62
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cultured in cages suspended offshore, using quantitative measurements and analyses, and sensory evaluation.
2. Materials and methods (1) Cage culture
A total of 500 adult M. nudus (47–54 mm diameter) were collected by scuba diving from a barren 2.5–3.0 m deep off Nojima Island, Shizugawa Bay, Miyagi Prefecture (38° 40′ N, 141° 30′ E) on 6 May 2016. Immediately after collection, sea urchins were placed in five cages (100 individuals per cage) suspended horizontally along a straight line at a depth of approximately 4.5 m at a sheltered site off Areshima Island (38° 40′
N, 141° 27′ E) until 19 July 2016. The cages were cuboid (90 × 87 × 45 cm) with 3 cm meshes made of polyethylene. The sea urchins were starved for 5 days before the start of the experiment. The culture experiment was conducted for 63 days from 11 May until 13 July 2016. Fresh cultivated S. japonica was fed to the sea urchins ad libitum every 7–10 days. Water temperature was measured every 15 min by three data loggers (HOBO UA00264, Onset) attached to the upper surface of three of the cages. Daily water temperature was calculated as an average of 96 data points over 24 h from the three loggers.
(2) Sea urchin sample collection
On 16 May, the gonad quality of 30 urchins (47–54 mm diameter) collected from the same barren where the urchins were collected for the culture experiment were examined to evaluate gonad quality prior to culture. On 13 July, at the end of culture period, the gonad quality of 30 randomly selected urchins from the culture cages, 30 urchins from
64
the barren, and 30 urchins from an Eisenia bicyclis kelp bed at depths of 2.2–3.0 m off Nojima Island were assessed. Thus, four different sea urchin groups were established:
urchins from the barren at the start of the culture period (BS), urchins from the culture cages at the end of the culture period (CE), urchins from the barren at the end of the culture period (BE), and urchins from the E. bicyclis bed at the end of the culture period (EE). Urchins from each of these groups were transported to the Marine Plant Ecology Lab, Tohoku University in Sendai (38° 28′ N, 140° 87′ E) within 2 h of collection. Measurements and analyses of TD, BW, gonad index, gonad hardness and color, and FAA contents in gonads were conducted same as Section 1 of Chapter 2. As gonad hardness changes with gonad development from the recovering to the mature stages (McBride et al. 2004; Section 1), when the gonad increases in size (Unuma and Walker 2009), the correlation between gonad indices and gonad hardness was analyzed across groups.
(3) Sensory evaluation
The sea urchins in the culture cages were not fed from 13 July. On 19 July, 20 urchins from the culture cages, 20 urchins (ca. 50 cm TD) from an E. bicyclis bed and 20 urchins from the barren were collected. After collection, the sea urchins were kept in two cool boxes with moist urethane mats immersed in seawater. The urchins were then transported to the Riken Food Co. Ltd. Factory in Tagajo, Miyagi Prefecture (38° 16′ N, 141° 0′
E). After the sea urchins were dissected, the gonads were soaked three times in sterile seawater at 5 °C. The gonads were then drained using cotton on some strainers at 5 °C for 30 min according to Kinoshita et al. (2009). Tasting panels consisted of three cooks and six people associated with the sea urchin fishery who are familiar with the taste of
65
sea urchin gonads. Gonad quality was evaluated for each group. Three to four pieces of gonad from an individual group was placed on a white dish for each tasting panel. The panel evaluated the desirability of gonad color, shape, and smell using five grades: −2 (undesirable), −1 (slightly undesirable), 0 (moderate), +1 (slightly desirable), and +2 (desirable). After eating the gonads, the panel evaluated the strength of gonad sweetness, umami, bitterness, and saltiness with the five grades: −2 (weak), −1 (slightly weak), 0 (moderate), +1 (slightly strong), +2 (strong), and the desirability of gonad texture, richness, overall taste, and overall quality (including appearance and taste) with the five grades: −2 (undesirable), −1 (slightly undesirable), 0 (moderate), +1 (slightly desirable), +2 (desirable). After cleansing their palates, the panel evaluated the gonads of the other groups in the same way.
(4) Statistical analysis
Statistical analyses were conducted using JMP 10 (SAS Institute Inc.), with the exception of general linear model (GLM) ANOVA and principle component analysis (PCA). GLM ANOVA was conducted using R ver. 3.4.0 (R Core Team 2017) using RStudio ver. 1.0.143 (RStudio Inc., USA). AIC values determined that the distribution of the GLM of gonad indices; gonad hardness; L*, a*, and b* values; and His content were Gaussian, and TD, BW, and FAA contents except for His content were Gamma.
