Skip to main content
PMC home page
PLOS ONE logoLink to PLOS ONE
. 2020 Apr 16;15(4):e0231673. doi: 10.1371/journal.pone.0231673

Odor-active compounds from the gonads of Mesocentrotus nudus sea urchins fed Saccharina japonica kelp

Satomi Takagi 1, Yoichi Sato 2,3, Atsuko Kokubun 4, Eri Inomata 1, Yukio Agatsuma 1,*
Editor: Filippo Giarratana5
PMCID: PMC7161986  PMID: 32298339

Abstract

Gonad size, color, texture and taste of Mesocentrotus nudus sea urchins collected from a barren can be improved by a short-term cage culture while being fed fresh Saccharina japonica kelp during May–July. We investigated the effect of S. japonica feeding during May–July on the improvement of gonad flavor in M. nudus collected from a barren. After feeding, we analyzed the odor-active volatile organic compounds (VOCs) from the gonads using gas chromatography-mass spectrometry and GC-sniffing analyses and compared to those from the gonads of wild sea urchins from an Eisenia kelp bed (fishing ground) and a barren. A total of 48 VOCs were detected from the gonads of cultured and wild sea urchins. Of them, a larger number of odor-active compounds were detected in the gonads of cultured sea urchins (25) than in those from the Eisenia bed (14) and the barren (6). Dimethyl sulfide from the gonads of sea urchins from the barren was described as having a strong, putrid odor. Sea urchin-like aromas were attributed to 2-butanol, 2-ethylhexanol, benzaldehyde and ethylbenzene from the gonads of cultured sea urchin and those of the Eisenia bed. Kelp feeding decreased the putrid odor from dimethyl sulfide, and enhanced pleasant, sweet aromas.

Introduction

Olfaction plays a dominant role in the taste of food [1]. In the human brain, the perception of flavor occurs in the neural system, which affects food preference and appetite [2]. Yamamoto et al. [3] demonstrated that the odor of the fragrant tea olive Osmanthus fragrans decreases appetite and food intake, resulting in a decrease in body weight. Furthermore, odor can change the taste of food [4].

Sea urchin gonads are a highly valued cuisine and, particularly, a favorite sushi ingredient in Japan [5]. Japan is the largest consumer of sea urchin gonads in the world, accounting for approximately 90% of the total world sea urchin landing [6]. Past studies revealed that gonads with large size, bright orange or yellow color, medium hardness, high sweet-tasting alanine and low bitter-tasting arginine contents are preferred [7–9]. The factors concerned with gonad quality have been studied [10–13]. Flavor is an important factor in the sensory quality of sea urchin gonads [10, 14]. Some components associated with the flavor differ among sea urchin species [15–18]. In Evechinus chloroticus, flavor and the associated volatile compounds in gonads vary between the north and south of New Zealand [15, 16]. To date, the odor-active volatile compounds in the gonads of E. chloroticus [15, 16], Paracentrotus lividus [17] and Mesocentrotus nudus [18] have been identified. Some of them were inferred to be associated with sea urchin aroma [17, 18], but the compounds with the “sea urchin-like” aroma have still not been determined.

Mesocentrotus nudus accounts for more than two-thirds of the total sea urchin landing in Japan together with Strongylocentrotus intermedius and is the most expensive sea urchin gonad in the world [19]. This species densely distributes on barrens [20], where the gonads show small size [21] and deteriorated color, texture, and taste [13, 22], therefore no commercial value [20]. Recently, research on the improvement of the gonad quality of M. nudus from barrens by short-term culture techniques has been concentrated on in northern Japan [13, 23]. Takagi et al. [8] first improved the whole gonad quality (size, color, texture and taste) of barren individuals to a more desirable level compared to that of individuals from an Eisenia kelp bed (fishing ground) by feeding them fresh cultivated Saccharina japonica during May–July. They evaluated that the gonad smell of sea urchins fed S. japonica and that of those collected from an Eisenia kelp bed were more desirable than that of those from a barren, as determined by a panel test [8]. The results suggested that the odor-active compounds in gonads could vary in accordance with their habitats.

In the present study, M. nudus were collected from a barren and cultured in cages suspended offshore and fed fresh cultivated S. japonica during May–July. After feeding, the odor-active volatile compounds of the gonads were analyzed by gas chromatography-mass spectrometry (GC-MS) and GC-sniffing methods and compared to those from the urchin gonads collected from an Eisenia kelp bed and a barren. Takagi et al. [8] conducted a sensory evaluation and measurements and analyses of gonad size, developmental stages, color, texture and free amino acid contents of sea urchins cultured and collected from the same algal beds by the same methods on the same day with the present study. The purpose of this study is to (1) clarify the effect of S. japonica feeding to improve gonad flavor of sea urchins from a barren and (2) identify the compounds with sea urchin like aroma.

Materials and method

Ethics statement

Mesocentrotus nudus were collected from a site in the Shizugawa Bay, Miyagi Prefecture, Japan that is not privately-owned or protected in any way. Field studies did not include endangered or protected species. All experimental procedures on animals were in compliance with the guidelines of Miyagi Fisheries Cooperative Association and Miyagi Prefectural Government.

Sea urchin samples

A total of 500 adult M. nudus (46–55 mm diameter) were collected by scuba divers from a barren at depths of 2.5–3.0 m off Nojima Island, Shizugawa Bay, Miyagi Prefecture (38°40´N, 141°30´E) on 6 May 2016. Immediately after collection, 100 sea urchins were placed in each of five cages suspended horizontally along a straight line at a depth of approximately 4.5 m at a wave-sheltered site off Areshima Island (38°40´N, 141°27´E) in the bay until 19 July 2016. The cages were cuboid (90 × 87 × 45 cm) with 3 cm mesh and made of polyethylene. The sea urchins were kept without food for 5 days until the start of the experiment. Fresh S. japonica cultivated were fed to sea urchins ad libitum every 7–10 days for 63 days from 11 May to 13 July. On 19 July, 20 cultured sea urchins were collected randomly from the cages, and 20 wild sea urchins were collected from each of the barren where we collected the sea urchins for the cage culture and an Eisenia bicyclis kelp bed at depths of 2.2–3.0 m off Nojima Island. Thus, three different groups, cultured sea urchins (CSU), and wild sea urchins collected from a barren (BSU) and an E. bicyclis bed (ESU), were designed. After collection, the sea urchins were kept in two cool boxes with moist urethane mats immersed in seawater and then transported to the factory of Riken Food Co. Ltd. in Tagajo (38°16´N, 141°0´E). These sea urchins were dissected, and the gonads were isolated and soaked 3 times in 5°C sterile seawater. Thereafter, they were patted dry on bleached cotton at 4°C for 30 minutes according to Kinoshita et al. [24]. No gamete released from the gonads was observed. A total of ca. 5 g gonads was randomly collected from one group of gonads and stored in a polystyrene storage container at 4°C until further analyses. Three containers were prepared for each group (n = 3). Headspace sampling, GC-MS analysis and GC-sniffing analysis were conducted according to Sato et al. [18] within 48 hours after dissection. Takagi et al. [8] showed no significant differences in test diameter among these sea urchins (CSU, ESU and BSU), and almost the gonads were in the growing stage.

