Pronounced effects of the basal frond portion of the kelp Saccharina japonica on gonad qualities of the sea urchin Mesocentrotus nudus from a barren

Journal Pre-proof Pronounced effects of the basal frond portion of the kelp Saccharina japonica on gonad qualities of the sea urchin Mesocentrotus nudus from a barren Satomi Takagi, Yuko Murata, Eri Inomata, Masakazu N. Aoki, Yukio Agatsuma PII:

S0044-8486(19)31959-3

DOI:

https://doi.org/10.1016/j.aquaculture.2019.734623

Reference:

AQUA 734623

To appear in:

Aquaculture

Received Date: 1 August 2019 Revised Date:

19 October 2019

Accepted Date: 20 October 2019

Please cite this article as: Takagi, S., Murata, Y., Inomata, E., Aoki, M.N., Agatsuma, Y., Pronounced effects of the basal frond portion of the kelp Saccharina japonica on gonad qualities of the sea urchin Mesocentrotus nudus from a barren, Aquaculture (2019), doi: https://doi.org/10.1016/ j.aquaculture.2019.734623. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

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Pronounced effects of the basal frond portion of the kelp Saccharina japonica on gonad qualities of the

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sea urchin Mesocentrotus nudus from a barren

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Satomi Takagia, Yuko Muratab, Eri Inomataa, Masakazu N. Aokia, Yukio Agatsumaa*

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468-1, Aza-Aoba, Aramaki, Aoba, Sendai, Miyagi 980-0845, Japan

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b

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Laboratory of Marine Plant Ecology, Graduate School of Agricultural Science, Tohoku University,

National Research Institute of Fisheries Science, Japan Fisheries Research and Education Agency,

2-12-4, Fukuura, Kanazawa, Yokohama, Kanagawa 236-8648, Japan

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*Corresponding author (Y. Agatsuma)

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Email address: [email protected]

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Tel: +81-22-757-4151

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Abstract

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The gonad quality of sea urchin (Mesocentrotus nudus) adults from a barren can be improved by feeding

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the animals fronds of the kelp Saccharina japonica from May–July. The improvement in taste is expected

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to depend on the amino acid composition of the kelp fronds, which varies among the frond portions. We

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investigated the effects of feeding apical, middle and basal frond portions of S. japonica on gonad size,

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hardness, color (L*a*b*) and taste (free amino acids) of M. nudus from a barren from May–July in a

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rearing experiment in which we analyzed the protein and total and free amino acid content of each frond

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portion. Feeding of each frond portion improved gonad size, hardness, color and taste. Feeding of the

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basal portion, which contained high levels of glutamic acid, alanine, and proline, markedly increased the

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alanine and glutamic acid content and decreased the content of bitter-tasting amino acids in gonads,

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implying greatly enhanced sweet and umami taste. Based on a comparison of alanine content with that

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reported in past studies of M. nudus, the gonad taste would be evaluated at a higher level. The basal

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portion of S. japonica fronds has the high potential to improve gonad taste.

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Keywords:

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Saccharina japonica, Frond portion, Total and free amino acid, Mesocentrotus nudus, Gonad taste

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1. Introduction

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The world food supply of fish and seafood has increased from 69,420,942 t in 1991 to 132,828,714 t

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in 2013 (FAO, 2019a). Sea urchin gonads are a premium delicacy. Japan consumes approximately 90% of

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the total sea urchin harvest in the world (Sun and Chiang, 2015). The Asahi Shimbun Company, a major

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national newspaper in Japan, published a preferred sushi ingredient ranking based on a questionnaire that

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was administered to people in Japan; the ranking indicated that sea urchin gonads were the fourth most

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preferred sushi ingredient, following medium fatty tuna, lean tuna and squid (Iwai, 2018). In Japan,

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Mesocentrotus nudus account for more than two-thirds of the total sea urchin landing together with

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Strongylocentrotus intermedius and is the most expensive source of sea urchin gonads in the world

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(Unuma, 2015). The wholesale price of M. nudus per wooden tray (250–300 g gonads) can exceed 30,000

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yen, whereas the average price of all imported sea urchins is approximately 6,000 yen/kg gonad (Unuma,

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2015).

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Catches of edible sea urchins have been decreasing worldwide from 109,736 t in 1995 to 70,833 t in

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2017 (FAO, 2019b) because of stock depletion due to overfishing (Andrew et al., 2002). As a means of

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meeting market demands, studies on the production of hatchery raised urchins in aquaculture systems

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have been conducted (McBride 2005; Pearce2010). Some studies reported that feeds containing 19–36%

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protein notably enhance gonad production (de Jong-Westman et al., 1995; Pearce et al., 2002), and

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β-carotene contained in feed improve gonad color (Robinson et al., 2002; Pearce et al., 2003; Shpigel et

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al., 2005). However, several studies indicate that feeds with high protein content, such as fish flesh and

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the red alga Pyropia yezoensis, lead to undesirable gonad color (Agatsuma, 1998), decrease the content of

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sweet-tasting amino acids (glycine and alanine), and increase the content of bitter-tasting amino acids

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(valine and lysine) in the gonads (Hoshikawa et al., 1998; Inomata et al., 2016).

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Sea urchins densely distributed on barrens, where communities of crustose coralline red algae

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without erect macrophytes dominate the subtidal rocky sea floors, have small gonads (e.g., Lang and

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Mann, 1976; Johnson and Mann, 1982; Keats et al., 1984) and exhibit inferior gonad color, texture, and

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taste compared with sea urchins growing in kelp beds (Agatsuma et al., 2005; Takagi et al., 2017). Takagi

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et al. (2017) reported that the taste of gonads obtained from M. nudus harvested from a barren was

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markedly improved by feeding of fresh cultivated Saccharina japonica kelp from late April to early June.

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Moreover, the feeding of S. japonica at the late sporophyte stage from May–July could improve the gonad

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taste to a level that was more desirable than that from an Eisenia kelp bed (fishing ground) (Takagi et al.,

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2019). In addition, feeding with S. japonica from December–March and December–May improved the

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gonad taste less markedly compared to feeding from spring–early summer (Takagi et al., 2018). The

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authors suggested that the improvement in gonad taste might be due to phenological changes in the free

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amino acid composition of cultivated S. japonica fed to sea urchins.

