Evolutionary Ecology Research, 2013, 15: 143–153

Divergence in thyroid hormone concentrations
between juveniles of marine and stream ecotypes of
the threespine stickleback (Gasterosteus aculeatus)
Jun Kitano1,2 and Sean C. Lema3
1

Ecological Genetics Laboratory, National Institute of Genetics, Mishima, Shizuoka, Japan,
2
PRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan and3
Center for Coastal Marine Science and Biological Sciences Department,
California Polytechnic State University, San Luis Obispo, California, USA

ABSTRACT
Background: Hormones regulate the expression of multiple phenotypic traits. Therefore,
divergence in hormone concentrations may lead to evolutionary changes in the coordinatedphysiological and behavioural traits that comprise an organism’s integrated phenotype. Adults
of marine ecotypes of threespine stickleback (Gasterosteus aculeatus) have higher concentrations of the thyroid hormone thyroxine (T4 ) than adults of stream-resident ecotypes (Kitano
et al., 2010). Thyroid hormones are well-established mediators of osmoregulation and migratory
behaviours in fish, and the difference in T4 concentrations indicates that changes in thyroid
hormone signalling may underlie the evolutionary and ecological divergence of migratory andnon-migratory ecotypes.
Questions: Is the variation in T4 concentrations present in earlier life stages where it
could contribute developmentally to differences in phenotype? Do T4 concentrations change inmarine ecotypes before seaward migration?
Organisms: A parapatric pair of marine and stream ecotypes of threespine stickleback
collected from British Columbia, Canada, and a marine ecotype collected from Washington
State, USA.
Methods: We collected juvenile fish of both marine and stream ecotypes on the same day in asingle river to compare the whole body concentrations of T4 using radioimmunoassay. We also
sampled juvenile fish of the marine ecotype in another river at three different times to determine
whether these fish exhibit temporal changes in T4 concentrations before seaward migration.
Results: Juvenile stickleback of the marine ecotype had higher T4 concentrations thanthe parapatric stream-resident juveniles. The T4 concentrations in another marine population
varied slightly across sampling times before seaward migration.
Conclusions: T4 concentrations differ consistently between marine and stream ecotypes
in both juvenile and adult life stages consonant with the hypothesis of evolutionary changes inthyroid signalling.
Keywords: endocrine, phenotypic correlation, physiology, stickleback, trade-offs.

Correspondence: J. Kitano, Ecological Genetics Laboratory, National Institute of Genetics, Yata 1111, Mishima,
Shizuoka 411–8540, Japan. E-mail: jkitano@lab.nig.ac.jp
Consult the copyright statement on the inside front cover for non-commercial copying policies.
© 2013 Jun Kitano