Large volume static headspace sampling

For analysis of volatile organic compounds (VOCs) of sea urchin gonads, headspace volatile compounds were collected in a large volume static headspace (LVSH) system (Entech 7100A series, Entech Instruments Inc., Simi Valley, CA, USA). Sea urchin gonads were analyzed within 48 hours after collection. From each container, ca. 5 g gonads were sealed in a 375 ml glass jar for measurement via LVSH, which was stored in an incubator (DK400, Yamato Scientific Co., Ltd., Tokyo, Japan) at 30°C for 10 min. After incubation, 150 ml of headspace gas was vacuum-extracted from the glass jar. The VOCs were desorbed by thermodesorption using a preconcentrator (Entech 7100A series, Entech Instruments Inc.) and injected into the GC-MS system.

GC-MS analysis

Quantification of the VOCs was performed using an Agilent 6890 series gas chromatograph (Agilent Technologies Inc., Palo Alto, CA, USA) equipped with an Agilent 5975B mass-selective detector and a sniffing port. One half of the column flow was directed to the MS system, while the other half was directed to the heated sniffing port. The GC-MS system was equipped with a DB-WAX column (60 m × 0.25 mm i.d., 0.5 μm film thickness; 122–7063; Agilent Technologies Inc.). The GC injector temperature was 250°C. Analyses were carried out using helium as the carrier gas at an average flow rate of 27 cm sec-1 with the following temperature program: 40°C for 5 min, an increase at 5°C min-1 to 240°C, followed by a final 5 min hold at 240°C.

Mass spectrometry was carried out in scan mode using an electron ionization voltage of 70 eV and a scan range from m/z 10 to 300 with a scan rate of 1.58 scans per sec. Analysis of VOCs was performed using Powered Pro software (Wiley 11N17main, Agilent Technologies Inc.). Each VOC was identified by similarity search [25] using the library of the software (Wiley 11N17main, Agilent Technologies Inc.). When a VOC was detected in triplicate analysis, this determined the presence of the VOC in the group. The VOCs detected in the present study were compared to those of Sato et al. [18]. The relative amounts of each VOC detected by GC-sniffing analysis were calculated based on the peak areas in the chromatograms.

GC-sniffing analysis

GC-sniffing analysis was conducted for each group. One half of the column flow was directed to a heated sniffing port (ODP2 olfactory detection port, Gerstel GmbH & Co. KG, Mülheim an der Ruhr, Germany). To the sniffing port, humidified air (50–75% relative humidity) was carried at 1.02 ml min-1 to prevent the nose from drying out. The three panelists, who are well versed in sea urchin gonad quality and share common perceptions, recorded the retention time and the related description of the aroma compounds by writing in a paper. They could record without taking off their nose from the sniffing port.

Statistical analyses

Statistical analyses were conducted using JMP 10 software (SAS Institute Inc., Cary, NC, USA). Differences in the peak areas of compounds detected by GC-MS analysis among the three groups were analyzed with one-way analysis of variance (ANOVA). The values of not detected compounds were inputted as 0.0001. Tukey’s multiple comparison test was performed as a post hoc test.

Results

Typical total ion chromatograms of the VOCs from sea urchin gonads of each group are shown in Fig 1. The VOCs we detected are shown in Table 1 and compared to those reported by Sato et al. [18]. A total of 48 VOCs were detected in the gonads of CSU (48), ESU (48) and BSU (46). Of them, S-methyl thioacetate and bromoform were not detected in the gonads of BSU. These compounds could be categorized into the following chemical families: alcohols (7), aldehydes (8), aromatic compounds (9), esters (4), halomethanes (4), hydrocarbons (7), ketones (6), and others (3; dimethyl sulfide (DMS), acetonitrile, and bis-(methylthio)-methane). Of them, 35 compounds were also detected by Sato et al. [18]. The peak areas of the odor-active VOCs detected by GC-sniffing analysis are shown in Table 2. Odors of 25, 14, and 6 compounds detected from the gonads of CSU, ESU and BSU, respectively, were described.

Fig 1. Chromatograms of volatile organic compounds (VOCs) in the gonads of Mesocentrotus nudus using gas chromatography-mass spectrometry (GC-MS) (N = 3).

Fig 1

Open in a new tab

Cultured, Eisenia and Barren indicate cultured sea urchins, and sea urchins collected from an Eisenia bicyclis bed and a barren, respectively. Compounds were identified by peak numbers, as shown in Table 1.

Table 1. Volatile organic compounds (VOCs) detected from gonads of Mesocentrotus nudus using gas chromatography-mass spectrometry (n = 3).

Detection
NO. Rt Compound Cultured Eisenia Barren Sato et al. [18]
Alcohols
8 12.05 Methanol + + + +
11 13.21 2-propanol + + + +
12 13.46 Ethanol + + + +
20 16.57 2-butanol + + + +
22 17.04 Propanol + + +
31 21.00 Butanol + + +
45 30.41 2-ethylhexanol + + + +
Aldehydes
1 5.87 Acetaldehyde + + + +
2 6.74 Dimethoxymethane + + + +
7 11.78 Acetal + + + +
33 21.97 2-ethylhexanal + + +
39 25.10 Octanal + + +
42 28.12 Nonanal + + + +
46 30.92 Decanal + + + +
47 32.01 Benzaldehyde + + + +
Aromatic compounds
4 7.79 Methylcyclohexane + + + +
13 13.63 Benzene + + +
23 17.26 Toluene + + + +
27 19.87 Ethylbenzene + + + +
28 20.34 Xylene + + + +
35 23.13 Ethyltoluene + + +
36 23.77 Trimethylbenzene + + + +
37 24.23 Styrene + + + +
44 29.74 Dichlorobenzene + + + +
Esters
6 11.56 Ethyl acetate + + + +
14 14.80 Propyl acetate + + + +
24 17.48 S-methyl thioacetate + + +
25 18.18 Butyl acetate + + +
Halomethanes
10 12.92 Dichloromethane + + + +
15 15.39 Trichloroethane + + +
19 16.26 Chloroform + + + +
43 29.63 Bromoform + +
Hydrocarbons
16 15.53 Decane + + + +
21 16.68 α-pinene + + + +
26 18.56 Undecane + + + +
29 20.43 Δ3-carene + + +
32 21.28 2,5,6-trimethyloctane + + +
34 22.15 Limonene + + + +
38 24.61 Tridecane + + + +
Ketones
5 9.12 Acetone + + + +
9 12.13 2-butanone + + + +
18 16.06 4-methyl-2-pentanone + + + +
30 21.00 3-heptanone + + +
41 26.59 6-methyl-5-hepten-2-one + + +
48 35.20 Acetophenone + + + +
Others
3 7.09 Dimethyl sulfide + + + +
17 15.72 Acetonitrile + + + +
40 25.42 Bis-(methylthio)-methane + + + +
Open in a new tab

An explanation of the terms Cultured, Eisenia and Barren is provided with Fig 1.

Rt indicates retention time (min).

Table 2. The peak areas × 10−5 (mean ± S.E.) and descriptions of odor-active volatile organic compounds in Mesocentrotus nudus gonads of each group (n = 3).

