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and total and free amino acid contents in each thallus part.
2. Materials and methods
(1) Sea urchin collection, experimental design and rearing conditions
Three treatments of sea urchins fed the sporophyll (SU) and the midrib (MU) of U.
pinnatifida and the basal frond portion of S. japonica (BS) were designed. A total of 45 adult M. nudus (46–54 mm diameter) were collected by scuba dive from a barren at depths of 2.5–3.0 m off Nojima Island, Shizugawa Bay, Miyagi Prefecture (38°40´N, 141°30´E) on 16 May 2016. Immediately after collection, the sea urchins were kept in a cool box for approximately 1 h with moist urethane mats immersed in seawater and then transported to Onagawa Field Center, Tohoku University in Onagawa (38°26΄N, 141°27΄E). The sea urchins were held in each of nine 10 L aquaria at density of 5 individuals/aquarium (three aquaria for each treatment). The aquaria for each treatment were designated randomly.
The sea urchins were reared in running filtered sea water that was pumped from offshore, filtered twice using a Myclean Filter (AF515, Tanaka Sanjiro Co., Ltd.), aerated and exchanged twice or three times per hour. The sea urchins were reared without food for 4 days until the start of the experiment. The feeding experiment was conducted from 20 May to 11 July 2016. I used fresh U. pinnatifida and S. japonica kelp cultivated off Fudai, Iwate Prefecture (40°01΄N, 141°54΄E) as the feed for the sea urchins. The basal frond portion of S. japonica, cut into five equal lengths from the base to the apical portion, and the sporophyll and midrib of U. pinnatifida were fed to sea urchins in aquaria ad libitum every 3–7 days. The midrib of U. pinnatifida and the basal frond portion of S. japonica were designated as negative and positive controls, respectively. In addition to the sea urchins for the feeding experiment, 30 individuals were collected from the same site on
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the same day to examine gonad quality prior to the feeding. The seawater temperature in the aquaria was measured every 10 minutes using a wireless data logger (RTR52A, T&D). Each an individual fed the sporophyll and midrib of U. pinnatifida died in the beginning of the experiment and was culled immediately after the observation.
TDs and BWs of sea urchins at the start and the end of the experiment were measured with a vernier caliper and an electronic balance, respectively. The other 30 sea urchins collected at the start of the experiment and all sea urchins by treatment at the end of the experiment were dissected and used for measurements and analyses of gonad size, development, and color and free amino acid content of the gonads same as those in Section 1of Chapter 2.
(2) Protein, carbohydrate, TAA and FAA contents in each kelp part
Five samples of each U. pinnatifida and S. japonica part (approximately 200 g) were frozen at −30°C on 21 June 2016, in the middle of the experimental period. These samples were freezedried and pulverized. The NaOHsoluble protein, TAA and FAA contents of the frond portions was analyzed by the same method in Section 1 of Chapter 3. The carbohydrate content was obtained by analyzing the total sugar content using the phenol
sulfuric acid method (Dubois et al. 1956). To extract sugar, ca. 2–5 mg samples of each of pulverized thallus parts were extracted in 1 ml of distilled water by autoclaving at 121 °C for 1 h. Dglucose was used as the standard.
(3) Statistical analysis
Data were tested for normality (ShapiroWilk Wtest) and homogeneity of variances (Levene’s test). If the normality and homogeneity of variances were not detected, the data
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were logtransformed. Significant differences in TD and BW between the sea urchins in each treatment and the other 30 sea urchins collected at the start of the experiment were analyzed by ttest. Significant differences in TD and BW among treatments at the start and the end of experiment and in gonad index, L*, a* and b* values, and FAA contents in gonads among treatments at the end of experiment were analyzed by nested ANOVA with R ver. 3.4.0 (R Core Team 2017) using RStudio ver. 1. 0.143 (RStudio Inc., Boston, MA, USA). Significant differences in protein, carbohydrate, TAA and FAA contents among three thallus parts were analyzed by ANOVA. Tukey’s multiple comparison test was performed as a post hoc test. Except for the nested ANOVA, all analyses were conducted using JMP 10 (SAS Institute Inc.).
3. Results
(1) Water temperature
The water temperature varied in May from a maximum of 16.4°C to a minimum of 14.7°C, before decreasing to an overall minimum of 14.0°C in early June (Figure 22).
