Chromatic interaction between egg pigmentation and skin chromatophores in the nuptial coloration of female two-spotted gobies
1 Department of Biology, Norwegian University of Science and Technology,
N-7491 Trondheim, Norway
2 Kristineberg Marine Research Station, Royal Swedish Academy of Science,
450 34 Fiskebäckskil, Sweden
* Author for correspondence (e-mail: andreas.svensson{at}bio.ntnu.no)
Accepted 12 October 2005
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Summary |
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Key words: sexual selection, nuptial signal, female ornament, courtship, Gobiusculus flavescens
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Introduction |
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The two-spotted goby, Gobiusculus flavescens, is a small,
semi-pelagic marine fish with paternal care of the eggs
(Gordon, 1983;
Skolbekken and Utne-Palm,
2001
; Bjelvenmark and Forsgren,
2003
). It is sexually dimorphic with both males and females
exhibiting ornamentation during the reproductive season. While males have
brightly coloured fins and iridescent blue spots, females develop increasingly
orange bellies as gonads mature. Amundsen and Forsgren
(2001
) hypothesized that the
female belly coloration is caused mainly by the pigmented eggs being visible
through the semi-transparent abdominal skin but also by pigment in the skin
itself. The development of nuptial coloration over the course of the
reproductive season suggests that steroids are involved in its regulation
(Burton, 1981
;
Fujii and Oshima, 1994
).
Notably, the degree of belly coloration varies even among fully mature females
(T. Amundsen, E. Forsgren, C. Pélabon and P. A. Svensson, unpublished),
and males show preference for females with the most colourful bellies
(Amundsen and Forsgren, 2001
).
Female courtship involves approaching the male and bending the body (sigmoid
display), a behaviour that seems to emphasize the round and colourful belly
(Amundsen and Forsgren, 2001
).
Both during courtship and in agonistic interactions, females can temporarily
attain strikingly orange bellies, often while performing repeated sigmoid
displays [U. Berger (2002), Diplomarbeit im Fach Biologie,
Westfälische-Willhelms-Universität, Münster, Germany].
Early in the breeding season there is a shortage of mature females, and
males court females actively (Forsgren et
al., 2004). However, the sex roles are dynamic, and later in the
season mature females increase in proportion, leading to a drastic increase in
female courtship as well as femalefemale agonism
(Forsgren et al., 2004
). The
belly coloration of female G. flavescens may therefore function as a
ready-to-spawn signal or an ornament, or both. Little is known, however, about
the relative contributions and possible interaction of gonad and skin
pigmentation to the female nuptial coloration. Likewise, no studies have been
performed on chromatophore regulation in this species.
In the present study, we tested the hypothesis that skin chromatophores interact with egg coloration in determining the externally visible belly coloration of female G. flavescens. Specifically, we investigated the potential of chromatophores to alter female belly coloration through pigment dispersion and aggregation. By doing so, we reveal whether live females have the potential to modify their belly colour, used in sexual signalling during courtship and competition.
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Materials and methods |
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Chromatophore assays
Preliminary light microscopy observations showed that the chromatophore
pigment dispersed after death. Therefore, rather than using potential
dispersing factors such as MSH (melanocyte stimulating hormone), ACTH
(adrenocorticotropic hormone) or prolactin, killing the fish was used as a
dispersing treatment. Although many factors may have aggregating effects on
the G. flavescens chromatophore pigments, noradrenaline is known to
be one of the most general regulators of melanophores and erythrophores in
fish, by binding to 2-adrenoceptors
(Fujii and Oshima, 1994
).
Noradrenaline was therefore used to aggregate the chromatophore pigment. These
two treatments were chosen to approach maximal and minimal contributions of
skin chromatophores to coloration and thus estimate the potential for colour
modification by chromatophores. For details regarding chromatophore
regulation, e.g. at the second messenger and the cytoskeleton level, see Fuji
and Oshima (1994), Nilsson Sköld et al.
(2002
) and Aspengren et al.
(2003
).
Experiment 1: whole fish
To investigate belly coloration, 10 G. flavescens females were
photographed three times: (1) when the fish were alive, (2) 60 min after the
fish were killed by decapitation, when the chromatophore pigment had fully
dispersed, and (3) after exposing the dead fish to 10 µmol
l1 noradrenaline for another 60 min to allow for pigment
aggregation.