Peak area (×10−5) Description
NO. Rt Compound name Cultured Eisenia Barren p Cultured Eisenia Barren
Alcohols
11 13.21 2-propanol 6.94 ± 1.09 8.25 ± 1.27 10.29 ± 0.61 0.076 Kelp, seaweed, hey smell
12 13.46 Ethanol 456.02 ± 212.64 559.31 ± 221.89 503.28 ± 187.53 0.922 Mint
20 16.57 2-butanol 1.72 ± 0.39 1.70 ± 0.41 2.80 ± 0.52 0.216 Seafood, hey smell Unpleasant, sea urchin
45 30.41 2-ethylhexanol 616.30 ± 284.38 526.13 ± 52.00 566.66 ± 102.69 0.938 Sea urchin, seafood Sea urchin
Aldehydes
1 5.87 Acetaldehyde 6.12 ± 1.93 3.95 ± 1.24 3.72 ± 0.75 0.392 Putrid
2 6.74 Dimethoxymethane 3.12 ± 0.54 2.65 ± 0.35 2.65 ± 0.65 0.755 Green, acrid Putrid
7 11.78 Acetal 1.77 ± 1.33 2.31 ± 2.57 0.93 ± 0.32 0.714 Sweet
33 21.97 2-ethylhexanal 0.49 ± 0.09 0.21 ± 0.18 0.36 ± 0.01 0.119 Sweet
39 25.10 Octanal 0.15 ± 0.01 0.14 ± 0.04 0.19 ± 0.02 0.193 Green Putrid Green
42 28.12 Nonanal 2.31 ± 0.16 2.54 ± 0.54 2.60 ± 0.29 0.723 Plastic, medicinal
46 30.92 Decanal 2.74 ± 0.39 2.51 ± 0.51 2.45 ± 0.29 0.813 Green
47 32.01 Benzaldehyde 0.22 ± 0.06 0.21 ± 0.05 0.23 ± 0.02 0.962 Sea urchin
Aromatic compounds
4 7.79 Methylcyclohexane 3.29 ± 0.27 2.74 ± 0.89 4.05 ± 0.39 0.150 Benzene
13 13.63 Benzene 0.62 ± 0.05 0.62 ± 0.17 0.66 ± 0.15 0.958 Tingle in nose, heave note Mint
23 17.26 Toluene 71.64 ± 16.58 54.25 ± 17.59 59.44 ± 7.81 0.606 Fruit Sweet
27 19.87 Ethylbenzene 54.44 ± 10.42 61.95 ± 9.12 58.98 ± 10.80 0.873 Sea urchin, baked fish Putrid fish, sour
36 23.77 Trimethylbenzene 1.03 ± 0.09 1.26 ± 0.41 1.07 ± 0.06 0.542 Hey smell
37 24.23 Styrene 0.68 ± 0.12b 0.86 ± 0.16b 1.91 ± 0.08a < 0.001 Hey smell Hey smell
44 29.74 Dichlorobenzene 4.43 ± 1.32 3.83 ± 1.50 6.62 ± 0.93 0.227 Green
Esters
6 11.56 Ethyl acetate 11.76 ± 1.40 10.51 ± 1.28 8.65 ± 0.75 0.171 Sweet
14 14.80 Propyl acetate 3.00 ± 0.25 2.74 ± 1.47 2.33 ± 0.24 0.616 Sweet, sweet candy
24 17.48 S-methyl thioacetate 10.75 ± 9.61a 0.38 ± 0.33a NDb < 0.001 Tingle in nose, heavy note, fishy
25 18.18 Butyl acetate 0.73 ± 0.13 0.62 ± 0.11 0.48 ± 0.12 0.327 Green Putrid
Halomethanes
19 16.26 Chloroform 8.17 ± 1.82 5.61 ± 1.01 6.55 ± 1.94 0.542 Green, hey smell Plastic, medicinal
43 29.63 Bromoform 0.12 ± 0.04a 0.04 ± 0.01b NDc < 0.001 Green
Hydrocarbons
16 15.53 Decane 2.02 ± 0.41 2.56 ± 1.47 2.32 ± 0.53 0.833 Fishy
26 18.56 Undecane 2.25 ± 0.17 2.10 ± 0.57 2.67 ± 0.50 0.545 Tingle in nose, heavy note Oxidized seaweed, hey smell
Ketones
5 9.12 Acetone 116.18 ± 45.05 103.60 ± 20.71 113.94 ± 9.31 0.926 Sweet
9 12.13 2-butanone 11.62 ± 2.92 9.41 ± 3.49 7.85 ± 1.45 0.521 Peach
41 26.59 6-methyl-5-hepten-2-one 2.61 ± 0.72 1.87 ± 1.13 2.29 ± 0.70 0.761 Sweet, citrus fruit
Others
3 7.09 Dimethyl sulfide 16.18 ± 7.39 9.47 ± 1.59 65.17 ± 48.88 0.261 Putrid
17 15.72 Acetonitrile 48.98 ± 20.79 44.59 ± 18.81 56.33 ± 17.72 0.887 Tree bark
40 25.42 Bis-(methylthio)-methane 2.32 ± 0.96 1.92 ± 0.92 0.97 ± 0.11 0.257 Fishy
Open in a new tab

Significance of values in peak areas among groups analyzed by ANOVA are provided. ND indicate no detection. An explanation of the terms Cultured, Eisenia and Barren is provided in Fig 1. Lower-case letters indicate significant differences in peak areas among groups (p < 0.05, Tukey’s test). An explanation of the term Rt is provided with Table 1.

Alcohols

Seven alcohols, methanol, 2-propanol, ethanol, 2-butanol, propanol, butanol and 2-ethyhexanol, were detected. The odors of four alcohols were described. There were no significant differences in the peak areas of each odor-active alcohol among each group. The odors of 2-butanol from the gonads of CSU and ESU were described as acceptable seafood and hey smells and as an unpleasant sea urchin-like odor, respectively. 2-Ethylhexanol from the gonads of CSU and ESU was described as having a sea urchin-like aroma. 2-Propanol from the gonads of CSU was described as having kelp and hey smells.

Aldehydes

Eight aldehydes, acetaldehyde, dimethoxymethane, acetal, 2-ethylhexanal, octanal, nonanal, decanal and benzaldehyde, were identified from the sea urchin gonads of each group. The odors of all aldehydes could be described. There were no significant differences in the peak areas of each aldehyde among the groups. Acetaldehyde, dimethoxymethane and octanal from gonads of ESU were described as having a putrid odor. Acetal and 2-ethylhexanal from the gonads of CSU were described as having a sweet aroma. Octanal from the gonads of CSU and BSU were described as having green scents. Benzaldehyde from the gonads of CSU was described as having a sea urchin-like aroma.

Aromatic compounds

Nine aromatic compounds, methylcyclohexane, benzene, toluene, ethylbenzene, xylene, ethyltoluene, trimethylbenzene, styrene and dichlorobenzene, were detected from the gonads of sea urchins of each group. The odors of seven compounds were described. Except for styrene, there were no significant differences in aromatic compound peak areas among each group. Benzene from the gonads of CSU was described as having a heavy note, although that from ESU was described as having a mint aroma. Toluene from the gonads of CSU and ESU were described as having fruit-like and sweet aromas, respectively. The peak area of toluene from the gonads of CSU was large compared to that of the other groups. Ethylbenzene from the gonads of CSU was described as having sea urchin and baked fish-like aromas compared to the noted putrid fish and sour odors from that of ethylbenzene from ESU gonads. The peak area of styrene from the gonads of BSU was significantly larger than in the other groups (P < 0.001). Styrene from the gonads of CSU and ESU was described as having a hey smell.