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Several studies have reported seasonal changes in the nutrient composition of Laminariaceae. The

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nitrogen and carbon content of Undaria pinnatifida (Gao et al., 2013), Macrocystis pyrifera (Zimmerman

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and Kremer, 1986), Alaria esculenta, Laminaria digitata, Laminaria hyperborea, Saccharina latissima

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(Schiener et al., 2015), Saccharina longissima (Li et al., 2009), S. japonica (Li et al., 2007), S. japonica

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var. ochotensis (Sato and Agatsuma, 2016) and S. japonica var. diabolica (Li et al., 2009) varies

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seasonally. The amino acid, protein and glucose content of S. latissima changes phenologically (Marinho

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et al., 2015; Sharma et al., 2018). For S. japonica, crude protein, crude alginic acid, ash, amino acid,

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chlorophyll a, iodine and mannitol contents were shown to change seasonally (Oishi and Kunisaki, 1970;

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Fukushi, 1988). In addition, the nitrogen content of Macrocystis pyrifera (Stephen and Hepburn, 2016)

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and S. japonica (Fukushi, 1988) varied among the sporophyte portions of the plants. The carbon and

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nitrogen contents of various portions of fronds of Laminaria solidungula, S. latissima, S. longissima, S.

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japonica, S. japonica var. ochotensis and S. japonica var. diabolica differ (Oishi et al., 1967; Dunton and

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Schell, 1986; Sjøtun, 1993; Henley and Dunton, 1995; Li et al., 2007, 2009; Sato and Agatsuma, 2016).

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Li et al. (2007) reported that the nitrogen and carbon content of S. japonica fronds increased as the fronds

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matured in summer; they were particularly high in the meristem of the basal portion compared to the

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apical portion, which is used for storage, in plants that persisted for a second year. Oishi and Kunisaki

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(1970) reported that the free amino acid content of the basal and central portions of S. japonica fronds is

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high compared to that of the apical portion. These findings suggest that feeding of different portions of S.

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japonica fronds to sea urchins could affect gonad taste. It is possible that gonad taste would be further

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improved by feeding of the basal or central portions of the kelp.

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In the present study, M. nudus were collected from a barren. They were reared in aquaria and fed the

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basal, middle or apical portions of fresh S. japonica fronds from May–July, when the gonad of the sea

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urchin is in the growing stage with large size (Agatsuma, 1997), desirable color (Borisovets et al., 2002)

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and hardness (Takagi et al., 2017), and high umami-tasting amino acid and low bitter-tasting amino acid

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contents (Takagi et al., 2017). The gonad quality (size, color, texture and taste) of the sea urchins was

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determined at the start and at the end of the feeding experiment. The protein and total and free amino acid

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content of each frond portion was also analyzed. This study aimed to (1) clarify whether the qualities of

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sea urchin gonads vary when the animals are fed different S. japonica frond portions and (2) verify the

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relationship between gonad qualities and the constituents of each frond portion. This study first reports

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the total and free amino acid content of individual frond portions of S. japonica.

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2. Materials and methods

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2.1. Experimental design and rearing conditions

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A total of 75 adult M. nudus (46–54 mm diameter) were collected from a barren by scuba diving at

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depths of 2.5–3 m off Nojima Island, Shizugawa Bay, Miyagi Prefecture (38º40' N, 141º30' E) on 16 May

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2016. After collection, the sea urchins were placed in a cool box containing moist urethane mats

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immersed in seawater and transported to Onagawa Field Center, Tohoku University, in Onagawa (38º 26'

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N, 141º27' E) (the transportation time was approximately 1 h). Of 75 sea urchins collected, 45 individuals

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were used in the feeding experiment. In this experiment, five sea urchins were held in each of nine 10 L

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aquaria. The sea urchins were reared in running seawater that had been filtered twice using Myclean Filter

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(AF-515, Tanaka Sanjiro Co., Ltd., Fukuoka, Japan) and aerated, and the water was exchanged two or

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three times per hour. The seawater was pumped from offshore waters. The sea urchins were reared

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without food for 4 days until the start of the experiment. The feeding experiment was conducted from 20

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May to 11 July 2016. We used fresh S. japonica kelp cultivated off Fudai, Iwate Prefecture (40º 01' N,

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141º 54' E) as feed for the sea urchins. The fronds of S. japonica were cut into five equal lengths from the

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basal to the apical portions (Figure 1). The basal, middle and apical portions of the fronds were fed to sea

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urchins (three groups for each treatment in nine aquaria) ad libitum every 3–7 days. The aquaria used for

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each treatment were randomly selected. Thus, the feeding experiment was designed as three treatments of

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sea urchins fed the apical (AP), middle (MD) or basal portions (BS) of S. japonica fronds. The remaining

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30 sea urchins that were collected were randomly divided into six groups of five individuals (ST) at the

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start of the experiment; at the end of the experiment, all of the sea urchins were used for measurements

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and analyses of gonad size, development, hardness and color and free amino acid content of the gonads.

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The temperature of the seawater in the aquaria was measured every 10 min using a wireless data logger

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(RTR-52A, T&D, Nagano, Japan). The survival rate of the sea urchins during the rearing experiment was

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100%.

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2.2. Measurements and histological observation

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The test diameters (TD) (0.1 mm accuracy) and body wet weights (BW) (0.1 g accuracy) of the sea

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urchins in each group were measured at the start and end of the experiment using a vernier calliper and an

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electronic balance, respectively. The gonadal wet weight was measured after blotting excess water, and

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the gonad index (gonad wet weight × 100/BW) was calculated. A portion of each individual gonad was

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preserved in 20% formalin. Using standard histological techniques, serial cross-sections (6 µm) were cut

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and stained with Mayer’s hematoxylin and eosin. Sections were classified based on the stage of

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development of the germinal cells and the nutritive phagocytes (NPs): stage I, recovering; stage II,

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growing; stage III, premature; stage IV, mature; stage V, partly spawned; stage VI, spent (Byrne, 1990;

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King et al., 1994).

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2.3. Gonad hardness

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Gonad hardness was measured using a creep meter (RE2-3005B, YAMADEN, Tokyo, Japan)

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according to Takagi et al. (2017). One of five pieces of each gonad was placed on the stand with the side

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attached to the test facing up. Compression by a flat-faced plastic cylinder (5 mm diameter) was measured

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as the force in Newtons (N) with the distance fixed at 5 mm (McBride et al., 2004). The velocity of

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pressurization and decompression was 5 mm/s. Withdrawal time to the original sample height was

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determined by setting the sensitivity to 0.02 N.