Fish were placed in a 7x4x2 cm aquarium containing either seawater (live and unexposed dead treatments) or 10 µmol l1 noradrenaline (noradrenaline-exposed treatment). Considerable attention was given to the standardization of photographic conditions. In a dark room, a Canon D30 digital camera with a Canon 50 mm f/2.5 EF Compact Macro lens (Canon Norge AS, Oslo, Norway) and a Senz stereo macro flash (Photax AB, Nybro, Sweden) were used to take an image of each side of every fish (Fig. 1A). Exposure time (1/60), aperture (f8) and flash power settings were kept constant for all photographs.
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Since no chromatophores were observed on the gonads or on the gonad epithelium, noradrenaline was not expected to affect the coloration of gonads themselves. To test this assumption, a control experiment was performed. All gonads were placed in Petri dishes containing 9 psu water, and a photograph was taken. The gonads were then placed in Petri dishes containing 10 µmol l1 noradrenaline for 60 min, and a second photograph was taken. The Petri dishes were photographed placed on a white background, and one image was taken of each gonad from directly above using the same camera, lens and flash. Exposure time (1/60), aperture (f32) and flash power settings were kept constant for all photographs.
The close-up photographs of skin biopsies shown in Fig. 2A were taken with a Canon D30 digital camera mounted on a Wild M3Z stereo dissecting microscope (Leica, Stockholm, Sweden).
Image analysis
All image analyses were performed using Adobe Photoshop 4.0 (Adobe Systems
Inc., Mountain View, CA, USA). The digital images were converted to CIE
L*a*b*, a colour space recommended by the Commission International de
l'Eclairage (CIE). CIE L*a*b* consists of three parameters: the L* value
(lightness) gives the relative lightness ranging from total black to total
white, the a* value (`redness') represents the balance between red and green,
and the b* value (`yellowness') represents the balance between yellow and
blue. In contrast to, for example, RGB colour space, CIE L*a*b* is a
standardized, perceptually uniform and device-independent colour space
(Chen et al., 2004). It has
been frequently used in fish colour quantifications, especially in connection
with carotenoid-based colorations (e.g.
Skrede and Storebakken, 1986
;
Smith et al., 1992
;
Hatlen et al., 1998
;
Craig and Foote, 2001
).
Colour systems based on human vision might be inappropriate when studying
colour signals in animals with unknown visual pigments, for instance if
animals are sensitive to UV light
(Bleiweiss, 2004). The results
of Amundsen and Forsgren (2001
)
show that G. flavescens females scored as `colourful' by human
observers were preferred by males over females scored as `drab'. Thus, there
seems to be an overall agreement between human vision and fish vision in this
species, at least in the yellowred part of the spectrum. In addition,
retinal absorbance data suggest that G. flavescens has tristimulus
vision similar to humans and lacks UV receptors (A. C. Utne-Palm, unpublished
data). Therefore, using CIE L*a*b* values from digital photographs is likely
to be an appropriate method for colour quantification in our case.
For the photographs of whole fish, the belly area was selected with the lasso tool of Photoshop. This was defined as the roughly elliptic area between the anal pore, the pectoral fin base and the blue spots below the lateral line (Fig. 1A). The mean values of lightness, redness and yellowness of the selected area were measured using the histogram tool. The average values from the two photographs of each fish were used in the analyses.
For the photographs of abdominal skin biopsies, only skin located directly lateral of the gonads was measured, i.e. the area below the lateral line and above an imagined line between the pelvic girdle and the anal pore (Fig. 2A). Lines were drawn on the images using landmarks to ensure that the same area was selected in both photographs, and the selection was done using the lasso tool. To remove any differences in light intensity between photographs, the L* channel was normalized by setting the opaque object as black (L*=0) and the background as white (L*=255), an operation that does not affect the values of redness and yellowness. The average values of lightness, redness and yellowness in the selected area were measured using the histogram tool. Since these photographs were taken on a light table where all light permeated through the skin, the L* of the skin biopsy was directly related to its transparency. As the concept of skin transparency is relevant to the understanding of this colour signal, we converted L* to a measurement of transparency. This was done by calculating the percentage of L* in the selected area relative to the L* of the background (transparency=100xL*/255).