Significant differences among groups, between sex, and interactions of the traits with sea urchin size and gonad quality were analyzed by GLM ANOVA, followed by SteelDwass multiple comparison test, when homoscedasticity of some data were not detected by Levene test. When GLM ANOVA showed no significant differences in traits between sex, and interactions between groups and sex, the data for both sexes combined were analyzed
66
by SteelDwass multiple comparison test. When there were significant sexual differences without interactions, the multiple comparison test among groups was conducted by sex.
When there were significant interactions in the traits between groups and sex, the sex
independent data were analyzed using the multiple comparison test. To evaluate the correlations between FAA content and association of groups by sex, FAA content data was analyzed by PCA using Canoco 5 (ter Braak and Šmilauer 2012). Statistical differences in sensory scores of the gonads among groups were analyzed by the Steel
Dwass multiple comparison test, following the KruskalWallis test because normality and homoscedasticity of some data were not detected by the ShapiroWilk W test and Levene test, respectively. To evaluate consistency of gonad quality among individuals, the coefficient of variation (standard deviation/average × 100) of the traits for gonad qualities were calculated. A correlation between gonad index and hardness was analyzed using combined data from all groups (n =120) using Spearman’s rank correlation.
3. Results
(1) Water temperature
Daily water temperature is shown in Figure 14. The average water temperature during the experimental period was 15.5 °C. The water temperature increased from 10.4 °C at the start of the experiment to 15.2 °C at the end of May, decreased rapidly in early June, and then increased to a peak of 19.4 °C in late June. Water temperate changed erratically from midJune until the end of the culture period in midJuly.
(2) Urchin body size and age
TD and BW of M. nudus from each group are shown in Table 11. No significant
67
Figure 14. Daily water temperatures at the upper surface of cultured cages.
68
Table 11. Test diameters (T.D.), body wet weight (B.W.), and gonad index (mean ± SE) for each group of Mesocentrotus nudus.
Significances among groups, between sex, and group and sex as determined by GLM ANOVA are shown. A, B, C and D indicate significant difference among groups (p < 0.05).
BS, CE, EE and BE indicate sea urchins collected from a barren at start of the culture period (BS), from the culture cage at the end of culture period (CE), Eisenia bicyclis kelp bed at the end of the culture period (EE) and the barren at the end of the culture period (BE).
BS CE EE BE p
Group Sex Group × Sex
T.D. (mm) 50.5 ± 0.4 51.4 ± 0.3 51.4 ± 0.4 50.3 ± 0.4 0.063 0.388 0.924 B.W. (g) 57.6 ± 1.5AB 62.7 ± 1.4A 63.6 ± 1.6A 56.0 ± 1.3B < 0.001 0.684 0.679 Gonad index 5.6 ± 0.5D 19.0 ± 0.4B 21.6 ± 0.7A 9.0 ± 0.7C < 0.001 0.984 0.847
69
difference in TD was detected among groups. BW differed significantly among groups.
There were no significant differences in TD and BW between sex, and significant interactions of group and sex. BW of the CE and EE groups were significantly higher than the BE group (p < 0.01). The BS, CE and BE, and EE groups comprised individuals of 3 and 4 years of age and 2 to 4 years of age, respectively (Table 12).
(3) Gonad development and gonad index
The gonad developmental stages of M. nudus by sex in each group are shown in Table 13. Ninety percent of gonads from the BS group were in the growing stage, with increasing numbers of spermatocytes or early vitellogenic oocytes along the acinal wall, and NPs filling the lumen. A few gonads were in the recovering stage with small numbers of primary spermatocytes or previtellogenic oocytes along the acinal wall, and NPs filling the lumen. At the end of the culture period, the majority of gonads from all groups were in the growing stage. Some gonads of the CE, EE, and BE groups were in the premature stage with spermatozoa or ova at the center of the lumen, and with spermatocytes and vitellogenic oocytes along the acinal wall.
Gonad indices differed significantly among groups (Table 11). There were no significant differences between sex or significant interactions between group and sex. The gonad index of the CE group was significantly higher than for BS and BE (p < 0.001), although it was lower than that of EE (p < 0.01). The coefficient of variation of the gonad index of CE was the lowest compared to other groups and was markedly lower than the gonads of the BS group (Table 14).
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Table 12. Age composition of each group of Mesocentrotus nudus.
An explanation of the terms BS, CE, EE and BE is provided in Table 11.
Group II III IV
BS 20 10
CE 17 13
EE 7 13 10
BE 23 7
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Table 13. Gonad developmental stages in each Mesocentrotus nudus group.
I, II and III indicate the recovering, growing and premature stages, respectively. An explanation of BS, CE, EE and BE is provided in Table 11.
Group Male Female
I II III I II III
BS 2 16 1 11
CE 12 1 17
EE 7 2 1 19 1
BE 14 5 11
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Table 14. Coefficient of variation of gonad indices, gonad hardness and L*, a*, b* values by sex, and of sexcombined in each Mesocentrotus nudus group.