Esters

Four esters, ethyl acetate, propyl acetate, S-methyl thioacetate and butyl acetate, were identified. S-methyl thioacetate was not detected in the gonads of BSU. The odors of all detected esters were described. Except for S-methyl thioacetate, there were no significant differences in peak areas among each group. Ethyl acetate and propyl acetate from the gonads of CSU were described as having sweet aromas. S-methyl thioacetate from the gonads of CSU showed a markedly larger peak area than those of other groups and was described as having a heavy note tingling in nose and fishy odor. Butyl acetate from the gonads of CSU and ESU were described as having a green aroma and putrid odor, respectively.

Halomethanes

Four halomethanes, dichloromethane, trichloroethane, chloroform and bromoform, were identified. Bromoform was not detected in the gonads of BSU. Chloroform and bromoform from the gonads of CSU were described as having a green aroma. The peak area of bromoform from CSU was significantly larger than that in the other groups (P < 0.05).

Hydrocarbons

Seven hydrocarbons, decane, α-pinene, undecane, Δ3-carene, 2,5,6-trimethyloctane, limonene and tridecane, were detected from each group. The odors of decane and undecane were described. There were no significant differences in hydrocarbon peak areas among each group. Decane and undecane from the gonads of ESU were described as having a fishy odor and oxidized seaweed-like and hey smells, respectively.

Ketones

Six ketones, acetone, 2-butanone, 4-methyl-2-pentanone, 3-heptanone, 6-methyl-5-hepten-2-one and acetophenone, were detected from the gonads of each group. The odors of three ketones were described. There were no significant differences in ketone peak areas among each group. 2-Butanone from the gonads of CSU was described as having a peach-like aroma. 6-Methyl-5-hepten-2-one from the gonads of ESU was described as having citrus-like and sweet aromas.

Other compounds

Three other compounds, dimethyl sulfide (DMS), acetonitrile and bis-(methylthio)-methane, were detected, and their odors were described. DMS from the gonads of BSU was described as having a putrid odor. The peak area of DMS from the gonads of BSU was markedly large compared to that from the other groups. Bis-(methylthio)-methane from the gonads of CSU was described as having a fishy odor.

Discussion

In the present study, 2-butanol, 2-ethylhexanol, benzaldehyde and ethylbenzene detected from the gonads of M. nudus were described as having sea urchin-like aromas for the first time. Benzaldehyde has pleasant almond, nutty and stony fruit like aromas of peach (Prunus persica) [26]. De Quirós et al. [17] suggested that this compound is associated with the pleasant aroma of sea urchin. Ethylbenzene is detected in the gonads of some sea urchin species [17, 18]. Phillips et al. [16] suggested a positive correlation between ethylbenzene concentration and marine odor. Sato et al. [18] reported that ethylbenzene from the gonads of M. nudus in Naburi Bay was described as having a fish oil scent, which would lead to the pleasant aroma of fresh sea urchin gonads. In the present study, ethylbenzene from the gonads of CSU was described as having sea urchin-like and baked fish aromas, and that from ESU was described as having putrid fish-like and sour odors with slightly larger peak areas than those of CSU.

A larger number of sweet and fruity pleasant odor descriptions (acetal, 2-ethylhexanal, toluene, ethyl acetate, propyl acetate, acetone, 2-butanone and 6-methyl-5-hepten-2-one described) detected from the gonads of cultured sea urchins than from those of wild sea urchins would reflect the sweet taste of their gonads [8]. Esters are associated with the sweet aroma of various fruits [27]. Propyl acetate detected from gonads of M. nudus in Naburi Bay was described as having a sweet aroma [18]. Toluene can be detected from some fruits: strawberry, apple, grape, mango and sapodilla [28]. Toluene from sapodilla fruit (Achras sapota) and mango (Mangifera indica) are described as having sweet and caramel aromas, and caramel and solvent odors, respectively [29, 30]. Ketones from crustaceans have sweet floral and fruity flavors [31]. 2-Butanone can be detected from pineapple [32]. This compound is also detected from yellow passion fruit and has fruity, moldy, woody, fresh and bitter aromas [33]. These past studies suggest that propyl acetate, toluene and 2-butanone influence the fresh and fruity sweet aromas of the gonads of CSU.

The odor descriptions of dimethoxymethane, octanal, benzene, ethylbenzene, butyl acetate, and undecane differed between gonads from CSU and ESU. Of them, octanal is contained in several seaweeds [34–36] and the octanal content differs among seaweed species [35]. Sato et al. [18] suggested that octanal in M. nudus gonads would be derived from their consumption of seaweeds as food. Differences in seaweed species consumed between CSU and ESU changed the odor description of octanal. The differences in odor descriptions of other compounds remain to be identified.

Sulfur-containing compounds affect the overall flavor because of their low thresholds [37]. Bis-(methylthio)-methane has a garlic-like odor, which is one of the off-flavor components of prawns and sand lobsters [38]. This compound is produced by a metabolite of Shewanella putrefaciens grown on high-pH pork [39]. This compound from the gonads of M. nudus in Naburi Bay was described as having ozone and sulfur odors [18] in comparison to having a fishy odor when extracted from the gonads of CSU in the present study. These results suggest that bis-(methylthio)-methane can be one of the factors affecting the unpleasant odor of decomposed shellfish. DMS is known to have a sulfur odor when extracted from mussels [40] and oysters [41]. This compound from the gonads of M. nudus in Naburi Bay was described as having marine and fish-like odors [18]. From the gonads of E. chloroticus, DMS is also associated with marine, seafood and sharp odors [16]. In the present study, this compound from the gonads of BSU was described as having a strong putrid odor, and the peak area was markedly large regardless of a lack of an odor description from the gonads of other groups. This compound is detected from several fish and shellfish species [42, 43]. Therefore, M. nudus from the barren would reflect the omnivorous food habit [20] because there is no erect algae on barrens, and the sea urchins would consume dead fish or other shellfish [18].

In conclusion, from the gonads of cultured M. nudus, a larger number of odor-active VOCs with a green scent and sweet aromas were detected than from those of wild sea urchins, which would enhance the pleasant aroma and richness of flavor, leading to high sensory evaluation [8]. The present study demonstrated the effect of Saccharina kelp feeding on the odor-active compound profile of gonads of edible sea urchin for the first time. The number of VOCs detected from the gonads of cultured and wild M. nudus was almost the same. However, kelp feeding decreased the strong, putrid odor from DMS, enhanced pleasant, sweet aroma, and would enhance the richness and sweetness of flavor. The appropriate feeding duration for improvement in gonad flavors must be further studied.

Supporting information

S1 Table. Raw data of chromatogram and peak area of volatile organic compounds from gonads of Mesocentrotus nudus of each group.

