Evolutionary Ecology Research, 2016, 17: 525–534

Comparison of freshwater tolerance during spawningmigration between two sympatric Japanese marine
threespine stickleback species
Asano Ishikawa1, Makoto Kusakabe2, Manabu Kume1 and Jun Kitano1
1

Division of Ecological Genetics, National Institute of Genetics, Shizuoka, Japan and2
Atmosphere and Ocean Research Institute, University of Tokyo, Chiba, Japan

ABSTRACT
Background: The colonization of empty niches can trigger phenotypic diversification. For
example, the colonization of newly formed freshwater environments by marine ancestors led tophenotypic diversification in threespine sticklebacks. However, not all lineages of threespine
stickleback have taken advantage of these ecological opportunities; all Japanese freshwater
populations are derived from the Pacific Ocean lineage rather than the Japan Sea lineage ofGasterosteus.
Hypothesis: Marine ancestors of these two lineages differed in their ability to survive infreshwater environments. Japan Sea and Pacific Ocean marine sticklebacks may differ in freshwater tolerance and transcript levels of the prolactin (PRL) gene, which encodes a hormone
important for freshwater osmoregulation.
Methods: We collected Japan Sea and Pacific Ocean marine sticklebacks migrating upstream
near the mouth of a brackish water lake in early May and challenged them with fresh water. We
then compared the survival rates, plasma sodium concentrations, and PRL expression levels of
the two species.
Results: When challenged with fresh water, Japan Sea fish showed significantly higher death
rates and a trend towards a greater reduction in plasma sodium concentration than thesympatric Pacific Ocean fish. Levels of PRL were consistently higher in the Pacific Ocean fish
both before and after the freshwater challenge.
Keywords: hormones, osmoregulation, physiology, prolactin, stickleback.

INTRODUCTION
An ecological opportunity can trigger phenotypic diversification (Glor, 2010; Losos, 2010; Yoder
et al., 2010). The colonization of new habitats, appearance of new resources, or extinction ofcompetitors can allow a lineage to adapt to a broad range of newly available ecologicalniches, leading to rapid speciation and/or adaptive diversification (Losos, 2010; Yoder et al., 2010).

Correspondence: J. Kitano, Division of Ecological Genetics, National Institute of Genetics, Yata 1111, Mishima,
Shizuoka 411–8540, Japan. email: jkitano@lab.nig.ac.jp
Consult the copyright statement on the inside front cover for non-commercial copying policies.
© 2016 Jun Kitano

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However, not all lineages have taken advantage of these ecological opportunities. Many
lineages fail to diversify even in the presence of ecological opportunities. For example,
Darwin’s finches and Hawaiian honeycreepers have acquired tremendous phenotypic diversity after their colonization of oceanic islands, while Galapagos mockingbirds andHawaiian thrushes have not (Losos, 2010).
Several potential factors can prevent some taxa from utilizing ecological opportunities(Glor, 2010; Losos, 2010). Early colonizers may preclude further colonization by later colonizers;
thus, extrinsic factors, such as differences in geographical accessibility, may explain variation in the exploitation of new niches. Alternatively, some taxa may lack intrinsic genetic
and physiological abilities to colonize and utilize new niches, or may be less capable in thisregard than others. Therefore, identification of the intrinsic constraints on exploitationof new empty niches is important to understand and predict patterns of evolutionary
diversification in nature.
Repeated glacial cycles provided a number of novel empty niches in freshwater environments during the Quaternary period. Multiple marine ancestors of cnidarians, annelids,
molluscs, arthropods, and teleosts have colonized these newly formed freshwater environments, resulting in rapid evolutionary diversification of these taxa (Schluter and Rambaut, 1996;
Lee and Bell, 1999). Threespine sticklebacks (Gasterosteus aculeatus) provide a notable example
of freshwater colonization followed by phenotypic diversification (Wootton, 1976, 1984; Bell andFoster, 1994). After the last glacial retreats, ancestral marine sticklebacks have colonized andadapted to a variety of newly formed freshwater environments throughout the northernhemisphere. Freshwater sticklebacks showed tremendous diversification of morphological,
physiological, and behavioural traits to adapt to diverse freshwater habitats (Bell and Foster, 1994;
McKinnon and Rundle, 2002; Kitano et al., 2012). Because of the presence of extant marine ancestors, we
can compare derived freshwater populations with their marine ancestors to investigate theecological and genetic mechanisms underlying adaptation to freshwater environments, suchas reduction of armour, trophic morphology, body shape, and hormonal signalling (Bell andFoster, 1994; Schluter, 1994; Colosimo et al., 2005; Chan et al., 2010; Kitano et al., 2010; Wark et al., 2012; Cleves et al., 2014;
O’Brown et al., 2015).