An explanation of BS, CE, EE and BE is provided in Table 11.
BS CE EE BE
Sex combined Male Female Sex combined Male Female Sex combined Male Female Sex combined Male Female
Gonad index 44.6 47.5 41.2 11.4 12.6 10.8 16.5 12.7 18.2 40.2 39.7 41.9
Hardness 45.4 47.8 40.0 20.5 18.0 20.8 40.5 17.5 48.0 46.2 35.4 63.8
L* 15.5 17.0 13.6 6.0 6.6 4.0 7.0 8.4 6.0 11.6 12.8 8.5
a* 20.2 11.5 15.9 17.9 15.1 18.8 12.7 13.0 12.6 12.1 12.2 11.5
b* 16.4 12.7 17.0 10.5 11.5 9.2 12.7 15.5 10.0 10.2 10.2 8.8
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Table 15. Gonad hardness (N), and L*, a*, and b* values (mean ± SE) of gonads in each Mesocentrotus nudus group by sex. Significance of values among groups, between sex, and group and sex as determined by GLM ANOVA are provided. A, B, C and D indicate significant differences among groups, when values for both sexes were combined (p < 0.05). X, Y and Z indicate significant difference among groups, independent of sex (p < 0.05).
An explanation of BS, CE, EE and BE is provided in Table 1.
Sex combined Male Female p
BS CE EE BE BS CE EE BE BS CE EE BE Group Sex Group × Sex
Hardness 0.43 ± 0.04A 0.14 ± 0.01C 0.14 ± 0.01C 0.24 ± 0.02B 0.46 ± 0.05 0.15 ± 0.01 0.17 ± 0.01 0.25 ± 0.02 0.39 ± 0.05 0.13 ± 0.01 0.13 ± 0.01 0.23 ± 0.04 < 0.001 0.125 0.848 L* 45.7 ± 1.3D 57.0 ± 0.6B 59.6 ± 0.8A 50.7 ± 1.1C 46.0 ± 1.8 55.0 ± 1.0 61.2 ± 1.7 49.5 ± 1.5 45.2 ± 1.8 58.6 ± 0.6 59.0 ± 0.8 52.7 ± 1.4 < 0.001 0.309 0.107 a* 10.8 ± 0.4 10.0 ± 0.3 10.6 ± 0.2 11.8 ± 0.3 12.1 ± 0.3X 10.6 ± 0.4XYZ 10.3 ± 0.4XYZ 12.1 ± 0.3X 8.7 ± 0.4Z 9.5 ± 0.4YZ 10.8 ± 0.3XY 11.4 ± 0.4XY < 0.001 < 0.001 < 0.001 b* 37.5 ± 1.1 34.0 ± 0.7 39.8 ± 0.9 39.1 ± 0.7 40.1 ± 1.2XY 32.8 ± 1.0Z 36.5 ± 1.9XYZ 38.1 ± 0.9XYZ 33.5 ± 1.6YZ 35.0 ± 0.8Z 41.2 ± 0.9X 41.0 ± 1.1XY < 0.001 0.411 < 0.001
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(4) Gonad hardness
The gonad hardness of the BS group was significantly higher than other groups (p <
0.001) (Table 15). There was no significant difference in gonad hardness between CE and EE. The coefficient of variation of gonad hardness of CE was markedly lower than the other groups. Gonad hardness had a significant negative correlation with gonad index (ρ
= − 0.77, p < 0.001).
(5) Gonad color
L*, a*, and b* values of gonads by sex in each group are shown in Table 15. Values of L*, a*, and b* differed significantly among each group. There was no significant difference in L* value between sex or group and sex. The L* values of gonads from the CE group were significantly higher than those of BS and BE (p < 0.001) and lower than EE (p < 0.05). A significant difference in a* values of gonads among groups and between sexes were detected, and there was a significant interaction between group and sex. The a* value of testes did not differ significantly among groups. Ovaries of the CE group showed significantly lower a* value than testes of BS and BE (p < 0.01). There was a significant difference in the a* value of gonads of BS between the two sexes (p < 0.001).
The b* values of gonads differed significantly among groups, and there was a significant interaction between group and sex. The b* values of testes and ovaries of the CE group were significantly lower than those of testes of BS and ovaries of EE and BE (p < 0.05).
The coefficient of variation of L* value was lowest in testes and ovaries of the CE group, markedly lower than BS (Table 14). The a* value of gonads for both sexes combined from the BS group was higher than that for each sex alone. The b* value of gonads for both sexes combined from the BS group showed high a coefficient of variation
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when compared to those of the CE group.