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2.4. Gonad color

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Gonad color was measured using a color meter (ZE–6000, Nippon Denshoku Industries Co. Ltd.,

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Tokyo, Japan) that detects tristimulus values directly through flicker photometry using a 12-V, 20-W

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halogen lamp. L* (lightness), a* (redness), and b* (yellowness) values based on the Commission

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Internationale de l’Eclairage colour measurement system (Robinson et al., 2002) were measured in three

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replicates per gonad.

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2.5. Free amino acid content of gonads

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Approximately 1.0 g of gonadal tissue from each animal was quickly frozen at −30 ºC, and the free

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amino acids (FAA) in the tissue were analyzed using the method of Murata et al. (1994) with a slight

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modification. The gonadal tissue (0.1−0.5 g) was homogenized in 5 mL of 10% perchloric acid, and the

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homogenate was centrifuged at 7500×g for 10 min. The precipitate was re-extracted twice with 2 mL of

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5% perchloric acid. The supernatants were combined and neutralized with 10 N and 1 N KOH, and the

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precipitates were removed by filtration. The volume of the filtrate was brought to 25 mL with water.

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FAAs in the perchloric acid extract were analyzed using an automatic amino acid analyzer (L-8900,

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Hitachi High-Technologies Corporation, Tokyo, Japan). The FAAs can be categorized into four groups:

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umami-tasting (aspartic acid (Asp) and glutamic acid (Glu)), sweet-tasting (alanine (Ala), glycine (Gly),

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serine (Ser), proline (Pro), and threonine (Thr)), bitter-tasting (arginine (Arg), histidine (His), isoleucine

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(Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), tyrosine (Tyr), and valine (Val))

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and others (taurine (Tau), cysteine (Cys), ornithine (Orn), and phosphoserine (P-Ser)) according to

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Kaneko et al. (2009).

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2.6. Protein and total and free amino acid content of kelp

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On 21 June 2016, the midpoint of the experiment, five samples of each frond portion (approximately

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200 g) of S. japonica were frozen at −30 ºC. The NaOH-soluble protein content of the frond portions was

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analyzed according to the method of Lowry et al. (1951) with the DC™ Protein Assay Kit (DC™ Protein

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Assay Kit II, BIO-RAD, Hercules, CA, USA). Bovine serum albumin was used as the standard. The FAA

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content of the kelp samples was analyzed using the same method that was used to measure the FAA

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content of the gonads. For total amino acid (TAA) content analyses (total of free amino acids and amino

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acids in proteins and peptides), freeze-dried samples (5 mg) were placed in a vacuum hydrolysis tube, and

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3 dL of 2-mercaptoethanol and 5 mL of 6 N HCl were added. The mixture was hydrolysed at 110 ºC for

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24 h in vacuo. The hydrolysate was evaporated to dryness and dissolved in 5 ml of lithium citrate buffer

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(pH 2.2). The solution was filtered through a 0.2-µm membrane filter (Millex-LG, Merck Millipore,

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Burlington, MA, USA). Twenty microliters of the filtrate were injected into an amino acid analyzer

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(L-8900, Hitachi High-Technologies Corporation, Tokyo, Japan).

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2.7. Statistical analyses

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The data were tested for normality (Shapiro-Wilk W-test) and for homogeneity of variance (Levene’s

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test). Data that were not normally distributed or did not show homogeneity of variance were

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log-transformed. Significant differences in the TD and BW of the sea urchins in each group at the start

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and ST were analyzed using the t-test. Significant differences in TD and BW among the groups at the start

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of the experiment and differences in TD, BW, gonad index, hardness, L*, a* and b* values and in the

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FAA content of gonads at the end of the experiment were analyzed using nested one-way analysis of

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variance (nested ANOVA) with R ver 3.4.0 (R Core Team., 2017) through RStudio ver 1.0.143 (Rstudio

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Inc., Boston, MA, USA). Significant differences in TD and BW of ST (n = 6) and in the protein, TAA and

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FAA contents of the frond portions of S. japonica were analyzed by one-way ANOVA. Tukey’s multiple

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comparison test was performed as a post hoc test. Significant differences in TD, BW, gonad index,

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hardness, L*, a* and b* values and FAA contents between sea urchins at the start and end of each

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treatment were tested using the t-test. To evaluate the correlations among the contents of each

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taste-associated FAA in the AP, MD and BS groups, the FAA content data were analyzed by principal

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component analysis (PCA) using Canoco 5 (ter Braak and Šmilauer, 2012). Except for nested ANOVA

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and PCA, all analyses were conducted using JMP 10 (SAS Institute Inc., Cary, NC, USA).

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3. Results

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3.1. Water temperature

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The water temperature varied erratically during the experiment; it decreased to its lowest level of

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14.0 ºC in early June and then increased sharply to its highest level (20.3 ºC) at the beginning of July

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(Figure 2).

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3.2. Protein, TAA and FAA content of kelp

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The NaOH-soluble protein contents of the apical, middle and basal portions of S. japonica fronds fed

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to M. nudus were 3.23 ± 0.35%, 3.23 ± 0.24% and 3.67 ± 0.12% (mean ± SE), respectively, indicating

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that there was no significant difference in this parameter among the portions (df = 2, MS = 0.32, F = 0.97,

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P = 0.406). The TAA and FAA content of each portion of S. japonica fronds fed to M. nudus are shown in

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Figure 3 and Table S1. There were significant differences in the content of nine TAAs and six FAAs

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among the portions (Table S2). The total TAA and total Glu contents of the basal portion were ca. 475

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mg/100 g and 100 mg/100 g higher, respectively, than those of the apical portion. The total Ala, Gly and

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Val contents of the basal and middle portions were significantly higher than those of the apical portion (P

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< 0.05). The total Pro content of the basal portion was significantly higher than that of the other portions

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(P < 0.001). The total Leu and Tyr contents of the basal portions did not differ significantly from those of

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the other portions, while the Leu and Tyr contents of the middle portion were significantly higher than

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those of the apical portion (P < 0.05). The total His content of the basal portion was significantly higher

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than that of the apical portion (P < 0.05).