From the pictures of gonads, the gonads were selected using the lasso tool and the average values for lightness, redness and yellowness in the selected area were measured using the histogram tool.
Reagents
Stock solutions of noradrenaline (Sigma Aldrich, St Louis, MO, USA) were
stored at 20°C and diluted to the experimental concentrations in
phosphate-buffered saline (PBS; 136.9 mmol l1 NaCl, 2.7 mmol
l1 KCl, 1.5 mmol l1
KH2PO4 and 8.0 mmol l1
Na2HPO4 at pH 7.4) just before use.
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Results |
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There was a considerable range in belly coloration among individual females (live fish: L*=120155, a*=138151, b*=155171). Noradrenaline exposure significantly increased the lightness (L*) compared with live fish (Tukey HSD, N=10, P=0.031) but not compared with untreated dead fish (Tukey HSD, N=10, P=0.26) (Fig. 1B). There was no significant difference in L* between live and untreated dead fish (Tukey HSD, N=10, P=0.49).
Redness (a*) did not differ between live and untreated dead fish (Tukey HSD, N=10, P=0.82), but was significantly reduced after noradrenaline exposure compared with live fish (Tukey HSD, N=10, P=0.003) and untreated dead fish (Tukey HSD, N=10, P=0.001) (Fig. 1B).
Yellowness (b*) increased significantly after death (Tukey HSD, N=10, P=0.001) and decreased significantly after noradrenaline exposure compared with untreated dead fish (Tukey HSD, N=10, P<0.0001) (Fig. 1B). However, there was no significant difference between live and noradrenaline-exposed fish (Tukey HSD, N=10, P=0.081). In conclusion, the results show that noradrenaline induced pigment aggregation, which caused increased lightness and reduced coloration of the bellies. This confirms a role of chromatophores in female belly coloration.
Abdominal skin and gonad coloration
Observations of the skin indicated the presence of melanophores
(black-brownish pigment), erythrophores (red pigment) and xanthophores (yellow
pigment) on a whitish background colour. All observed females had highly
transparent abdominal skin and largely lacked the silvery peritoneum that in
many fishes surrounds the abdominal cavity. On the part of the skin directly
overlying the gonads, there were mainly erythrophores and xanthophores present
(Fig. 2A). Gonads and skin
biopsies were subject to two treatments only: untreated and noradrenaline
exposed. Therefore, gonad coloration and abdominal skin coloration and
transparency were analysed with pairwise t-tests. Percentages were
arcsine square root transformed prior to analyses.
Aggregation of chromatophore pigments following noradrenaline exposure caused the transparency to increase from 69% to 74% (transparency, paired t-test N=10, t=5.02, P<0.001) while decreasing redness (a*, paired t-test N=10, t=5.74, P<0.001) and yellowness (b*, paired t-test N=10, t=15.62, P<0.0001) (Fig. 2B). Compared with erythrophores and xanthophores, melanophores appeared to respond slower to noradrenaline, but this was not further investigated.
In an experiment aimed to verify methodology, it was confirmed that the observed changes in coloration and transparency were indeed an effect of noradrenaline, rather than postmortem paling (data not shown). Even after several hours, unexposed tissue was much darker and more colourful compared with tissue exposed to noradrenaline.
Exposing excised gonads to noradrenaline did not affect any of the three CIE L*a*b* colour parameters (pairwise t-tests, N=10, all P>0.34). No coloration or chromatophores were observed on the gonad epithelium, which indicates that the gonad coloration originates solely from pigments deposited in the eggs.
Relationships between belly colour, abdominal skin colour and gonad colour
Regression analyses were performed to analyse the relationship between
gonad coloration and belly coloration at different levels of chromatophore
pigment aggregation. Since gonads were not affected by noradrenaline exposure,
only data from unexposed gonads were used in the regressions. Redness (a*) was
chosen for these analyses, since this parameter produced the best correlation
between belly and gonad colour. Redness also produces the best correlations
between gonad colour and egg carotenoid concentration (T. Amundsen, J. D.
Blount, C. Pélabon and P. A. Svensson, unpublished). There was a
significant relationship between gonad redness and belly redness, regardless
of whether the fish were alive, dead or treated with noradrenaline (live fish,
N=10, r2=0.80, F=31.45,
P<0.001; untreated dead fish, N=10,
r2=0.75, F=23.98, P=0.001; noradrenaline
exposed fish, N=10, r2=0.49, F=7.67,
P=0.024) (Fig. 3).