(6) FAA content
The FAA content in the gonads of each group is shown in Figure 15. There were significant differences in the content of the 18 FAAs and total FAAs among groups (Table 16). There were no significant differences in the content of Glu, Ala, Pro, Ser, His, ornithine, and total FAAs between sexes. Significant interactions in the content of Asp, Ala, Ser, Thr, Arg, and total FAAs were detected between group and sex. Of the umami
tasting amino acids, there was no significant difference in Asp content in testes among groups. Glu content in the gonads of both sexes in the CE and BS groups did not differ significantly. Of the sweettasting amino acids, Ala content in the gonads both sexes in the CE group were > 400 mg/100 g and were significantly higher than those of other groups (p < 0.01). Gly content in the gonads of both sexes of the CE group were significantly higher than those of the BS and BE groups (p < 0.05). The levels of Pro and Ser in the CE group were significantly higher than those of the EE group (p < 0.01). The content of Thr in the testes and ovaries of the CE group was significantly higher than those in testes of other groups and in the ovaries of the EE and BE groups (p < 0.01). Of the bittertasting amino acids, the content of Arg in the testes and ovaries of the CE group was significantly lower than those in the ovaries of other groups (p < 0.05) and in the testes of the BS and BE groups (p < 0.001). The content of Lys in the testes and ovaries of the CE group were significantly lower than those of the BS group (p < 0.01). The content of Ile, Leu, Met, Phe, and Val in the testes and ovaries of the CE group were significantly higher than those of the EE group (p < 0.01). With the exception of Arg and Lys, there was no significant difference in the content of other bittertasting FAAs in the
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Figure 15. Free amino acid contents (mg/100 g) in sex combined gonads, testes and ovaries of Mesocentrotus nudus (mean ± SE). A and B indicate significant differences among sexcombined groups (p < 0.05). V, W, X, Y and Z indicate significant differences among groups independent of sex (p < 0.05). a, b and c, and x, y and z indicate significant differences among groups by sex, respectively (p < 0.05). An explanation of BS, CE, EE and BE is provided in Table 11. 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; Orn, ornithine. An explanation of BS, CE, EE and BE is presented in Table 11.
Sex combined
Male
Female
BS CE EE BE
B B B A
ABB AABA
WWY X W W
W
Z Z
Y X
a a
b b
Z Y V
X W V
Z Y XW
ZZY V
Y Y X WWV
b a a
c c b c b c b a a
b a a
c c b
b a
a c c b b b a a
aa b b
a a cbc
Z X W
Z YZY
X
Z Y W
X Z Y X
y y
xx
Z Y X W WV
Y X
Z X W
Z WYXZY
X W
Z XY
X
xx yy
y x x
zzy x
yyy x
xxyy yx xzz
y yx y x x
y xxyy xzyz
1000
1000 800 600 400 200 0
1000 800 600 400 200 0
1000 800 600 400 200 0
FAA c on ten t (m g/ 100 g )
BBA A A B
Glu
Asp Ala Gly Pro Ser Thr Arg His Ile Leu Lys Met Phe Tyr Val Tau Orn
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Table 16. Free amino acid content in the gonads of Mesocentrotus nudus of each group (mg/100 g) (mean ± SE) by sex. Significances among groups, between sex, and group and sex as determined by GLM ANOVA are provided. A and B indicate significant differences among groups, using data of both sexes combined (p < 0.05). V, W, X, Y and Z indicate significant differences among groups, independent of sex (p < 0.05). a, b and c, and x, y and z indicate significant differences among groups by sex (p < 0.05).
The abbreviations for the amino acids are defined in the legend to Figure 15.
An explanation of BS, CE, EE and BE is provided in Table 11.
