Fisheries science JSFS , tập 77, số 3, 2011 5

Fish Sci (2011) 77:289
DOI 10.1007/s12562-011-0346-7

OBITUARY

In Memoriam: Professor Michizo Suyama (1923–2011)

Ó The Japanese Society of Fisheries Science 2011

Professor Michizo Suyama, an Honorary Member of the
Japanese Society of Fisheries Science and Former Professor of Tokyo University of Fisheries, passed away on
January 19, 2011. He was 87 years old.
Professor Suyama was born in April 1923 in Tokyo. He
graduated from the Imperial Fisheries Institute in 1943 and
became a Research Associate at the Imperial Fisheries
Institute in 1945. In 1949, the National School Establishment
Law created the Tokyo University of Fisheries by incorporating the Imperial Fisheries Institute and the Faculty of
Fisheries. This new institute was placed under the jurisdiction of the Ministry of Agriculture and Forestry. Professor
Suyama remained at the new Tokyo University of Fisheries,
becoming an Associate Professor in 1960 and a Full Professor in 1974. He retired from the university in 1987.

The focus of Professor Suyama’s research activities
was marine food chemistry. His various fields of research
and educational interest included studies on the amino
acid composition of fish protein, extractive components,
taste-active components, and smells of volatile substances.
His contributions to these fields and those on the
improvement and modification of an amino acid analyzer
are excellent and have contributed to developments in
fisheries science.

Professor Suyama also contributed to the Japanese
Society of Fisheries Science, serving first as a member of
the Board of Directors, then as Vice President and finally
President. He received the Japanese Society of Fisheries
Science Award of Merit in the fields of nitrogenous
extractive components from aquatic animals in 1987. His
social contributions as a scientist were invaluable.
In 1999, he received The Order of the Sacred Treasure,
Gold Rays with Neck Ribbon.
Professor Suyama educated and inspired many young
people with his profound knowledge and warm personality.
He was especially fanatical in his desire to improve the
taste and palatability of food.
We offer heartfelt our condolences to the family of
Professor Suyama.
Takaaki Shirai
Associate Professor, Tokyo University of Marine Science
and Technology

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Fish Sci (2011) 77:291–299
DOI 10.1007/s12562-011-0326-y

ORIGINAL ARTICLE

Fisheries

Growth and fatness of 1975–2002 year classes of Japanese sardine

in the Pacific waters around northern Japan
Atsushi Kawabata • Hirotsune Yamaguchi
Seigo Kubota • Masayasu Nakagami

•

Received: 3 March 2010 / Accepted: 23 December 2010 / Published online: 26 February 2011
Ó The Japanese Society of Fisheries Science 2011

Abstract We examined individual growth and fatness in
the 1975–2002 year classes of Japanese sardine. Samples
were collected at the feeding grounds in the Pacific waters
off northern Japan during drastic fluctuations in the population in the 1970s to 2000s. Growth rates for ages 1–3 of
the 1979–1988 year classes, which included low-recruitment year classes subsisting during the high population
levels of the 1980s, were apparently slower than for other
year classes. There was no obvious trend when comparing
year classes, growth during the first year of life (age 0), and
maximum body length (BL) at age C5. The condition
factors (CF, indicating fatness) for adult sardines of BL
C19 cm in the 1979–1983 year classes during the maximum population level of the mid-1980s were significantly
lower than for other year classes. However, there were no
apparent trends in CF variations for small sardines of BL
\19 cm. The apparent decreases in growth rate and fatness
were strongly related to the cumulative sum of population
abundance that each year class experienced. It is thought
that insufficient food owing to the density-dependent effect
of an abundant population at feeding grounds resulted in a

A. Kawabata (&)
National Research Institute of Fisheries Science,

Fisheries Research Agency, Japan, 2-12-4 Fukuura,
Kanazawa, Yokohama, Kanagawa 236-8648, Japan
e-mail:
H. Yamaguchi
Japan Fisheries Information Service Center,
4-5 Toyomi-cho, Chuo-ku, Tokyo 104-0055, Japan
S. Kubota Á M. Nakagami
Tohoku National Fisheries Research Institute,
Fisheries Research Agency, Japan, 25-259 Shimomekurakubo,
Samemachi, Hachinohe, Aomori 031-0841, Japan

decrease in the growth rate for small-bodied sardines that
are investing their energy intake in body growth, and a
decrease in fatness for large-bodied adults that are accumulating fat for the next reproduction.
Keywords Age Á Body length Á Condition factor Á Density
effect Á Growth Á Population Á Sardinops melanostictus

Introduction
Japanese sardines Sardinops melanostictus spawn in the
coastal waters along the south of Japan, near Kuroshio, in
winter–spring, and migrate northward in summer–autumn
to forage in the Pacific waters [1–4]. The population biomass has fluctuated drastically in the past, in synchronization with global climate change, and this fluctuation has
impacted Japanese fisheries and Japanese society [5]. The
Japanese sardine population in the Pacific waters increased
in the 1970s due to the occurrence of comparatively high
recruitment levels in 1973, 1977 and 1978. They became
very abundant in the 1980s due to very high sequential
recruitment from 1980 to 1987 (Fig. 1). These population
increases were thought to be due to favorable environmental conditions for reproduction and an increase in the
number of spawned eggs. The increased number of eggs

was due to the accumulation of spawners from several
generations due to the comparatively long life span of this
species. In the 1980s, over one million tons of sardine were
fished, mainly by purse seiner, in the feeding grounds off
Sanriku and the eastern Hokkaido regions of northern
Japan. However, the population declined through the
1990s, reaching a very low level in the 2000s. The sardine
catch in the Pacific waters off northern Japan decreased to
only 0–4000 tons in the 2000s.

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292

Fish Sci (2011) 77:291–299

Abundance (x10 9 ind.)
600

Catch (x10 6 t)
1.6

500
400
300

1.2

Materials and methods

0.8

From 1975 to 2005, we collected samples of Japanese
sardine during the fishing season from commercial landings
at the Hachinohe Port, an important fishing port in northern
Japan (Fig. 2). These were caught by purse seiner off the
Sanriku coast. The fishing season off the Sanriku coast
varied with sardine population abundance, extending to
10 months (from late April to early February) during the
1980s due to the high population levels at that time, but
shortening to only a few summer months in recent years
due to low population levels. Vessel survey samples taken
by drift net or pelagic trawl were also collected off the
Pacific coast of northern Japan in May–January from 1998
to 2003 in order to supplement the decreasing commercial
catch sample (Fig. 2).
We measured scaled body length [BL (cm)] and determined age in years (t) by the method of counting annual
rings on the scales [16] for a total of 12,161 specimens.
These specimens were assigned to the 1975–2002 year
classes. We measured body weight [BW (g)] and gonad
weight [GW (g)] for a subset of 11,918 specimens. Internal
abdominal fat [FW (g)] was also weighed for a subset of
7,633 specimens. The condition factor (=coefficient of
fatness, CF) was calculated from the obtained biometric
data using the equation CF = 103 (BW – GW)/BL3. Fat
weight index was also calculated (=105 FW/BL3). Having
examined the correlation between BL and CF and also the
seasonal variation of CF, as described later, we used the
mean CF for separate BL ranges of samples during May–

November (BL \19 cm) or May–December (BL C19 cm)
to examine the effect of density.
To estimate the BL at the start of each age, the von
Bertalanffy growth curve was fitted by the method of
least squares to the age and BL data of each year class:
Lt = L? [1 - exp(-K (t - t0))], where Lt is the BL at the
start of age t, L? is the asymptotic BL, K is the rate at
which BL tends toward the asymptote, and t0 is the age
when BL = 0. The beginning of each age, the birthday,
was set to 1st March, as this was midway through the main
spawning season. The age data used to fit the curve was
t ? d/365, where d is the number of days from 1st March
to the sampling date. In fitting the curve, L? was limited to
less than 30 cm, which is the length of the largest sardine
that has been caught [17].
For the catch of Japanese sardines in the Pacific waters
off northern Japan, we referred to the fishery statistics
collected by the Fisheries Research Agency and to previous
reports [18–20], and used them as an index of abundance at

200
0.4
100
0
1970

on variations in body length, growth rate and fatness
among year classes.