Then, it sharply increased in early June and reached a maximum of 20.3°C in the beginning of July.
(2) Protein, carbohydrate, TAA and FAA content of kelp
The protein and carbohydrate contents in the basal frond portion of S. japonica and in the sporophyll and midrib of U. pinnatifida fed to M. nudus are shown in Table 23. The protein and carbohydrate contents in the sporophyll of U. pinnatifida were significantly higher than those in the midrib of U. pinnatifida and the basal frond portion of S. japonica (p < 0.01). The protein content in the basal frond portion of S. japonica was significantly
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Figure 22. Daily water temperatures in aquaria.
Water temperature (°C)
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Table 23. Protein and carbohydrate contents (%) in the basal frond portion of Saccharina japonica and the sporophyll and midrib of Undaria pinnatifida (mean ± SE). Significant values among thallus parts as determined by ANOVA are shown. Lowercase letters indicate significant differences among the three thallus parts (p < 0.05).
Saccharina japonica Basal frond portion
Undaria pinnatifida
Sporophyll Midrib df MS F P
Protein 3.7 ± 0.1b 6.8 ± 0.2a 3.0 ± 0.1c 2 20.13 169.26 < 0.001 Carbohydrate 22.1 ± 1.0b 34.4 ± 2.2a 21.3 ± 2.5b 2 269.57 13.43 < 0.001
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higher than that in the midrib of U. pinnatifida (p < 0.05). However, there was no significant difference in carbohydrate content between the basal frond portion of S.
japonica and the midrib of U. pinnatifida.
Results of TAA and FAA analyses of each kelp part fed to M. nudus are shown in Figure 23. There were significant differences in the contents of 16 TAAs and 14 FAAs among the thallus parts (Table 24). Of the TAAs, the total Glu content in the basal frond portion of S. japonica and the sporophyll of U. pinnatifida were significantly higher than that in the midrib of U. pinnatifida (p < 0.001). The TAA content in the basal frond portion of S. japonica was ca. 109 mg/100 g higher than that in the sporophyll of U. pinnatifida.
The total Ala and Gly contents in the sporophyll of U. pinnatifida were significantly higher than those in other kelp parts (p < 0.05). The total Ala content in the sporophyll was ca. 205 mg/100 g, whereas those in the basal frond portion (ca. 78 mg/100 g) of S.
japonica and in the midrib (ca. 45 mg/100 g) were lower. The total Thr, Arg, His, Ile, Leu, Lys, Met, Phe, Tyr, Val and taurine contents in the sporophyll of U. pinnatifida and the basal frond portion of S. japonica were significantly higher than those in the midrib of U.
pinnatifida (p < 0.01). Of the FAAs, the Glu content in the basal frond portion of S.
japonica was ca. 6.6 and 29.0 times higher than those in the sporophyll and midrib of U.
pinnatifida, respectively. The Ala and Gly contents in the sporophyll of U. pinnatifida were significantly higher than those in the other kelp parts (p < 0.001). The Ala content in the sporophyll of U. pinnatifida was ca. 184 mg/100 g, markedly higher than those in the basal frond portion of S. japonica (30.3 mg/100 g) and in the midrib of U. pinnatifida (12.8 mg/100 g).
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Figure 23. Total and free amino acid contents (mg/100 g) in the basal frond portion of Saccharina japonica (filled bars), and sporophyll (open bars) and midrib (gray bars) of Undaria pinnatifida (mean ± SE). Lowercase letters indicate significant differences among the three thallus parts (P < 0.05). Asp, aspartic acid;
Glu, glutamic acid; Ala, alanine; Gly, glycine; Pro, proline; Ser, serine; Thr, threonine; Arg, arginine; His, histidine; Ile, isoleucine; Leu, leucine; Lys, lysine; Met, methionine; Phe, phenylalanine; Tyr, tyrosine; Val, valine; Tau, taurine; Cys, cysteine; Orn, ornithine; Phosphoserine, PSer.
