Bulletin of the National Salmon
Resources Center, No. 2: 15-20
December 1999

Seawater Tolerance of Lacustrine Sockeye Salmon(Oncorhynchus nerka) from Lake Toya
Masatoshi Ban*', Hiroyuki Haruna**, and Hiroshi Ueda*'
Research Division, National Salmon Resources Center, Fisheries Agency of Japan,
2-2 Nukanoshima, Toyohira-ku, Sapporo 062-0922, Japan(dukeban@ salmon.affrc.go.jp)*' Toya Lake Station for Environmental Biology, Faculty of Fisheries, Hokkaido University,
122 Tsukiuru, Abuta-cho, Abutu-gun, 049-5723, Japan*I

Abstract.- External changes and seawater tolerance related to smoltification of yearling lacustrine
sockeye salmon (Oncorhynchus nerka) originating from Lake Toya (LSS) were investigated monthly from March to July 1993. Ocean-run sockeye salmon (OSS) originating from the Bibi River were
examined for comparison. As an external index of smolting, dorsal and caudal fin margin blackening was observed in more than 80% of LSS and OSS during May and June, while it was below 55%
during other months. The serum sodium concentration of LSS and OSS 24 h after transfer to seawater decreased below 165 mEqL from April to June and from April to May, respectively. The gill
Na', K-ATPase activity of both groups in fresh water showed a rapid increase from March andreached the maximum level of about 18 pmols Pi/mg proh in May, followed by a gradual decrease.
These results indicate that yearling LSS smolt, like OSS, are maximally seawater tolerant in May.
Key words : sockeye salmon, seawater tolerance, smoltification

Introduction
Smoltification in salmonids is a very complex
phenomenon that includes morphological, physiological, and behavioral changes (Hoar 1976). External
body silvering and fin margin blackening are themost dramatic characteristics used to distinguishsmolts from parr (Kubo 1980; Gorbman et al. 1982;
Yamauchi et al. 1984). The major physiologicalprocess of smoltifying salmonids is the increase in
the gill Na', K-ATPase activity and gill chloride cellnumber concurrent with increased seawater tolerance
preceding life in seawater (McCormick et al. 1987).
Salmonid smolts decrease aggressive behavior andtend to form aggregates or schools. There have also
been observations that migrating smolts will actively
swim downstream to reach seawater (Solomon1978). Most salmonids smolt in the natal river orlake prior to seaward migration.
Sockeye salmon (Oncorhynchus nerka) exhibit agreater variety of life history patterns than chum (0.
Contribution A No. 12 from the National Salmon
Resources Center.
0 1999 National Salmon Resources Centerketa) or pink salmon (0.gorbuscha). The sockeye
salmon is typically anadromous and usually migrates
to the ocean after smolting. The kokanee is a formof the sockeye salmon that remains in fresh water tomature and reproduce. They show the seawater tolerance cycle typical of sockeye salmon, despite nothaving experienced an anadromous life history for asmuch as 10,000years (Foote et al. 1994). In Japan,
lacustrine sockeye salmon occur, but not native
anadromous sockeye salmon. Lacustrine sockeye
salmon living in Lake Toya (LSS), which are a very
important fishery resource of this lake, have reproduced for many generations without oceanic migration. They were probably planted approximately 100
years ago into Lake Toya from Lake M a n , however,there are no detailed records on the origin of LSS.
The life cycle of LSS is similar to that of kokanee,
however, there have been few investigations about
their physiological condition, especially in relation tosmoltification. Recently, releases of LSS into rivers
have been performed to produce a new fisheryresource of anadromous sockeye salmon in Japan. In
this study, changes in seawater tolerance related tosmoltification of yearling LSS were examined andcompared with ocean-run sockeye salmon.