0

1975

1980

1985

1990

1995

2000

2005

Year

Fig. 1 Annual total population abundance (all ages, available after
1977, gray and unfilled bars) and recruitment (age 0, gray bars) of
Japanese sardine in Pacific waters (after Wada and Jacobson [6],
Nishida et al. [7]), and catch in the Pacific off the Sanriku and the
eastern Hokkaido areas of northern Japan (closed diamonds and solid
line)

It is known that the individual growth rates of Japanese
sardine in the coastal waters off eastern Hokkaido
decreased in the 1980s compared with the 1970s [8–10]. A
similar phenomenon was observed at the past population
peak in the 1930s [11, 12]. This slow growth was considered to be subject to a density-dependent effect of the
abundant population [8–10]. However, in these studies,
sampling was restricted to the coastal waters of eastern

Hokkaido and so they did not examine differences in
growth rates among year classes. There has not been
enough discussion about whether the growth rate in a year
class is affected by its own year class recruitment abundance or rather by the population abundance (standing
stock of all ages) in Japanese Pacific waters. The growth
rate of sardines recovered after the 1990s, but recent
changes in growth related to population abundance have
not yet been examined in depth.
Variations in fatness relating to both growth and population abundance have also not been examined in detail in
previous studies. It was reported that fatness in the 1980s
year classes was lower than in the 1970s year classes when
comparing same-age fish [10]. However, there is a positive
correlation between fatness and body length, as described
later in this study. The decrease in the fatness of same-age
fish in the 1980s probably reflected shorter body lengths,
rather than fatness per se. The effect of fish density on
fatness should be examined clearly by comparing fish of
the same body size.
In this study, we examine the growth and fatness of
1975–2002 sardine year classes in relation to the drastic
fluctuations in the population abundance of sardines in the
Japanese Pacific waters, based on biometric data collected
over 31 years. We also discuss the density-dependent
effects of recruitment abundance or population abundance

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Fish Sci (2011) 77:291–299
°N

45

°N
50
Oyashio

Hokkaido
40
Kuroshio Extension

Pacific Ocean

Spawning ground

Sanriku

40

Hachinohe Port

Fig. 2 Location of the
sampling area, and schematic
view of the oceanographic
current systems and the
spawning ground of the
Japanese sardine population in
Pacific waters (after Watanabe
[13], Kondo [2], Kuroda [14],
Kobayashi and Kuroda [15]).
Cross and diamond symbols

indicate the locations of purse
seiner sampling (n = 340) and
survey vessel sampling
(n = 126), respectively

293

35
140

the feeding grounds. For the total population abundance
(standing stock of all ages) and the recruitment of each year
class (cohort abundance at age 0), we referred to the estimates gained from the virtual population analysis performed in Wada and Jacobson [6] and Nishida et al. [7].
We examined variations in both the Lt at each age and the
CF for different BLs with respect to population abundance
and year-class recruitment among the 1975–2002 year
classes. To examine the effect of population abundance on
the growth of a year class, we used the cumulative sum of
population abundance for the period from age 0 to the
previous age (t - 1) as an index of the abundance that the
P
year class born in year x experienced: xþtÀ1
Ni ; where Ni
i¼x
is the total population abundance in year i. Population
abundance estimates were available for years after 1977
(Fig. 1), so year classes after 1977 were examined.

Results
Growth

The BLs and ages of the specimens ranged from 6.4 to
24.4 cm and from 0 to 9 years, respectively. Figure 3
shows age and BL observations and the fitted von Bertalanffy growth curves for typical year classes. The number
of observations for each year class depended on the ease of
sampling (i.e., year-class abundance) and ranged from 53
(1990 year class) to 2,332 (1986 year class).

130

145

140

150

30

160°E

150°E

Figure 4 shows the estimated BL of each year class at
the start of each age from 1 to 9, as estimated from the
fitted growth curves. Gaps are due to a lack of observations
for some ages in some year classes. The maximum BL at
age 5 or older was about 21–22 cm for all year classes,
although there was a lack of observations for some year
classes. Estimated BL at age 1 for each year class was
13–16 cm. Small BLs (13–14 cm) at age 1 occurred in
high-recruitment (more than 150 billion individuals, as

occurred in 1980–1987) year classes during high population levels, and in low-recruitment (less than 50 billion
individuals, as occurred in 1979, 1998, 1999 and 2001)
year classes during low population levels (Fig. 1). Also,
large BLs (15–16 cm) at age 1 occurred in both the comparatively high recruitment (94 billion individuals) year
class of 1978 and the low-recruitment year classes in the
1990s–2000s. There was no obvious relation between
estimated BL at age 1 and either recruitment of year classes
or total population abundance.
Estimated BLs at ages 2–4 differed distinctly between
year classes. Estimated BLs of the 1979–1987 year classes
were 15–16 cm at age 2, 17–18 cm at age 3, and 18–19 cm
at age 4 (Fig. 4). These BLs at ages 2–4 were smaller than
those of the 1975–78 year classes by 1–2 cm and smaller
than those of 1989–2002 year classes by 2–3 cm.
Table 1 shows the estimated BLs, the growth rates at
age t (increase in BL (Lt?1 - Lt)/Lt) and the fitted von
Bertalanffy equation parameters for typical year classes living
during different population abundance levels. Growth rates

123


294
Fig. 3 Relationships between
age and body length as well as
fitted von Bertalanffy growth
curves for typical year classes

Fish Sci (2011) 77:291–299

Body length (cm)
25
20
15
10
5

1979

n = 169

n = 1,728

1987

1989

n = 559

0
25
20
15
10
5

1982

n = 210

n = 271

1988

1995

n = 54

0
0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10

Observation

Growth curve

during age 1 in the 1979 and 1982 year classes (i.e., for
high population levels of more than 300 billion individuals)
were apparently slower than those for the 1987–1989 year
classes (i.e., when the population was decreasing) and for
the 1995 year class (i.e., for low population levels of less
than 30 billion individuals) (Fig. 1). However, the estimated BLs at age 1 for the 1979 and 1982 year classes
were not very different to those for other year classes. The
growth rates during the early ages for the year classes
corresponding to high population levels (e.g., 1979 and
1982) were slower than those for year classes corresponding to decreasing or low population levels, as indicated by the comparatively small K parameters.
Furthermore, the estimated BL at age 2 for the
1988 year class was smaller than those for the 1975–1978
and 1989–2002 year classes (Fig. 4; Table 1). The growth
rate during age 1 of the 1988 year class should have been
slow, as it is for the 1979–1987 year classes, although there

was a lack of age-1 BL observations. However, the estimated BLs at ages 3 and 4 of the 1988 year class were
similar to those for the 1975–1978 and 1989–2002 year
classes (Fig. 4; Table 1). The BL of the 1988 year class
improved with age. The 1987 year class showed a similar
improvement in BL with age (Fig. 4; Table 1).
Recruitment in these slow-growing year classes was not
high. Recruitment was high (more than 150 billion individuals) in the 1980–1987 year classes, but was low (less
than 50 billion individuals) in the 1979 and 1988 year
classes (Fig. 1; Table 1). Significant correlations were
found between the recruitment and the estimated BLs at
ages 2–4 for the year classes (P \ 0.01). However, there
were some outliers, such as the 1979 year class (Fig. 5).
The 1979 year class subsisted during the high-level populations of the 1980s.
On the other hand, sardine populations in the Japanese
Pacific waters were at very high levels during 1980–1989

123

Age

Body length: L t (cm)

24
Age t
9
8
7
6
5
4

3
2
1

22
20
18
16
14
12
1975

1980

1985

1990

1995

2000

Year class
Fig. 4 Estimated body length at the beginning of each age for the
1975–2002 year classes

(306–554 billion individuals). The 1979–1986 year classes
were at ages 1–3 at this time, and had small BLs at ages
2–4 (Figs. 1, 4). Figure 6 shows the relationships between
the estimated BL of each year class at ages 1–4 and the

cumulative sum of the population abundance (as explained
in ‘‘Materials and methods’’) in Japanese Pacific waters.
Significant negative correlations were noted between the
BL of each age group from 2 to 4 and the cumulative sum
of population abundance, though the BL at age 1 was not
significantly correlated. This suggests that the total population abundance affected the growth rates during ages 1–3.
To examine the effects of the recruitment and total
population abundance on the growth of sardines, we conducted multiple regression analysis for the estimated BL at
ages 2–4 for the year classes against recruitment and the
cumulative sum of population abundance (Table 2). For
BLs at all ages from 2 to 4, the population abundance
exhibited a large absolute value for the partial regression
coefficient and was a significant explanatory variable,
whereas the recruitment was not a significant variable. It


Fish Sci (2011) 77:291–299

295

Table 1 Estimated body length (Lt, cm) at the beginning of each age (t), increase in body length [(Lt?1 - Lt)/Lt, %], and the von Bertalanffy
equation parameters L?, K and t0 for typical year classes
Year class

L1

1979

13.7

13.7%

15.6

1982

14.1

12.1%

1987

14.0

17.3%

1988

ND

1989
1995

L2

L3

L4

L5

L6

L?

K

t0

10.7%

17.3

8.5%

18.7

6.9%

20.0

5.7%

21.2

30.0

0.12

-3.98

15.8

9.3%

17.3

7.3%

18.5

5.8%

19.6

4.7%

20.5

26.1

0.15

-4.06

16.5

10.3%

18.1

6.5%

19.3

4.2%

20.1

2.8%

20.7

22.0

0.36

-1.81

ND

16.6

13.8%

18.9

6.5%

20.1

3.2%

20.7

1.7%

21.1

21.5

0.63

-0.34

15.5

16.7%

18.0

7.9%

19.5

4.0%

20.3

2.1%

20.7

ND

ND

21.2

0.59

-1.20

15.1

25.9%

18.9

8.1%

20.5

3.0%

21.1

1.1%

21.3

ND

ND

21.5

0.93

-0.30

Body length (cm): Lt

Body length (cm): Lt
17

t=1
16

n = 20
r = -0.524

15

t=1

t=2

19

16

n = 27
r = -0.824**

18

15

12
21

17
14

16

13

1979

15

12

14
22

20

n = 26
r = -0.829**

18

16

1979
13
2001

t=2

19

n = 20
r = -0.468

15

17 1988
14

20

17

20

14

0

t=3

t=4

n = 28
r = -0.838**

n = 25
r = -0.851**

21

200

400

600

0

400

800

1200

22

21
20

19

t=3

t=4

n = 26
r = -0.953**

n = 25
r = -0.940**

21

19

20

20

18
17

18

19

1979

16
0

100

200

18
300 0

19

17

1979
100

200

300

Recruitment (109 ind.)
Fig. 5 Relationships between the estimated body length (Lt) at the
beginning of each age (t) and recruitment for the 1975–2002 year
classes (**P \ 0.01)

was the total population abundance rather than the
recruitment that strongly affected the BLs of sardines at

ages 2–4.
Fatness
Almost all specimens, including adults near the maximum
BL, had small gonads. Most specimens had large amounts
of subcutaneous and internal abdominal fat. Fat weight
index was positively correlated to the CF (r = 0.560,
n = 7,633, P \ 0.01). The CFs of all specimens were in
the range 7.9–17.2, with an average of 12.6. There was a
weak but significant trend for sardines with a larger BL to
have a higher CF (r = 0.395, n = 11,918, P \ 0.01). A
comparison of the CF at the same age between year classes
could reflect the differences in BL mentioned above.