However, not all stickleback lineages have colonized newly formed freshwater environments. In Japan, there are two genetically distinct marine Gasterosteus species: the Japan
Sea sticklebacks (Gasterosteus nipponicus), which are distributed mainly in the Sea of Japanand Sea of Okhotsk (Higuchi and Goto, 1996), and Pacific Ocean populations of G. aculeatus(hereafter referred to as Pacific Ocean sticklebacks), which are distributed along the Pacificcoast (Kitano et al., 2007). These two species diverged about 1.5–2 million years ago when the Sea
of Japan was isolated from the Pacific Ocean by sea-level changes (Kitano et al., 2007). Previous
phylogenetic analysis demonstrated that all Japanese freshwater populations, including
recently introduced populations, are derived from Pacific Ocean sticklebacks, not fromJapan Sea sticklebacks (Higuchi and Goto, 1996; Cassidy et al., 2013; Ravinet et al., 2014). Because there aremany freshwater lakes and rivers near the Sea of Japan as well as the Pacific Ocean, theJapan Sea sticklebacks may have lower intrinsic ability to exploit freshwater niches than the
Pacific Ocean sticklebacks.
In eastern Hokkaido, Japan, the distribution of Japan Sea sticklebacks overlaps with thatof the Pacific Ocean sticklebacks (Higuchi and Goto, 1996; Kitano et al., 2007). In sympatric conditions,these two sticklebacks are reproductively isolated by multiple barriers, including spatial
isolation of spawning grounds (Kitano et al., 2009). Most Pacific Ocean sticklebacks migrate to
upstream freshwater regions for spawning, while a large portion of Japan Sea fish remain inFreshwater tolerance of Japanese sticklebacks

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brackish water for breeding (Kume et al., 2005, 2010). This is consistent with the idea that theJapan Sea sticklebacks may have lower intrinsic ability to exploit freshwater niches than
the Pacific Ocean sticklebacks.
Environmental differences between marine and freshwater habitats include differencesin osmotic pressure and ionic concentration (Lee and Bell, 1999; Willmer et al., 2005; Hill et al., 2008).
Because the ionic concentration of fresh water is low, freshwater teleosts need to counteract
the passive gain of water and loss of ions by several physiological mechanisms, such as the
production of dilute urine and the active intake of ions across the gill epithelium (McCormick,2001). We therefore hypothesized that the Japan Sea sticklebacks possess a lower osmoregulation ability in low-salinity environments than do Pacific Ocean sticklebacks, resulting indiffering abilities to colonize freshwater environments.
Osmoregulatory adaptation is generally regulated by the neuroendocrine system(McCormick, 2001). In a number of euryhaline teleosts, prolactin (PRL), a multifunctional peptide hormone, plays a central role in osmoregulation in low-salinity environments (Manzon,
2002; Sakamoto and McCormick, 2006). Prolactin has been demonstrated to increase plasma ion
concentrations and decrease the permeability of osmoregulatory organs, such as gills,
kidneys, intestines, the urinary bladder, and skin. After freshwater exposure, PRL gene
expression, synthesis, secretion, and plasma levels increased (Manzon, 2002); treatment with
PRL was also shown to decrease the ion and water permeability of osmoregulatory surfaces(Manzon, 2002). Injection of PRL markedly increased the survival rate of threespine sticklebacks when transferred from salt to fresh water (Lam and Leatherland, 1969; Leatherland and Lam, 1969),
changed their kidney structure (Wendelaar Bonga, 1976), and increased their preference for low
salinities (Audet et al., 1985). Thus, we hypothesized that different PRL levels may underlie thediffering abilities of Japan Sea and Pacific Ocean sticklebacks to exploit freshwater habitats.
To test whether these two marine sticklebacks differ in osmoregulation ability, we
first conducted freshwater challenge experiments in which we observed survival rate andmeasured changes in plasma sodium concentrations. Next, to compare the patterns of PRLlevels between the two species, we quantified the expression of the PRL gene in the two
stickleback species both before and after freshwater exposure.
MATERIALS AND METHODSFish sampling and freshwater challenge experimentThreespine sticklebacks of the Japanese sympatric pair, Pacific Ocean fish (G. aculeatus)
and Japan Sea fish (G. nipponicus), were collected with seine nets in Akkeshi Bay,
Hokkaido, Japan on 11 May 2012. Details of the sampling are described elsewhere (Kumeet al., 2005). Adult fish were collected on the same day and from the same location to minimize
potential environmental effects: mean (± SD) standard length was 86.57 ± 4.00 mm and66.72 ± 3.51 mm for the Pacific Ocean and Japan Sea sticklebacks, respectively. To compare
freshwater tolerance of Pacific Ocean and Japan Sea fish, we conducted a freshwater challenge experiment immediately after collection. Pacific Ocean and Japan Sea fish (N = 15 and13, respectively) were exposed to fresh water for up to 24 hours. Fresh water used in theexperiment was obtained from groundwater at the Akkeshi Waterfowl Observation Center(salinity = 0.01%). As controls, some Pacific Ocean and Japan Sea fish (N = 15 and 13,
respectively) were maintained in the water of Akkeshi Bay (salinity = 1.40–1.44%) fromwhich they were caught. Four or five fish of each species were held in a bucket containing

