophore (red) pigments, the combination of melatonin and prolactin resulted in dispersed erythrophore pigment together with aggregated melanophore pigment. Scale bar 0.2 mm. C: Presence and regulation of the inside (peritoneal) xanthophores are illustrated by increased yellowness by prolactin treatment (PRL) compared to its control biopsy which displays xanthophores with aggregated pigment among other chromatophores. Scale bar 0.2 mm.

peritoneum, there was an overall enhancement of the orange coloration during courtship may be regulated by the combined effects of in combination with melanophore pigment aggregation either by the multiple hormonal stimuli, with MSH, melatonin and prolactin as combinations of melatonin+MSH or by melatonin+prolactin (Fig. 3a). likely candidates. We are not aware of any other studies analysing The regulation of internal chromatophores is exemplified by melano- the chromatic effects caused by combinations of different hor- phore, erythrophore and xanthophore responses to melatonin and mones. Given our results, it is quite possible that simultaneous prolactin (Fig. 3b,c). regulation of multiple hormones could account for complex color changes in two-spotted gobies and potentially also in many other Discussion animal species.

Observations and photography of female G. flavescens during The roles and regulations of orange belly coloration courtship displays indicate that they become overall pale and transparent with increased coloration of the orange belly. These The transparency of the skin may serve two simultaneous combined effects make the female belly ‘glow’ intensely (Fig. 1). In functions in female two-spotted goby coloration. First, increased order to better understand the regulation of this rapid color change transparency of the belly may more clearly reveal the colorful and that optimizes the orange belly coloration, we examined chromatic carotenoid-rich gonads, which are part of the nuptial signal (Svensson effects of multiple neurohumoral regulation of ventral and side et al., 2005, 2006). Second, increased transparency of the side and skin chromatophores, as well as on the chromatophores lining the back would allow more light to penetrate the body and thus con- peritoneum of the fish. We found that the opposing effects of tribute to the belly “glow” effect. We found that the pigment ag- reduced black pigmentation (melanophore pigment aggregation) gregating hormones noradrenaline and melatonin increased the and increased orange coloration (erythrophore and xanthophore transparency of the abdominal skin. This effect is in agreement with pigment dispersion) that likely take place during ‘glow’ could be previous work (Fujii and Oshima, 1994; Svensson et al., 2005). reproduced by a combination of melatonin and MSH, and to a lesser Melanocyte stimulating hormone has been reported to disperse degree also by combining melatonin and prolactin. Thus, the melanophores in many other fish species (Fujii, 2000), but did not conspicuous color display used by female two-spotted gobies show such an effect here. However, most melanophore pigment was H.N. Sköld et al. / Hormones and Behavior 54 (2008) 549–556 555 nearly fully dispersed in our controls, so a further dispersal could be female two-spotted gobies actively engage in courtship and intrasexual difficult to detect. competition, and would benefit from enhancing and rapid regulation of Xantophores and erythrophores, especially common in the ab- its nuptial signal. Interestingly, the baseline coloration of the abdominal dominal area of the female two-spotted goby skin, can have at least skin also increases over the mating season (Svensson, 2006). This could two functions. First, they may cause a gradual increase in belly co- be a response to the increased female–female competition that late in loration as gonads mature, which could signal a general readiness to the breeding season (Forsgren et al., 2004), but it could also be affected spawn. Second, a rapid increase in color could signal willingness to by changes in availability of carotenoid-rich prey. mate, or aggression toward a female competitor. We found that MSH and prolactin increased the orange belly coloration by dispersing Future perspectives erythrophore and xanthophore pigments. MSH has been described to disperse melanophore, erythrophore and xanthophore pigment in Although we found a chromatic response to MSH and melatonin other fishes (Fujii, 2000), and MSH plasma levels has been reported to and to some extent prolactin and melatonin that matches the ‘glow’ correlate with skin darkness in some fishes (Höglund et al., 2002), during courtship, we cannot conclude from the present study that but little is known about MSH in the context of nuptial coloration. these combinations of hormones are fully responsible for the rapid Interestingly, recent work indicates that MSH also increases female colour change observed in live females under mating circumstances. sexual behaviour in rats (Pfaus et al., 2004). Prolactin is far less well Future work should aim to confirm the presence of MSH, melatonin studied than MSH in the general context of pigment cell research but and/or prolactin in the skin or in the abdominal cavity during has also been described to stimulate courtship (Toyoda et al., 2005). It courtship or female–female agonistic behaviours of the species, even has also been reported to have a dispersing effect on erythrophore and if this is not easily done in such a small fish and for such short-term xanthophore pigments of some