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The total FAA content of the basal portion was 2.0 and 2.3 times higher than those of the apical and

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middle portions, respectively. Except for Asp, Glu, Ala and Gly, the content of FAAs was markedly lower

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than the content of TAAs. The free Glu content of the basal portion was 2.4 and 3.2 times higher than

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those of the apical and middle portions, respectively. The free Gly and Met contents of the apical portion

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were significantly higher than those of the other portions (P < 0.01).

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3.3. Body and gonad size and gonad development

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At the start of the experiment, there were no significant differences in TD or BW between ST and the

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sea urchins in the treatment groups in the aquaria (TD, df = 1, MS = 4.52, F = 1.07, P = 0.304; BW, df = 1,

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MS = 19.09, F = 0.32, P = 0.574) or among the sea urchins in the various treatment groups (Table 1).

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There were no significant differences in TD or BW among the five specimens randomly allotted to each

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of the six ST groups (TD, df = 5, MS = 2.75, F = 0.62, P = 0.683; BW, df = 5, MS = 81.08, F = 1.36, P =

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0.273). No significant differences in TD, BW or gonad indices were detected in the animals in the various

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treatment groups at the end of the experiment (Table S3). The gonad indices of sea urchins that were fed

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portions of S. japonica fronds were significantly higher than those of the animals in the ST group (P <

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0.001) (Table 2).

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The gonadal developmental stages (according to sex) of the M. nudus used in the experiment are

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shown in Table 3. The gonads of most sea urchins were in the growing stage, with increasing numbers of

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spermatocytes or early vitellogenic oocytes along the acinal wall and with NPs filling the lumen. Three

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individuals in the ST group and two individuals in the MD group had recovering gonads. A male

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individual in the BS group had premature gonads.

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3.4. Gonad qualities

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No significant differences in gonad hardness were detected among the animals in the treatment

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groups (Table S3). Gonad hardness in each treatment group at the end of the experiment was significantly

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lower than in ST (P < 0.01) (Table 2).

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A significant difference in b* value was found among treatments by nested ANOVA (Table S3),

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while Tukey’s test showed no significant difference (Table 2). No significant differences in L* or a*

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values were detected among treatments (Table S3). The L* values of the gonads of sea urchins fed any of

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the frond portions were significantly higher than the L* values in the ST group (P < 0.001) (Table 2).

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The FAA content of the gonads is shown in Figure 4 and Table S4. There were significant

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differences among the treatment groups in total FAA content and in the content of 13 individual FAAs

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(Table S3). The total FAA content of the gonads was significantly higher in the MD and BS groups than

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in the AP group (P < 0.05). The Glu content of the gonads in the BS group was significantly higher than

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that in the ST group (P < 0.05). The Ala and Ser contents of the gonads in all treatment groups were

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significantly higher than those in the ST group (P < 0.001). The Ala content of the gonads in the BS group

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was significantly higher than that in the AP group (P < 0.01). The Gly content of the gonads in the MD

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and BS groups was significantly higher than that in the AP group (P < 0.01). The His, Met and Tyr

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contents of the gonads in the MD group were higher than those in the BS and/or AP groups (P < 0.05).

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The Arg, Lys and Tau contents of the gonads of the animals in each treatment group were significantly

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lower than those in the ST group (P < 0.01). The Ile, Phe and Tyr contents of the gonads in the BS group

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were significantly lower than those in the ST group (P < 0.05).

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The results of PCA of the FAA content of the gonads of the animals in each treatment group are

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shown in Figure 5. The FAA contents of AP were separated from MD and BS along principal component

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(PC) 1, which explained 49.7% of the variance in the data. The plots of BS were shifted slightly in the

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negative direction of PC 2 compared to those of MD. With the exception of Asp, Ser and Ile, all FAAs

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showed significant negative correlations with PC 1 (P < 0.05). Ala, Glu and Pro had significant negative

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correlations with PC 2 (P < 0.05). Only Gly showed a significant positive correlation with PC 2 (P <

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0.05).

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4. Discussion

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In this study, the feeding of different portions of S. japonica fronds to M. nudus did not affect sea

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urchin somatic (TD and BW) growth, gonadal growth (gonad indices), or gonad hardness or color

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(L*a*b*). Feeding of whole fronds of S. japonica enhance gonad production, increase L* value and

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improve gonad color (Agatsuma, 1997; Takagi et al., 2017; 2018; 2019). In the animals in the AP, MD

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and BS groups, the values of these traits were similar to those of M. nudus fed whole fronds of S.

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japonica from May–July (Takagi et al., 2019). Improvement in gonad size and color of each treatment

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was partly attributed to the growing stage, which increase gonad size (Agatsuma 1997; Byrne 1990) and

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echinenone content in gonads (Borisovets et al. 2002; Shpigel et al. 2005; Rocha et al. 2019). The lack of

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significant differences in protein content, which enhances gonad production (de Jong-Westman et al.,

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1995; Pearce et al., 2002; Eddy et al., 2012), among the frond portions of S. japonica is consistent with

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the similarities in these gonad indices. The color of sea urchin gonads can be improved by β-carotene

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contained in feed (Robinson et al., 2002; Pearce et al., 2003; Shpigel et al., 2005). Henley and Dunton

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(1995) reported that the carotenoid content of yearling fronds of S. latissima was lower in April than in

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July. In May, the β-carotene content of the apices of fronds of A. esculenta, L. digitata, L. hyperborea and

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Saccorhiza polyschides is higher than that of the basal portions (Schmid and Stengel, 2015). In contrast,

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in S. latissima, the β-carotene content of the apex, middle and base of the frond does not differ (Schmid

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and Stengel, 2015). In the present study, the finding of no significant difference in gonad color in M.

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nudus in the different treatment groups might be due to a lack of difference in β-carotene content among

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the frond portions of S. japonica. Gonad hardness, which is affected by gonad development and size

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(McBride et al., 2004; Takagi et al., 2017; 2019), also showed no significant differences among the

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treatment groups, likely due to the similarities in gonadal development and size in the three groups. The

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FAA content of gonads is closely associated with their taste (Komata et al., 1962; Lee and Haard, 1982;

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Liyana-Pathirana et al., 2002). In M. nudus gonads, high alanine content increases the sweetness and

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results in preferable taste (Takagi et al. 2017). In the present study, the Ala content of the gonads of M.