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INTRODUCTION
Adaptation to contrasting environments is often accompanied by divergence in multiple
phenotypic traits (Lande and Arnold, 1983; Schluter, 2000). When selection favours multiple phenotypic traits in combination and a certain permutation of traits conveys a fitness advantage,
genetically based trait correlations can evolve resulting in an adaptive, integrated phenotype(Pigliucci, 2003). Alternatively, phenotypic correlations may exist as a non-adaptive consequenceof inherent genetic and developmental constraints (Pigliucci, 2003). Given such opposing
mechanisms, it has been proposed that phenotypic correlations can either facilitate adaptive
evolution to divergent environments or constrain independent evolution of different phenotypic traits (Lande, 1979; Schluter, 2000; Agrawal and Stinchcombe, 2009; Kirkpatrick, 2009). Understanding
the distinction between these two possibilities, therefore, requires not only investigationof the multiple phenotypic traits that have evolved in divergent environments, but also
identification of the genetic and physiological mechanisms that underlie correlations
between those traits, and how those mechanisms may create evolutionary trade-offs for
populations occupying dissimilar environments.
Since animal migration involves many different phenotypic attributes, including morphological, physiological, and behavioural characters (Dingle, 1996), evolutionary divergence
between migratory and non-migratory ecotypes requires divergence in a suite of phenotypic
traits. Migratory and non-migratory ecotypes can evolve within a single species, andexamples of such ecotypes are known across diverse taxa (McKeown, 1984; McDowall, 1988; Berthold,
1993; Hendry et al., 2004; Roff and Fairbairn, 2007). Previous theoretical and ecological studies
have demonstrated trade-offs associated with animal migration. Migration not only has
advantages such as optimal foraging, avoidance of adverse environments, and selection of
better spawning sites, but also has disadvantages such as high energetic costs and increased
predation (McKeown, 1984; Dodson, 1997; Hendry et al., 2004). The optimal trade-offs between
these advantages and disadvantages may vary in different environments, which can lead todiversity in migratory behaviours.
In fish and other vertebrates, hormones have been shown to coordinate the expression of
migration-related characters as organisms prepare for and undertake migration. Hormones
act on a variety of target tissues to regulate the expression of multiple phenotypic traits atthe organismal level and, therefore, have been proposed to be key mediators of phenotypic
integration (Finch and Rose, 1995; Ketterson and Nolan, 1999; Flatt et al., 2005; Zera et al., 2007; McGlothlin
and Ketterson, 2008; Ketterson et al., 2009). Thyroid hormones mediate many physiological andbehavioural functions related to fish migration, including locomotor activity (Castonguay andCyr, 1998; Katzman and Cech, 2001; Edeline et al., 2005; Kitano et al., 2010), rheotactic behaviour (Edeline et al.,2005; Imbert et al., 2008), swimming speed (Katz and Katz, 1978), metabolic rate (Kitano et al., 2010), salinity
preference (Baggerman, 1957), osmoregulation (Gutz, 1970), reproduction (Costadinos et al., 1994; Swapna
and Senthilkumaran, 2007), growth (Power et al., 2001), olfactory imprinting (Nevitt and Dittman, 1998; Lema and
Nevitt, 2004), and silvering (Ura et al., 1994). Therefore, studying the evolutionary changes inhormone signalling that underlie phenotypic divergence between migratory and nonmigratory ecotypes may not only inform how hormones mediate the morphological, physiological, and behavioural changes necessary for migration (McKeown, 1984; McDowall, 1988; Roff andFairbairn, 2007), but also provide new insights into the genetic changes underlying the evolutionof hormonally mediated trait correlations under dissimilar environmental conditions.
The threespine stickleback (Gasterosteus aculeatus) provides a tractable model system forelucidating the genetic mechanisms of variation in migratory behaviour (Kitano et al., 2012).

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Tremendous diversification of threespine stickleback has occurred during the last few
million years and resulted in the evolution of multiple phenotypically and ecologicallydivergent ecotypes, including sea-run migratory and non-migratory ecotypes (Wootton, 1976,
1984; Bell and Foster, 1994). Even within sea-run migratory ecotypes, some breed in estuaries, whileothers breed in fresh water (see Kitano et al., 2012 and references therein). In this paper, we call bothmarine ecotypes. Marine and stream-resident ecotypes have diverged in many morphological, physiological, and behavioural traits. For example, the marine ecotypes have higher
tolerance of seawater (Heuts, 1946, 1947; Gutz, 1970), higher metabolic rate (Tudorache et al., 2007; Kitano
et al., 2010; Dalziel et al., 2012a), and higher swimming endurance (Taylor and McPhail, 1986; Dalziel et al.,2012b) than stream ecotypes. Interestingly, hormonal manipulations have demonstrated thatthyroid hormones improve performance on most of these physiological and behavioural
measures of migratory ability (Gutz, 1970; Kitano et al., 2010), suggesting that thyroid hormones
may play a key role in mediating the phenotypic changes necessary for migration in sticklebacks, as these hormones do in other teleost fish taxa (McCormick et al., 1998). Previous studies
provide evidence to support this idea, as adult marine ecotypes have higher plasma concentrations of the thyroid hormone thyroxine (T4 ) than adult stream ecotypes (Kitano et al.,2010). These differences in thyroid hormone concentrations appear to have a genetic basis,
as laboratory-raised fish continue to show the same ecotypic differences in plasma T4
concentrations (Kitano et al., 2010).
Currently, however, we do not know whether the variation in T4 concentrations between
marine and stream ecotypes of threespine stickleback is also present in earlier life stages
where it could contribute developmentally to differences in phenotype. Thyroid hormone
signalling pathways can regulate skeletal development in animals (Power et al., 2001; Abe et al., 2003;
Waung et al., 2012), and developmental variation in thyroid hormones has been shown in teleostfishes to result in differences in body morphology, fin growth, and behaviour (Brown, 1997;
Power et al., 2001; Lema and Nevitt, 2006; Shkil et al., 2012). In this study, we collected juvenile fish of aparapatric pair of marine and stream ecotypes on the same day to compare the whole bodytitres of T4 using radioimmunoassay.
We also do not know whether T4 concentrations change in fish of the marine ecotype
before seaward migration. If expression of the traits important for migration has somecosts, titres of these hormones may increase only before and during migration. In
salmonids, for instance, plasma T4 concentrations increase only before and/or during
downstream migration (Dickhoff et al., 1978; Grau et al., 1981; Youngston and Simpson, 1984). To determine
whether juvenile marine stickleback exhibit temporal changes in T4 concentrations before
seaward migration, we sampled juvenile fish of the marine ecotype in another river at threedifferent time points and analysed temporal changes in T4 concentrations.
MATERIALS AND METHODS
Juvenile fish of threespine stickleback were collected from the estuary (n = 18) and upstream(approximately 7 km from the sea) (n = 14) of the Little Campbell River in British
Columbia in Canada on 20 August 2006, and from the estuary of Duwamish River in
Washington State, USA on 1 August (n = 12), 17 August (n = 19), and 11 September 2006
(n = 8) (Fig. 1). No stickleback were caught after early September in the Little Campbell
estuary (Hagen, 1967) and no stickleback were caught after 20 September in the Duwamish
River estuary (J. Kitano, unpublished observation), so all fish in these sampling sites are likely marine
ecotypes with seasonal migration. In addition, previous studies demonstrated that all adults