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approximately 6 litres of fresh water or brackish water (three buckets for each experimentalgroup). Except salinity, all buckets were treated in the same way: they were maintained atambient temperature, which ranged from 0.4 to 8.0⬚C during the experiment. We counted
the number of survivors 24 hours after freshwater transfer. Fisher’s exact test was used totest the difference in the number of survivors between species using R v.3.1.0. We alsocollected plasma and whole brain from the survivors. For sampling, fish were killed using an
overdose of buffered MS222 (300 mg · L−1). Blood samples were collected from the caudal
veins using heparinized capillary tubes (Fisher Scientific, Waltham, MA, USA), as
described previously (Kitano et al., 2010, 2011). After centrifugation for 10 minutes at 2000 g, theplasma was frozen and transported to the laboratory at −20⬚C, and stored at −80⬚C until
use. Each whole brain was immersed in RNAlater solution (Ambion, Austin, TX, USA)overnight at 4⬚C, before being stored at −80⬚C until RNA extraction.
Plasma sodium measurement
Plasma samples were thawed on ice, and 0.5-µL sub-samples were used for the measurementof Na+ concentration using an atomic absorption spectrophotometer (Z-5300 Hitachi,
Tokyo, Japan), as described previously (Kusakabe et al., 2014). Na+ measurements were performedin duplicate when possible. Sufficient plasma for duplicate measurements could not be
obtained from three Japan Sea fish experiencing the freshwater challenge and one Japan Sea
fish from the control experiment. For statistical analysis, a two-way ANOVA was conductedwith species, salinity, and their interaction as explanatory variables using R v.3.0.2(R Development Core Team, 2013).
Quantification of PRL gene expression
To assess whether expression of the PRL gene differed between the Pacific Ocean andJapan Sea fish, we analysed PRL expression levels using the quantitative real-time PCR
method (qPCR). Total RNA was extracted from whole-brain samples with the AllPrep
DNA/RNA Micro Kit (Qiagen, Valencia, CA, USA) (N = 12, 7, 4, and 12 for Pacific Ocean
and Japan Sea fish in the freshwater challenge, and Pacific Ocean and Japan Sea fish in
the control group, respectively). One microgram of total RNA was treated with DNase I(Invitrogen, Waltham, MA, USA) to eliminate genomic DNA contamination, and wasthen used for cDNA synthesis using High-Capacity cDNA Reverse Transcription Kits
(Applied Biosystems, Foster City, CA, USA). As an endogenous control, we conducted
qPCR of a housekeeping gene encoding L13A ribosomal binding protein, which shows
stable expression in the context of immune system function (Hibbeler et al., 2008). Our analysisof potential reference genes encoding beta-actin, eukaryotic elongation factor I, L13A
ribosomal binding protein, and ubiquitin, also showed that transcript levels of the L13A
ribosomal binding protein gene was the most stable in the brain among different salinity
conditions in the North American sticklebacks (A. Ishikawa, unpublished data). Therefore, thegene encoding L13A ribosomal binding protein was used as a reference gene to quantify
relative expression levels of the PRL gene. Primers of PRL were designed with Primer
Express 3.0 Software (Applied Biosystems). Primer sequences used were as follows:
PRL forward primer, 5⬘-TTTGTCCACCTCTGCCAACTT; PRL reverse primer, 5⬘-TCT
TGTTGGATATGGCGTTTTG; L13A forward primer, 5⬘-CACCTTGGTCAACTTGA-Freshwater tolerance of Japanese sticklebacks