Sex combined Male Female P
BS CE EE BE BS CE EE BE BS CE EE BE Group Sex Group×Sex
Asp 6.3 ± 0.5 4.0 ± 0.3 3.8 ± 0.5 5.2 ± 0.4 6.5 ± 0.7W 5.7 ± 0.3W 5.1 ± 0.6WXY 6.2 ± 0.5W 6.1 ± 0.6WX 2.7 ± 0.2Z 3.2 ± 0.7YZ 3.6 ± 0.4XYZ < 0.001 < 0.001 0.017
Glu 125.2 ± 5.2B 116.3 ± 12.4B 160.4 ± 16.1B 240.1 ± 25.0A 122.7 ± 7.5 111.3 ± 18.3 167.4 ± 29.0 285.4 ± 33.0 128.9 ± 6.5 120.2 ± 17.3 157.3 ± 19.8 162.0 ± 24.7 < 0.001 0.059 0.143
Ala 124.8 ± 10.1 433.8 ± 12.2 235.9 ± 11.8 149.4 ± 9.7 110.7 ± 10.2Z 452.6 ± 19.8W 244.5 ± 17.1XY 153.5 ± 10.2Z 145.9 ± 19.1YZ 419.4 ± 14.7W 232.2 ± 15.4X 142.3 ± 20.3XYZ < 0.001 0.841 <0.001
Gly 806.6 ± 20.3 997.4 ± 21.0 977.7 ± 22.1 782.5 ± 28.6 769.5 ± 26.8b 965.9 ± 36.5a 932.4 ± 45.4a 772.0 ± 29.0b 862.4 ± 24.1y 1021.4 ± 23.7x 997.2 ± 24.4x 800.5 ± 61.8y < 0.001 0.015 0.741
Pro 24.2 ± 2.5A 31.1 ± 3.2A 7.3 ± 2.2B 7.4 ± 1.6B 22.3 ± 3.1 38.7 ± 5.6 14.2 ± 3.9 8.0 ± 2.0 27.1 ± 4.3 25.3 ± 3.3 4.3 ± 2.5 6.4 ± 2.9 < 0.001 0.358 0.131
Ser 35.6 ± 3.8 90.5 ± 6.2 46.9 ± 3.8 21.3 ± 1.9 28.3 ± 2.7YZ 104.4 ± 10.8V 56.4 ± 6.8VWX 22.9 ± 2.3Z 46.4 ± 7.8WXYZ 80.0 ± 6.3VW 42.8 ± 4.4XY 18.6 ± 3.4Z < 0.001 0.211 0.003
Thr 30.7 ± 4.0 51.9 ± 2.6 15.2 ± 2.5 17.7 ± 2.0 21.3 ± 2.4XY 47.7 ± 3.3W 11.3 ± 1.2Z 15.8 ± 2.3YZ 44.9 ± 7.6WX 55.2 ± 3.8W 16.8 ± 3.5YZ 21.0 ± 3.5XYZ < 0.001 0.004 0.049
Arg 468.8 ± 19.8 215.3 ± 8.0 322.0 ± 14.5 396.1 ± 26.4 523.9 ± 20.5V 245.7 ± 11.1Y 338.8 ± 40.0WXY 466.5 ± 30.1VW 386.2 ± 23.8WX 192.0 ± 7.4Z 314.9 ± 12.3X 274.5 ± 18.8XY < 0.001 < 0.001 0.003
His 25.5 ± 1.8A 24.5 ± 1.4A 17.7 ± 1.5B 27.9 ± 1.5A 25.9 ± 2.3 23.6 ± 2.0 17.7 ± 3.4 28.1 ± 2.1 25.1 ± 2.9 25.2 ± 1.9 17.7 ± 1.6 27.5 ± 2.0 < 0.001 0.960 0.949
Ile 61.8 ± 6.3 57.6 ± 3.5 25.7 ± 3.6 34.6 ± 3.1 48.8 ± 5.8ab 50.7 ± 4.8a 16.7 ± 4.2c 31.8 ± 3.8bc 81.3 ± 11.1x 62.8 ± 4.7x 29.6 ± 4.6y 39.4 ± 5.4y < 0.001 < 0.001 0.227
Leu 103.0 ± 10.7 105.3 ± 6.3 49.0 ± 6.4 62.6 ± 5.4 82.8 ± 9.7ab 93.1 ± 8.8a 31.6 ± 7.4c 58.8 ± 6.7bc 133.4 ± 20.0xy 114.7 ± 8.3x 56.4 ± 8.1z 69.1 ± 9.3yz < 0.001 < 0.001 0.175
Lys 267.1 ± 13.7 168.4 ± 6.6 171.3 ± 9.2 234.6 ± 15.4 309.2 ± 14.2a 194.8 ± 8.8c 194.9 ± 23.1bc 276.0 ± 17.6ab 203.8 ± 12.7x 148.2 ± 6.2y 161.2 ± 8.2y 163.1 ± 9.9xy < 0.001 < 0.001 0.184
Met 29.4 ± 3.1 28.1 ± 1.5 16.0 ± 2.0 18.1 ± 1.3 20.4 ± 2.3ab 25.3 ± 2.3a 10.9 ± 2.3c 16.9 ± 1.2bc 42.9 ± 4.9x 30.3 ± 2.0x 18.2 ± 2.6y 20.2 ± 3.0y < 0.001 < 0.001 0.071
Phe 42.0 ± 4.0 48.0 ± 3.0 23.4 ± 2.9 24.8 ± 2.2 34.3 ± 4.5a 39.1 ± 3.0a 13.5 ± 2.6b 21.3 ± 2.4b 53.7 ± 6.3xy 54.8 ± 4.2x 27.7 ± 3.7z 30.7 ± 3.9yz < 0.001 < 0.001 0.077
Tyr 82.9 ± 7.0 72.5 ± 3.5 55.6 ± 5.9 66.2 ± 5.0 71.7 ± 7.8 70.6 ± 5.2 41.7 ± 9.7 64.9 ± 6.1 99.7 ± 11.6x 73.9 ± 4.8xy 61.5 ± 7.0y 68.5 ± 9.1xy 0.005 0.022 0.296
Val 96.2 ± 7.6 101.4 ± 5.2 52.0 ± 5.6 64.4 ± 4.7 83.7 ± 8.8a 89.6 ± 7.5a 36.7 ± 9.3b 62.8 ± 5.5ab 114.9 ± 12.1x 110.4 ± 6.6x 58.6 ± 6.5y 67.1 ± 9.0y < 0.001 0.004 0.251
Tau 89.6 ± 4.1 44.7 ± 1.6 59.1 ± 2.6 45.4 ± 2.7 99.7 ± 4.8a 50.3 ± 2.4c 65.3 ± 3.5b 51.4 ± 2.9c 74.6 ± 4.8x 40.4 ± 1.4z 56.4 ± 3.2y 35.1 ± 3.6z < 0.001 < 0.001 0.060
