Aquacult Int (2013) 21:53–64

DOI 10.1007/s10499-012-9533-5

Effect of nitrate fertilization of gametophytes of the kelp

Undaria pinnatifida on growth and maturation
of the sporophytes cultivated in Matsushima Bay,
northern Honshu, Japan

Xu Gao • Yukio Agatsuma • Kazuya Taniguchi

Received: 1 September 2010 / Accepted: 14 April 2012 / Published online: 6 May 2012
Ó Springer Science+Business Media B.V. 2012

Abstract In northern Japan, the early production of high-quality specimens of kelps

Undaria pinnatifida prior to the outbreak of pinhole disease is needed. To address this
need, we tested the effect of nitrate fertilization of gametophytes on the growth and
maturation of sporophytes using experimental and control gametophytes derived from
sporophytes cultivated in Matsushima Bay, northern Honshu, Japan. From mid-August to
October 2008, experimental gametophytes were cultured on a rope indoors at a nitrate
concentration of 1 mg/L at the optimal seawater temperature of 20 °C, whereas control
gametophytes were cultured without the addition of nitrate in normal seawater at an
average temperature of 23.5 °C during this period in the past 30 years. Next, the seedlings
were cultivated in Matsushima Bay until March 2009. For the experimental kelps, the total
length, stipe length, stipe width, sporophyll length, sporophyll width, and dry weight of the
blade, midrib, and sporophyll increased significantly from December to January compared
to those of control kelps. Matured sporophylls of the experimental kelps were more than
those of the control kelps from early December to late January. Significant higher pho-
tosynthesis occurred at seawater temperatures of 10 and 15 °C, as well as higher uptake
rates of NO3–N of the experimental kelps in December and January. These results show
that nitrate fertilization of the gametophytes makes it possible to harvest the high-priced
blade, midrib, and sporophyll in January, which is earlier than the traditional harvest date.

Keywords Cultivation  Gametophyte  Growth  Nitrate fertilization  Maturation 

Sporophyte  Seawater temperature  Kelp  Undaria pinnatifida

Introduction

The large brown kelp, Undaria pinnatifida (also called Wakame), belongs to the Alaria-
ceae in the Laminariales and is endemic to Japan, the Korean peninsula, and China

X. Gao (&)  Y. Agatsuma  K. Taniguchi

Laboratory of Marine Plant Ecology, Graduate School of Agricultural Science, Tohoku University,
1-1, Tsutsumidori-amamiya, Aoba, Sendai, Miyagi 981-8555, Japan
e-mail: gaoxu69@126.com

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54 Aquacult Int (2013) 21:53–64

(Ohno 2004). U. pinnatifida is distributed widely in Japan, where it grows mainly in