16

18
0

500

1000

1500

0

500 1000 1500 2000

Cumulative sum of population abundance (10 9 ind.)
Fig. 6 Relationships between the estimated body length (Lt) at the

beginning of each age (t) for the 1977–2002 year classes and the
cumulative sum of population abundance for the period from age 0 to
age (t - 1) (**P \ 0.01)

Therefore, the variation in CF between year classes was
examined for separate BL ranges. Also, to examine seasonal variations in CF, monthly mean CFs for each year
class was determined. The means for all year classes were
then averaged (Fig. 7). The mean CF in each month can be
seen to decrease in winter: December–February for BLs
\19 cm or in January for BLs C19 cm. The foraging
season for Japanese sardine in the Pacific waters is summer
and autumn [21]. To examine the variations in CF among
year classes, we used the comparatively high CF values
obtained from samples during May–November for BL
\19 cm or during May–December for BL C19 cm as the
values that were considered to occur in the foraging season.
The mean CFs by BL range in each year class are shown
in Fig. 8. The mean CFs for BL C19 cm were about 13–14

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296

Fish Sci (2011) 77:291–299

Table 2 Results from a multiple regression analysis of the estimated body length (Lt) at the beginning of each age from 2 to 4 against the
recruitment (1011 ind.) of each year class and the cumulative sum of population abundance for the period from age 0 to age (t - 1) (1011 ind.)
Dependent
variable

Explanatory variable

L2 (n = 26)

Recruitment

Partial regression
coefficient
0.0362

Standard
error

Standard partial
regression coefficient

T value

P value

0.354

0.024

0.10

0.920

Population abundance

-0.352

0.089

-0.933

3.98

0.001**

L3 (n = 26)

Recruitment
Population abundance

0.0311
-0.219

0.218
0.037

0.023
-0.975

0.14
5.96

0.888
0.000**

L4 (n = 23)

Recruitment

-0.111

0.180

-0.104

0.62

0.545

Population abundance

-0.117

0.023

-0.847

5.01

0.000**

for most of the year classes, but were significantly lower
(11.7–12.4) for the 1979–1983 year classes. The mean CFs
of each year class for BL = 17–19 cm and BL = 15–17 cm

were about 12–13 and about 11–12, respectively, and those for
BL \15 cm ranged from 10 to 12. There were no apparent
trends in CF variation for BL\19 cm, which was similar to
what was seen for BL C19 cm.
The mean CFs of large-bodied sardines with BL
C19 cm during each year are shown along with the catch in
Fig. 9. The CF values in 1983–1988 were in the range
11.4–12.2, lower than in most other years. During this time,
population levels in the Japanese Pacific waters were high
(Fig. 1), and the catch levels around northern Japan (an
index of abundance in the feeding grounds) were at a
maximum. There was a significant negative correlation
between CF and the catch (r = 0.578, n = 26, P \ 0.01).
These large-bodied sardines of low CF mainly consisted of
1979–1983 year classes. The high abundance in the feeding grounds must have affected the fatness of large-bodied
sardines.

Discussion
We compare our results with growth patterns for other
small pelagic fishes that show biomass fluctuations like
Japanese sardine. Previous studies have reported densitydependent growth for some small pelagic fishes. The
western North Pacific chub mackerel (Scomber japonicus)
undergoes a seasonal migration similar to that of Japanese
sardine. Their body lengths at age C1 are dependent on
their growth in age 0 and are negatively correlated with the
year-class strength [22]. The body size (length, mass) at
age 3 of the Pacific herring (Clupea pallasii) from the
southwest coast of Vancouver Island is negatively related
to parental biomass, owing to a pre-recruit effect at age 0
[23]. The body length at age of Pacific Hokkaido spring

spawning herring is highly dependent on growth during age
0, and exhibited weak density-dependent effects of yearclass strength [24]. Thus, the body sizes at older ages in
these species are determined by their growth early in life,

123

Mean CF
14

BL

>19 cm

13

17-19 cm
12

15-17 cm

<15 cm
11
10
9

May Jun Jul Aug Sep Oct Nov Dec Jan Feb

Fig. 7 Monthly mean CF values for the 1975–2002 year classes

which is in turn affected by own recruitment level or

parental biomass (i.e., egg abundance).
Our results indicate different growth patterns for Japanese sardine from these species. Year classes subsisting
during high population levels showed apparent decreases in
the growth rate of small-bodied sardines and in the fatness
of large-bodied sardines (Figs. 4, 8). These decreases were
more strongly related to the cumulative sum of population
abundance that each year class experienced, rather than the
recruitment level (Fig. 6; Table 2). Significant correlations
that were found between the recruitment and the estimated
BLs were unlikely to indicate a direct effect of recruitment
abundance on the growth (Fig. 5).
Density-dependent growth was not apparent during the
first year of life (age 0); there were no obvious relationships between estimated BL at the beginning of age 1 and
recruitment or total population level (Figs. 5, 6). Insufficient food due to the density effect during age 0 would not
be serious compared with age C1. Juvenile sardines of age
0 have less swimming ability and are thought to be
entrained passively by ocean currents such as the Kuroshio
Extension. During their denatant feeding migration, they
are distributed broadly from the coastal area to around the
Shatsky Rise and far off the Kuril Islands to about 165°E in
the Pacific Ocean. This is regardless of the population level
or the recruitment level [25–27]. Age 0 sardines are well
dispersed and able to use the broad waters as foraging areas


Fish Sci (2011) 77:291–299
Fig. 8 Mean CF in each BL
range for the 1975–2002 year
classes

297

CF
14

BL
>19 cm

13

17-19 cm
15-17 cm

<15 cm

12

SD

11
14

13

12
13
12

11

10

9
1975

1980

1985

1990

1995

2000

Year class

(even if they are not in the best habitat), so an obvious
density effect would not occur. After wintering, age C1
sardines actively migrate, looking for good feeding
grounds to forage in. Consequently, the sardines compete
intra-specifically in favorable feeding grounds and have to
migrate further to find more food. In such a situation, the
high population abundance caused an obvious density
effect. It is suggested that decreases in growth rates or
fatness were caused by a decrease in energy intake due to
high density at the feeding grounds.
Lower CF was observed in large-bodied sardines (BL
C19 cm) during the period of maximum abundance at the
feeding grounds in the mid-1980s (Figs. 8, 9). Large-bodied sardines of BL C19 cm are near to the maximum body

size for this species and have a slow growth rate in terms of
body length. They are adults and accumulate the food
energy taken in at the feeding ground during summerautumn as body fat for reproduction in the following
winter-spring [21]. Small-bodied sardines of BL \19 cm
have higher growth rates than large-bodied sardines and
would invest their energy intake into increasing their body
lengths. During the period of maximum population, the
effect of high density on small sardines emerged as a
decrease in growth rate for 10 year classes (1979–1988)
over a comparatively long period: the effect on largebodied sardines appeared as a decrease in fatness for only

Catch in numbers (x109 ind.)

CF

30
14

25
20

13
15
10

12

5
11
1975

1980

1985

1990

1995

2000

0
2005

Year
CF of BL >19 cm

Catch in numbers

SD

Fig. 9 Annual changes in mean CF of BL C19 cm sardines and catch
in the Pacific waters off Sanriku and the eastern Hokkaido areas of
northern Japan (after Murakami and Kobayashi [18], Wada [10],
Yamaguchi and Kawabata [20], fisheries statistics of FRA)

5 year classes (1979–1983). It seems that small-bodied
sardines were more affected by the density than largerbodied sardines. The mean BL of sardines caught during
the 1980s in the waters off eastern Hokkaido (further from
the spawning ground) was greater than that of sardines

caught in the coastal waters off Sanriku [20]. This may
mean that larger-bodied sardines can forage farther afield
than smaller sardines and so reduce the density effect.