Orn 15.6 ± 0.5A 12.1 ± 0.7B 17.1 ± 1.2A 13.2 ± 0.8AB 15.5 ± 0.7 11.4 ± 1.5 17.8 ± 1.7 13.9 ± 1.0 15.7 ± 0.8 12.6 ± 0.5 16.9 ± 1.6 12.0 ± 1.2 < 0.001 0.728 0.545
Total 2435.3 ± 49.6 2603.0 ± 38.4 2256.1 ± 62.2 2211.5 ± 88.7 2396.9 ± 55.6XY 2620.5 ± 53.4X 2216.8 ± 143.8XY 2356.1 ± 99.9XY 2492.8 ± 92.5XY 2589.5 ± 55.3X 2272.9 ± 66.6Y 1961.7 ± 145.8Y < 0.001 0.309 0.011
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testes and ovaries of the CE and BS groups. The content of Tau in the gonads of both sexes of the CE and BE groups were significantly lower than those of the BS and EE groups (p < 0.05). There was no significant difference in total FAA content in the testes of the different groups. However, total FAA content in the ovaries of the CE group was significantly higher than those of EE and BE groups (p < 0.05).
The result of PCA analysis of FAA content in the gonads of both sexes in each group is shown in Figure 16. The FAA content in the ovaries and testes of the CE group, and those of the BS and BE groups were separated along principal component (PC) 1, which explained 53.2% of the variance in the data. The EE group was located between the CE, and BS and BE groups. Plots of the gonads from the BS and BE groups varied and differed with sex. Asp, Glu, Arg, His, Ley, and Tau had a significant positive correlation with the horizontal axis (p < 0.05). In contrast, Ala, Gly, Pro, Ser, Thr, and Phe had a significant negative correlation with the horizontal axis (p < 0.05). Glu, Gly, Arg, Lys, taurine, and ornithine had a significant negative correlation with the vertical axis (p < 0.05). Overall, the PCA biplot showed an increase in the content of sweettasting amino acids and a decrease in the content of bittertasting amino acids in the gonads of the CE group.
(7) Sensory evaluation
The scores of the sensory evaluation of the gonads of each group are shown in Table 17. There were significant differences in the scores obtained for each group, with the exception of scores for bitterness and saltiness (Table 17). There was no significant difference in the scores for all traits reported for the CE and EE groups. All scores for the CE group were high compared with those of the EE group, and, with the exception of smell and saltiness, were significantly higher than those of the BE group (p < 0.05).
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Figure 16. Principal component analysis biplot of compounds detected by free amino acid content analysis of Mesocentrotus nudus gonads. Arrows indicate the position of each free amino acid. Explanation of each free amino acid is indicated in the title of Figure 15.
An explanation of BS, CE, EE and BE is presented in Table 11.
80
Table 6. Sensory evaluation scores of Mesocentrotus nudus gonads from different groups at the end of culture period in July (mean ± SE) and results of Kruskal–Wallis test of the scores among groups. A and B indicate significant differences among groups.
An explanation of BS, CE, EE and BE is provided in Table 11.