Asp Glu Ala Gly Pro Ser Thr Arg His Ile Leu Lys Met Phe Tyr Val Tau Cys Orn P-Ser
Total amino acids
Free amino acids
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Am ino Ac id C ont en t (m g/ 100 g)
a a
b a
b b a
a
b b
a
b b b b b b a a a b a b a b a b b a
b b a b a a b
b b a b a b a
a
a
b a
b b
b a
c a
b c
a a
b a a
b a a b a a b
a a
b a a
b a b a a a
b a a
b b
a a
a b a 150
200 250 300 350
50 0
a b b
100 150 200
50 0
ND a
ND ND
Basal frond portion of S. japonica Sporophyll of U. pinnatifida Midrib of U. pinnatifida
a a b
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Table 24. Results of ANOVA of total and free amino acid contents (mg/100 g) among treatments of the basal frond portion of Saccharina japonica and the sporophyll and midrib of Undaria pinnatifida fed to Mesocentrotus nudus.
TAA FAA
df MS F P df MS F P
Aspartic acid Treatment 2 5581.09 3.63 0.059 2 16.51 44.47 < 0.001
Error 12 1539.29 12 0.37
Glutamic acid Treatment 2 0.91 22.96 < 0.001 2 9.25 8.50 0.005
Error 12 0.04 12 1.09
Alanine Treatment 2 35687.80 26.92 < 0.001 2 10.65 32.36 < 0.001
Error 12 1325.60 12 0.33
Glycine Treatment 2 6943.83 22.42 < 0.001 2 4976.68 36.92 < 0.001
Error 12 309.67 12 134.80
Proline Treatment 2 0.99 88.12 < 0.001 2 21.67 6.79 0.011
Error 12 0.01 12 3.19
Serine Treatment 2 0.34 1.58 0.246 2 5.89 13.06 0.001
Error 12 0.22 12 0.45
Threonine Treatment 2 2551.36 22.86 < 0.001 2 7.96 16.30 < 0.001
Error 12 111.59 12 0.49
Arginine Treatment 2 0.46 48.56 < 0.001 2 4.99 30.46 < 0.001
Error 12 0.01 12 0.16
Histidine Treatment 2 207.13 17.88 < 0.001
Error 12 11.59
Isoleucine Treatment 2 1087.20 16.99 < 0.001 2 4.87 3.14 0.080
Error 12 63.98 12 1.55
Leucine Treatment 2 3627.64 17.79 < 0.001 2 0.55 3.38 0.069
Error 12 203.87 12 0.16
Lysine Treatment 2 2204.75 18.42 < 0.001 2 4.44 5.11 0.025
Error 12 119.70 12 0.87
Methionine Treatment 2 0.45 17.51 < 0.001 2 4.47 4.10 0.044
Error 12 0.03 12 1.09
Phenylalanine Treatment 2 1353.17 15.74 < 0.001 2 1.52 3.31 0.071
Error 12 85.97 12 0.46
Tyrosine Treatment 2 743.87 20.82 < 0.001 2 6.90 2.87 0.096
Error 12 35.73 12 2.40
Valine Treatment 2 2122.14 22.51 < 0.001 2 3.92 14.19 < 0.001
Error 12 94.26 12 0.28
Taurine Treatment 2 1.02 132.46 < 0.001 2 4.69 29.33 < 0.001
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Significant (p< 0.05) results are shown in bold.
Error 12 0.01 12 0.16
Cysteine Treatment 2 11.01 7.40 0.008
Error 12 1.49
Ornithine Treatment 2 0.38 2.24 0.149
Error 12 0.17
Phosphoserine Treatment 2 4.29 631.96 < 0.001 2 7.70 6.48 0.012
Error 12 0.01 12 1.19
(Continued)
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(3) Body and gonad size and gonad development and color
At the start of the experiment, the TD and BW of the sea urchins in the feeding treatments were 49.9 ± 0.3 cm and 58.0 ± 1.2 g (mean ± SE), respectively, which were not significantly different from those of the other 30 sea urchins collected (TD, df = 1, MS = 6.60, F = 1.53, p = 0.220; BW, df = 1, MS = 2.31, F = 0.04, p = 0.850). There were no significant differences in TD and BW among treatments (Table 25). The TD, BW and gonad indices of M. nudus at the start and the end of the experiment are shown in Table 26. There were no significant differences in TD and BW among treatments at the end of the experiment (Table 25). The gonad index increased from 5.6 to 21.9 in SU, which was significantly higher than that of MU (p < 0.05). At the end of the experiment, the gonads of almost all sea urchins were in the growing stage, with increasing numbers of spermatocytes or early vitellogenic oocytes along the acinal wall and NPs filling the lumen (Table 27). One and two individuals of BS and SU had premature gonads, respectively. The gonad color (L*, a* and b* values) of the gonads from each treatment is shown in Table 28 Although significant differences in L* and a* values among treatments were detected by nested ANOVA (Table 25), they were not detected by Tukey’s test.