subtidal zones up to a depth of 4 m (Saito 1975). This kelp exhibits an annual life history
pattern and a heteromorphic life cycle with alternating sporophytic and gametophytic
generations. The sporophytes mature in spring and produce zoospores in sporophylls on the
basal part of the stipe; they then wither in summer when the seawater temperature increases
(Akiyama and Kurogi 1982; Lee and Sohn 1993; Oh and Koh 1996).
Undaria pinnatifida has long been used as a major food resource for humans (Saito
1972) and is one of the major commercially cultivated seaweeds in Asia. The kelp is also in
demand as a supplement food in the world (Taira 2006). In Japan, cultivation of this kelp
began in 1953 in Onagawa Bay, Miyagi Prefecture (Taira 2006). In 1970s, the cultivation
method shifted from the hanging type to the horizontal long-line type, and the development
of salting technology increased the annual production from 50,000 to 120,000 tons (Taira
2006). Currently, the production from cultivation accounts for 97 % of the total of this kelp
in Japan (Taira 2006). The largest-scale cultivation of this species was carried out in
Sanriku district and Tokushima Prefecture comprising about 90 % of the total production
(Tokuda et al. 1994). Although the crop increased remarkably in recent years due to the
progress of cultivation technology and management, it still could not meet the demands of
consumers. The annual consumption of U. pinnatifida in Japan has reached
350,000–400,000 tons, of which 60 and 20 % are imported from China and Korea,
respectively (Taira 2006). On the Pacific coast of northeastern Honshu, the high-quality
fresh thalli and sporophyll of U. pinnatifida, which were harvested during growth phase (in
January), are called ‘‘Haru-ichiban,’’ and its price is five times higher than the normal.
Due to its great commercial importance, more and more people started to draw close
attention to the good way of increasing production in recent years. However, there were
indeed some problems in the process of cultivation, which had a negative impact on crop
yields. Among them, pinhole disease is a bacterial infection that occurs in U. pinnatifida in
Japan constantly from late January. It also has been reported for U. pinnatifida in south-
western Korea (Park et al. 2008). This disease decreased the yields severely, suggesting the
need for early production of high-quality kelps. Additionally, U. pinnatifida has been
cultivated with traditional methods for a long time, in which gametophytes were cultured
in an aquarium on land only using natural seawater from Matsushima Bay. The high
average seawater temperatures from August to October and lack of nutrients in natural
seawater may have a negative impact on the growth and maturation of gametophytes and
delay the harvest date.
The growth and maturation of kelp gametophytes may be affected by seawater tem-
peratures and nutrient conditions. The gametophytes of U. pinnatifida generally grow from
late spring to summer or autumn when seawater temperatures are relatively high, and the
optimum seawater temperature for growth ranges from 15 to 20 °C (Akiyama 1965).
Yamanaka and Akiyama (1993) reported that maturation of the gametophyte of U. pin-
natifida ceased and that gametophytes went dormant or died at warm seawater temperature
in summer. Similar findings were also reported by Izquierdo et al. (2002) and Lee and
Brinkhuis (1988) that seawater temperature is the principal factor affecting the survival and
growth of gametophytes of Laminarian species. A study of Macrosystis pyrifera demon-
strated that the addition of extra nutrients to gametophytes in the field in California resulted
in increased densities of sporophytes (Deysher and Dean 1986), indicating that nutrients
can be an important factor for recruitment success. However, no studies have clarified the
effect of nitrate concentration of gametophytes at the optimal temperature on the growth
and maturation of sporophytes of U. pinnatifida.

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Aquacult Int (2013) 21:53–64 55

Thus, the goal of the present study was to determine the effect of nitrate fertilization of
the gametophytes on the growth and maturation of the sporophytes and to advance culti-
vation method for earlier production.

Materials and methods

Culture of gametophytes and cultivation of sporophytes

Gametophytes of U. pinnatifida were obtained by the induction of zoospores from mature

sporophytes cultivated in Matsushima Bay (38°220 N, 141°030 E) in April 2008. After the
zoospores were released into vessels with seawater, homogeneous stir was conducted to
make them attach to plastic fiber threads 2 mm in diameter in order to obtain similar initial
settlement density. Then, these stationary cultures were conducted indoors until mid-
August at an average seawater temperature of 18.5 °C. For the experimental group, the
gametophytes were cultured in nitrate-enriched (1 mg/L) seawater at the optimum sea-
water temperature of 20 °C in control (Morita et al. 2003) from mid-August to October.
Aeration was conducted to mix nitrate and seawater evenly. For the control group, the
stationary culture was continued without temperature control. The average seawater
temperature during this period in the past 30 years was 23.5 °C. No nutrients were added.
The cultivation of the experimental and control kelps (young germinated sporophytes)
began in October 2008 at a depth of 2 m in Matsushima Bay. The young sporophytes that
had germinated on the plastic fiber threads had been immersed at a depth of 0.5 m in
Matsushima Bay. The threads on which the young sporophytes grew were cut into 3–5 cm
sections. Fifty sections of experimental kelps were tucked into a rope 50 m in length and
15 mm in diameter at intervals of 0.5 m for cultivation; 50 sections of control kelps were
tucked into a second rope. The two ropes were deployed at the sea surface with floats at
intervals of 3 m.

Measurements of morphological characteristics

The kelp specimens were cultivated until March 2009. The kelps derived from one thread
section were regarded as one stock. In early December 2008, both experimental and
controlled kelps were thinned out into 15 sporophytes per stock. To examine the changes in
morphological characteristics, three stocks of each treatment were collected every 2 weeks
and transported to the laboratory within 1 h. Total length, stipe length, stipe width, number
of pinnate blade, length of the longest pinnate blade, sporophyll length, and sporophyll
width of 30 sporophytes in sequence from the largest were measured from December to
February (Fig. 1). In March, only 10 sporophytes were measured because their number had
decreased due to withering. At each time point, the number of sporophytes with sporophyll
was also counted. Finally, the sporophytes were carved into midrib, blade, and sporophyll,
and each part was weighed after drying in a convection oven (NDO–600ND, Eyela, Tokyo,
Japan) at 80 °C for 7–10 days to ensure them dry out completely.