16

2 If . 3 ?&%@B+

Materials and Methods
Fish sampling
Yearling LSS and ocean-run sockeye salmon(OSS) from Lake Toya and Bibi River, respectively,
were used for this experiment. LSS were hatched
and reared at Toya Lake Station for Environmental
Biology, Faculty of Fisheries. Hokkaido University.
OSS were hatched and reared at the Chitose Branchof the National Salmon Resources Center. In June
1992, 1,000 individuals of LSS from stock the group
and OSS transferred from the Chitose Branch wereplaced in several fiberglass tanks (1 ton capacity) at
the Toya Lake Station as underyearlings. Mean fork
length of LSS and OSS in June was 55.8 mm and40.1 mm, respectively. Both groups were fed commercial pellet at a ration of 3% body weight per day.
The photoperiod was natural and the temperature of
supplying well water was 9- 10°C throughout theexperiment. Samplings were conducted monthly
from March to July, 1993.
Biological measurements and collection of gill jilaments
At each sampling time, fork length (FL, mm),body weight (BW, g) and gonad weight (GW, g)
were measured for 30 fish in fresh water, after anesthetization by ethyl 3-aminobenzoate methanesulfonic acid salt (Acros Co., 100 mg/L). Gill filaments
were dissected from 10 of the 30 fish mentioned
above, rinsed in ice-cold homogenizing buffer (250mM sucrose, 6 mM EDTA 2Na, 20 mM imidazole,p H 6.8) and kept frozen in buffer (20 times
weight/volume) at -40°C for the analysis of Na+,KATPase activity.
External smolt characters
External smolt characters were estimated visually, based on the appearance of distinct blackening on
the dorsal and caudal fin margins, for the 30 fishused for biological measurement. A smolt ratio (%)
was calculated as the proportion of externally smolted fish among 30 fish.
Seawater tolerance
To estimate seawater tolerance, 20 fish weretransferred directly into artificial seawater (salinity
33%, 10.1 f l.0"C) for 24 h and serum sodium concentration was measured in survivors. For thisassay, serum was separated from blood samples
obtained from the caudal vessels using a micro test
tube with centrifugation at 2,000 g for 15 min andwas stored at -40°C. After dilution of the thawed

2 9 -68% Bull. Natl. Salmon Resources Center, No. 2 , 1999

serum with distilled water (1 : lOOO), serum sodium
concentration was measured using an Atomic
Absorption and Flame Emission Spectrometer (Shimadzu, AA-640-13).
The Na+,K+-ATPaseactivity of the gill filaments
was determined using the method of Folmar andDickhoff (1979), modified by Ban and Yamauchi(1991). The enzyme activity was expressed as
amount of inorganic phosphate transformed (Pi) per
milligram protein per hour (pmols Pi/mg proh).
Precocious males
A gonad somatic index was calculated as GSI =lOOXGW/BW. Males which showed a GSI over
0.I % were classified as precocious males; immature
males showed GSI under 0.1% throughout the experiment. Data for precocious males were removed
from the mean serum sodium concentration and gill
Na+,K+-ATPase activity analysis.
Statistical analysis
The data presented were expressed as mean+
SEM. The data were subjected to one-way analysis
of variance (ANOVA) followed by Student t-test or
Cochran-cox test to determine significant differences
between LSS and OSS, and monthly changes withineach group. A probability level of less than 0.05 wasconsidered significant in all tests.

Results
Mean fork length of LSS and OSS increased
from 114.8 mrn and 105.3 mm in March to 158.0
mm and 144.2 mm in July, respectively (Fig. 1).
The fin margin blackening in both groups began in
April and the smolt ratio was greater than 80% in
May and June (Fig. 2). Most unsmolted fish during
this period were precocious males. The rate of occurrence of precocious males in LSS was 20% in May
and 10% in June, and in OSS it was 6.7% in bothmonths. Mean fork lengths of the precocious males
and immature males of LSS were 153.8 mm and
140.8 mm in May and 161.7 mm and 154.1 mm inJune, respectively. In the case of OSS, mean fork
lengths of precocious and immature males were 137
mm and 129.6 mm in May and 145.3 mm and 138.6
mm in June, respectively. A significant difference(p<0.05) in mean fork length was observed only
between precocious and immature males of LSS in
May. In July, the smolt ratio of LSS and OSS
reduced to 55% and lo%, respectively, due to fadingof fin margin blackening.
All fish transferred to seawater survived for 24 h.
Serum sodium concentrations of LSS and OSS 24 hBan et al. - Smoltification of lacustrine sockeye salmon from Lake Toya

17

100,
1

7

0

1

160n

150U

5 140w-5 130-g 120LL

110100I

Fig. 1. Changes in mean fork length of yearling LSS (closed
circles) and OSS (open circles). The vertical bars show
standard errors. Some standard errors are hidden by sym-

I

I

I

Mar. Apr. May Jun. Jul.
Month

Mar. Apr. May Jun. Jul.
Month

Fig. 2. Changes in smolt ratio of yearling LSS (closed circles)
and OSS (open circles).

bols.