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nudus in all treatment groups increased significantly compared to the Ala content at the start of the

310

experiment; Ala content was particularly high in the gonads of the BS group. In addition, the higher Gly

311

content in the gonads of BS and MD compared with that of AP and the low Ser content of the gonads of

312

the animals in all of the treatment groups (below the threshold value of 150 mg/dl) (Kirimura et al., 1969)

313

suggest that the sweetness of the gonads in the treatment groups ranked as BS > MD > AP. The higher

314

content of the bitter-tasting amino acids His, Met and Tyr in the gonads of the MD group compared to the

315

BS group would tend to further decrease the relative bitterness of the gonads of the BS animals. The PCA

316

results indicated that the FAA compositions of the gonads varied among the treatment groups. The FAA

317

contents of AP were separated from those of MD and BS along PC 1. In addition, low total FAA content

318

in the gonads of AP would lessen taste enhancement. The PCA biplots also suggest high Ala, Pro and Glu

319

content and low Gly content in the gonads of the BS group compared with the MD group, although no

320

significant differences in Gly, Pro or Glu content were found among the treatment groups. These results

321

suggest that feeding of the basal portion of S. japonica fronds largely enhanced umami and sweet taste,

322

while feeding of the middle portion relatively enhanced bitter taste, and feeding of the apical portion

323

lessened taste compared with other portions. High levels of bitter-tasting Arg in gonads make the taste

324

undesirable (Komata, 1964). The increased Ala and decreased Arg content found in the gonads of the

325

animals in all treatment groups in the present study would be expected to lead to high evaluation of the

326

taste (Takagi et al., 2019). The Ala content of gonads of the animals in the BS group (536.1 mg/100 g)

327

was higher than that reported in previous studies of M. nudus (38.0–379.4 mg/100 g) (Hirano et al., 1978;

328

Hoshikawa et al., 1998; Nabata et al., 1999; Inomata et al., 2016; Takagi et al., 2017). Takagi et al. (2019)

329

reported that gonads of M. nudus fed whole fronds of S. japonica from May–July were evaluated as

330

having more desirable taste than those from a fishing ground. In that study, the Ala content of testes was

331

452.6 mg/100 g, and that of ovaries was 419.4 mg/100 g. In contrast, the Arg content of testes was 245.7

332

mg/100 g and that of ovaries was 192.0 mg/100 g, values that are approximately similar to the level of

333

245.3 mg/100 g found in the gonads of the animals in the BS group. Therefore, the taste of gonads

334

obtained from animals in the BS group would be evaluated at a higher level in this study than in the

335

previous study.

336

The FAA content of the basal portions of S. japonica fronds is high compared to that of the apex

337

(Oishi et al., 1967). Oishi and Kunisaki (1970) reported that the free Asp, Glu, Ala and Pro contents of the

338

central and basal portions of S. japonica fronds are high compared to those in the apex from April–July.

339

In the present study, the free Asp, Glu and Pro contents of the basal portion were high compared to those

340

of the apical portion. The TAA content of the basal portion was also high. Gonads of Evechinus

341

chloroticus fed feeds containing Glu (glutamate) and Gly are sweet compared to those fed feeds

342

containing Val and Met (Phillips et al., 2009). Takagi et al. (2019) suggested that high Ala content in

343

gonads of M. nudus fed S. japonica fronds from May–July would be affected by changes in the FAA

344

content of the mature fronds at the late sporophyte stage. Glu can be converted to Ala by alanine

345

aminotransferase (Brosnan and Brosnan, 2009). The genome sequence of this enzyme in

346

Strongylocentrotus purpuratus has been recorded (NCBI: LOC580780). Black (1964) identified the

347

enzyme in Lytechinus variegatus from the egg to the pluteus larva stage. These studies suggest that Ala

348

can be produced from Glu in the gonad. Furthermore, the correlated ranking of total Glu content in the

349

basal > middle > apical portions of S. japonica fronds could contribute to the Ala content of gonads.

350

Likewise, correlations of the His, Met and Tyr contents of the gonads of the animals in each treatment

351

group and in the frond portions were not found. The associated factors remain unclear.

352

353

5. Conclusions

354

In the present study, feeding of the basal, middle and apical portions of S. japonica fronds to M.

355

nudus from a barren during May–July improved gonad size, hardness, color and taste. There were no

356

significant differences in gonad size, hardness or color among sea urchins fed different portions of the

357

fronds. The basal portion, which contained high levels of glutamic acid, alanine and proline, markedly

358

increased the alanine and glutamic acid content and decreased the bitter-tasting amino acid content of the

359

gonads of sea urchins; this is expected to greatly enhance the sweet and umami taste of the product. In

360

particular, the gonadal alanine content was higher than that reported in previous studies of M. nudus,

361

inferring that the gonad taste would be evaluated at a higher level. The results of FAA analysis suggest

362

that feeding the middle portion of the fronds relatively enhanced bitter taste and that feeding of the apical

363

portion decreased the desirability of the taste compared to feeding other portions. Alanine may be

364

synthesized from total glutamic acid present in S. japonica fronds fed to M. nudus. The basal portion of

365

the frond has the high potential to improve gonad taste. An investigation when the gonad taste can be

366

improved during May–July is needed to clarify the adequate culture duration.

367

368

Acknowledgements

369

We sincerely thank Dr Y. Sato and D. Saito of Riken Food Co., Ltd and S. Ogami, Operations Chief

370

of the Fudai Fisheries Cooperative Association, for providing S. japonica. We also thank Professor A.

371

Kijima and the staff members of the Onagawa Field Center of Tohoku University for their cooperation in

372

the feeding experiment. We are grateful to Associate Professor T. Yamaguchi and Assistant Professor N.

373

Nakano of Tohoku University for their technical support in the use of the texture analyzer and to

374

Associate Professor K. Takahashi and Assistant Professor K. Nagasawa for their guidance with

375

histological techniques. We also thank M. Oshima and S. Kodama of Diving Stage Ariel, Head of Youth

376

Division Y. Sugawara and other staff of the Shizugawa Branchi of the Miyagi Fisheries Cooperative

377

Association for their cooperation in sea urchin collection. A portion of this study was financially

378

supported by JSPS KAKENHI for JSPS Research Fellow [grant number 17J02308].