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Fig. 1. Map showing the collection sites in the Little Campbell River and the Duwamish River. Solidcircles indicate the sampling sites of the marine ecotype, while the open circle indicates the collection
site of the stream ecotype.

caught in the Little Campbell estuary and the majority of adults caught at the Duwamish
estuary were completely plated (Hagen, 1967; Kitano et al., 2008): armour plate morph is often wellcorrelated with ecotypes in threespine stickleback (Bell and Foster, 1994). In contrast, we consider
that all fish caught 7 km upstream of the Little Campbell River were stream-resident
ecotypes, because all fish caught at this site were low plated and several falls exist between
the estuary and the sampling site (Hagen, 1967; Kitano et al., 2008). Fish were collected under
the permits WA06-159 and NA/SUS06-21454. The fish were caught with a hand-net,
euthanized with lethal doses of MS222, frozen immediately on dry ice, and stored at −70⬚Cuntil use.
Whole-body concentrations of total T4 were measured as described previously (Lema and
Nevitt, 2006). Fish were homogenized in ice-cold 100% ethanol (0.9 mL) containing 1 m
5-propyl-2-thiouracil (PTU) to block endogenous deiodinase activity, and the homogenate
was spiked with 10 µL of I125-labelled T4 (1240 counts per minute; Perkin-Elmer, Waltham,
MA, USA) to permit subsequent calculation of extraction efficiency. After sonication for
20 s followed by centrifugation at 3000 g for 20 min at 4⬚C, the supernatant was removed
and saved for T4 quantification. The tissue pellet was resuspended in 0.3 mL of ice-coldStickleback ecotypes differ in thyroid hormone concentration