However, after noradrenaline exposure (when chromatophore pigment was
aggregated), gonad redness explained less of the variation in belly redness
(49%) compared with untreated dead fish and live fish where pigment was more
dispersed (75% and 80%, respectively). To investigate whether the estimated
slopes differed between regressions, a* was defined as the difference
in belly redness between two treatments. If the slope of
a* plotted
against gonad redness was significantly different from zero, this would
correspond to a difference in slope between those two treatments. The slope
from the regression using fish exposed to noradrenaline was significantly
lower than the slope in the regression using live fish (N=10,
r2=0.64, F=14.24, P=0.005). This result
shows that the belly coloration is formed by the additive effects of skin
chromatophore pigment and gonad coloration. The regression slope using
untreated dead fish did not differ from the slope using live fish
(N=10, r2=0.08, F=0.71, P=0.42)
or using fish exposed to noradrenaline (N=10,
r2=0.26, F=2.39, P=0.13). Thus,
chromatophore pigment aggregation caused belly colour to reveal gonad colour
less efficiently, both by reducing the r2 (increasing the
variation) and by significantly reducing the slope of the relationship
(Fig. 3).
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Discussion |
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Although death caused pigment to disperse compared with live fish, the effects of death on coloration were equivocal in that yellowness but not lightness nor redness increased significantly. This weak response may be due to black/brown and, in particular, red pigment being almost fully dispersed also in live fish.
Observations of courtship suggest that female G. flavescens regulate chromatophores in specific parts of the body to modify the coloration of, for example, the belly area [U. Berger (2002), Diplomarbeit im Fach Biologie, Westfälische-Willhelms-Universität, Münster, Germany]. It is inherently difficult to study such ephemeral patterns in a quantifiable manner, since constraining the fish is necessary for accurate colour measurements, while females confined in too small volumes will not court. Instead, death was used as a treatment to obtain full pigment dispersion. Because death seemed to affect all types of chromatophores in all parts of the fish, it is unlikely that the `death treatment' mimics the colour increase observed during courtship [U. Berger (2002), Diplomarbeit im Fach Biologie, Westfälische-Willhelms-Universität, Münster, Germany], and the effects of death should be interpreted cautiously.
Colouration of abdominal skin biopsies and gonads
There are two interesting characteristics of the abdominal skin in female
G. flavescens. The first is its transparency, which seems to function
as an `abdominal window' (cf. Baird,
1988) that allows direct assessment of gonad coloration. The
second characteristic is the presence of chromatophore pigments (mainly red
and yellow; Fig. 2A), which may
be viewed more as a traditional secondary sexual character.
The transparency of the abdominal skin biopsies was high and increased
after noradrenaline had caused chromatophore pigment to aggregate. This
corresponds to previous studies where aggregation of all chromatophores made
the skin more transparent (Fujii and
Oshima, 1994). Darwin
(1871
) distinguished between
primary sexual characters, which are the reproductive organs themselves, and
secondary characters such as ornaments. The high degree of transparency of
abdominal skin in our study is interesting in its own right, since a direct
display of the gonad coloration blurs the distinction between primary and
secondary sexual characters. Partially transparent abdominal skin in mature
females has previously been described in some teleost species, for instance
brook stickleback (Culaea inconstans;
McLennan, 1995
) and
straight-tailed razorfish (Xyrichthys matinicensis;
Baird, 1988
). One could argue
that the observed transparency in these relatively small fishes is a mere side
effect of the skin being stretched by large gonads. This seems unlikely for
G. flavescens, however, since all mature females investigated (even
with small gonads) lacked the opaque silvery peritoneum common to most fishes.
In comparison, females of closely related and similarly sized
Pomatoschistus species retain the opaque peritoneum as gonads mature
(P. A. Svensson, personal observation). It is therefore possible that the
transparency of abdominal skin in female G. flavescens has evolved as
a signal or a component of a signal, aiming to attract males by displaying the
colourful gonads (Amundsen and Forsgren,
2001
).