T
200 -

190180-

170-

160-

-.-

L
Q)

150

Mar. Apr. May Jun. Jul.
Month

a

0

1

Mar. Apr. May Jun. Jul.
Month

Fig. 3. Changes in mean serum sodium concentration 24h
after transfer to seawater of yearling LSS (closed circles)
and OSS (open circles). The vertical bars show standard
errors. Some standard errors are hidden by symbols.
Asterisks above point show significant difference(**p<O.OOl) between LSS and OSS.

Fig. 4. Changes in mean gill Na',K-ATPase activity of yearling LSS (closed circles) and OSS (open circles). The
vertical bars show standard errors. Some standard errors
are hidden by symbols. An asterisk above point shows
significant difference (*p<O.Ol) between LSS and OSS.

after transfer to seawater in March were 175.8
mEq/L and 191.7 mEq/L, respectively (Fig. 3). Bothgroups decreased significantly (p<O.OOl) below
165.0 rnEq/L in April. LSS maintained this low level until June, followed by a significant increase(p<O.OOl) to 188.3 mEq/L in July. The serum sodi-

um concentration of OSS was elevated significantly(p<O.OOl) after June, averaging 201.9 mEq/L in July.
The difference in serum sodium concentration
between the groups in June w a s significant(p<O.OOl). The gill Na', K-ATPase activity of bothgroups increased from about 4.0 pmols Pi/mg pro/h

218. 3 $@'iRgB+2 9 -@f@ Bull. Natl. Salmon Resources Center, No. 2, 1999

in March to about 18.0pmols Pi/mg pro/h in May(Fig. 4). The enzyme activity of LSS and OSS was
reduced to 13.9pmols Pi/mg pro/h and 8.7,umols
Pi/mg pro/h in July, respectively. The difference inenzyme activity between LSS and OSS in July wassignificant (p<O.Ol).

Discussion
Yearling ocean-run sockeye salmon display distinct pigmentation on the dorsal and caudal fin margin (Ban and Yamauchi 1991). In this study, morethan 80% of LSS showed clear fin margin blackening from May to June and the degree of this coloration was similar to that of OSS. This result
demonstrates that LSS externally smolted during
May and June. However, the smolt ratio of LSS during this period was slightly lower than that of OSS as
a result of a difference in appearance rate of precocious males. Larger males usually mature earlier
than smaller ones, and this sexual maturation inhibits
smoltification (Saunders et al. 1982; Aida et al.
1984; Foote et al. 1991). We also found that precocious males were larger than immature males, andthat precocious males did not smolt. Therefore, wesuggest that high growth rate of juveniles may
obstruct smoltification of male LSS.
Clarke and Blackbum (1978) suggested that the
optimal serum sodium concentration of smolts after a
24 h transfer to seawater was actually in the range of150-160 mEq/L. To regulate serum sodium concentration of fish in seawater, the gill Na', K-ATPasesystem is involved in excretion of sodium ionsdirectly from the blood to the external environment(Kamiya 1972). Some salmonids increase their gill
Na', K-ATPase activity during smoltification (Banand Yamauchi 1991; Ewing and Rodgers 1998;
Pirhonen and Forsman 1998). In the present study,
LSS transferred to seawater showed serum sodium
concentrations below 165 mEq/L from April to June.
Furthermore, the gill Na', K-ATPase activity of LSS
increased rapidly from March and peaked in May,
like OSS. These results indicate that LSS maintains
the capacity to develop seawater tolerance in thespring. The serum sodium excretion ability in thegills of LSS was highest in May. This capacity has
been preserved for many generations with or without
an oceanic migration.
Johnston and Eales (1970) found that, althoughtemperature is a key factor for external changes during smoltification in Atlantic salmon (Sulmo sulur),its effect is highly dependent on the size of fish considered. Smolting occurs sooner at higher temperatures, and changing temperature seems to be morestimulatory than constant temperature to Atlantic
salmon and steelhead trout (0. mykiss) (Wagner
1974; Jonsson and Ruud-Hansen 1985). In the present study, LSS showed dramatic changes in fin margin blackening and seawater tolerance, despite theirhaving been reared under stable food conditions andambient temperature from the underyearling stage.
Thus, it is likely that the trigger for smoltification in
LSS was stimulated by environmental factors otherthan water temperature or growth pattern. Photoperiod may be particularly important as a regulating
factor (McCormick et al. 1987; Bjornsson et al.
1998; Duncan and Bromage 1998).
If smolts are retained in fresh water past the normal downstream migration period, external characters and seawater tolerance usually decrease (Zaugg
and McLain 1970; Lasserre et al. 1978). Thisdecrease has been termed desmolting. Stefansson etal. (1998) reported that temperature significantly
affects desmoltification. In this study, LSS and OSS
demonstrated fading in fin margin blackening anddecreased seawater tolerance, despite they werereared under stable water temperature conditions.
This indicates that desmoltification in LSS is not initiated by changing water temperatures. Mortensen
and Damsgard (1998) suggested that Atlantic salmon
kept at salinities of 15%0or higher could maintain ahigh and stable hypoosmoregulatory capacity. A
moderate influx of sodium ion into the body may benecessary to keep seawater tolerance at a high level.
Thus, desmoltification seems to be a fresh water
adaptation process occurring in smolts whose seaward migration is delayed or prevented.
The magnitude of desmoltification in LSS wasless than OSS in the present study. These more gentle reversions may be a feature of the lacustrine stock
which is adapted to a more stable environment (lake)
than the pelagic ocean. Unfortunately, we have done
this comparison only in one year. To ascertain if thisobservation is significant, further investigations are
required.
In conclusion, yearling LSS originating from
Lake Toya smolt and develop maximum seawater
tolerance in May. This capacity to smolt may be an
inherited character unrelated to their lack of experience of ocean life

