Escolar Documentos
Profissional Documentos
Cultura Documentos
DOI 10.1007/s10499-012-9533-5
Received: 1 September 2010 / Accepted: 14 April 2012 / Published online: 6 May 2012
Ó Springer Science+Business Media B.V. 2012
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
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54 Aquacult Int (2013) 21:53–64
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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.
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.
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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56 Aquacult Int (2013) 21:53–64
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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Aquacult Int (2013) 21:53–64 57
temperature in Matsushima Bay from 1979 to 2004 were obtained from the Miyagi
Prefecture Fisheries Technology Institute.
Statistical analysis
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
20
15
10
0
D J F M A M J J A S O N
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58 Aquacult Int (2013) 21:53–64
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.
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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Aquacult Int (2013) 21:53–64 59
*
B
control kelps among months, BC
A
respectively. Asterisks indicate 0
statistically significant 6 d
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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60 Aquacult Int (2013) 21:53–64
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
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
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)
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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62 Aquacult Int (2013) 21:53–64
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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Aquacult Int (2013) 21:53–64 63
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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