(4) FAA contents of the gonad
The FAA contents in gonads are shown in Figure 24. Significant differences among treatments in all FAA contents except ornithine content were detected by nested ANOVA (Table 25). The Glu content in SU was significantly higher than those in other treatments (p < 0.01). The Ala content in the gonads of each treatment markedly increased from ca.
124 mg/100 g to > 400 mg/100 g. The Ala contents in BS and SU were significantly
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Table 25. Results of nested ANOVA on test diameter, body weight, gonad index and color, and free amino acid contents in the gonads of Mesocentrotus nudus among treatments.
df MS F P
At the start of the experiment
Test diameter Treatment 2 1.44 0.295 0.746
Treatment: Aquarium 3 0.88 0.180 0.909
Body weight Treatment 2 27.98 0.394 0.677
Treatment: Aquarium 3 7.85 0.110 0.954
At the end of the experiment
Test diameter Treatment 2 2.19 0.390 0.680 Treatment: Aquarium 3 0.29 0.052 0.984
Body weight Treatment 2 75.82 1.266 0.294
Treatment: Aquarium 3 44.86 0.749 0.530 Gonad index Treatment 2 3.17 104.029 < 0.001
Treatment: Aquarium 3 0.02 0.646 0.590
L* Treatment 2 1822.10 30.843 < 0.001
Treatment: Aquarium 3 29.10 0.493 0.689
a* Treatment 2 26.70 5.561 0.008
Treatment: Aquarium 3 3.88 0.808 0.498
b* Treatment 2 34.83 0.507 0.607
Treatment: Aquarium 3 31.24 0.454 0.716 Aspartic acid Treatment 2 50.33 11.511 < 0.001
Treatment: Aquarium 3 1.82 0.416 0.743 Glutamic acid Treatment 2 366155.00 140.570 < 0.001
Treatment: Aquarium 3 3622.00 1.390 0.261 Alanine Treatment 2 87649.00 166.785 < 0.001
Treatment: Aquarium 3 7881.00 1.506 0.229 Glycine Treatment 2 177406.00 11.790 < 0.001
Treatment: Aquarium 3 13391.00 0.890 0.455 Proline Treatment 2 84.61 28.324 < 0.001
Treatment: Aquarium 3 1.77 0.594 0.623 Serine Treatment 2 13007.00 38.237 < 0.001
Treatment: Aquarium 3 534.00 1.568 0.214 Threonine Treatment 2 461.20 13.318 < 0.001
Treatment: Aquarium 3 19.20 0.556 0.648
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Arginine Treatment 2 126114.00 10.312 < 0.001 Treatment: Aquarium 3 3194.00 0.261 0.853
Histidine Treatment 2 349.10 4.992 0.012
Treatment: Aquarium 3 39.10 0.558 0.646
Isoleucine Treatment 2 333.00 4.977 0.012
Treatment: Aquarium 3 32.60 0.487 0.694 Leucine Treatment 2 4048.00 14.222 < 0.001
Treatment: Aquarium 3 96.00 0.336 0.799 Lysine Treatment 2 108224.00 18.924 < 0.001
Treatment: Aquarium 3 4353.00 0.761 0.523 Methionine Treatment 2 300.95 18.012 < 0.001
Treatment: Aquarium 3 14.05 0.841 0.480 Phenylalanine Treatment 2 593.10 14.476 < 0.001
Treatment: Aquarium 3 27.30 0.667 0.577 Tyrosine Treatment 2 2249.20 8.900 < 0.001
Treatment: Aquarium 3 114.90 0.455 0.716 Valine Treatment 2 3197.00 9.540 < 0.001
Treatment: Aquarium 3 187.00 0.557 0.647 Taurine Treatment 2 25364.00 32.890 < 0.001
Treatment: Aquarium 3 108.00 0.140 0.935
Ornithine Treatment 2 3.16 1.996 0.150
Treatment: Aquarium 3 1.47 0.928 0.437 Significant (p < 0.05) results are shown in bold.