379

380

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381

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Table S1. Total and free amino acid content (mg/100 g wet weight) of the apical, middle and basal portions of Saccharina japonica fronds (N = 5) (mean ± S.E.). Lower-case letters indicate significant differences among the portions according to Tukey’s test (P < 0.05).

Total amino acids

Free amino acids

Apical portion

Middle portion

Basal portion

Apical portion

Middle portion

Basal portion

670.6 ± 109.4

870.6 ± 67.2

1155.3 ± 142.5

105.3 ± 32.4

93.4 ± 29.1

215.8 ± 76.0

Aspartic acid

93.5 ± 15.1

47.4 ± 4.3

79.7 ± 21.3

14.7 ± 3.4

29.7 ± 5.9

54.0 ± 17.9

Glutamic acid

145.7 ± 57.6

179.9 ± 52.9

250.0 ± 71.6

49.7 ± 24.0

37.5 ± 21.0

118.8 ± 46.8

10.1 ± 3.0

6.6 ± 1.2

12.8 ± 4.5

Total

b

52.3 ± 3.9

Glycine

39.8 ± 2.6b

59.1 ± 6.5a

56.6 ± 5.6a

5.8 ± 0.6a

1.3 ± 0.7b

Proline

28.2 ± 2.6c

41.9 ± 3.0b

85.9 ± 7.2a

7.6 ± 2.3

6.3 ± 1.1

17.9 ± 7.0

Serine

35.5 ± 2.4

45.7 ± 6.4

53.9 ± 6.1

0.8 ± 0.1

0.9 ± 0.1

1.2 ± 0.3

Threonine

34.3 ± 2.2

45.3 ± 5.9

50.6 ± 5.2

0.8 ± 0.1

0.6 ± 0.1

0.8 ± 0.3

Arginine

33.9 ± 2.3

47.4 ± 4.0

45.6 ± 4.7

1.0 ± 0.2a

0.3 ± 0.1b

0.5 ± 0.1ab

12.9 ± 1.0ab

14.8 ± 1.7a

ND

ND

39.8 ± 3.5

37.7 ± 5.3

0.7 ± 0.1

Isoleucine

9.0 ± 0.8b 25.8 ± 1.8

Leucine

50.7 ± 3.8

Lysine

34.2 ± 2.4

Methionine

11.9 ± 2.8

Phenylalanine

34.0 ± 2.9

b

74.7 ± 7.1

a

47.3 ± 4.3

22.9 ± 1.9

Valine

36.0 ± 2.6b

33.6 ± 2.6

0.3 ± 0.0

43.9 ± 5.4 a

54.5 ± 5.0a

31.2 ± 3.2

1.1 ± 0.1

ab

50.5 ± 4.9a

0.4 ± 0.1

0.7 ± 0.2 b

0.5 ± 0.1 a

0.1 ± 0.0

0.7 ± 0.2b

ND

0.3 ± 0.1 a

1.0 ± 0.1

15.5 ± 2.1

47.6 ± 4.4

Tyrosine

67.0 ± 8.2

ab

48.8 ± 5.6

14.9 ± 1.5

b

78.2 ± 8.2

a

Alanine

Histidine

73.6 ± 6.6

a

0.7 ± 0.2ab 1.3 ± 0.7

b

0.1 ± 0.0b

1.8 ± 0.3

1.1 ± 0.2

1.0 ± 0.4

0.7 ± 0.1

0.6 ± 0.1

0.8 ± 0.1

1.6 ± 0.2

1.0 ± 0.2 a

2.3 ± 0.2

1.4 ± 0.4 a

1.2 ± 0.2b

Taurine

1.5 ± 0.4

4.8 ± 1.5

3.5 ± 0.4

3.5 ± 0.7

Cysteine

ND

ND

ND

0.4 ± 0.0a

0.1 ± 0.0ab

0.1 ± 0.0b

Ornithine

ND

ND

ND

0.5 ± 0.1

0.5 ± 0.1

0.8 ± 0.2

Phosphoserine

ND

ND

41.9 ± 5.6

3.1 ± 0.3

3.3 ± 0.9

2.2 ± 0.6

Table S2. Results of ANOVA of the total and free amino acid contents (mg/100 g) of the apical, central and basal portions of Saccharina japonica fronds (N = 5). Total amino acids

Total

Aspartic acid

Glutamic acid

Alanine

Glycine

Proline

Serine

Threonine

Arginine

Histidine

Isoleucine

Leucine

Lysine

Methionine

Phenylalanine

Tyrosine

df

MS

Portion

2

185043

Error

12

61290

Portion

2

1512.82

Error

12

1656.43

Portion

2

0.07

Error

12

0.08

Portion

2

0.04

Error

12

0.01

Portion

2

0.04

Error

12

0.01

Portion

2

0.30

Error

12

0.01

Portion

2

0.04

Error

12

0.01

Portion

2

347.83

Error

12

110.77

Portion

2

271.54

Error

12

72.99

Treatment

2

44.93

Error

12

7.40

Portion

2

282.99

Error

12

71.68

Portion

2

0.04

Error

12

0.01

Portion

2

0.03

Error

12

0.01

Portion

2

18.21

Error

12

23.60

Portion

2

0.03

Error

12

0.01

Portion

2

158.32

Error

12

34.77

Free amino acids F 3.02

0.91

0.90

5.63

5.62

48.56

3.03

3.14

3.72

P

df

MS

0.087

2

0.09

12

0.15

2

0.30

12

0.10

2

0.34

12

0.39

2

47.97

12

51.07

2

1.38

12

0.07

2

0.13

12

0.14

2

0.01

12

0.05

2

0.06

12

0.21

2

0.61

12

0.12

2

1.532

12

1.543

2

0.65

12

0.12

2

1.69

12

1.64

2

0.09

12

0.004

2

0.19

12

0.07

2

0.05

12

0.12

0.427

0.431

0.019

0.019

<0.001

0.086

0.080

0.055

6.07

0.015

3.95

0.048

3.92

3.84

0.77

2.88

4.55

0.049

0.051

0.484

0.095

0.034

F

P

0.59

0.574

3.17

0.078

0.86

0.446

0.94

0.418

19.23

<0.001

0.87

0.443

0.13

0.882

0.29

0.751

5.07

0.025

0.99

0.399

5.34

0.022

1.03

0.387

20.43

<0.001

2.59

0.116

0.40

0.680

Valine

Taurine

Cysteine

Ornithine

Phosphoserine

Portion

2

0.04

Error

12

0.01

Portion

2

0.32

Error

12

0.09

2

0.53

12

0.35

2

0.27

12

0.03

Portion

2

7.04

Error

12

1.47

Portion

2

0.90

Error

12

1.63

2

0.05

12

0.04

Portion

2

Error

12

45.14

6.47

3.71

85626.74

Significance levels (P < 0.05) are shown in bold type.