(XLSX)

Click here for additional data file. (651.7KB, xlsx)

Acknowledgments

We are grateful to the Operative Chief, N. Sasaki, the Head of the Youth Division, Y. Sugawara, and other staff of the Shizugawa Branch of the Miyagi Fisheries Cooperative Association for their full cooperation with managing the sea urchin cage cultures. We sincerely thank S. Owada of the Hirota Fisheries Cooperative Association for providing S. japonica. We also thank M. Oshima and S. Kodama of Diving Stage Ariel for collecting sea urchins for culturing.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This study was financially supported by the Grand-in-Aid for JSPS Fellow [grant number 17J02308] from Japan Society for the Promotion of Science [S.T.], and basic science research funding of Riken Food Co., Ltd. [Y.S.] and RIKEN VITAMIN Co., Ltd. [A.K.]. The funders provided financial support in the form of salaries for authors [S.T., Y.S., A.K.] and research materials, but did not have any additional role in the study design, data collection and analysis, decision for publication, or preparation of the manuscript.

References

  • 1.Spence C. Just how much of what we taste derives from the sense of smell? Flavour 2015; 4: 30. [Google Scholar]
  • 2.Shepherd GM. Smell images and the flavour system in the human brain. Nature 2006; 444: 316–321. 10.1038/nature05405 [DOI] [PubMed] [Google Scholar]
  • 3.Yamamoto T, Inui T, Tsuji T. The odor of Osmathus fragrans attenuates food intake. Sci. Rep. 2013; 3: 1518 10.1038/srep01518 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Bakalar N. Sensory science: Partners in flavour. Nature 2012; 486: S4–S5. 10.1038/486S4a [DOI] [PubMed] [Google Scholar]
  • 5.Iwai T. Favorite sushi ingredient ranking. Asahi Shimbun newspaper. 2018 Jul 28; be on Saturday: 2. Available from: https://www.asahi.com/articles/DA3S13606955.html. Japanese
  • 6.Sun J, Chiang FS. Use and exploitation of sea urchins In Brown NP, Eddy SD, editors. Echinoderm aquaculture. Hoboken: Wiley Blackwell; 2015. p. 25–45. [Google Scholar]
  • 7.Kelly MS, Symonds RC. Carotenoids in sea urchins In Lawrence JM, editor. Sea urchins: biology and ecology. 3rd ed. San Diego: Academic Press; 2013. p. 171–177. [Google Scholar]
  • 8.Takagi S, Murata Y, Inomata E, Aoki MN, Agatsuma Y. Production of high quality gonads in the sea urchin Mesocentrotus nudus (A. Agassiz, 1864) from a barren by feeding on the kelp Saccharina japonica at the late sporophyte stage. J. Appl. Phycol. 2019; 31: 4037–4048. [Google Scholar]
  • 9.Walker CW, Böttger SA, Unuma T, Watts SA, Harris LG, Lawrence AL et al. In Brown NP, Eddy SD, editors. Echinoderm aquaculture. Hoboken: Wiley Blackwell; 2015. p. 263–286. [Google Scholar]
  • 10.McBride SC, Price RJ, Tom PD, Lawrence JM, Lawrence AL. Comparison of gonad quality factors: color, hardness and resilience, of Strongylocentrotus franciscanus between sea urchins fed prepared feed or algal diets and sea urchins harvested from the Northern California fishery. Aquaculture 2004; 233: 405–422. [Google Scholar]
  • 11.Pearce CM, Daggett TL, Robinson SMC. Effects of starch type, macroalgal meal source, and β-carotene on gonad yield and quality of the green sea urchin, Strongylocentrotus droebachiensis (Müller), fed prepared diets. J. Shellfish Res. 2003; 22: 505–519. [Google Scholar]
  • 12.Robinson SMC, Castell JD, Kennedy EJ. Developing suitable colour in the gonads of cultured green sea urchins (Strongylocentrotus droebachiensis). Aquaculture 2002; 206: 289–303. [Google Scholar]
  • 13.Takagi S, Murata Y, Inomata E, Endo H, Aoki MN, Agatsuma Y. Improvement of gonad quality of the sea urchin Mesocentrotus nudus fed the kelp Saccharina japonica during offshore cage culture. Aquaculture 2017; 477: 50–61. [Google Scholar]
  • 14.Phillips K, Bremer P, Silcock P, Hamid N, Delahunty C, Barker M et al. Effect of gender, diet and storage time on the physical properties and sensory quality of sea urchin (Evechinus chloroticus) gonads. Aquaculture 2009; 288: 205–215. [Google Scholar]
  • 15.Niimi J, Leus M, Silcock P, Hamid N, Bremer P. Characterisation of odour active volatile compounds of New Zealand sea urchin (Evechinus chloroticus) roe using gas chromatography–olfactometry–finger span cross modality (GC–O–FSCM) method. Food. Chem. 2010; 121: 601–607. [Google Scholar]
  • 16.Phillips K, Niimi J, Hamid N, Silcock P, Delahunty C, Barker M, et al. Sensory and volatile analysis of sea urchin roe from different geographical regions in New Zealand. LWT-Food Sci. Technol. 2010; 43: 202–213. [Google Scholar]
  • 17.De Quirós ARB, López-Hernández J, González-Castro MJ, de la Cruz-García C, Simal-Lozano J. Comparison of volatile components in fresh and canned sea urchin (Paracentrotus lividus, Lamarck) gonads by GC–MS using dynamic headspace sampling and microwave desorption. Eur. Food Res. Technol. 2001; 212: 643–647. [Google Scholar]
  • 18.Sato Y, Takagi S, Inomata E, Agatsuma Y. Odor-active volatile compounds from the gonads of the sea urchin Mesocentrotus nudus in the wild in Miyagi Prefecture, Tohoku, Japan. Food and Nutrition Sciences 2019; 10: 860–875. [Google Scholar]
  • 19.Unuma T. Introduction: Sea urchin fisheries in Japan, In Brown NP, Eddy SD, editors. Echinoderm aquaculture. Hoboken: Wiley Blackwell; 2015. p. 77–85. [Google Scholar]
  • 20.Agatsuma Y. Strongylocentrotus nudus In Lawrence JM, editor. Sea urchins: biology and ecology. 3rd ed. San Diego: Academic Press; 2013. p. 449–460. [Google Scholar]
  • 21.Agatsuma Y. Ecological studies on the population dynamics of the sea urchin Strongylocentrotus nudus. Sci. Rep. Hokkaido Fish. Exp. Stn. 1997; 51: 1–66. Japanese with English abstract. [Google Scholar]
  • 22.Agatsuma Y, Sato M, Taniguchi K. Factors causing brown-colored gonads of the sea urchin Strongylocentrotus nudus in northern Honshu, Japan. Aquaculture 2005; 249: 449–458. [Google Scholar]
  • 23.Unuma T, Murata Y, Hasegawa N, Sawaguchi S, Takahashi K. Improving the food quality of sea urchins collected from barren grounds by short-term aquaculture under controlled temperature. Bull. Fish. Res. Agen. 2015; 40: 145–153. [Google Scholar]
  • 24.Kinoshita Y, Yoshioka T, Miyazaki S, Kato S, Konno K. Storage of sea urchin gonad in oxygenated sea water. Nippon Suisan Gakk. 2009; 75: 237–243. Japanese with English abstract [Google Scholar]
  • 25.Zhu X, Gao Y, Chen Z, Su Q. Development of a Chromatogrophic fingerprint of tobacco flavor by use of GC and GC-MS. Chromatographia 2009; 69: 735–742. [Google Scholar]
  • 26.Narain N, Hsieh TCY, Johnson CE. Dynamic headspace concentration and gas chromatography of volatile flavor components in peach. J. Food Sci. 1990; 55: 1303–1307. [Google Scholar]
  • 27.Shalit M, Katzir N, Tadmor Y, Larkov O, Burger Y, Shalekhet F et al. Acetyl-CoA: alcohol acetyltransferase activity and aroma formation in ripening melon fruits. J. Agric. Food Chem. 2001; 49: 794–799. 10.1021/jf001075p [DOI] [PubMed] [Google Scholar]
  • 28.Hui YH. Handbook of fruit and vegetable flavors. Hoboken: John Wiley & Sons, Inc.; 2010. [Google Scholar]
  • 29.MacLeod AJ, de Troconis NG. Volatile flavor components of sapodilla fruit (Achras sapota L.). J. Agric. Food Chem. 1982; 30: 515–517. [Google Scholar]
  • 30.MacLeod AJ, Snyder CH. Volatile components of two cultivars of mango from Florida. J. Agric. Food Chem. 1985; 33: 380–384. [Google Scholar]
  • 31.Cha YJ, Cadwallader KR, Baek HH. Volatile flavor components in snow crab cooker effluent and effluent concentrate. J. Food Sci. 1993; 58: 525–530. [Google Scholar]
  • 32.Montero-Calderón M, Rojas-Graü MA, Martín-Belloso O. Pineapple (Ananas comosus [L.] Merril) flavor In: Hui YH editor. Handbook of fruit and vegetable flavors. Hoboken: John Wiley & Sons, Inc.; 2010. p. 391–414. [Google Scholar]
  • 33.Narain N, Nigam N, de Sausa Galvão M. Passion Fruit In: Hui YH editor. Handbook of fruit and vegetable flavors. Hoboken: John Wiley & Sons, Inc.; 2010. p. 345–389. [Google Scholar]
  • 34.Le Page MA, Grua-Priol J, Prost C, Demaimay M. Optimization of dynamic headspace extraction of the edible red algae Palmaria palmata and identification of the volatile compounds. J. Agric. Food Chem. 2004; 52: 550–556. 10.1021/jf030478x [DOI] [PubMed] [Google Scholar]
  • 35.López-Pérez O, Picon A, Nuñez M. Volatile compounds and odour characteristics of seven species of dehydrated edible seaweeds. Food Res. Int. 2017; 99: 1002–1010. 10.1016/j.foodres.2016.12.013 [DOI] [PubMed] [Google Scholar]
  • 36.Tamura H, Nakamoto H, Yang R, Sugisawa H. Characteristic aroma compounds in green algae (Ulva pertusa) volatiles. Nippon Shokuhin Kagaku Kogaku Kaishi 1995; 42: 887–891. Japanese with English abstract [Google Scholar]
  • 37.Buttery RG, Guadagni DG, Ling LC, Seifert RM, Lipton W. Additional volatile components of cabbage, broccoli, and cauliflower. J. Agric. Food Chem. 1976; 24: 829–832. [Google Scholar]
  • 38.Whitfield FB. Microbiology of food taints. Int. J. Food Sci. Tech. 1998; 33: 31–51. [Google Scholar]
  • 39.Edwards RA, Dainty RH. Volatile compounds associated with the spoilage of normal and high pH vacuum-packed pork. J. Sci. Food Agric. 1987; 38: 57–66. [Google Scholar]
  • 40.Le Guen S, Prost C, Demaimay M. Characterization of odorant compounds of mussels (Mytilus edulis) according to their origin using gas chromatography–olfactometry and gas chromatography–mass spectrometry. J. Chromatogr. A 2000; 896: 361–371. 10.1016/s0021-9673(00)00729-9 [DOI] [PubMed] [Google Scholar]
  • 41.Cruz-Romero M, Kerry JP, Kelly AL. Changes in the microbiological and physicochemical quality of high-pressure-treated oyster (Crassostrea gigas) during chilled storage. Food Control 2008; 19: 1139–1147. [Google Scholar]
  • 42.Miyasaki T, Kitamura Y. Suppression of tilapia muscle odor by seaweed supplementation to diet. Aquaculture Sci. 2012; 60: 349–357. Japanese with English abstract [Google Scholar]
  • 43.Senger-Emonnot P, Rochard S, Pellegrin F, George G, Fernandez X, Lizzani-Cuvelier L. Odour active aroma compounds of sea fig (Microcosmus sulcatus). Food Chem 2006; 97: 465–471. [Google Scholar]