(Continued)
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Table 26. Test diameters (mm), body weights (g) and gonad indices of Mesocentrotus nudus of each treatment at the start and end of the experiment (mean ± SE). Lowercase letters indicate significant differences among treatments (p < 0.05). The initial gonad index indicates that of 30 sea urchins dissected at the start of the experiment.
Initial, BS, SU and MU indicate sea urchins at the start of the experiment, sea urchins fed the basal portion of the Saccharina japonica frond, and sea urchins fed the sporophyll and midrib of Undaria pinnatifida, respectively.
Initial BS SU MU
Test diameter 49.9 ± 0.3 50.2 ± 0.5 49.9 ± 0.2 50.6 ± 0.3 Body weight 58.0 ± 1.2 60.9 ± 1.1 58.4 ± 1.2 57.0 ± 2.6 Gonad index 5.6 ± 0.5 19.1 ± 0.2ab 21.9 ± 0.1a 17.5 ± 1.3b
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Table 27. Gonad developmental stages of Mesocentrotus nudus of each treatment at the start and end of the experiment, by sex.
An explanation of Initial, BS, SU and MU is provided in Table 26. I, II and III indicate the recovery, growth and premature stages.
I II III
Male Female Male Female Male Female
Initial 2 1 16 11
BS 8 6 1
SU 6 6 2
MU 6 8
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Table 28. Gonad color (L*, a* and b* values) of gonads of Mesocentrotus nudus of each treatment at the start and end of the experiment (mean ± SE).
An explanation of Initial, BS, SU and MU is provided in Table 26.
Initial BS SU MU
L* 45.7 ± 1.3 56.0 ± 0.5 57.5 ± 0.6 56.0 ± 1.3 a* 10.8 ± 0.4 10.3 ± 0.6 8.6 ± 0.3 10.2 ± 0.6 b* 37.5 ± 1.1 36.8 ± 0.9 33.7 ± 0.5 35.8 ± 1.3
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Figure 24. Free amino acid (FAA) contents (mg/100 g) in gonads of Mesocentrotus nudus of each treatment at the start and end of the experiment (mean
± SE). Lowercase letters indicate significant differences among treatments (p < 0.05). An explanation of Initial, BS, SU and MU, and amino acids is provided in Table 26 and Figure 22, respectively.
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higher than that in MU (p < 0.05). The Arg and Lys contents in the gonads of each treatment decreased to almost half of its initial value. The Pro and Thr contents in BS were significantly higher than those in the other treatments (p < 0.05). Ile, Leu, Met, Phe, Tyr and Val contents in SU and MU were significantly lower than those in BS (p < 0.05).
4. Discussion
In the present study, the SU treatment had the highest gonad index, which could reflect the higher protein and carbohydrate levels in the sporophyll of U. pinnatifida than those in other thallus parts. This is similar to the results of past studies (e.g., de Jong-Westman et al. 1995; Pearce et al. 2002b; Heflin et al. 2012; 2016). βcarotene in feed affects the gonad color of sea urchins (Robinson et al. 2002; Pearce et al. 2003; Shpigel et al. 2005; 2018). Robinson et al. (2002) reported that an artificial feed containing approximately 250 mg/kg (dry weight) βcarotene produced more yellow gonads than those containing 50 and 100 mg/kg βcarotene. The gonad color of sea urchins fed artificial feeds containing 27.1 µg/g(dry weight) and 33.0 µg/gcarotenoids did not differ (Baião et al. 2019). The lack of difference in gonad color among treatments might be attributable to the small difference in βcarotene content among the thallus parts.