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ethanol with PTU (1 m) by vortexing and sonication for 20 s, and centrifuged (3000 g for
20 min at 4⬚C). The resulting supernatant was then combined with the previously saved
supernatant. After centrifugation at 64 g for 5 min at 4⬚C, the supernatant was vacuum
dried at 37⬚C under N2 gas, and then reconstituted in 100 µL of ice-cold phosphate bufferedsaline (0.1 m, pH 7.4) containing 1 m PTU. For 20 µL volumes of each sample, T4
concentrations were then quantified in duplicate using radioimmunoassay as described
previously (Dickhoff et al., 1982). Samples were incubated for 2 h at 37⬚C in 0.11  sodium
barbital buffer with anti--T4 antiserum (1:5000; Accurate Chemical & Scientific Corp.,
Westbury, NY, USA) and I125-labelled T4 (Perkin-Elmer, Waltham, MA, USA). Sodium
barbital buffer containing 20% polyethylene glycol was then added to each sample, andsamples were centrifuged at 1400 g for 20 min at 4⬚C. The supernatants were removed toseparate free and bound hormone, and the remaining pellets were assayed for radioactivity(Cobra II gamma counter, Packard, Downer’s Grove, IL, USA). The intra-assay coefficientof variation was 11.3%. The T4 extraction efficiency from whole-body tissues was
69.04 ± 0.01% (mean ± ..), and the resulting T4 value for each sample was corrected using
the respective extraction efficiency for that same sample. Standards of 1.25 to 60 ng · mL−1
were used to define the sensitivity of the T4 assay. All data were normal log (Ln)transformed before statistical analysis.
RESULTSIn the Little Campbell River, juvenile fish of marine and stream ecotypes collected on
20 August 2006 did not differ in body weight (ANOVA, F1,30 = 2.5, P = 0.124; body weightof the marine ecotype = 0.67 ± 0.02 g; body weight of the stream ecotype = 0.71 ± 0.02 g;
mean ± ..). However, the whole-body T4 titres were significantly higher in marine juveniles
than in stream juveniles (Fig. 2; ANOVA, F1,30 = 51.2, P < 0.001). Even after inclusion ofbody weight as a covariate, the difference remained significant (ANCOVA, effect of ecotype:F1,28 = 51.7, P < 0.001; effect of body weight: F1,28 = 1.7, P = 0.205; effect of interaction:
F1,28 = 0.6, P = 0.449).
In the Duwamish River, body weight differed significantly between sampling times(Fig. 3; ANOVA, effect of date: F2,36 = 25.7, P < 0.001), which might be due to samplingbias. Therefore, to compare T4 concentrations among the sampling times, body weight was
included as a covariate. T4 concentrations did not differ significantly among the samplingtimes when the interaction term between date and body weight was included (ANCOVA,
effect of date: F2,33 = 0.50, P = 0.613; effect of body weight: F1,33 = 1.5, P = 0.228; effect ofinteraction: F2,33 = 0.3, P = 0.737). After excluding the interaction term, however, wefound significant differences among sampling dates (ANCOVA, effect of date: F2,35 = 4.3,
P = 0.021; effect of body weight: F1,35 = 1.0, P = 0.319).
DISCUSSION
In the present study, we found that pre-migratory juvenile marine stickleback in the Little
Campbell River had higher whole-body T4 concentrations than the juvenile stream-resident
stickleback in that same watershed, suggesting that divergence in thyroid hormone
physiology already exists between marine and stream-resident ecotypes at the juvenilestage before seaward migration. Treatment of adult stickleback with exogenous T4 has beenshown to increase metabolic rate (Kitano et al., 2010), basal swimming activity (Kitano et al., 2010),

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Fig. 2. Relationships between Ln-transformed body weight and Ln-transformed T4 concentrations in
the juvenile fish collected in the Little Campbell River. Solid and open circles indicate the T4 concentrations of marine and stream ecotypes, respectively. Solid and broken lines indicate the regression
lines for marine and stream ecotypes, respectively.

Fig. 3. Relationships between Ln-transformed body weight and Ln-transformed T4 concentrationsin the juvenile fish collected on 1 August (open circles and broken lines), 17 August (grey circles andgrey lines), and 11 September (solid circles and black solid lines) in the Duwamish River.