Measurements of photosynthetic rates and uptake rates of NO3–N

In December 2008 and January 2009, a stock from each treatment was collected and 10
sporophytes in sequence from the largest were selected to measure photosynthetic rates and

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Fig. 1 Morphological
measurements of Undaria
pinnatifida. TL, total length; STL,
stipe length; STW, stipe width;
LBL, the length of longest
pinnate blade; SPL, sporophyll
length; SPW, sporophyll width

nutrient uptake rates of NO3–N. For each sporophyte, sixty discs with the area of 2.01 cm2
were cut from the pinnate blades close to the margins of the meristem using a 1.6-cm-
diameter cork borer. The discs were incubated in a flowing seawater system for 12 h to
allow recovery from cutting damage. An improved ‘‘Product-meter’’ (Yokohama et al.
1986) was used to measure photosynthetic rates and nutrient uptake rates. To measure
photosynthetic rate, each disc was placed in a reaction vessel containing 10 mL of filtered
1/4 PESI (Tatewaki 1966) seawater (GF/F, Whatman, Maidstone, UK) and incubated in a
water bath at 150 rpm min-1 under saturated irradiance of Laminaria japonica of
180 lmol/m2/s (Narita et al. 2008) at seven different temperatures (2.5, 5, 10, 15, 20, 25,
and 30 °C). Four replicates were tested at each temperature. The discs were incubated for
30 min to allow them to adapt to each experimental temperature, and then the oxygen
evolution produced by the discs was measured 6–10 times at 3-min interval.
The uptake rate of NO3–N was measured at 10 °C and 180 lmol/m2/s using enriched
seawater at 1/4PESI, 1/8PESI, 1/16PESI, and 1/32PESI and was obtained from the dif-
ferences between the concentrations in the blanks and those measured by the auto-analyzer
(TRAACS 800; Bran—Luebbe, Tokyo, Japan) after a 60-min incubation of the discs.
Seawater used in this experiment was collected off Sasunohama, Ishinomaki, which was
near the study site and had similar environmental conditions to Matsushima Bay. The
nutrient concentrations in the seawater were almost below the limit of detection, and the
water was used in the experiment after filtration (GF/F, Whatman).

Environmental parameters

In parallel with the collection of the cultivated kelps, surface seawater was collected
adjacent to the study site using a 5-L plastic bucket. The concentrations of NH4–N, NO3–
N, and PO4–P were measured using an auto-analyzer (TRAACS 800; Bran-Luebbe) after
filtration through a glass fiber (GF/F, Whatman). The seawater temperature at the study site
was also monitored using a normal thermometer. Data for the average bimonthly

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temperature in Matsushima Bay from 1979 to 2004 were obtained from the Miyagi
Prefecture Fisheries Technology Institute.

Statistical analysis

Significant differences in morphological characteristics among months were analyzed

using the Kruskal–Wallis test followed by the Steel–Dwass multiple comparison test.
Significant differences in morphological and physiological characteristics between the
experimental and the control groups were analyzed using the Mann–Whitney U test.

Results

Environmental factors

Changes in surface seawater temperature at the study site and the averages in Matsushima
Bay are shown in Fig. 2. The seawater temperatures at the study site reached the lowest
value (4.1 °C) in early January, slightly higher than the average in Matsushima Bay from
February to mid-March. The concentrations of NH4–N and NO3–N changed erratically,
ranging 0.03–0.1 mg/L and 0.03–0.07 mg/L, respectively. The concentration of PO4–P
ranged from 0.02 to 0.03 mg/L during the study (Fig. 3).