Decision Letter 0

Filippo Giarratana

24 Feb 2020

PONE-D-20-02508

Odor-active compounds from the gonads of Mesocentrotus nudus sea urchins fed Saccharina japonica kelp

PLOS ONE

Dear Yukio Agatsuma,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

ACADEMIC EDITOR:

Please verify carefully all the comments of the reviewers expecially on the "number of pannelist".

We would appreciate receiving your revised manuscript by 25 March 2020. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Filippo Giarratana

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating in your Funding Statement:

"A part of this work was financially supported by the Grand-in-Aid for JSPS Fellow [grant number 17J02308] from Japan Society for the Promotion of Science to S.T."

Please provide an amended statement that declares *all* the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now.  Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement.

Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf.

3. Thank you for stating the following in the Competing Interests section:

"The authors have declared that no competing interests exist."

We note that one or more of the authors are employed by a commercial company: RIKEN VITAMIN Co., Ltd. and Riken Food Co., Ltd

 

a.     Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

b. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.  

Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and  there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is an interesting study. It addresses a little studied aspect of the attractiveness of sea urchin gonads, i.e. its aroma. It asks whether aroma is caused by aromatic compounds in the gonads and that these are affected by the food the sea urchin eats. The documentation that the aromatic compounds of gonads of sea urchins from different environments and individuals fed kelp differ is excellent. This is of interest in itself. It has implications for intermediary metabolism of sea urchins.

.However I have some questions.

My major concern is the analysis of the aroma of the gonads. The authors state “141 GC-sniffing analysis The 146 panelist recorded the retention time and the related description of the aroma compounds.

How many panelists were there? The statement implies there was only one. If so, this is inadequate, and the paper should be rejected. One person is not a panel.

Other concerns and questions are less important.

1. 90 prefecture (38°40´N, 141°30´E) on 6 May 2016

What was the gonadal stage? Did it differ between the groups? Could gonadal stage affect odor?

2. 107 gonads were randomly collected from one group of gonads and stored in a polystyrene

108 storage container at 4 °C until further analyses.

How long before “further analyses”?

3. 108 Three containers were prepared for each group (n = 3).

Table 1. Volatile organic compounds (VOCs) detected 181 from gonads of Mesocentrotus

182 nudus using gas chromatography-mass spectrometry (n = 3). The table does not indicate variability. The results are given only as + or –. Were the results the same for all three samples?

4. The descriptions are qualitative.

Some descriptions are not understandable: green, gas, thinner, heavy and other descriptive terms must be given.

Sweet, sweet candy is not descriptive of aroma. Sweet is a qualitative indicator of taste, not aroma.

Unpleasant, putrid, sour, fishy are good descriptions of aroma.

5. The authors use both “aroma” and “odor”. They should be consistent. The words are synonyms, although the dictionary states “odor” is considered to indicate unpleasant in the US.