tropical fish species, especially in the behaviours. If this can be done, however, it would also be interesting mating season (Kitta et al., 1993; Fujii, 2000). These pigment dis- to analyze whether the timing of this regulation is at the level of persing effects are in line with our results for female two-spotted hormone production and release or via changes in receptor binding gobies. It has also been suggested that steroids, which have a pivotal affinity in the skin. role in reproduction and reproductive behaviour in vertebrates, are Independent of chromatophore type, it has been shown that a involved in the development of nuptial coloration in fishes over the decrease in intracellular cAMP levels in combination with increase in reproductive season (Fujii and Oshima, 1994; Toft and Baatrup, 2001; calcium leads to pigment aggregation, while an increase in cAMP leads Larsson et al., 2002). However, we observed no short-term effect of to pigment dispersion (Fujii, 2000; Aspengren et al., 2003). In order to oestrogen, testosterone or 11-ketotestosterone on skin coloration. allow for the fine tuning of skin pigmentation patterns it is likely that Thus, sex steroids are unlikely to play a role in the rapid changes in different receptors are located on different chromotophore types. nuptial coloration of two-spotted gobies. It is possible that steroids Such hypothesis can be tested by immunocytochemistry using anti- increase the amount of certain chromatophores over longer time bodies against candidate receptors. periods, as oestrogens and testosterone are known to increase skin As daytime production of melatonin by the gastrointestinal tract in redness in numerous animals after treatments lasting weeks or teleost fishes and in other higher vertebrates is significant (Bubenik, months (Rhodes et al., 1997; Noriega and Hayes, 2000; Larsson et al., 2002), it is plausible that melatonin can be involved in daytime re- 2002; Toft and Baatrup, 2003). Given that prolactin increases orange gulation of the female coloration. It is noteworthy that melatonin also coloration of the skin and that prolactin production has been functions as an antioxidant (Filadelfi and Castrucci,1996). Recent work described to be extensively induced by oestrogens (Elango et al., has shown that melatonin supplementation enhances carotenoid- 2006), prolactin could be a down-stream mediator of the oestrogenic based sexual signals, as for example beak redness in zebra finch males color effect seen in vivo. Whether prolactin may have different effects (Bertrand et al., 2006). It is therefore possible that melatonin also on male versus female ornamentation or if its effect relates to certain enhances sexually selected nuptial signals in female two-spotted sex roles, remains to be shown. gobies. Also prolactin may have other beneficial effects than color Interestingly, the chromatophores present on both sides of the skin modulation, since studies on fish have shown that prolactin enhances biopsies (i.e., epidermal and peritoneal chromatophores) responded to the immune system by increasing phagocytosis as well as increasing the hormonal stimuli. Little work has been done on the regulation of the amount of leukocytes and antibodies (Harris and Bird, 2000). A internal chromatophores such as those lining the peritoneum on similar positive effect on the immune system is also described for MSH the inside of the skin biopsies. Goda and Fujii (1996) reported that (Harris and Bird, 2000). To further understand why certain hormones melanophores in the peritoneum of the ice goby, Leucopsarion petersii, have been selected to regulate the nuptial signal, systemic and be- are responsive to background adaptation, but regulation of inner havioural effects should be investigated by in vivo experiments. erythrophores and xanthophores has to our knowledge not been We believe that investigations of potential multiple hormone color described before. For two-spotted goby females that have relatively regulation, such as effects of MSH, melatonin and prolactin, in a transparent skin, the two layers of chromatophores may both play a variety of species, could provide important new insights into the role in enhancing and regulating their nuptial signal. Given the limited complex social color regulation in fishes and other animals. information available concerning the presence and possible regulation of internal chromatophores, our finding may also be of ethological Acknowledgments importance for other fish species. Internal chromatophores could potentially be important both in species where females have colorful This study was conducted at Kristineberg Marine Research Station patches on the skin that covers the gonads, for example the convict in Sweden and financially supported by the Royal Swedish Academy of cichlid (Cichlasoma nigrofasciatum; Beeching et al., 1998), the stream Science, the Research Council of Norway, the Nordic Research Board, goby (Rhinogobius brunneus; Takahashi et al., 2000) and the lagoon the Swedish Research Council, the C. F. Lundström's foundation and by goby (Knipowitschia panizzae; Massironi et al., 2005), but also for the Gothenburg University Integrative Physiology platform. certain male ornaments or for general camouflage as in the ice goby (Goda and Fujii, 1996). The two-spotted goby has dynamic sex roles (Forsgren et al., 2004), References with predominant male mating competition (conventional sex roles) Amundsen, T., 2000. Why are female birds ornamented? 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