Morphological changes

The temporal changes in morphological characteristics of the experimental and control

kelps are shown in Fig. 4. All of these morphological parameters differed significantly
among months for both experimental and control kelps (Table 1). Mean total length of
experimental kelps was 95.6 cm in early December 2008 and reached 228.0 cm in late
January, whereas mean total length of control kelps was 57.3 cm in early December 2008
and reached 189.1 cm in late January. Mean stipe lengths of both experimental and control
kelps ranged between 15.4 and 38.0 cm, 5.9 and 29.1 cm in early December and early
January, respectively. Also, mean stipe widths of both experimental and control kelps
ranged between 1.1 and 3.9 cm, 0.7 and 3.2 cm in early December and late January,

Fig. 2 Changes in surface 30

seawater temperature at the study
site during this study (solid
25
circles), bimonthly averages in
Matsushima Bay from 1979 to
2004 (empty circles)
Seawater temperature

20

15

10

0
D J F M A M J J A S O N

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Fig. 3 Temporal changes in the 0.12

concentrations of NH4–N (empty
circles), NO3–N (solid circles), 0.1
and PO4–P (solid squares) in the

Concentration mg/L
surface layer at the study site
0.08

0.06

0.04

0.02

0
Dec. Jan. Feb. Mar.
2008 2009

respectively. The number of pinnate blade and length of the longest pinnate blade of both
experimental and control kelps increased from December to January, and they were also
greater at the collection in January compared to the initial one. Undaria sporophytes
bearing sporophylls were found in both experimental and control kelps in early December.
From early December to late January, the length and width of sporophylls of experimental
kelps increased from 5.6 to 21.4 cm and from 2.3 to 8.6 cm, respectively, whereas the
length and width of sporophylls of control kelps increased from 2.3 to 14.2 cm and from
1.3 to 7.8 cm, respectively. The morphological parameters of the experimental kelps were
significantly greater than those of the control kelps in early December and in January,
except for the length of the longest pinnate blade, the number of pinnate blades, and the
sporophyll length and width. In late February, the sporophyll length of the experimental
kelps was significantly greater than that of the control, whereas the number of pinnate
blades of the experimental kelps was less than that of the control.
The temporal changes in dry weight of the blade, midrib, and sporophyll of the
experimental and control kelps are shown in Fig. 5. These parameters differed significantly
between months (Table 1). Mean dry weight of blade of experimental and control kelps
were 4.4 and 1.4 g in early December, respectively, and then increased to 26.1 and 17.4 g
in late January. Mean dry weight of midrib of experimental and control kelps ranged
between 1.4 and 16.0 g, 0.4 and 8.9 g in early December and late January, respectively.
Also, mean dry weight of sporophyll of experimental and control kelps ranged between
0.2 and 9.0 g, 0.1 and 5.4 g in early December and late January, respectively. These were
significantly greater in the experimental kelps than in the control kelps in January and early
December except for sporophyll weight. In late February, the blade weight of the exper-
imental kelps was significantly lower than that of the control.
The percentages of the experimental kelps with sporophyll were 21.7–97.1 %, higher
than those of the control (10.0–77.5 %) from early December to late January. All sporo-
phytes in both treatments had sporophyll beginning in early February.

Photosynthetic rates and uptake rates of NO3–N

The photosynthetic rates of both treatments in December increased with increased seawater
temperature from 2.5 to 20 °C and decreased thereafter. In January, the photosynthetic
rates at 5 °C were lower than those at 2.5 °C, and they increased from 5 to 25 °C (Fig. 6).
The temperature response curves of photosynthesis in December and January showed that

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Fig. 4 Temporal changes in 400

Total length (cm)

total length, stipe length, stipe 300 d
width, length of longest pinnate c
CD D abc
b
blade, number of pinnate blades, 200 a
sporophyll length, and sporophyll CD
100 B BC b b
width of the experimental (solid A
circles) and control (empty 0
circles) Undaria pinnatifida 60 c c

Stipe length (cm)

c D c
cultivated in Matsushima Bay.
Different small and large letters 40 ab
indicate statistical significance a D
bc CD
(P \ 0.01) of experimental and 20

*
B
control kelps among months, BC
A
respectively. Asterisks indicate 0
statistically significant 6 d