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seawater tolerance (Gutz, 1970), and seawater preference (Baggerman, 1957), indicating that theelevated thyroid hormone concentrations in juvenile marine stickleback may mediate theexpression of physiological and behavioural traits that increase fitness in marine environments. The presence of higher T4 concentrations in juvenile marine stickleback also points
to a possible role for thyroid hormones as developmental mediators of phenotypic
differences between ecotypes. For example, the marine and stream ecotypes have divergedin body size and body shape (Wootton, 1976, 1984; Bell and Foster, 1994; Rogers et al., 2012). Thyroid
hormones are involved in growth and skeletal development in fishes (Power et al., 2001; Shkil et al.,2012), and variation in developmental thyroid status can result in differences in body shape(Brown, 1997; Lema and Nevitt, 2006). Although our common garden experiments indicatedthat divergence in T4 concentrations between adults of marine and stream ecotypes are
genetically determined (Kitano et al., 2010), we do not know whether the differences between
juveniles of different ecotypes are determined by genetic factors, environmental factors, or a
genotype × environment interaction. Studies exploring the effects of thyroid hormone
treatment on juvenile stickleback and the genetic mechanisms causing the divergencein thyroid hormone concentrations should help to resolve how this divergence in thyroid
hormone signalling links to the divergence in integrated physiological, behavioural, andmorphological phenotype that characterizes the marine and stream ecotypes of this species.
In many fishes, including salmonid species (Dickhoff et al., 1978; Grau et al., 1981; Youngston andSimpson, 1984), migratory populations exhibit a surge in the production of thyroid hormone
concentrations with migration. The thyroid hormone surge in salmonid species plays a keyrole in coordinating the physiological and behavioural changes required for freshwater
individuals to undertake the downstream migration and transition to seawater, and can betriggered by environmental factors such as lunar cycles (Grau et al., 1981), temperature variation(Youson et al., 1994), and dissimilar or changing water currents (Youngston, 1989; Youngston and McLay,
1989). In the present study, we found a trend that the juvenile fish caught earlier in time
were larger in body size and had higher T4 concentrations. Because size thresholds for thedecision to migrate have been found in salmonids (Nicieza et al., 1994), our data may indicatethat larger fish with higher T4 concentrations leave the estuary first, followed by smaller
individuals who delay their migration decision due to either their sub-threshold body size or
sub-threshold T4 concentrations. In addition, some fish caught at the Duwamish estuary on
17 August had similar T4 concentrations to the stream-resident fish of the Little Campbell
River (Figs. 2 and 3), suggesting that some environmental or temporal factors may influence
the T4 concentrations of marine ecotypes. However, we cannot exclude the possibility thatsignificant effects of sampling date on body size and T4 concentrations observed in thisstudy are due to our sampling bias and small sample sizes, so more thorough sampling of
pre-migratory fishes should be conducted as part of future studies.
Peripheral regulation of thyroid hormones may also be important. Diverse functions of
thyroid hormones are mediated by the action of thyroid hormones on thyroid hormone
receptors in a variety of peripheral target tissues (e.g. liver, brain, gills, gonads), and tissue
specificity in thyroid hormone action is determined both by the abundance and type ofreceptors in a given tissue, as well as by tissue-specific expression of converting enzymes(Ishikawa and Kitano, 2012). For example, iodothyronine deiodinase enzyme types 1 and 2 convert
thyroid hormones to more active forms, while iodothyronine deiodinase enzyme type 3
primarily converts thyroid hormones to less active forms (Orozco and Valverde-R, 2005; Orozco et al.,2012). In other teleost fishes, the genes encoding thyroid hormone receptors and deiodinase
enzymes exhibit tissue-specific patterns of transcriptional regulation (Lema et al., 2009; Johnson and150

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Lema, 2011), suggesting that the many physiological, behavioural, and morphological traits
regulated by thyroid hormones could be evolutionarily decoupled by peripheral tissuespecific changes in receptor or deiodinase enzyme expression. In salmonids, the expressionof thyroid hormone receptors changes during preparation for seaward migration (Harada et al.,2008). Although such peripheral regulation has not yet been examined in sticklebacks, themarine ecotype was found to exhibit both elevated type 1 deiodinase mRNA concentrations
and lower concentrations of type 3 deiodinase mRNA abundance in the gills compared withstream-resident fish (Kitano et al., 2010), indicating that ecotypic variation in thyroid hormone
physiology may extend beyond upstream hormone production, and include downstream
differences in the regulatory mechanisms for thyroid hormone action in peripheral tissues.
Given that we now know that thyroid hormone status differs between marine and streamresident ecotypes in both the juvenile and adult life stages, future work examining ecotypicvariation in mechanisms of thyroid hormone action in peripheral target tissues may lend
fundamental insights into how hormonally mediated traits are coupled and decoupled as
integrated phenotypes evolve.
In conclusion, we found that migratory and non-migratory ecotypes within a species candiffer in the levels of systemic hormone titres. Because hormones regulate the expressionof many morphological, physiological, and behavioural traits, hormones can mediate thetrade-offs between migratory and non-migratory ecotypes. For a better understanding of
the genetic and physiological mechanisms of adaptive evolution of multiple traits, it willbe essential to know whether such hormone-mediated phenotypic integration can either
facilitate or constrain adaptive evolution of divergent life-history ecotypes.

ACKNOWLEDGEMENTS
We thank the Semiahnoo First Nations for permission to collect fish in the Little Campbell River,Katie Peichel and Penny Swanson for the use of facilities and discussion, and Dolph Schluter forassistance with fish collection. Andrew Hendry, Katie Peichel, and an anonymous reviewer madeconstructive comments on the manuscript. This research is supported by the JST PRESTO programand Grant-in-Aid for Scientific Research on Innovative Areas (23113007 and 23113001) from the
Ministry of Education, Science, Sports, and Culture.

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