0.012

0.056

<0.001

1.49

0.265

10.19

0.003

4.80

0.029

0.55

0.590

1.21

0.332

Table S3. Results of nested ANOVA of the data for test diameter, body weight, gonad index, gonadal moisture, gonad hardness, gonad colour and free amino acid content of the gonads of Mesocentrotus nudus in different treatment groups. df Test diameter

Body weight

Gonad index

Hardness

L*

a*

b*

Total FAA

Aspartic acid

Glutamic acid

Alanine

Glycine

Proline

Serine

Threonine

Arginine

MS

F

P

Treatment

2

0.005

0.001

0.999

Treatment: aquarium

3

0.585

0.130

0.941

Treatment

2

5.920

0.095

0.909

Treatment: aquarium

3

15.59

0.251

0.860

Treatment

2

0.393

0.053

0.949

Treatment: aquarium

3

11.734

1.569

0.212

Treatment

2

0.002

2.970

0.063

Treatment: aquarium

3

0.002

2.865

0.049

Treatment

2

6.203

0.734

0.487

Treatment: aquarium

3

17.626

2.085

0.118

Treatment

2

2.795

1.621

0.211

Treatment: aquarium

3

3.570

2.071

0.120

Treatment

2

54.03

3.649

0.035

Treatment: aquarium

3

9.95

0.672

0.574

Treatment

2

1,193,950

12.631

<0.001

Treatment: aquarium

3

77,668

0.822

0.490

Treatment

2

0.007

0.239

0.789

Treatment: aquarium

3

0.042

1.480

0.235

Treatment

2

3277

0.958

0.392

Treatment: aquarium

3

2856

0.835

0.483

Treatment

2

0.096

15.537

<0.001

Treatment: aquarium

3

0.002

0.272

0.845

Treatment

2

166,313

20.150

<0.001

Treatment: aquarium

3

9642

1.168

0.334

Treatment

2

823.0

3.262

0.049

Treatment: aquarium

3

52.4

0.207

0.891

Treatment

2

3419

4.219

0.022

Treatment: aquarium

3

684

0.844

0.478

Treatment

2

1977.5

8.702

<0.001

Treatment: aquarium

3

506

2.227

0.100

Treatment

2

8102

1.719

0.193

Treatment: aquarium

3

1023

0.217

0.884

Histidine

Isoleucine

Leucine

Lysine

Methionine

Phenylalanine

Tyrosine

Valine

Taurine

Ornithine

Treatment

2

398.3

9.896

<0.001

Treatment: aquarium

3

39.5

0.982

0.411

Treatment

2

2176.1

8.102

0.001

Treatment: aquarium

3

680.7

2.534

0.071

Treatment

2

0.165

6.263

0.004

Treatment: aquarium

3

0.04

1.527

0.223

Treatment

2

3311

2.453

0.099

Treatment: aquarium

3

755

0.559

0.645

Treatment

2

687.4

10.042

<0.001

Treatment: aquarium

3

72.3

1.056

0.379

Treatment

2

1191.2

7.454

0.002

Treatment: aquarium

3

283.1

1.772

0.168

Treatment

2

4295

10.028

<0.001

Treatment: aquarium

3

753

1.758

0.171

Treatment

2

4111

5.442

0.008

Treatment: aquarium

3

1864

2.468

0.076

Treatment

2

112.42

1.932

0.159

Treatment: aquarium

3

175.24

3.012

0.042

Treatment

2

0.185

11.729

<0.001

Treatment: aquarium

3

0.017

1.048

0.382

Significance levels (P < 0.05) are shown in bold type

Table S4. Free amino acid content (mg/100 g) of Mesocentrotus nudus gonads at the start (n = 30, N = 6) and end of the experiment (n = 15, N = 3) (mean ± S.E.). ST, AP, MD and BS are defined in Table 2. Lower-case letters indicate significant differences among treatments according to Tukey’s test (P < 0.05). Asterisks indicate significant differences between ST and AP, MD, and BS according to the t-test. *, P<0.05; **, P<0.01; ***, P<0.001.