Stipe width (cm)

differences (P \ 0.01) between 5 cd cd E
c
experimental and control kelps. 4
As only two plants with 3 b cd
DE
sporophyll were found in early 2 a CD
December, no statistical B BC
1
significance in the sporophyll 0
A

parameter was analyzed

80
pinnate blade (cm)
Length of longest

d
60 cd D d
bc
ab
40
a d
CD D
20 B BC

A
0
cd
Nnumber of pinnate blade

100
Sporophyll width (cm) Sporophyll length (cm) (ind./oneside of a plant)

C C
80 B
e
60 C C bd
a be
be
40

20 A

0
40
d

30 cd
cd cd
c
20
b

10 a C C
BC
A AB
0
16 c
C
C
12 c c

8 b
c c

4 a B
B
A
0
Dec. Jan. Feb. Mar.
2008 2009

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Table 1 Statistical significance of morphological parameters of the experimental (E) and control (C)
Undaria pinnatifida cultivated in Matsushima Bay compared among months using the Kruskal–Wallis test
Morphological parameters v2 df p

Total length E: 77.7079 6 0.0000

C: 72.4993 5 0.0000
Stipe length E: 64.3255 6 0.0000
C: 79.0018 5 0.0000
Stipe width E: 87.5073 6 0.0000
C: 95.6761 5 0.0000
Length of longest pinnate blade E: 77.1040 6 0.0000
C: 79.7921 5 0.0000
Number of pinnate blades E: 82.5966 6 0.0000
C: 70.6893 5 0.0000
Sporophyll length E: 65.7399 6 0.0000
C: 45.1755 4 0.0000
Sporophyll width E: 68.1972 6 0.0000
C: 65.3935 4 0.0000
Blade weight E: 80.2602 6 0.0000
C: 89.8898 5 0.0000
Midrib weight E: 97.3420 6 0.0000
C: 104.5990 5 0.0000
Sporophyll weight E: 76.7840 6 0.0000
C: 65.3816 4 0.0000

photosynthetic rates of experimental and control kelps were highest at a water temperature
of 20 °C in December and 25 °C in January, respectively. In January, the photosynthetic
rates of the experimental kelps were significantly higher than those of the control at 10 and
15 °C.
The uptake rates of NO3–N in December and January increased with increased NO3–N
concentration (Fig. 7). In December, the uptake rate of the experimental kelps was sig-
nificantly higher than that of control kelps at 1/8 PESI. In January, the uptake rate of the
experimental kelps was significantly higher than that of the control kelps at 1/4 PESI.

Discussion

In the present study, the continuous culture of U. pinnatifida gametophytes under the
optimal temperature in control provided favorable conditions for their growth and matu-
ration and laid a solid foundation on the rapid growth and maturation of sporophytes.
Compared to relatively high temperature of above 20 °C, the gametophytes of Ecklonia
radiata, which also belongs to the Laminariales, exhibited optimal development, maximal
fecundity, and reproductive success at temperatures of 12 and 20 °C in saturating light
(Novaczek 1984). The gametophytes of Laminaria abyssalis, a brown kelp species from
the southern hemisphere, grew and became fertile at 18 °C, but they did not survive at
23 °C (Yocie 1990). Similarly, the formation of oogonia and spermatogonia in Eisenia
bicyclis gametophytes could not be observed when seawater temperature exceeded 20 °C

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Aquacult Int (2013) 21:53–64 61

Fig. 5 Temporal changes in dry 60

b
weight of blade, midrib, and 50
sporophyll of the experimental
(solid circles) and control (empty 40 b E

Blade (g)
b
circles) Undaria pinnatifida b
30
cultivated in Matsushima Bay.
Different small and large letters 20 a
a b
indicate statistical significance 10 D
(P \ 0.01) of experimental and B C CD
A
control kelps among months, 0
respectively. Asterisks indicate
25
statistically significant d d
D
differences (P \ 0.01) between 20
D
experimental and control kelps.
Midrib (g)

As only two sporophytes with 15 c

sporophyll were found in early
December, no statistical 10 d
b d
significance in the sporophyll
parameter was analyzed 5 a C
A B C
0
40
d
35
30
Sporophyll (g)