Acknowledgements
We wish to express our appreciation to the staffof the Chitose Branch, National Salmon Resources
Center for their effort supplying the experimental
fish. Thanks also to Dr. Jill B. K. Leonard for revision of this manuscript.

Ban et al. - Smoltification of lacustrine sockeye salmon from Lake Toya

References
Aida, K., T. Kato, and M. Awaji. 1984. Effects ofcastration on the smoltification of precocious male
m a w salmon Oncorhynchus masou. Bull. Jap.
SOC.Sci. Fish., 50: 565-571.
Ban, M., and K. Yamauchi. 1991. Seasonal changes
in seawater adaptability of the hatchery reared
juvenile sockeye salmon, Oncorhynchus nerka.
Sci. Rep. Hokkaido Salmon Hatchery, 45: 25-33.
Bjornsson, B. T., G. V. Stefansson,
I. Berge, T.
Hansen, and S. 0. Stefansson. 1998. Circulating
growth hormone levels in Atlantic salmon smolts
following seawater transfer: effects of photoperiod
regime, salinity, duration of exposure and season.
Aquaculture, 168: 121- 137.
Clarke. W. C., and J. Blackburn. 1978. Seawater
challenge tests performed on hatchery stocks ofchinook and coho salmon in 1977. Can. Fish. Mar.
Sew. Tech. Rep., 761: 19 p.
Duncan, N., and N. Bromage. 1998. The effect ofdifferent periods of constant short days on smoltification in juvenile Atlantic salmon (Salmo salar).
Aquaculture, 168: 369-386.
Ewing, R. D., and J. D. Rodgers. 1998. Changes inphysiological indices of smolting during seaward
migration of wild coho salmon, Oncorhynchus
kisutch. Aquaculture, 168: 69-83.
Folmar L. C., and W. W. Dickhoff. 1979. Plasma
thyroxine and gill Na+-K+ATPase changes during
seawater acclimation of coho salmon, Oncorhynchus kisutch. Comp. Biochem. Physiol., 63A: 329332.
Foote, C. J., W. C. Clarke, and J. Blackburn. 1991.
Inhibition of smolting in precocious male chinook
salmon, Oncorhynchus tshawytscha. Can. J. Zool.,69: 1848-1852.
Foote, C. J., I. Mayer, C. C. Wood, W. C. Clarke,
and J. Blackburn. 1994. On the developmental
pathway to nonanadromy in sockeye salmon,
Oncorhynchus nerka. Can. J. Zool., 72: 397-405.
Gorbman, A., W. W. Dickhoff, J. L. Mighell, E. F.
Prentice, and F. W. Waknitz. 1982. Morphologicalindices of developmental progress in the parrsmolt coho salmon, Oncorhynchus kisutch. Aquaculture, 28: 1-19.

A.

Hoar, W. S. 1976. Smolt transformation: evolution,
behavior, and physiology. J. Fish. Res. Board
Can., 33: 1233-1252.
Johnston, C. E., and J. G. Eales. 1970. Influence ofbody size on silvering of Atlantic salmon (Salrno
salar) at parr-smolt transformation. J. Fish. Res.
Board Can., 27: 983-987.