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ACAGTG; L13A reverse primer, 5⬘-TCCCTCCGCCCTACGAC. The Fast SYBR Green
Master Mix (Applied Biosystems) was used for qPCR reaction, and qPCR was run on
StepOnePlus™ (Applied Biosystems). Relative expression levels were calculated fromstandard curves drawn from serially diluted cDNA pools of all analysed fish. We analysed
expression levels of the PRL gene by a two-way ANOVA with species, salinity, and theirinteraction as explanatory variables. Correlations between expression levels of the PRL
gene and plasma sodium levels were tested by Spearman’s rank correlation test using R
v.3.0.2 (R Development Core Team, 2013).
RESULTSJapan Sea fish showed higher death rates in the freshwater challenge experiments thanPacific Ocean fish. Specifically, 7 of 13 Japan Sea fish died during the freshwater challenge,
while only 1 of 15 Pacific Ocean fish died (Fisher’s exact test: P = 0.011). All fish survived in
the control experiments (N = 15 and 13 for the Pacific Ocean and Japan Sea fish,
respectively).
Freshwater exposure significantly reduced the plasma sodium level of both sticklebacks(two-way ANOVA, effect of salinity: F = 168.323, P < 0.001; effect of species: F = 0.002,
P = 0.965) (Fig. 1). Japan Sea fish showed a trend towards a greater reduction in plasma
sodium concentration than did Pacific Ocean fish. However, the difference was not
significant (two-way ANOVA, interaction between species and salinity: F = 2.341,
P = 0.133) (Fig. 1).
PRL levels were consistently higher in Pacific Ocean fish than Japan Sea fish regardless of
salinity (two-way ANOVA, effect of species: F = 20.267, P < 0.001) and tended to increase
in response to the freshwater challenge (two-way ANOVA, effect of salinity: F = 3.145,
P = 0.089) (Fig. 2). In freshwater challenge experiments, PRL levels were positively

Fig. 1. Box plots of plasma sodium concentration in control and freshwater-challenged fish. Whiteand black boxes indicate the Japan Sea and the Pacific Ocean sticklebacks, respectively.

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Fig. 2. Box plots of relative expression levels of PRL in the brains of control and freshwaterchallenged fish. White and black boxes indicate the Japan Sea and the Pacific Ocean sticklebacks,
respectively.

Fig. 3. Relationship between plasma sodium concentration and relative expression levels of PRL. The
grey band indicates 95% confidence intervals around the fitted line. White and black circles indicate
the Japan Sea and Pacific Ocean sticklebacks, respectively.

correlated with plasma sodium levels when data from both species were pooled andanalysed together (Spearman’s rank correlation test: ρ = 0.74, P = 0.008). The same trend
was observed even when data were analysed separately for each species (Spearman’s rankcorrelation test: ρ = 0.71 and 0.9, P = 0.081 and 0.083, for Pacific Ocean and Japan Sea fish,
respectively) (Fig. 3).