Of the TAA and FAA contents, Ala was the highest in the sporophyll and midrib of U.
pinnatifida. Nisizawa et al. (1987) reported that Leu is the most abundant TAA in the whole frond of U. pinnatifida followed by Val, Glu and Asp. The midrib had low TAA and FAA contents overall, but a high Ala content was found in the midrib for the first time in this present study. Gao et al. (2013) reported that nitrogen and carbon contents in the sporophyll of U. pinnatifida cultivated in Matsushima Bay, Miyagi Prefecture, Japan, increased between April and May, and resources were translocated from blades to sporophylls for maturation after the cessation of growth. The seawater temperature at a
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depth of 2.5 m in Kamaishi Bay and Ofunato Bay, Iwate Prefecture, near Fudai, where the U. pinnatifida used in this study were cultivated, exceeded 13°C, which initiated the release of the zoospore from the sporophyll (Saito 1962) after June in 2016 (Iwate Fisheries Technology Center 2017). Ala would have been translocated from the blades to the sporophylls though the midrib and the stipe for maturation as a highconcentrated amino acid during the experimental period. Maturation occurs in the sporophyll of U.
pinnatifida and in the frond of S. japonica in summer. In addition, carbon and nitrogen are accumulated in the basal frond portion of S. japonica for regrowth to form a second plant (Li et al. 2007). The total and free Glu contents in the basal frond portion, which were higher than those in the apical and middle frond portions (Section 1 of Chapter 3), were also high compared to those in the sporophyll and midrib of U. pinnatifida. Thus, the amino acids required for the maturation and growth of Laminariales might differ.
The present study showed equally high Ala contents in the gonads of SU and BS and higher Glu content in the gonads of SU than in the gonads of BS. In Section 1, Chapter 3, I suggested that the high content of free Ala in the gonads of M. nudus fed the basal frond portion of S. japonica was synthesized from the high content of total Glu in the basal frond. However, the markedly high total Glu content in the basal frond portion of S.
japonica compared to that in the sporophyll of U. pinnatifida resulted in equal amounts of free Ala in the gonads of BS and SU. The free Ala content in the sporophyll of U.
pinnatifida was higher than that in other thallus parts. Alanine aminotransferase, which synthesizes Glu and pyruvic acid from Ala and 2oxoglutarate and reacts reversibly (Brosnan and Brosnan 2009), has been identified from the egg to the pluteus larva in L.
variegatus (Black 1964). The genome has been sequenced in some sea urchin species (HpBase: HPU_17238; NCBI: LOC580780). This evidence raises the possibility that
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total Ala, if sufficiently provided by the feed, would be synthesized to Glu, and the surplus would be directly accumulated in the gonads. Past studies revealed high bittertasting FAA contents in the gonads of sea urchins fed high protein containing feeds (Hoshikawa 1998; Osako et al. 2006; Inomata et al. 2016). However, bittertasting FAA contents in the gonads of SU were lower than those of BS regardless of higher protein content in the sporophyll of U. pinnatifida than that in the basal frond portion of S. japonica in the present study.
Feeding the whole frond of S. japonica to M. nudus from a barren from May–July improved gonad quality and surpassed the gonad taste of sea urchins from a fishing ground (Section 3, Chapter 2). The desirable taste was caused by the sweetness from the high Ala and low Arg contents and the strong umami flavor. I found the highest Ala content in the gonads of M. nudus fed the basal frond portion of S. japonica in Section 1, Chapter 3. In the present study, the Ala and Arg contents in the gonads of SU were the same as those in the gonads of BS. In addition, the higher Glu and lower bittertasting FAA contents in the gonads of SU than in those of BS suggest that the sporophyll of U.
pinnatifida would improve the gonad taste more than the basal frond portion of S.
japonica.
In the present study, feeding the sporophyll of U. pinnatifida, with its high protein, carbohydrate, and total and free alanine contents, to M. nudus from a barren improved M.
nudus gonad size and color, and increased Ala content and decreased Arg content, which was the same result as feeding the basal frond of S. japonica to M. nudus. Feeding the sporophyll increased free glutamic acid and decreased bittertasting free amino acid contents in the gonads of sea urchins compared to the contents after feeding the sea
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urchins the midrib and basal frond portion of S. japonica. Feeding the sporophyll to M.
nudus improved gonad taste more than feeding the basal frond portion of S. japonica.
High Ala content in the sporophyll might contribute to high Glu and Ala contents in the gonads of sea urchins. The appropriate period when gonad quality can be improved must be confirmed in order to shorten the culture duration to reduce the cost.
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