CE EE BE χ2 df p
Preference
Color 1.33 ± 0.17A 0.67 ± 0.44A −1.44 ± 0.24B 15.81 2 < 0.001 Shape 1.11 ± 0.20A 0.56 ± 0.38A −1.44 ± 0.34B 14.38 2 < 0.001 Smell 0.33 ± 0.17A 0.50 ± 0.31A −0.78 ± 0.22B 10.99 2 0.004 Texture 1.22 ± 0.22A 0.56 ± 0.38AB −0.22 ± 0.22B 9.55 2 0.008 Richness 1.11 ± 0.26A 0.67 ± 0.33AB −0.22 ± 0.36B 6.41 2 0.041 Overall taste 1.00 ± 0.17A 0.56 ± 0.29AB −0.56 ± 0.29B 11.71 2 0.003 Overall quality 1.11 ± 0.11A 0.33 ± 0.37A −1.00 ± 0.24B 16.38 2 < 0.001
Strength
Sweetness 1.33 ± 0.17A 0.33 ± 0.44AB −0.44 ± 0.34B 11.34 2 0.004 Umami 1.33 ± 0.24A 0.33 ± 0.44AB −0.44 ± 0.38B 9.22 2 0.010 Bitterness −0.44 ± 0.24 −0.89 ± 0.26 −0.56 ± 0.29 1.19 2 0.552 Saltiness −0.67 ± 0.24 −0.67 ± 0.29 −0.67 ± 0.29 0.03 2 0.986
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In contrast, the scores for texture, richness, overall taste, sweetness, and umami taste of the gonads form the EE group were not significantly different from those of the BE group.
4. Discussion
Previous research has shown that feeding S. japonica to M. nudus (50 mm diameter) increased gonad indices from 7.6 in early April to 21.0 in late July (Agatsuma et al. 2002).
Experimental feeding of S. japonica var. religiosa to M. nudus (ca. 50 mm diameter) from a barren increased gonad indices from 6.5 in early April to 17.5 in early June, 20.2 in late June and 22.3 in early August (Agatsuma 1999). These two studies show that it takes over 2 months to increase the gonad index to more than 18 from early April, which is the minimum size required for commercial landing (Agatsuma 1999). Mesocentrotus nudus (ca. 48 mm diameter) from a barren cultured in offshore cages that were fed on S. japonica exhibited an increase in gonad index from 5.6 in early December to 16.9 in midMay (Section 2), indicating low gonad growth. In contrast, the results of the current study indicate that feeding S. japonica to M. nudus (ca. 51 mm diameter) resulted in an increase in gonad index from5.6 in early May to 19.0 in midJuly. Agatsuma (1997) reported that consumption of S. japonica var. religiosa by M. nudus increased from February and reached a maximum in June, when water temperature increased from 7 to 16 °C (Agatsuma 1997). Gonads of M. nudus increase in size during the growing stage (Agatsuma 1997), because nutrients are accumulated in NPs (Walker et al. 2015). In addition, the content of carbon and nitrogen increases in the fronds of cultivated S.
japonica from May or June as zoospores are forming (Li et al. 2007), suggesting an increase in the nutrient value as sea urchin food. Therefore, it is likely that commencing the culture of M. nudus in May enhanced gonad growth, due to the increased accumulation of nutrients in the gonads through high consumption of the kelp. In the
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current study, there was no sexual difference in gonad index in the recovering, growing and premature stage, which is in keeping with previous studies (Fuji 1960b; Section 1 of Chapter 2). The CE group had the lowest coefficient of variation, indicating uniformity of gonad size. Section 1 showed that the coefficient of variations of gonad indices of male and female M. nudus cultured during late April–early June were 17.0 and 20.3, respectively, higher than the values in the present study. Comparing the results of the current study with those of Section 1 further indicate an increased uniformity in gonad size.
Gonad hardness is associated with gonad texture, and past studies have revealed that gonad hardness varies during gonad development (McBride et al. 2004; Section 1 and 2 of Chapter 2). I suggested that gonads in the growing stage exhibit the most preferable hardness because they are harder than those in the maturation stage and softer than those in the recovering stage in Section 1. In the present study, most gonads from all groups were in the growing stage, showing that gonad hardness had a significant negative correlation with gonad index. Nutrient accumulation in NPs (Unuma and Walker 2009) and the increase in gonad size in the growing stage may be partly responsible for the soft texture.
Kinoshita et al. (2013) reported that undesirable colored gonads increased with a decrease in gonad indices during the maturation of M. nudus to postspawning stages.
Brown colored gonads were found in M. nudus with low gonad indices from barrens (Agatsuma et al. 2005). L* values of brown colored gonads in M. nudus from a barren and starved for 3 months were low (Section 1 and 2). L* values of gonads of M. nudus in the current study that were fed S. japonica from late April to early June (Section 1 of Chapter 2), and from early May to midJuly, increased significantly. The same finding
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concludes that the increase in L* values of M. nudus gonads fed on S. japonica contributed to an improvement of gonad color.
There was a significant different a* values between sexes and a significant interaction in a* and b* values between group and sex. In addition, the higher coefficient of variation of a* value of both sexes in the BS group compared with each sex individually indicates a large variation of sexual difference among groups. Section 1 of Chapter 2 showed that a* and b* values of M. nudus testes from a barren in February and June, and from an E. bicyclis bed in February were higher than those of M. nudus ovaries.