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298

According to previous studies based on analyses of
ancient documents relating to Japanese regional fisheries,
extra big catch years due to explosive increases in the
Japanese sardine population in Pacific waters have occurred only five or six times since the sixteenth century [3, 11,
28]. These previous periods of population increase seemed
to last no longer than a few decades. The recent explosive
population increase in the 1980s was considered to have
lasted for about a decade. Only 8 year classes (1980–1987)
had outbreaks contributing to the explosive increase in
population during the 1980s (Fig. 1). This explosive population increase (outbreak) caused an obvious density
effect, as mentioned above. Changes in biological characteristics owing to the density effect, such as an apparent
decrease in growth rate or fatness, were conspicuous and
seemed to be unusual for Japanese sardine. Additionally,
the condition of spawning sardines (i.e., accumulation of
body fat during the previous summer’s feeding migration)
affects their gonadal development and the quality and
quantity of eggs [29–31]. Kawasaki and Omori [32] state
that the density effect at the feeding ground in the 1980s
caused the condition of the spawners and the egg quality at
the spawning ground to deteriorate. The outbreak was not
necessarily a favorable situation for the sardine population.

During the long intervals between outbreaks of the population, the year classes that occur show relatively low
recruitment and biological characteristics that might be
regarded as normal. The outbreak of Japanese sardines was
a phase variation that coincided with global climate change
[33]. This does not indicate a recovery of the sardine
population; instead it may be a short-term, disadvantageous
state for the population.
Acknowledgments We would like to thank the editor and
two anonymous reviewers for their constructive comments and
suggestions. We also thank Ms. J. Momosawa, Ms. N. Kubo,
Mr. M. Kawamura and other staff at the Tohoku National Fisheries
Research Institute for their assistance and support in this study. This
study was funded by the Program of Marine Fisheries Stock
Assessment and Evaluation for Japanese Waters from the Fisheries
Agency of Japan. This paper is a contribution to the Study for the
Prediction and Control of Population Outbreak in Marine Life in
Relation to Environmental Change (POMAL) of the Agriculture,
Forestry and Fisheries Research Council (AFFRC).

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DOI 10.1007/s12562-011-0330-2

ORIGINAL ARTICLE

Biology

Expression patterns of type II and III iodothyronine deiodinase
genes in the liver of the goldlined spinefoot, Siganus guttatus
Nina Wambiji • Yong-Ju Park • Ji-Gweon Park
Se-Jae Kim • Sung-Pyo Hur • Yuki Takeuchi •
Akihiro Takemura

•

Received: 17 November 2010 / Accepted: 23 January 2011 / Published online: 10 March 2011
Ó The Japanese Society of Fisheries Science 2011

Abstract Iodothyronine deiodinases play an important
role in thyroid hormone regulation in vertebrates. The aim
of this study was to clone type II (SgD2) and type III
(SgD3) iodothyronine deiodinase cDNA from the goldlined
spinefoot (Siganus guttatus) using 30 - and 50 -rapid amplification of cDNA ends and then to assess their expression
patterns in the liver under several experimental conditions
by using quantitative real-time PCR. SgD2 (1013 bp) and
SgD3 (1492 bp) contained open reading frames of 810 and

804 bp and encoded 270 and 269 amino acids, respectively. They were characterized by an in-frame TGA codon
that was considered to be selenocysteine. An abundance of
SgD2 and SgD3 mRNA was expressed in several tissues,
with an increase at 1200 hours and a decrease at
2400 hours. Food deprivation suppressed the expression of
SgD2, but not SgD3. Higher SgD2 and SgD3 mRNA levels
in the liver were found in fish reared at 25°C than in those
reared at 20 and 30°C. These results suggest that exogenous factors influence the mRNA levels of iodothyronine
deiodinase genes in the liver and that transcription of the

N. Wambiji Á S.-P. Hur Á Y. Takeuchi Á A. Takemura (&)
Department of Chemistry, Biology and Marine Sciences,
Faculty of Science, University of the Ryukyus, Nishihara,
Okinawa 903-0213, Japan
e-mail:
J.-G. Park Á S.-J. Kim
Department of Biology, Jeju National University,
66 Jejudaehakno, Jeju City, Jeju Special Self-Governing
Province 690-756, Republic of Korea
Y.-J. Park
Marine and Environmental Research Institute, Jeju National
University, 3288 Hamduk, Jocheon, Jeju Special Self-Governing
Province 695-814, Republic of Korea

genes in certain tissues is partially regulated in a circadian
manner.
Keywords Cloning Á Day–night variations Á Food
availability Á Rabbitfish Á Quantitative real-time PCR Á
Temperature

Introduction
There are two types of thyroid hormones (THs), namely,
3,5,30 ,50 -tetraiodothyronine (T4) and 3,5,30 -triiodothyronine (T3), both of which play important roles in the physiological aspects of growth, development, and reproduction
[1, 2]. T3 is the potent and biologically active form of TH
and is produced by the enzymatic outer-ring deiodination
(ORD) of T4 in extrathyroidal tissues. In contrast, the
generation of 3,30 ,50 -triiodothyronine (reverse T3 or rT3),
the inactive form of TH, is produced by inner-ring deiodination (IRD) [3–5]. ORD and IRD are also active in the
metabolic pathways that form the inactive compound 3,30 diiodothyronine (T2) from T3 and rT3, respectively [2]. The
deiodination processes that occur during ORD and IRD are
considered to be tissue specific and to regulate intracellular
TH availability and disposal [6]. Iodothyronine deiodinases, which are members of the selenoprotein family, are
the enzymes responsible for TH deiodination [4, 7]. Three
types of iodothyronine deiodinases have been identified in
vertebrates [7]: type-I (D1) enzymes possess ORD and IRD
activities, while type-II (D2) and type-III (D3) only have
ORD and IRD activity, respectively [4, 8]. It appears that
the expression patterns of iodothyronine deiodinases in
respective organs are species specific and can vary in the
same species depending on the organism’s physiological
status [9].

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302

In certain teleost fishes, deiodination activities of TH
occur primarily in the liver [10, 11]. A deiodination assay
using radiolabeled iodine demonstrated high levels of

activity of low-Km T4ORD (the functional equivalent of D2)
in the liver of a large number of teleost fishes [6, 11–15].
Plasma T3 levels have been found to be highly correlated to
T4ORD activity in the liver of the Atlantic salmon Salmo
salar, suggesting that this organ is a major source of circulating T3 in teleosts [16]. On the other hand, D3-like
activity has also been reported in the liver of salmonids
[17, 18], sturgeon Acipenser fulvescens [19], walleye
Sander vitreus [20], American plaice Hippoglossoides
platessoides [21], and Nile tilapia Oreochromis niloticus
[22]. Feeding Nile tilapia and rainbow trout Oncorhynchus
mykiss with T3-supplemented food resulted in an increase in
D3 activity in the liver and gills but not in the brain and
kidney [23–25], while rainbow trout immersed in a solution
supplemented with T4 showed induced D3 activity in the
brain, liver, and retina [26]. These findings for D2 and D3
indicate that alternations in hepatic iodothyronine deiodinase activity impact on the TH-based status in certain
peripheral organs.
Acclimation to low temperature conditions was
observed to decrease D2 activity in the liver of the Atlantic
cod Gadus morhua [14]. The enzymatic activities of D2
and D3 in fish are also affected by nutritive and stress
conditions [27, 28], and D2 activity in the liver has been
shown to respond to sex steroids and pituitary hormones
[29–31]. These results demonstrate that the hepatic deiodination processes are directly or indirectly affected by
endogenous and exogenous factors [2]. To date, most
studies on THs in fish have focused on the effects of
environmental factors on deiodinase enzyme activities, and
few studies have employed molecular approaches to evaluate the effects of such factors on the expression of iodothyronine deiodinase genes in fish, although those genes
have been fully cloned and characterized in Nile tilapia [3]
and rainbow trout [32]. The aim of the study reported here

was to assess the molecular characteristics of iodothyronine deiodinases in the liver of the goldlined spinefoot
(formerly referred to as the golden rabbitfish or orangespotted spinefoot), Siganus guttatus, a common coral reefs
species and an important fish resource in Southeast Asian
countries. Since TH is closely related to reproductive and
nutritive conditions, understanding the status of D2 and D3
in the liver may lead to improved aquaculture technologies
for this species. We first cloned the cDNA for D2 and D3
from this species (SgD2 and SgD3, respectively). After
establishing an assay system to measure D2 and D3 mRNA
levels by using quantitative real-time PCR (qPCR), we
assessed day–night differences in SgD2 and SgD3 mRNA
abundance in several tissues, as well as the effects of food

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Fish Sci (2011) 77:301–311

deprivation and temperature on their expression in the
liver.