In this regard, perhaps this statement in the abstract should be changed:

Kelp feeding decreased the strong, putrid odor from 34 dimethyl sulfide, increased the number of odor-active compounds, particularly those with 35 a sweet aroma, and enhanced the richness and sweetness of flavor

The abstract should concern only the results found in the study. The reference to “enhanced “enhanced the richness and sweetness of flavor” is from other studies and should be in the Discussion where it is pertinent.

Reviewer #2: The MS is on the whole clear and easy to read, however there is some essential information missing as well as information and discussion that would greatly enhance the paper.

1) Details on the GCMS method are missing. How were the RI of the volatiles calculated ? what standards were used ? Presumably these were then matched to the RI’s of the compounds obtained by the GC-O

2) How were the panellists trained ? How many panellists were used ? How many replicate sniffs ? The descriptors they used seem to be very limited. Odour descriptors such as “sweet, Gas, thinner, heavy, dry are not very informative. What is meant by these? Also “oxidised seaweed” what the standard odour used to train panellist on this ? In fact all odours used for panel training should be described. Panel training is best practice for GC-O

3) How was retention time assessed ? i.e. what actions did the panellist take – key pad ? finger span?

4) What were the size of the urchins ? roe weight ? yield – presumably the diet affected these values ?

5) What was the gender of the urchins ? is this a male / female difference rather than a diet difference being reported

6) What was the appearance – shape, colour of the roe obtained from the urchins on the three diets.

7) L 280. I find the comparison between sea urchin and Benzaldehyde as almond, nutty and stone fruit to be perplexing – are these typical descriptors for urchin roe.

8) L287 – Describing the same compound from two different sources as too different odours, could be due to a concentration affect, but I am also concerned about compound identification and panellist training – please include an explanation for this observation in the text.

9) L294 – “reflect the sweet taste of their gonads??? Were the gonads tasted.

10) Table 2 define ND – also does ND mean that it was not present ? what was the detection limit. Why it <0.001 used in the Sato et al data – presumably this also means that it is ND.

11) I found the discussion in general to be a little limited, particularly with reference and published literature.

12) Why / how do the diets impact on aroma – what is in the diets – what is the mechanism that generates different compounds – do compounds differ or do the differences in odour reflect differences in concentration – is so what are trends in VOC.

13) Did sampling only occur at a single timepoint ? Sampling over time would have provided useful information

14) L 330 – larger number of VOC with unpleasant odours …. Were detected …. which enhanced the pleasant aroma and richness of flavour, leading to high sensory evaluation. First of all what would more unpleasant odours improve taste ? How was taste assessed ? If this MS is part of a larger study – this neds to be made clearer in the MS and some data produced to support the statements.

15) The MS seemed to lack a conclusion

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Apr 16;15(4):e0231673. doi: 10.1371/journal.pone.0231673.r002

Author response to Decision Letter 0

27 Mar 2020

Response to Reviewers

Reviewer #1: This is an interesting study. It addresses a little studied aspect of the attractiveness of sea urchin gonads, i.e. its aroma. It asks whether aroma is caused by aromatic compounds in the gonads and that these are affected by the food the sea urchin eats. The documentation that the aromatic compounds of gonads of sea urchins from different environments and individuals fed kelp differ is excellent. This is of interest in itself. It has implications for intermediary metabolism of sea urchins.

・We appreciate your comments.

However I have some questions.

My major concern is the analysis of the aroma of the gonads. The authors state “141 GC-sniffing analysis The 146 panelist recorded the retention time and the related description of the aroma compounds.

How many panelists were there? The statement implies there was only one. If so, this is inadequate, and the paper should be rejected. One person is not a panel.

・Certainly. We had three panelists. The “panelist” was not correct. We changed the sentence “The panelist recorded the retention time and the related description of the aroma compounds” to “The three panelists, who are well versed in sea urchin gonad quality and share common perceptions, recorded the retention time and the related description of the aroma compounds by writing in a paper” on the lines 152–156 in Materials and methods.

We apologize for the mistake.

Other concerns and questions are less important.

1. 90 prefecture (38°40´N, 141°30´E) on 6 May 2016

What was the gonadal stage? Did it differ between the groups? Could gonadal stage affect odor?

・Takagi et al. (2019) showed the gonad developmental stages of the sea urchins which were collected and cultured in the same manner with sea urchins used in the present study. Almost gonads of the sea urchins from all groups were in the growing stage. Therefore, we think there is no need to consider the effects of gonad developmental stages. We added the sentence “Takagi et al. [8] showed no significant differences in test diameter among these sea urchins (CSU, ESU and BSU), and almost the gonads were in the growing stage” on the lines 113–115 in Materials and Methods.

2. 107 gonads were randomly collected from one group of gonads and stored in a polystyrene

108 storage container at 4 °C until further analyses.

How long before “further analyses”?

・Certainly, we should mention about that. Thank you for the comment. GC-MS and GC-sniffing analyses were conducted within 48 hours after dissection. We added “within 48 hours after dissection” at the end of the sentence “Headspace sampling, GC-MS analysis and GC-sniffing analysis were conducted according to Sato et al. [18]” on the lines 112–113 in Materials and methods.

3. 108 Three containers were prepared for each group (n = 3).

Table 1. Volatile organic compounds (VOCs) detected 181 from gonads of Mesocentrotus

182 nudus using gas chromatography-mass spectrometry (n = 3). The table does not indicate variability. The results are given only as + or –. Were the results the same for all three samples?

・As you mentioned, we prepared three containers for each group and conducted analyses per container. When a VOC was detected in triplicate analysis, this determined the presence of the VOC in the group as we mentioned on the lines 142–143 in Materials and methods.

4. The descriptions are qualitative.

Some descriptions are not understandable: green, gas, thinner, heavy and other descriptive terms must be given.

・Thank you for your comments. Green was used for the description of odor of sea urchin gonads in a past study (Niimi et al. 2010). We changed “gas” to “plastic, medicinal”, “thinner” to “benzene”, “heavy” to “tingle in nose, heavy note” in Table 2. In addition, we changed the sentences “Benzene from the gonads of CSU was described as having a heavy odor, although that from ESU was described as having a mint aroma” to “Benzene from the gonads of CSU was described as having a heavy note, although that from ESU was described as having a mint aroma” on the lines 224–225, “S-methyl thioacetate from the gonads of CSU showed a markedly larger peak area than those of other groups and was described as having a heavy and fishy odor” to “S-methyl thioacetate from the gonads of CSU showed a markedly larger peak area than those of other groups and was described as having a heavy note tingling in nose and fishy odor” on the lines 239–241 in Results.

Sweet, sweet candy is not descriptive of aroma. Sweet is a qualitative indicator of taste, not aroma.

・Sweet was also used as a description of aroma in past studies (e.g., Niimi et al. 2010; Lehtinen & Veijanen 2011).

Unpleasant, putrid, sour, fishy are good descriptions of aroma.

・We appreciate.

5. The authors use both “aroma” and “odor”. They should be consistent. The words are synonyms, although the dictionary states “odor” is considered to indicate unpleasant in the US.

In this regard, perhaps this statement in the abstract should be changed:

Kelp feeding decreased the strong, putrid odor from 34 dimethyl sulfide, increased the number of odor-active compounds, particularly those with 35 a sweet aroma, and enhanced the richness and sweetness of flavor

The abstract should concern only the results found in the study. The reference to “enhanced “enhanced the richness and sweetness of flavor” is from other studies and should be in the Discussion where it is pertinent.