25
C
20 C
bc
15 b
10
5 a a
B bc cd
B
0
A
Dec. Jan. Feb. Mar.
2008 2009

(Taniguchi and Akiyama 1982). Based on these conclusions, the gametophytes culture at
the temperature of 20 °C in control may promote the growth and decrease the mortality.
Therefore, the technology of temperature control should be applied widely in gametophyte
culture of U. pinnatifida.
In general, nutrient concentration is inversely correlated with seawater temperature,
which has been documented by Jackson (1977) and Zimmerman and Kremer (1984). The
concentrations of NH4–N and NO3–N in Matsushima Bay during our study were lower
than those in 2002 (Sasaki et al. 2002). Previous researches have shown that nutrients were
considered to be a significant ecological factor affecting the growth and maturation of
gametophytes (Hoffman and Santelices 1982; Kinlan et al. 2003). Other studies have
demonstrated that the decline in early development (Wheeler and North 1980) from
summer to autumn and the large-scale decline in algal productivity (Gerard 1997) results
from nitrogen limitation. However, the addition of nutrients to gametophytes has been
shown to promote the growth and production of M. pyrifera even at relatively high sea-
water temperatures (Deysher and Dean 1986). Nutrient limitation resulted in delayed
development and inhibition of reproduction of the giant kelp M. pyrifera gametophytes, but
these small gametophytes could then rapidly and consistently produce sporophytes once
nutrients increase (Carney and Edwards 2010). Therefore, the adequate nutrient supply to

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Fig. 6 Temperature response 60

curves of photosynthetic rates of December
50
the experimental (solid circles)
and control (empty circles) 40
Undaria pinnatifida cultivated in

Oxygen evolution (µL/cm 2/h)

Matsushima Bay in December 30
2008 and January 2009. Asterisks 20
indicate statistically significant
differences (P \ 0.01) between 10
experimental and control kelps
0
60
January
50

40

30

20

10

0
0 5 10 15 20 25 30 35
Temperature ( )

December January
200
Uptake rate (µg/L/cm2/h)

150

100

50

0
0 1000 2000 3000 0 1000 2000 3000
Initial concentration (µg/L)

Fig. 7 Uptake rates of NO3–N of the experimental (solid circles) and control (empty circles) Undaria
pinnatifida cultivated in Matsushima Bay in December 2008 and January 2009. Asterisks indicate
statistically significant differences (P \ 0.01) between experimental and control kelps

gametophytes could play a vital role in the acceleration of growth and maturation of
sporophytes afterward, so as to shorten the time to complete their life cycles.
In the present study, compared to the control kelp, significantly greater physiological
parameters of the experimental kelps in December or January and higher percentages of the
experimental kelps with sporophyll until late January were shown. Furthermore, signifi-
cantly higher photosynthesis at seawater temperatures of 10 and 15 °C and significantly
greater uptake rates of NO3–N of the experimental kelps compared to the control kelp
occurred in December and January. All of these were due to the higher growth rate of the
experimental kelps. The decrease in the number of pinnate blades and no increase in blade
weight of the experimental kelps were due to the promotion of maturation of sporophytes

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and subsequent early withering. The improvement of photosynthetic rates in the experi-
mental kelps in January likely was caused by higher chlorophyll concentrations affecting
the photosynthetic activity (Dring 1982). Increases in sporophyll weight of the experi-
mental kelps until mid-March suggest resource storage and translocation of resources from
the blades via photosynthesis and nutrient uptake.
In conclusion, the results of this study show that nitrate fertilization of gametophytes
makes it possible to harvest the high-priced blade, midrib, and sporophyll of U. pinnatifida
in January under poor nutrient conditions. The improved cultivation method should be
popularized vigorously for higher income. The harvest prior to the outbreak of pinhole
disease can minimize a loss. In addition, other commercial kelps, such as Laminaria
japonica and Gracilaria chorda, can be cultivated after harvesting of U. pinnatifida owing
to the shortening of cultivation period, which increases the rate of utilization of marine
ranching.

Acknowledgements We sincerely thank Professor M. Sato of the Graduate School of Agricultural Sci-
ence, Tohoku University for his critical comments on this manuscript. We also thank Mr. M. Ise of the
Shiogama Daiichi Fisheries Cooperative Association for providing the seedlings used in the present study
and for his kind cooperation with kelp cultivation and collection, the staff of the Miyagi Prefecture Fisheries
Technology Institute for using seawater for culture experiment and for providing the long-term water
temperature data, and Professor O. Nishimula and Mr. Chiba for helping with the analysis of nutrient
concentration.

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