Materials and methods
Fish
Juvenile goldlined spinefoot with a body mass of
0.08–0.15 g were caught using small-mesh nets from the
mangrove estuary of the Teima River, Northern Okinawa,
Japan, during daytime, at low tide, around the time of the
new moon in July and August. They were reared under
natural photoperiodic conditions in holding tanks (capacity 1 or 5 t) containing constantly flowing seawater at
ambient temperature at the Sesoko Station, Tropical
Biosphere Research Center, University of the Ryukyus,

Okinawa, Japan. The fish were fed daily at 1000 hours
with commercial pellets (EP1 and then EP2; Marubeni
Nisshin, Tokyo, Japan). Immature fish with a mean body
mass of 200 ± 0.5 g (age 1) and mature fish with a mean
body mass of 346 ± 0.5 g (age 3 and 4) were used in the
study.
All experiments were conducted in compliance with the
Animal Care and Use Committee guidelines of the University of the Ryukyus and with the regulations for the care
and use of laboratory animals in Japan.
Sample collections for molecular cloning and tissue
expression
The fish were transferred to outdoor polyethylene tanks
(capacity 300 l) containing running seawater and acclimated to the rearing conditions with a fixed food provision at 1000 hours for 1 week. The fish were taken from
the tanks at 1200 hours, anesthetized with 2-phenoxyethanol (Kanto Chemical, Tokyo, Japan), and immediately
killed by decapitation. The whole brain was taken from
the mature fish (n = 3) for the molecular cloning of SgD2
and SgD3 cDNA. For the analysis of the tissue distribution of SgD2 and SgD3 mRNA, the whole brain, retina,
gills, liver, spleen, kidney, gonads, heart, and skin were
collected at 1200 hours (n = 7) and 2400 hours (n = 7)
from immature fish that were acclimated under the same
conditions. The samples were immediately immersed in
RNAlater (Applied Biosystems, Foster City, CA) at 4°C
and then stored at -20°C until further analysis. During
the dark period, samples were collected under a dim red
light. Since the day/night variations of both deiodinases
among the tissues were higher during the day than at
night, we subsequently collected all our samples at
1200 hours.

Fish Sci (2011) 77:301–311

Effects of food deprivation and temperature on gene
expression
For the food deprivation experiment, immature fish were
transferred to two polyethylene tanks (capacity 300 l)
containing running seawater at 25 ± 1.0°C under natural
photoperiodic conditions. The fish (n = 7) in one tank
were fed daily at 1000 hours with commercial pellets at 5%
of their body mass, while the fish (n = 7) in the other tank
were not fed after the initiation of the experiment. After
rearing under these conditions for 1 week, the fish from
each tank were anesthetized and sacrificed at 1200 hours.
After weighing, blood was collected from the caudal vein
using heparinized syringes and centrifuged at 10,000 g for
10 min at 4°C to obtain plasma samples that were stored at
-20°C until their glucose levels could be measured. The
liver was then taken from the body cavity and weighed.
Pieces of the liver were immersed in RNAlater at 4°C and
stored at -20°C until analysis. The hepatosomatic index
(HSI) was calculated using the following formula:
HSI = (liver mass/body mass) 9 100.
For the temperature experiment, immature fish (8 fish
per aquarium) were transferred to three glass aquaria
(capacity 60 l) with running seawater at 25°C, maintained
under LD = 12:12 by placing a fluorescent lamp (20 W)
above each aquarium that provided illumination at 1200 lx
(light intensity 2.23 W/m2), which was measured using a
quantum photoradiometer (model HD 9021, Delta OHM,
Padova, Italy). After acclimation for 1 week, the temperature of each aquarium was gradually changed using a

temperature control system with a programmable set point
to 20°C (lowest in winter), 25°C (temperature during
spawning season), and 30°C (highest in summer). The fish
were fed with commercial pellets daily at 1000 hours at 5%
of their body mass. One week after rearing the fish under
these conditions, samples were collected as mentioned
above.
Measurement of glucose levels
Plasma glucose levels were determined using the Glucose
CII Test Wako kit (Wako Pure Chemical Industries, Osaka,
Japan) according to the manufacturer’s instructions.
Extraction of RNA and cDNA synthesis
Total RNA was extracted from the tissues using the TriPure Isolation Reagent (Roche Applied Science, Hague
Road, IN) according to the manufacturer’s instructions.
When necessary, the samples for qPCR were treated with
deoxyribonuclease (RT grade; Nippon Gene, Tokyo,
Japan) at 37°C for 15 min to avoid contamination with
genomic DNA. The amount of RNA was measured at

303

260/280 nm, and samples with an absorbance ratio (A260/
A280) of 1.8–2.0 were used for cDNA synthesis.
Complementary DNA (cDNA) was reverse-transcribed
from 0.5 lg of total RNA using the High Capacity cDNA
Reverse Transcription kit (Applied Biosystems) for qPCR
and molecular cloning according to the manufacturer’s
instructions. For cloning, the first strand cDNA was synthesized from 1 lg of total RNA using PrimeScript 1st
strand cDNA Synthesis kit (Takara Bio, Otsu, Japan).
Cloning of the iodothyronine deiodinase genes

The SgD2 and SgD3 cDNA fragments were amplified
using degenerate oligonucleotide primers (SgD2-F2 and
SgD2-R1 for SgD2, and SgD3-F1 and SgD3-R2 for SgD3)
that were designed on the basis of the highly conserved
regions of the target genes using Primer3 software
(Whitehead Institute/Massachusetts Institute of Technology, Boston, MA) (Table 1). Oligonucleotide primers were
designed on the basis of the D2 sequences of the tiger
pufferfish Takifugu rubripes (GenBank accession no.
AB360768), bastard halibut Paralichthys olivaceus
(AB362422), and mummichog Fundulus heteroclitus
(FHU70869), and on the basis of D3 sequences of the Nile
tilapia (Y11111), tiger pufferfish (AB360769), and bastard
halibut (AB362423) (Table 1). The PCR was performed in
Table 1 Primer sequences used for cDNA cloning and expression of
SgD2 and SgD3 of goldlined spinefoot (Siganus guttatus)
Primers

Sequence

SgD2-F2

50 -CGCTCCATCTGGAACAGYTT-30

SgD2-R1

50 -CTGATGAAKGGGGGTCAGGT-30

SgD3-F1

50 -CGGTGTGCGTCTCBGACTCY-30

SgD3-R2

50 -RTGCTTGGGGATCTGATASG-30

SgD2-GSP2

50 -CCGAGCCAAAGTTGACCACCAGAG-30

SgD2-NGSP2

50 -TGTAGGCATCCAGCAGGAAGCTGTT-30

SgD2-F1-GSP1

50 -CGCCCAACTCCAAAGTGGTGAAGGT-30

SgD2-F3-NGSP1 50 -CCTCTGGTGGTCAACTTTGGCTCAG-30
SgD3-GSP2

50 -CGCAATGTCTGCGTACTGACTCACG-30

SgD3-NGSP2

50 - CGGTCTCTTCCCTTTCATGCAGTCC-30

SgD3-GSP1

50 -GTGTGGTACGGCCAGAAACTGGACT-30

SgD3-NGSP1

50 -CGTCGTGAGCCAGTACGCAGACATT-30

SgD2-qPCR-F

50 -GATCTGCTCGTCACACTCCA-30

SgD2-qPCR-R

50 -TTCACCAGCACCACAGAGTC-30

SgD3-qPCR-F
SgD3-qPCR-R

50 -GTGTGCGTCTCTGACTCCAA-30
50 -GATGCGCCGATTTTAGAAAG-30

b-actin-qPCR-F

50 -TCCTCCCTGGAGAAGAGCTA-30

b-actin-qPCR-R

50 -CAGGACTCCATACCGAGGAA-30

E2E-SgD2-F1

50 -GGGGAGAGCTCTTGCGGCCC-30

E2E-SgD2-R1

50 -AGTGTTTGTCCTTCAAGACTCCTACCG-30

SgD2, D3, Siganus guttatus iodothyronine deiodinase type II, III,
respectively

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304

25 ll of sample with Go Taq Green Master Mix (Promega,
Madison, WI) under the following cycling conditions: 1
cycle of initial denaturation for 2 min at 94°C; 35 cycles of
denaturation at 94°C for 45 s, annealing at 58°C for 45 s,
and 72°C for 1 min. The PCR products were separated on a
1% agarose gel with an appropriate molecular weight
marker, stained with ethidium bromide, and visualized
under UV illumination (ATTO, Tokyo, Japan). When the
PCR products of the predicted sizes were obtained, these
were purified using the Wizard SV Gel and PCR Clean-up
System kit (Promega) and ligated. The purified products
(233 and 281 bp for SgD2 and SgD3, respectively) were
then cloned into the pGEM T-Easy Vector (Promega) and
sequenced.
Rapid amplification of cDNA ends (RACE) was carried
out using the SMART RACE cDNA Amplification kit
(Clontech Laboratories, Mountain View, CA) according to
the manufacturer’s instructions. On the basis of the

sequence of the partial cDNA fragments described above,
the specific primers and nested primers for the RACE of
SgD2 (SgD2-GSP2, SgD2-NGSP2, SgD2-F1-GSP1, and
SgD2-F3-NGSP1) and those for the RACE of SgD3
(SgD3-GSP2, SgD3-NGSP2, SgD3-GSP1, and SgD3NGSP1) were designed for the 50 - and 30 -ends, respectively
(Table 1). RACE reactions in the first PCR were performed
using the Universal Primer A Mix (UPM) and the genespecific primer in a three-step touchdown PCR program:
(1) 5 cycles of 94°C for 30 s and 72°C for 3 min; (2) 5
cycles of 94°C for 30 s, 70°C for 30 s, and 72°C for 3 min;
(3) 25 cycles of 94°C for 30 s, 68°C for 30 s, and 72°C for
3 min. Nested PCR was performed using the 20-fold
diluted first PCR products as a template with the Nested
Universal Primer A (NUP) and each gene-specific nested
primer at the following cycling conditions: 94°C for 2 min;
25 cycles of 94°C for 30 s, 68°C for 30 s, and 72°C for
2 min; a final step of 72°C for 3 min [33]. The cDNA
fragments amplified by RACE were cloned into the pGEMT Easy vector and then sequenced.
Sequence analysis
The nucleotide and deduced amino acid sequences were
analyzed using the BLAST program (.
gov/BLAST). Multiple alignments for phylogenetic analysis were performed using the full-length deiodinase
sequences of several vertebrates by the ClustalW program
( />Real-time quantitative PCR (qPCR)
The expression levels of SgD2 and SgD3 mRNA were
assessed using the CFX96 Real-Time System C1000
thermal cycler (Bio-Rad, Hercules, CA). The forward and