The red and yellow coloration of abdominal skin biopsies decreased after
noradrenaline exposure, confirming that abdominal skin coloration is caused by
chromatophore pigments. Several studies have described teleost species where
females have colourful patches on the skin covering the gonads, e.g. in the
convict cichlid (Cichlasoma nigrofasciatum;
Beeching et al., 1998), the
stream goby (Rhinogobius brunneus;
Takahashi and Kohda, 2004
),
the lagoon goby (Knipowitschia panizzae;
Massironi et al., 2005
) and
the pink belly wrasse (Halichoeres margaritaceus; L. H. LaPlante,
personal communication). Interestingly, G. flavescens females thus
seem to use a combination of two previously described phenomena: a nuptial
signal that involves a transparent, yet highly pigmented, abdomen.
Furthermore, because the skin pigmentation is chromatophore based, it is
adjustable and may be used to temporarily modify the belly colour intensity,
for instance during courtship and agonistic behaviours.
Relationship between belly and gonad coloration
Amundsen and Forsgren (2001)
hypothesized that the male preference for colourful females can be adaptive if
females with colourful bellies have more carotenoids in the eggs and therefore
provide the males with higher quality offspring. Our results show a strong
relationship between belly coloration and gonad coloration. This could not be
explained as a correlation between skin and gonad pigmentation but instead
suggests that belly colour reflects gonad pigmentation directly. If highly
pigmented eggs are of higher quality
(Pettersson and Lignell, 1999
;
Blount et al., 2002
), male
G. flavescens may therefore be able to assess the quality of their
potential offspring directly by observing the female belly coloration.
There was a rather small difference in redness between live and untreated dead fish (Fig. 3), possibly because live fish already had almost fully dispersed red pigment (cf. a* in Fig. 1B). The aggregation effect of noradrenaline was, on the other hand, considerable. Although most of the 10 females produced the weakest belly colour when pigment was aggregated, the effect of aggregation varied. Interestingly, the strongest effect of pigment aggregation occurred on the most colourful females (Fig. 3). This implies that colourful females have a larger potential of modifying their appearance, and possibly attractiveness, through the use of chromatophores.
Surprisingly, the positive relationship between belly colour and gonad
colour was weakened when noradrenaline caused skin pigment to aggregate. So,
despite the fact that less pigment was `obstructing the view' of the gonads
and despite increased transparency, pigment aggregation caused belly
coloration to be a poorer predictor of gonad coloration. This pattern could be
interpreted as abdominal skin chromatophores acting as amplifiers of gonad
coloration (sensu Hasson,
1989), i.e. the skin pigment allows males to more accurately
estimate gonad coloration (which may be a more costly and therefore more
honest trait). In comparison, Berglund
(2000
) suggested that in female
pipefish, an ephemeral barred pattern functions as an amplifier by aiding the
males in correctly assessing female size (which is a female trait preferred by
males). On the other hand, skin and gonad coloration of female G.
flavescens may be viewed as two components of the same signal, which
together determine a female's attractiveness
(Candolin, 2003
). A third
alternative is that skin and gonad pigmentation convey different types of
information. For example, the gradual increase in belly coloration as gonads
mature could signal a general readiness to spawn, while the quick change
caused by chromatophores could signal interest in a specific mate.
When interpreting the somewhat counterintuitive effect of pigment
aggregation, it is important to recall that the treatments affected pigment in
the entire fish and not only the quantified belly area. Furthermore, females
may differ in the degree of pigment dispersion when photographed alive and may
respond to noradrenaline to different extents. The methodological limitations
inherent in this study thus call for future more-detailed investigations of
the role and the regulation of abdominal skin chromatophores in the female
nuptial signal of G. flavescens, especially during natural
behaviours. An interesting question is whether females with colourful eggs use
chromatophores differently in, for instance, courtship compared with females
with paler eggs. Kodric-Brown
(1998) proposed that ephemeral
colour changes can be used in both mate attraction and intrasexual conflict
and should therefore be subjected to sexual selection. She also suggested that
such colour changes can be combined with behavioural displays to increase the
efficacy of a given signal. Since all of these aspects seem to be present in
G. flavescens, it is an excellent model system for further
investigations, both in the field of pigment cell physiology and in
behavioural ecology. There is clearly a need for studies illuminating the
relationships between female coloration, carotenoid based egg pigmentation and
female quality.
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Acknowledgments |
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