However, the a* and b* values of the gonads of male and female urchins from an E.
bicyclis bed in June exhibited no difference. The content of echinenone and βcarotene in the testes of M. nudus are higher than those in ovaries from the recovering to the mature stages (Borisovets et al. 2002). This difference may be due to variation in food availability and the quantity of each carotenoid synthesized by each sex. In particular, significant differences in a* values of male and female urchins from the BS group would be due to low nutrient accumulation in the gonads.
Degree of sweetness of Ala is quite strong (Schutz and Pilgrim 1957). The organoleptic threshold value of Ala is 60 mg/dL, although that of Gly is 130 mg/dL (Kirimura et al. 1969). Solms et al. (1965) quantified that the taste of 0.3% LAla and L
Gly are equivalent to 0.54% and 0.45% sucrose, respectively. Of the bittertasting FAAs, Arg produces an undesirable taste in sea urchin gonads (Komata 1964). I suggested that increased Ala content and decreased Arg content in sea urchin gonads contributes to an improvement of taste in Section 1 of Chapter 2. The researchers showed that the Ala content per 100 g in the gonads of M. nudus fed S. japonica from late April to early June was 339.3 mg in testes and 379.4 mg in ovaries. The Arg content was 378.2 mg in testes
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and 288.2mg in ovaries. The Ala and Arg content in the gonads of both sexes of M. nudus fed fresh S. japonica from December to May were 270.2 mg and 351 mg, respectively (Section 2 of Chapter 2). The present study demonstrated an even higher increase in Ala content and decrease in Arg content in the gonads by fresh S. japonica feeding during early May–early July. In S. japonica, levels of Asp, Glu, Ala, and Pro are high compared with other FAAs (Oishi and Kunisaki 1970). Glu content in the frond of kelp cultivated in Hakodate, Hokkaido, increased gradually from April and sharply from May to July. In parallel with this, nitrogen and carbon levels in S. japonica fronds increased from April to July during maturation (Li et al. 2007). The taste of E. chloroticus gonads fed with feeds containing Glu and Gly is sweeter compared with those containing LVal and L
Met (Phillips et al. 2009). Previous studies suggest that high levels of alanine in the gonads of the CE group are a result of changes in FAAs in S. japonica at the late sporophyte stage.
The contents of other sweet tasting FAAs (Pro, Ser, and Thr) in the gonads of the CE group did not reach threshold values (Kirimura et al. 1969), although those FAA values were higher than those in wild urchins. Therefore, it is concluded that the strong sweet flavor of gonads of the CE group could be attributed to the high Ala content in the gonads.
Section 2 of Chapter 2 showed an increased level of umamitasting Glu and lower levels of bitter tasting Tyr and Val in the gonads of M. nudus fed fresh Saccharina kelp during December–May compared to those during December–March. In the gonads of the CE group, levels of Tyr and Val were low compared to those in the previous study (Section 2 of Chapter 2). Higher contents of bittertasting FAAs in the CE group, with the exception of Arg, His, Lys, and Tyr compared to those of the EE and BE groups, and the absence of significant differences in bitterness among groups by sensory evaluation, is likely due to
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offset of bittertasting FAAs.
PCA analysis showed a drastic change in FAA composition and taste uniformity of the CE group, which are distinctively different from those of the BS and BE groups. The separate plotting of ovaries and testes of BS and BE, and significant interactions in some FAA contents between group and sex suggested possible differences in gonad taste between sex on barrens.
The intensity of the umami taste is enhanced when Glu and 5′ribonucleotides are mixed together (reviewed by Yamaguchi and Ninomiya 2000). In gonads, the umami flavor is weak without nucleotides (inosinic acid and guanylic acid) (Komata 1964). In addition, the umami taste of inosinic acid can be enhanced by adding Ala, Gly, Ser, Thr, and Met (Kawai et al. 2002). The higher umami score in gonads of the CE group compared with the EE group, despite the absence of any significant difference in the content of the umamitasting FAAs, suggest that the difference in taste was due to a difference in nucleotides and/or these amino acids contents. The higher overall taste score in the gonads of the CE group compared with the EE group can be attributed to the sweetness produced by high Ala content, strong umami taste, and low Arg content.
Furthermore, improvement in all commercial gonad traits (size, color, hardness, taste, appearance, mouth feel, and their whole balance and uniformity) of the CE group would result in the highest score of overall quality. This study first demonstrated that a higher level of commercial gonad traits and their consistency can be achieved in individuals of M. nudus from a barren compared with urchins from a fishing ground, by feeding with fresh S. japonica at the late sporophyte stage during May–July.