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Fish Sci (2011) 77:301–311

reverse primers for the qPCR (SgD2-qPCR-F and SgD2qPCR-R for SgD2; SgD3-qPCR-F and SgD3-qPCR-R for
SgD3; b-actin-qPCR-F and b-actin-qPCR-R for b-actin)
were designed as shown in Table 1. b-actin mRNA levels
in the same sample were determined using qPCR to normalize the expression data [33]. The qPCR reaction mixture (10 ll) contained 5 ll Express SYBR GreenER qPCR
Supermix Universal (Invitrogen, Carlsbad, CA), 0.3 lM
forward primer, 0.3 lM reverse primer, 2 ll cDNA template, and 2.4 ll RNase-free water. The following PCR
cycling conditions were used: 95°C for 30 s; 40 cycles of
95°C for 5 s and 60°C for 34 s. To ensure the specificity of
the PCR amplicons, a melting curve analysis was carried
out by raising the temperature of the sample slowly from
60 to 95°C until the final step of the PCR. The expression
levels of SgD2, SgD3, and b-actin mRNA were measured
in triplicate. Data were normalized relative to the mean
expression level of each gene and analyzed using the
normalized gene expression [DDC(t)] method [34].
Statistical analysis
All data were expressed as the mean ± standard error of
the mean (SEM). Normality was tested using the Kolmogorov–Smirnov method. Student’s t test and the Mann–
Whitney U test were used to analyze the statistical differences between two sets of data. A one-way analysis of
variance (ANOVA) was performed for the temperature
experiment. Probabilities of P \ 0.05 and P \ 0.01 were
considered to be statistically significant.

Results
Cloning and properties of SgD2 and SgD3
The RACE analyses of SgD2 cDNA yielded a 1013 bp
fragment with an open reading frame (ORF) of 810 bp
(Fig. 1). The predicted amino acid sequence was 270 residues long. The ORF was interrupted by an in-frame TGA
codon at position 591 that was likely to encode for selenocysteine (Sec); however, the sequence did not contain a

SECIS element that has been reported to be responsible for
the incorporation of Sec into the protein during translation.
The SgD3 cDNA consisted of a 1492 bp fragment with an
ORF of 804 bp. The Sec residue was at position 504. The
predicted amino acid sequence of SgD3 was 269 residues
long (Fig. 2).
In terms of the amino acid sequence, SgD2 showed a
high similarity with D2 from several teleosts, such as the
gilthead seabream Sparus aurata (90%), bastard halibut
(85%), medaka Oryzias latipes (83%), and tiger pufferfish
(80%). It showed a moderately high similarity with D2


Fish Sci (2011) 77:301–311

305

Fig. 1 Nucleotide and deduced
amino acid sequence of the
Siganus guttatus iodothyronine
deiodinase type II (SgD2)
cDNA clone. The complete
mRNA spans 1013 bp with an
open reading frame (ORF) of
810 bp (270 amino acids). Sec
in bold Selenocysteine residue
at position 591. The start and
stop codons are indicated in
bold, with the stop codon
denoted by an asterisk

from the rat Rattus norvegicus (69%) and chicken Gallus
gallus (70%). SgD3 also showed a high similarity with D2
from gilthead seabream and Nile tilapia (90%), Senegalese
sole Solea senegalensis (86%), bastard halibut (83%), and
Atlantic halibut Hippoglossus hippoglossus (79%) and had
equally high similarities with D2 from the cow Bos taurus
(67%) and chicken (72%) (data not shown).

in the liver, skin, brain, heart, and gonads were significantly
higher at 1200 hours than at 2400 hours. A significantly
higher expression of SgD3 mRNA during the daytime was
also observed in the liver, retina, brain, gills, heart, gonads,
and skin. Negligible levels of SgD2 mRNA were observed
in the gonads and heart, while the smallest amount of SgD3
mRNA was detected in the kidney followed by the gills and
the heart at 2400 hours (Fig. 3a, b).

Tissue distribution of iodothyronine deiodinase genes
Food deprivation
The tissue distribution of SgD2 and SgD3 was examined at
1200 and 2400 hours using qPCR. SgD2 and SgD3 mRNA
were detected in all of the tissues tested. A comparison of
the expression of SgD2 mRNA in various tissues at
1200 hours revealed high expression in the liver, brain,
skin, and spleen (Fig. 3a). High levels of SgD3 mRNA
were observed in the liver, brain, retina, spleen, and skin
(Fig. 3b).
Day–night differences in the abundance of SgD2 and
SgD3 mRNA were observed. The expression levels of SgD2

The effects of food deprivation on HSI and glucose were
examined at 1200 hours. Following food deprivation, HSI
and plasma glucose levels significantly decreased (Fig. 4a,
b) and glycogen levels in the liver dropped (data not
shown). The effects of food deprivation on SgD2 and SgD3
mRNA expression in the liver at 1200 hours are shown in
Fig. 5. Food deprivation significantly lowered the abundance of SgD2 mRNA (P \ 0.05) (Fig. 5a), but not of
SgD3 (Fig. 5b).

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Fish Sci (2011) 77:301–311

Fig. 2 Nucleotide and deduced
amino acid sequence of the S.
guttatus iodothyronine
deiodinase type III (SgD3)
sequence of the full-length
cDNA clone. The complete
mRNA spans 1492 bp with an
ORF of 804 bp (269 amino
acids). Sec in bold
Selenocysteine residue at
position 504. The start and stop
codons are indicated in bold,
with the stop codon denoted by

an asterisk. The putative SECIS
element determined by the
SECISearch ver. 2.19 in the 30
untranslated region is
underlined. The
polyadenylation signal is in
italics and underlined

Temperature
Effects of SgD2 and SgD3 mRNA expression were
examined in the liver for the range of water temperatures
encountered in the habitats of the goldlined spinefoot
(Fig. 6). We observed that temperature significantly
affected the expression of SgD2 and SgD3 mRNA
(P \ 0.01). The levels of SgD2 transcription were significantly higher (P \ 0.01) at 25°C than at 20 and 30°C
(Fig. 6a). A similar temperature effect was observed for
SgD3 mRNA levels (P \ 0.01) (Fig. 6b).

Discussion
The first step of this study was the cloning and characterization of cDNA encoding type II (SgD2) and type III
(SgD3) iodothyronine deiodinase of the goldlined spinefoot, with the aim of evaluating the effects of food deprivation and temperature on SgD2 and SgD3 mRNA
abundance in the liver. The ORF of SgD2 and SgD3 contained an in-frame TGA stop codon that is characterized by
the presence of selenocysteine. The incorporation of an
essential selenocysteine residue within the catalytic domain

123

requires the presence of a premature stop codon (TGA) in
the ORF and a SECIS element located in the 30 untranslated region (UTR) of the cDNA [35]. We identified a
SECIS element in the 30 UTR of SgD3 located between

nucleotide positions 1267 and 1367, but we failed to
identify a SECIS element in SgD2 and may therefore have
sub-cloned a fragment without a 30 UTR and poly (A) signal in this study. The difficulty in obtaining such a fragment may be partially attributable to the occurrence of long
introns; it has been reported in mammals that the 30 UTR of
D2 has long introns (8.1–8.5 kb) within two exons [36].
Similar incomplete sequences have been reported for D2 of
the Senegalese sole [37] and mummichog [38] and considered to be due to either the lack of an extended 30 UTR
(up to 7.5 kb in length) including the SECIS structure [39]
or the fragment being a splice variant [37]. The full
sequence of mummichog D2 cDNA was later cloned and a
SECIS element was found within the 4652 bp region with
an intron divided by a 4.8 kb exon [40].
SgD2 mRNA was highly expressed in the liver, skin,
brain, and spleen, and SgD3 mRNA was highly expressed
in the liver, retina, brain, and skin, although the expression
of both genes was, to some extent, detected in all of the
tissues tested. These results suggest that the organs and


Fish Sci (2011) 77:301–311

307

Fig. 3 Tissue distribution of
iodothyronine deiodinase gene
abundance. a SgD2 and b SgD3
mRNA expression in goldlined
spinefoot kept under natural
conditions for 1 week and
sampled at 1200 hours (white

bars) and at 2400 hours (black
bars). Data are given as the
mean ± standard error of the
mean (SEM) (n = 7/group).
Asterisks significant differences
according to Student’s t test
(P \ 0.05)

tissues with a high expression of SgD2 and SgD3 play a
role in metabolism of thyroid hormones. Exceptionally
high SgD3 mRNA levels were detected in the retina, followed by the brain. The transcription pattern of both genes
was different in the respective tissues, as has also been
observed in some mammals and birds [41, 48] and fishes
[20, 22] where the deiodinases were found to be regulated
in relation to growth and development, hormonal treatment, thyroid status, pollution biomarkers, and food
availability. A simultaneous comparison of D2 and D3
mRNA levels has been carried out in walleye using reverse
transcription-PCR; D2 mRNA abundance in the liver was
significantly higher than in all other tissues, while D3
mRNA was highly expressed in the liver and whole eye,
followed by the brain, gills, and skin [20]. In terms of
deiodination activities, high levels of T4ORD and T4IRD
were observed in the liver and brain, respectively, of the
blue tilapia O. aureus [42], salmonids [43], and Atlantic
cod [14]. In rainbow trout under physiological conditions,
the predominant deiodinase pathways in the brain were
observed to autoregulate T3 levels through the degradation
of T4 and T3, while the liver generated T3 [12]. A positive
correlation between hepatic D2 activity and plasma T3
levels has been found in the Nile tilapia [27] and red drum