19

Jonsson, B., and J. Ruud-Hansen. 1985. Water temperature as the primary influence on timing of seaward migrations of Atlantic salmon (Salrno salar)smolts. Can. J. Fish. Aquatic. Sci., 42: 593-595.
Kamiya, M. 1972. Hormonal effect on Na-K-ATPase
activity in the gill of Japanese eel, Anguilla japonica, with special reference to seawater adaptation.
Endocrinol. Jpn., 19: 489-493.
Kubo, T. 1980. Studies on the life history of "Masu"
salmon (Oncorhynchus masou) in Hokkaido. Sci.
Rep. Hokkaido Salmon Hatchery, 34: 1-95. (InJapanese with English summary.)
Lasserre, P., G. Boeuf, and Y. Harache. 1978.
Osmotic adaptation of Oncorhynchus kisutch Walbaum. l: Seasonal variations of gill Na+-K'
ATPase activity in coho salmon, 0-age and yearling, reared in freshwater. Aquaculture, 14: 365382.
McCormick, S. D., R. L. Saunders, E. B. Henderson,
and P. R. Harmon. 1987. Photoperiod control ofparr-smolt transformation in Atlantic salmon(Salrno salar): changes in salinity tolerance, gill
Na+,K+-ATPaseactivity, and plasma thyrjoid hormones. Can. J. Fish. Aquatic. Sci., 44: 1462-1468.
Mortensen, A., and B. Damsgird. 1998. The effectof salinity on desmoltification in Atlantic salmon.
Aquaculture, 168: 407-41 1.
Pirhonen, J., and L. Forsman. 1998. Relationshipbetween Na+,K+-ATPaseactivity and migration
behaviour of brown trout and sea trout (Salrno
trutta L.) during the smolting period. Aquaculture,
168: 41-47.
Saunders, R. L., E. B. Henderson, and B. D. Glebe.
1982. Precocious sexual maturation and smoltification in male Atlantic salmon (Salmo salar).
Aquaculture, 28: 21 1-229.
Solomon, D. J. 1978. Some observations on salmon
smolt migration in a chalk stream. J. Fish. Biol.,
12: 571-574.
Stefansson, S. O., p\. I. Berge, and G. S. Gunnarsson.
1998. Changes in seawater tolerance and gill
Na+,K+-ATPaseactivity during desmoltification in
Atlantic salmon kept in freshwater at different
temperatures. Aquaculture, 168: 27 1-277.
Wagner, H. H. 1974. Photoperiod and temperature
regulation of smolting in steelhead trout (Salrno
gairdneri). Can. J. Zool., 52: 219-234.
Yamauchi, K., N. Koide, S. Adachi, and Y. Nagahama. 1984. Changes in seawater adaptability andblood thyroxine concentrations during smoltification of the masu salmon, Oncorhynchus masou,
and amago salmon, Oncorhynchus rhodurus.
Aquaculture, 42: 256-274.
Zaugg, W. S., and L. R. McLain. 1970. Adenosinet-

20

此sCenter,No

さけ・ます資源管理センター
所報 Bul1.Nat1.SalmonResour

riphosphatase activity fn gills Of salmonids: sea‑sonal variations and salt water influence fn coho

salmon, Oncorhynchus kisutch.COmp. Biochem.
PhysioL, 35: 587‑596.

． 2， 1999

照群 として， 美々 州産の降海理べ二ザケを 用いた．
外見的変化として ，背鰭と 尾鰭先端のつま 黒の状態を観察した結果， 5 月から 6 月にかけて両群の 80%以上の個体に 発現が認められた．海水適応能を 調べるために，海水移行 24 時間後の血清ナトリウム
洞爺湖産 べ二ヴケの海水適応能
伴 真俊
洞爺湖産 べ二ザケ(Na) 濃度と ，鯛のNa ・， K ‑ATPaSe 活性を調べた ．
ヒメマスと降悔型 べ二ザケの Na 濃度は，各々 4‑6
月および 4‑5 月にかけて低値を 示し 鯛 の酵素活性・・

春名寛幸 ・上田 宏(ヒメマス ) 1 年魚を飼育し ，スモルト化にともなう 外見的変化と 海水適応能の変化を， 3 月より 7 月までの間毎月 I 回調べた ．対は両 群 とも 5 月に最高値に 達した． これらの結果から， 洞爺湖産ヒメ マス @ 年魚は降海理べ二ザケと 同様に スモ ルト化すること ，その際，海水適応能は 5
月に最も高まることが 分かった ．