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DISCUSSION
In the present study, when challenged with fresh water, Japan Sea sticklebacks showedsignificantly higher death rates and a trend towards a greater reduction in plasma sodium
concentration compared with sympatric Pacific Ocean sticklebacks. This suggests thatJapan Sea sticklebacks have lower freshwater tolerance than Pacific Ocean sticklebacks,
which may partially explain why there are no freshwater populations derived from the Japan
Sea lineage.
Furthermore, we found significantly higher expression levels of the PRL gene in Pacific
Ocean sticklebacks than in Japan Sea sticklebacks. Although PRL expression levels
did increase in response to freshwater transfer in both species, expression levels wereconsistently higher in Pacific Ocean fish than in Japan Sea fish. Prolactin is generally
important for freshwater osmoregulation in euryhaline fishes (Manzon, 2002; Sakamoto and
McCormick, 2006). Injection of ovine PRL was reported to reduce freshwater mortality of a
Canadian marine threespine stickleback population (Lam and Leatherland, 1969). Furthermore,
our present study revealed that PRL expression levels were correlated with plasma sodium
levels. Taken together, lower PRL expression levels may be one of the physiologicalmechanisms underlying the lower freshwater tolerance in the Japan Sea fish than in the
Pacific Ocean fish during spawning migration.
Previous studies of both marine and freshwater ecotypes of G. aculeatus indicated that
PRL activity shows seasonal variation associated with migration between salt and fresh
water. Specifically, it decreases in early-winter fish (around the time of sea-run migration)and becomes maximally active in spring fish (around the time of spawning migration) (Lamand Hoar, 1967; Leatherland, 1969; Benjamin, 1974). If similar seasonal changes in PRL levels occur inJapan Sea sticklebacks, Japan Sea sticklebacks may show a much greater reduction in PRL
expression levels and lower freshwater tolerance in other seasons such as early winter, which
may function as an additional constraint for freshwater colonization.
Hormones generally perform multiple functions, and hormonal divergence can hence
lead to changes in multiple traits (Ketterson and Nolan, 1999; Kitano et al., 2014). Prolactin is amultifunctional hormone and plays important roles not only in freshwater adaptation, butalso in freshwater preference behaviour (Audet et al., 1985). Therefore, divergent PRL levels may
contribute to the divergent choice of breeding habitats – relative upper reaches for Pacific
Ocean fish and lower reaches for Japan Sea fish – possibly leading to spatial isolation
between the two species (Kume et al., 2005, 2010). Prolactin is also known to be involved inpaternal behaviours in G. aculeatus (Pall et al., 2004), although differences in paternal
behaviours between the Japan Sea and Pacific Ocean sticklebacks have not yet been
investigated. Further studies on PRL levels across multiple developmental stages andtheir roles in multiple functions will help to clarify the contribution of PRL levels as aconstraint for freshwater colonization, reproductive isolation, and other possible
behavioural differences between Japan Sea and Pacific Ocean sticklebacks.
In the present study, we only analysed freshwater tolerances of adult fish migrating tospawning grounds. Other developmental stages were not analysed. Furthermore, freshwater
challenge experiments were limited to transfer from brackish water to fresh water, andfreshwater acclimatization was not assessed for other salinity regimes such as gradualsalinity reduction. Future studies should assess acclimatization abilities of these fish at
multiple developmental stages using other salinity regimes. Because the fish analysed inthese experiments were collected from the same location on the same day, environmental

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effects were minimized, suggesting that the observed differences in freshwater tolerance may
have a genetic basis. However, we cannot exclude the possibility of other environmental
factors. For example, early exposure to different salinities is known to influence salinity
tolerance at later developmental stages in Nile tilapia (Watanabe et al., 1985). The spawning
grounds of the Pacific Ocean fish are generally lower in salinity than those of the Japan
Sea fish (Kume et al., 2005, 2010), and Pacific Ocean juvenile fish remain longer in the spawning
sites than Japan Sea juvenile fish (Kitamura et al., 2006; Kume and Mori, 2009). Therefore, different
durations of exposure to low-salinity environments at the juvenile stages could affectsalinity tolerance and PRL expression levels in the adult stages. Testing with laboratoryraised fish will be essential to assess genetic contributions to the differing freshwater
tolerances between the two species. Finally, we tested only one sympatric pair collected from
Akkeshi Bay in the present study. Since some populations of Japan Sea sticklebacks spawn
in freshwater environments (Higuchi et al., 2014), further studies on other Japan Sea populations
are necessary to confirm that low ability to tolerate fresh water is general across the
distribution of Japan Sea sticklebacks.
In conclusion, this study demonstrates that an intrinsic difference may exist in the abilitiesof these two marine sticklebacks to survive in freshwater environments, and that thisdifference may be a result of divergent expression levels of the PRL gene. These differences
may underlie their differing ability to colonize freshwater environments and/or the spatial
isolation between the two lineages. Further molecular and genetic studies on the different
abilities of these two lineages to survive in freshwater environments may suggest why somelineages can colonize and utilize empty niches to achieve adaptive radiation, while others
cannot.
ACKNOWLEDGEMENTS
We thank Kohta Yoshida, Mark Ravinet, and the Baba family of the Akkeshi Fisheries Cooperative
Association for help with fish sampling and Katie Peichel and two anonymous reviewers for helpfulcomments on the manuscript. All animal experiments were approved by the Institutional Animal
Care and Use Committee of the National Institute of Genetics (25-15 and 25-18). This research
was supported by the Japanese Society for the Promotion of Science Research Fellowship for
Young Scientists (PD) to A.I. and JST PRESTO program, Grant-in-Aid for Scientific Research on
Innovative Areas from the Ministry of Education, Science, Sports, and Culture (23113007 and
23113001), Asahi Glass Foundation, Takeda Science Foundation, and the Cooperative Program ofAtmosphere and Ocean Research Institute, The University of Tokyo to J.K.

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