Sciaenops ocellatus [44]. This situation appears to vary
among vertebrates; D1 inactivation and D3 activation
coincidentally occur in the mammalian liver [45–47], while
the activation of hepatic D3 is one of the main factors
responsible for decreasing plasma T3 levels in chicken
[48].
To the best of our knowledge, daily variations of iodothyronine deiodinase transcript levels have only been
reported in the late metamorphic stages of the Senegalese
sole [37]; D3 transcript levels in larval homogenates were
measured using qPCR and found to significantly increase
from Zeitgeber time (ZT) 7 to ZT12 and then decrease
from ZT12 to ZT24. The results of our study clearly show
that the abundance of SgD2 and SgD3 mRNA in several
tissues was higher at 1200 hours than at 2400 hours, suggesting that TH levels fluctuate daily. In contrast to T4,
little or no daily variation in plasma T3 levels has been
reported in certain teleosts [49, 50]. However, the plasma
levels of total triiodothyronine (TT3) were found to
increase during the scotophase in juvenile Atlantic salmon
parr when they were reared under LD = 8:16 in the winter,
while in the spring the TT3 levels were higher in smolts,
but there was no daily rhythm. The opposite effect was
observed for total thyroxine in parrs and smolts [51]. Daily

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308

Fig. 4 Effect of food deprivation on nutritive parameters of goldlined

spinefoot. Hepatosomatic index (a) and plasma glucose levels (b) in
fed and unfed fish (n = 7 per group) after a 1-week experimental
period. The liver was collected from the fish at 1200 hours. Data are
given as the mean ± SEM. Asterisks significant differences according
to Student’s t test (P \ 0.05)

fluctuations of plasma T4 and T3 levels has also been
reported in juvenile red drum, with an increase during the
photophase in fish fed 1 h before the light were turned off,
dusk-fed fish, and dawn-fed fish kept under LD = 12:12 at
23°C [42]. Similar increases in plasma TH levels during
the photophase were observed in goldfish Carassius
auratus reared under LD = 12:12 [52] and in channel
catfish Ictalurus punctatus reared under a natural photoperiod in July [53]. A free-running circadian rhythm of
circulating T4 levels was also noted in the juvenile red
drum reared under constant photoperiod conditions with
and without feeding [54]. These findings imply that an
endogenous circadian clock regulates TH levels. Concurrent variations of T3 with T4 in certain teleost species may
mean that the activity of D2 and D3 is regulated by the
circadian system and that it influences the intercellular and
extracellular levels of TH. Our data may imply that melatonin is directly or indirectly related to daily variations in
SgD2 and SgD3 mRNA in the liver because this hormone

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Fish Sci (2011) 77:301–311

Fig. 5 Effect of food deprivation on iodothyronine deiodinase gene
abundance in the liver of goldlined spinefoot. SgD2 (a) and SgD3
(b) mRNA expression levels in fed and unfed fish after a 1-week

experimental period. The liver was collected from the fish at
1200 hours and measured for SgD2 and SgD3 mRNA abundance by
qPCR. Data are given as the mean ± SEM (n = 7/group). Asterisks
significant differences according to Student’s t test (P \ 0.05)

increased during nighttime (peak at 2400 hours) and
decreased during daytime. In fact, the expression of SgD2
in the hypothalamus was down-regulated by melatonin
administration (Wambiji, Hur, Takeuchi, and Takemura
unpublished data). Similar effects of melatonin on the
expression of iodothyronine deiodinase genes may occur in
the liver.
Our results demonstrate that food deprivation lowered
plasma glucose and hepatic glycogen levels, similar to
results reported for channel catfish [55], suggesting that
stored nutrients in the liver are mobilized after food
deprivation. Concomitant with these metabolic changes,
the mRNA abundance of hepatic SgD2 decreased following food deprivation. It was reported that hepatic D2
activity decreases after starvation, with concurrent increases in plasma T3 levels and hepatic D2 activity after refeeding [27]. Food deprivation was observed to lower the


Fish Sci (2011) 77:301–311

309

juvenile marbled spinefoot (S. rivulatus), a relative species
to the goldlined spinefoot; when the fish were reared at 17,
22, 27, and 32°C, fish reared at 27 and 32°C were significantly larger. In addition, the specific growth rate was
higher in fish reared at 27 than at 32°C [58]. A similar
temperature effect was observed in the reproductive

activity of the sapphire devil Chrysiptera cyanea, a common species in the coral reefs of the West Pacific Ocean
[59, 60]. Overall, these results show that tropical fish have
a suitable range of temperature for an optimal physiological state, including the deiodination activities in the liver.
Based on these results, we conclude that the abundance
of SgD2 and SgD3 mRNA in the spinefoot is subject to the
exogenous factors they are exposed to. It is possible that
the impacts of exogenous factors are transduced to the liver
through endogenous factors, such as melatonin for day–
night difference and growth hormone–insulin-like growth
factor for nutrition status. Further studies are needed to
elucidate the involvement of endogenous factors in the
alternation of SgD2 and SgD3 mRNA levels in the goldlined spinefoot.
Acknowledgments This study was supported in part by a Grant-inAid for Scientific Research from the Japan Society for the Promotion
of Science (JSPS) and a Joint Research Project under the Japan–Korea
Basic Scientific Cooperation Program from JSPS to AT.

References
Fig. 6 Effect of chronic temperature regimes on iodothyronine
deiodinase gene abundance in the liver of goldlined spinefoot.
SgD2 (a) and SgD3 (b) mRNA expression levels in fish kept at 20, 25,
and 30°C under LD = 12:12 for 1 week. The liver was collected from
the fish at 1200 hours and subjected measured for SgD2 and SgD3
mRNA abundance by qPCR. Data are given as the mean ± SEM.
n = 7/group. Different letters indicate significant differences after
one-way ANOVA (P \ 0.01)

plasma TH levels of Nile tilapia [27, 56], rainbow trout
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of both genes being observed in fish reared under an
intermediate temperature (25°C), but not at lower (20°C) or
higher (30°C) temperatures. Since our study parameters
mimic the minimum winter and maximum summer temperatures, the abundance of these genes in the liver is likely
to reflect their physiological responses. In this regard, there
was a crucial effect of temperature on the growth of the

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Fish Sci (2011) 77:313–320
DOI 10.1007/s12562-011-0335-x

ORIGINAL ARTICLE

Biology

Migration history of Sakhalin taimen Hucho perryi captured
in the Sea of Okhotsk, northern Japan, using otolith Sr:Ca ratios
Kyoko Suzuki • Tomoyasu Yoshitomi •
Yoichi Kawaguchi • Masaki Ichimura •
Kaneaki Edo • Tsuguo Otake

Received: 24 August 2010 / Accepted: 4 February 2011 / Published online: 16 March 2011

Ó The Japanese Society of Fisheries Science 2011

Abstract Sakhalin taimen Hucho perryi populations have
decreased drastically to near extinction. It is urgent to
establish an effective conservation strategy based on an
understanding of the characteristics of migration and habitat use of this species. We examined the migration history
of anadromous Sakhalin taimen captured off the Sarufutsu
coast, northern Hokkaido, Japan, using otolith Sr:Ca ratios
and also examined the relationship between their otolith
Sr:Ca ratios during freshwater and seawater residence in a
rearing experiment. Otolith Sr:Ca ratios of some fish from
the Sarufutsu coast showed freshwater levels (0.5–4.0 9
10-3) near the core, which thereafter increased to brackish
The author Kyoko Suzuki is Research Fellow of the Japan Society for
the Promotion of Science.
K. Suzuki (&) Á T. Otake
International Coastal Research Center,
Atmosphere and Ocean Research Institute,
The University of Tokyo, 2-106-1 Akahama,
Otsuchi, Iwate 028-1102, Japan
e-mail:
T. Yoshitomi
Field Studies Institute for Environmental Education,
Tokyo Gakugei University, 4-1-1 Nukuikitamachi,
Koganei, Tokyo 184-8501, Japan
Y. Kawaguchi
Division of Ecosystem Design, Institute of Technology
and Science, The University of Tokushima,
2-1 Minami-josanjima, Tokushima 880-8506, Japan
M. Ichimura