ST Total FAA Aspartic acid Glutamic acid

AP

2435.3 ± 46.4 6.3 ± 0.7 125.2 ± 6.0

MD b

2070.5 ± 107.7 ** 5.6 ± 0.9 142.5 ± 12.1

BS a

2625.7 ± 59.5 *

2435.0 ± 54.5 a

5.1 ± 0.1

5.0 ± 0.6

146.0 ± 22.2 b

169.7 ± 17.8*

Alanine

124.8 ± 8.9

376.1 ± 14.3 ***

460.1 ± 31.5 ***

536.1 ± 1.8 a***

Glycine

806.6 ± 31.5

704.0 ± 20.7 b

908.6 ± 17.5 a

849.5 ± 21.8a

Proline

24.2 ± 4.0

27.6 ± 2.2

41.2 ± 5.2*

39.4 ± 3.2*

b

Serine

35.6 ± 3.9

73.5 ± 3.5 ***

Threonine

30.7 ± 4.3

40.4 ± 4.3

Arginine

468.8 ± 16.2

ab

a

102.0 ± 3.2 *** 51.5 ± 9.5

204.6 ± 23.9*** b

28.5 ± 1.8

244.5 ± 20.6*** a

245.3 ± 11.5*** 19.1 ± 0.9 b

Histidine

25.5 ± 2.5

20.6 ± 2.3

Isoleucine

61.8 ± 6.4

42.7 ± 4.7

57.3 ± 9.7

33.4 ± 1.5*

Leucine

103.0 ± 11.0

77.5 ± 7.7

111.9 ± 16.8

72.0 ± 1.6

Lysine

267.1 ± 16.0

119.7 ± 11.7*** ab

Methionine

29.4 ± 3.3

22.0 ± 2.3

Phenylalanine

42.0 ± 3.9

27.3 ± 2.7* ab

Tyrosine

82.9 ± 7.0

61.0 ± 7.7

Valine

96.2 ± 7.4

84.7 ± 10.1

Taurine

89.6 ± 4.2

32.9 ± 1.8***

Ornithine

15.6 ± 0.6

b

7.8 ± 0.2 ***

28.7 ± 1.9

79.1 ± 8.0 ab***

149.3 ± 5.5** 32.3 ± 3.9

a

19.6 ± 0.3 b

42.4 ± 7.5

26.7 ± 1.2*

83.6 ± 10.4

a

110.5 ± 14.3 37.6 ± 4.8*** 13.0 ± 1.6

132.2 ± 8.0***

a

50.5 ± 1.2 b** 79.7 ± 2.5 37.7 ± 2.7*** 11.5 ± 0.5 a**

Table 1. Results of nested ANOVA of test diameters and body weights of Mesocentrotus nudus that received different treatments at the start of the experiment.

df Test diameter Body weight

MS

F

P

Treatment

2

2.27 0.488 0.618

Treatment: aquarium

3

0.94 0.202 0.895

Treatment

2

9.23 0.149 0.862

Treatment: aquarium

3 33.84 0.547 0.653

Table 2. Test diameters (mm), body weights (g), gonad indices, gonad hardness (N), and L*, a* and b* values of Mesocentrotus nudus gonads at the start (N = 30, n = 6) and end of the experiment in each treatment group (N = 15, n = 3) (mean ± S.E.). ST indicates the values obtained at the start (ST) of the experiment, and AP, MD, and BS indicate the values obtained at the end of the experiment for M. nudus fed the apical, middle and basal portions, respectively, of Saccharina japonica fronds. Asterisks indicate significant differences between ST and AP, MD, and BS according to the t-test. *, P < 0.05; **; P < 0.01; ***, P < 0.001. Test diameter and body weight of ST were replaced by the data obtained from 45 sea urchins in the three treatment groups in aquaria at the start of the experiment.

Test diameter Body weight

Gonad index

L*

a*

b*

0.43 ± 0.04

45.7 ± 1.2

10.8 ± 0.3

37.5 ± 1.2

ST

50.0 ± 0.5

58.7 ± 2.0

AP

50.2 ± 0.5

61.0 ± 1.6

18.9 ± 0.9***

0.13 ± 0.01**

56.8 ± 0.7***

9.8 ± 0.4

33.3 ± 0.9

MD

50.2 ± 0.1

62.0 ± 1.2

18.8 ± 1.4***

0.14 ± 0.01***

57.3 ± 1.3***

9.5 ± 0.3*

36.3 ± 1.1

BS

50.2 ± 0.5

60.9 ± 1.1

19.1 ± 0.2***

0.12 ± 0.01**

56.0 ± 0.5***

.

5.6 ± 0.4

Hardness

10.3 ± 0.6

36.8 ± 0.9

Table 3. Gonadal development stages of Mesocentrotus nudus in each treatment group at the beginning and end of the experiment by sex. ST, AP, MD and BS are defined in Table 2. I, II and III indicate the recovering, growing and premature stages.

Male

ST

Female

I

II

2

16

AP

8

MD

8

BS

8

III

I

II

III

1

11 7

2 1

5 6

Figure Legends Figure 1. Morphology of individual portions of Saccharina japonica fronds fed to Mesocentrotus nudus Figure 2. Daily water temperatures in aquaria. Figure 3. Total and free amino acid content (mg/100 g) of the apical, middle and basal portions of Saccharina japonica fronds (mean ± S.E.). Lower-case letters indicate significant differences among the portions according to Tukey’s test (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; P-Ser, phosphoserine. Figure 4. Free amino acid (FAA) content (mg/100 g) of Mesocentrotus nudus gonads at the start of the experiment and in each treatment group at the end of the experiment (mean ± S.E.). Lower-case letters indicate significant differences among treatment groups by Tukey’s test (P < 0.05). Asterisks indicate significant differences between the values obtained at the start and end of each treatment according to the t-test (P < 0.05). The abbreviations ST, AP, MD and BS are defined in Table 2. The abbreviations for the amino acids are defined in the legend to Figure 3. Figure 5. Principal component analysis biplot of the umami, sweet- and bitter-tasting free amino acid content of Mesocentrotus nudus gonads. Each arrow indicates the position of a free amino acid. The abbreviations AP, MD and BS are defined in Table 3. The abbreviations for the amino acids are defined in the legend to Figure 3.

Figure 1

Water temperature (°C)

21 20 19 18 17 16 15 14 13 12

May

Figure 2

June

July

Total amino acids 1200 300 1000

Apical portion Middle portion Basal portion

250

800

200

100

a

aa

a aa

b

50

b

b

b

400

a b

c

a b ba

b

a ab

aa

a

b

200

bb

0

0

Free amino acids 300 150 200 100 100

50 0

ab

Asp

Figure 3

Glu

Ala

Gly

a abb

a a bb

b

Pro

Ser

Thr

Arg

His

Ile

Leu

abb

Lys

Met

Phe

Tyr

Val

aa b

a abb

Tau

Cys

Orn P-Ser

Total

0

Total amino acid content (mg/100 g)

Amino acid content (mg/100 g)

600 150

1000

ST

AP

MD

*a

BS

a

a

2500

*b

800 b

600

2000

* a* a b

1500

*b

400

1000

200

*

*

**

* ba b

** 0

Asp

Figure 4

Glu

Ala

Gly

Pro

Ser

500

* * *

a *b a* b

Thr

Arg

His

a bab

* Ile

Leu

Lys

Met

aa b b *

* * Phe

Tyr

Val

* **

*b a *a

Tau

Orn

0

Total

Total FAA content (mg/100 g)

FAA content (mg/100 g)

a

Figure 5

Highlights

Apical, middle and basal frond portion of Saccharina kelp were fed to Mesocentrotus nudus.

Feeding of each frond portion improved gonad size, hardness, color and taste.

Basal portion markedly increased alanine and glutamic acid contents in gonads.

Basal portion showed high potential to improve gonad taste.

Conflict of interest

To the best of our knowledge, the named authors have no conflict of interest.