Shibetsu Salmon Museum, 1-1 Kita 1-Jo Nishi 6-Chome,
Shibetsu, Shibetsu-gun, Hokkaido 086-1631, Japan
K. Edo
Monuments and Sites Division, Agency for Cultural Affairs,
Marunouchi 2-5-1, Chiyoda-ku, Tokyo 100-8959, Japan

water levels (4.0–6.0 9 10-3), and then to seawater levels
(6.0–10.0 9 10-3) in the outermost regions. Those findings
indicate that specimens from the Sarufutsu coast migrated to
the brackish water region or the sea and spent most of their
lives there. The anadromous migration pattern including the
timing of downstream migration seems to be flexible among
individuals in the species. They migrate between freshwater
and seawater or brackish water several times during their lives,
showing extensive habitat use. It is essential to secure the
continuity among the freshwater, brackish water, and seawater areas for their effective conservation.
Keywords Sakhalin taimen Á Migration Á Rare species Á
Anadromous Á Otolith Sr:Ca ratios Á Conservation

Introduction
Sakhalin taimen Hucho perryi is a species of the genus
Hucho, which is composed of five species [1, 2]. The
distribution is limited to the far northeastern part of Asia,
from the Primorye region of Siberia to Sakhalin Island, the
southern Kurile Islands, and the northern area of Hokkaido
[1]. Unlike the other species of the genus, which are strictly
freshwater residents, the Sakhalin taimen has been known
to perform an anadromous migration [2–4]. The population
of this species has decreased remarkably and is close to the
extinction level [5]. This situation is thought to result from

various human impacts such as indiscriminate fishing,
water pollution by development of agricultural land, and
habitat destruction. Takami et al. [6] reported that the
decrease of Sakhalin taimen was probably the result of the
loss of riparian forests and riverine habitats associated with
extensive development of agricultural land during the
1960s and 1970s. The construction of dams, barrages, and

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banks also caused habitat destruction including the loss of
spawning grounds. In particular, the straightening of the
rivers at middle to lower reaches conducted in the last
several decades has made the rivers markedly monotonous
with a loss of rapids and deep pools, and has damaged the
river environment for the reproduction of H. perryi [7]. The
life history, including migration and habitat use of this
species, should be urgently studied for the establishment of
an effective conservation strategy.
However, information about the life history of anadromous Sakhalin taimen is still limited with only a few
studies being done on ecological aspects of their freshwater
phases [8–11], the morphology of anadromous Sakhalin
taimen [12], or on the microchemistry of the otoliths and
scales [13–15]. Fish otoliths are metabolically inert with
the aragonite mineralogy remaining unaltered after deposition [16], so the elemental composition of the otolith
reflects to some degree the environment of the water in
which the fish lives [17]. The strontium content in otoliths,

in particular, varies with fluctuations in ambient salinity,
allowing the reconstruction of the anadromous migration
history of each fish [18–24]. Regarding Sakhalin taimen,
Arai et al. [13] used otolith analysis to report downstream
migration of Sakhalin taimen collected at Lake Aynskoye
in Sakhalin Island. Honda et al. [14] analyzed otolith Sr:Ca
ratios of Sakhalin taimen caught from Lake Akkeshi
(brackish water lake) in Hokkaido, Japan, and suggested
that the specimens had migrated into brackish waters, but it
was unlikely they went into the ocean. The present study is
the first report on detailed migration history of anadromous
Sakhalin taimen, actually captured in the Sea of Okhotsk,
the Sarufutsu coast, northern Hokkaido in Japan. The
population of Sakhalin taimen in the Sarufutsu River system is one of the stable populations of this species in Japan
[25]. Furthermore, there seems to be a genetic difference
among the stocks of several river systems (K. Edo, pers.
comm., 2007, 2009). Therefore the life history and migration
traits of Sakhalin taimen should be investigated in each river
system unit. This knowledge is vital for establishment of an
effective conservation strategy for the species whose local
population is declining. The Sr:Ca ratio levels of Sakhalin
taimen otoliths from fish reared in seawater and freshwater
were also examined to verify the affect of salinity on the
Sr:Ca ratios in the otoliths of this species.

Materials and methods
Rearing experiment
A total of 12 Sakhalin taimen used for this experiment were
provided by the Shibetsu Salmon Museum. We do not
report the river lineage from which these specimens were

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Fish Sci (2011) 77:313–320

taken to help protect this critically endangered species.
They were artificially hatched and kept in freshwater at
16.0–17.0°C under natural light conditions for 6 months
before the experiment. The average fork length and body
weight at the start of the rearing experiment were
424 ± 41 mm and 838 ± 250 g, respectively. The rearing
experiment was carried out in the Shibetsu Salmon
Museum. Six of 12 fish remained in a freshwater environment for another 6 months, and the other six were
transferred to seawater and also reared for 6 months. The
freshwater fish were reared in a 500 L tank supplied with
well water at 10–20 L/min. The temperature of the well
water was ca. 16.8°C. The seawater rearing was in filtration
tank (5,000 L) of the Shibetsu Salmon Museum, which has
a water recycling system. The seawater used for the rearing
was pumped up from the Shibetsu port and circulated in the
system at the rate of 180–240 L/min. The salinity of seawater used for the rearing was 30 psu. The seawater temperature varied from 12 to 17°C, and the average
temperature was ca. 15°C during the experiment. Lighting
conditions of each rearing tank were based on natural light.
Fish of both freshwater and seawater groups were fed
4 days per week with dry pellets (Hokuren). We could not
carry out the rearing experiment under a variety of salinities because of the limited numbers of this locally protected
fish species.
Wild fish used for the analyses of otolith Sr:Ca ratio
A total of seven wild Sakhalin taimen were used for the
analysis of otolith Sr:Ca ratios. All fish were unintentionally caught in a set net fishery mainly targeting pink salmon

(Oncorhynchus gorbuscha), chum salmon (Oncorhynchus
keta), and masu salmon (Oncorhynchus masou) conducted
by the Sarufutsu Fishery Cooperative and Fujimoto Fisheries Company along the Sarufutsu coast of northern
Hokkaido, Japan (the Sea of Okhotsk) (Fig. 1).
The fork length, body weight, sex, age, capture date, and
number of retained ovulated eggs of each fish are shown in
Table 1. Scales were used for the age determination of
each fish. Scales were removed from areas above and
below the lateral line of the fish body.
Otolith preparation and Sr:Ca ratio analyses
Sagittal otoliths were extracted from each fish and were
embedded in epoxy resin (Epofix, Struers, Denmark). They
were mounted on glass slides by epoxy bond and ground to
expose the core using a grinding machine equipped with a
diamond-cup wheel of 13 lm (Discoplan-TS, Struers,
Denmark). The ground surface of otolith was polished with
6 and 1 lm diamond paste on a polishing wheel (PlanopolV equipped with PdM-Force, Struers, Denmark), Pt-Pd


Fish Sci (2011) 77:313–320

315

Fig. 1 a Map of Hokkaido, northern Japan and Sarufutsu coast. b Map of Sarufutsu coast showing the location of the set net
Table 1 Characteristics of the
individual Sakhalin taimen
captured on the coast of
Sarufutsu

Fish no.

Capture date

Sex

Age
(years)

Fork
length (cm)

Body
weight (g)

Number of
retained eggs

1

1997.6.26

Male

13

75.2

6,180

0

2

1997.6.26

Female

8

74.4

5,500

2

3

1997.6.26

Female

8

69.4

4,600

0

4

2007.7.4

Male

11

73.2

4,888

0

5

2007.7.4

Male

12

76.5

5,692

0

6

2007.7.4

Female

13

78.6

5,789

0

7

2007.7.4

Female

12

77.6

5,820

0

coated with a high vacuum evaporator (E-1030, Hitachi,
Japan) after washing with deionized water.
For life history transect analysis, the profiles of Sr and
Ca concentrations were analyzed from the core to the edge
along the radius of each otolith using a wave-length dispersive X-ray electron probe microanalyzer (EPMA; JEOL

JXA-8900, Jeol, Japan). The accelerating voltage and beam
current were 15 kV and 1.2 9 10-8 A, respectively. The
electron beam for the rearing experiment samples was
focused on a point of 10 lm diameter, and the measurements were spaced at 10 lm intervals. The beam for wild
fish was focused on a point of 5 lm diameter, and the
measurements were spaced at 5 lm intervals. The outermost 100 lm of the otolith of fish no. 3 (Table 1) was
further examined to determine the Sr:Ca ratio deposited
under seawater conditions. For this analysis beam diameter
and measurement interval were both set to 1 lm with the
accelerating voltage and beam current the same as mentioned above.
Two-dimensional X-ray intensity maps of Sr and Ca
were also examined by EPMA under the following

measurement conditions: accelerating voltage, 15 kV;
beam current, 5.0 9 10-7 A; pixel size, 10 9 10 lm.
Calcite (CaCO3) and strontianite (SrCO3) were used as
standards.

Results
The rearing experiment showed that there was a remarkable difference in the Sr:Ca ratios between the otoliths of
the fish reared in the freshwater and seawater tanks
(Fig. 2). The otolith Sr:Ca ratios of individuals reared in
freshwater (freshwater sample) maintained lower levels
with an average ratio of 1.1 9 10-3 (range 0–3.4 9 10-3)
throughout the rearing period. In contrast, the ratios of
individuals transferred into seawater from freshwater
(seawater sample) sharply increased to a high level with an
average of 5.6 9 10-3 (range 4.0–7.8 9 10-3) at about
500 lm from the otolith edge. These facts are clearly
supported by two-dimensional X-ray intensity maps of Sr

content for otoliths of freshwater and seawater samples

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