・We appreciate your comments. As you mentioned we changed the sentence “Kelp feeding decreased the strong, putrid odor from dimethyl sulfide, increased the number of odor-active compounds, particularly those with a sweet aroma, and enhanced the richness and sweetness of flavor” to “Kelp feeding decreased the putrid odor from dimethyl sulfide, and enhanced pleasant, sweet aromas” on the line 33–34 in the abstract, and changed the sentence “However, kelp feeding decreased the strong, putrid odor from DMS, increased the number of odor-active compounds, particularly those with a sweet aroma, and enhanced the richness and sweetness of flavor” to “However, kelp feeding decreased the strong, putrid odor from DMS enhanced pleasant, sweet aroma, and would enhance the richness and sweetness of flavor” on the lines 332–333 in Discussion.

Thank you very much.

Reviewer #2: The MS is on the whole clear and easy to read, however there is some essential information missing as well as information and discussion that would greatly enhance the paper.

1) Details on the GCMS method are missing. How were the RI of the volatiles calculated? what standards were used? Presumably these were then matched to the RI’s of the compounds obtained by the GC-O

・There are past studies of GC-MS analysis published without RI (e.g., De Quirós et al. 2001; Zhu et al. 2009; de Alencar et al. 2017). We identified each VOC from retention time by similarity search (Zhu et al. 2009) using the library of Powered Pro software (Wiley 11N17main, Agilent Technologies Inc.). We added the sentence “Each VOC was identified by similarity search [25] using the library of the software (Wiley 11N17main, Agilent Technologies Inc.)” on the lines 141–142 in Materials and methods, and added Zhu et al. (2009) to the reference list. According to the reference addition, the reference NO. of other references was changed.

2) How were the panellists trained? How many panellists were use? How many replicate sniffs? The descriptors they used seem to be very limited. Odour descriptors such as “sweet, Gas, thinner, heavy, dry are not very informative. What is meant by these? Also “oxidised seaweed” what the standard odour used to train panellist on this? In fact all odours used for panel training should be described. Panel training is best practice for GC-O

・Thank you for your advices. One of the purposes of this study is to identify the compounds with sea urchin like aroma. Therefore, we prepared three panelists who are well versed in sea urchin gonad quality and share common perceptions. We added the sentence “The purpose of this study is to (1) clarify the effect of S. japonica feeding to improve gonad flavor of sea urchins from a barren and (2) identify the compounds with sea urchin like aroma” on the lines 78–80 in Introduction, and changed the sentence “The panelist recorded the retention time and the related description of the aroma compounds” to “The three panelists, who are well versed in sea urchin gonad quality and share common perceptions, recorded the retention time and the related description of the aroma compounds by writing in a paper” on the lines 152–155 in Materials and Methods.

“Sweet” is used as a description of odor in past studies (e.g., Phillips et al. 2010; Niimi et al. 2010; Lehtinen & Veijanen 2011). We changed “gas” to “plastic, medicinal”, “thinner” to “benzene”, “heavy” to “tingle in nose, heavy note”, and “dry” to “hey smell” in Table 2. In addition, we changed the sentences “Benzene from the gonads of CSU was described as having a heavy odor, although that from ESU was described as having a mint aroma” to “Benzene from the gonads of CSU was described as having a heavy note, although that from ESU was described as having a mint aroma” on the lines 224–225, “S-methyl thioacetate from the gonads of CSU showed a markedly larger peak area than those of other groups and was described as having a heavy and fishy odor” to “S-methyl thioacetate from the gonads of CSU showed a markedly larger peak area than those of other groups and was described as having a heavy note tingling in nose and fishy odor” on the lines 239–241, “The odors of 2-butanol from the gonads of CSU and ESU were described as acceptable seafood and dry odors and as an unpleasant sea urchin-like odor, respectively” to “The odors of 2-butanol from the gonads of CSU and ESU were described as acceptable seafood and hey smells and as an unpleasant sea urchin-like odor, respectively” on the lines 203–204, “2-Propanol from the gonads of CSU was described as having kelp and dry odors” to “2-Propanol from the gonads of CSU was described as having kelp and hey smells” on the lines 206–207, " Styrene from the gonads of CSU and ESU was described as having a dry odor” to “Styrene from the gonads of CSU and ESU was described as having a hey smell”, on the lines 231–232, and “Decane and undecane from the gonads of ESU were described as having a fishy odor and oxidized seaweed-like and dry odors, respectively” to “Decane and undecane from the gonads of ESU were described as having a fishy odor and oxidized seaweed-like and hey smells, respectively” on the lines 256–257 in Results. Thank you again.

3) How was retention time assessed? i.e. what actions did the panellist take – key pad? finger span?

・The panelists could watch the screen which shows elapsed time during the sniffing analysis. They recorded the retention time by writing in a paper. They could conduct the record without taking off their nose from the sniffing port. We added the sentence “They could record without taking off their nose from the sniffing port” on the lines 155–156 in Materials and methods, and changed the sentence “The panelist recorded the retention time and the related description of the aroma compounds” to “The three panelists, who are well versed in sea urchin gonad quality and share common perceptions, recorded the retention time and the related description of the aroma compounds by writing in a paper” on the lines 152–155 in Materials and Methods.

4) What were the size of the urchins? roe weight? yield – presumably the diet affected these values?

・The sea urchins on barrens have small and low quality gonads, showing low commercial value. For that reason, the gonads size per urchin varies among the three groups. One of the purposes of the culture experiment is to clarify the effect of S. japonica feeding on improvement in gonad flavor of sea urchins from barrens. In this study, we used the sea urchins with same size (ca. 50 mm test diameter) for each treatment, and ca. 5 g gonads were collected randomly from 20 individuals of each group and repeated three times.

We added the sentences “Takagi et al. [8] conducted a sensory evaluation and measurements and analyses of gonad size, developmental stages, color, texture and free amino acid contents of sea urchins cultured and collected from the same algal beds by the same methods on the same day with the present study. The purpose of this study is to (1) clarify the effect of S. japonica feeding to improve gonad flavor of sea urchins from a barren and (2) identify the compounds with sea urchin like aroma” on the lines 75–80 in Introduction, and “Takagi et al. [8] showed no significant differences in test diameter among these sea urchins (CSU, ESU and BSU), and almost the gonads were in the growing stage” on the lines 113–115 in Materials and methods. We also added “therefore no commercial value [20]” at the end of the sentence “This species densely distributes on barrens [20], where the gonads show small size [21] and deteriorated color, texture, and taste [13, 22]” on the lines 59–60 in Introduction.

5) What was the gender of the urchins? is this a male / female difference rather than a diet difference being reported

・In the present study, the gonads of sea urchins were in the growing stage (Takagi et al. 2019), but were not observed by sex because of no histological observation in the present study Because we had to conduct GC-MS and GC-sniffing analyses immediately after dissection to analyze fresh gonads. Furthermore, because gonads of sea urchins from the barren were too small to conduct the analyses individually, some individuals of each group have to be combined for the analyses (therefore we collected 5 g of gonads from 20 individuals of each group with three replication). Thus, it was impossible to separate sex in the present study.

6) What was the appearance – shape, colour of the roe obtained from the urchins on the three diets.

・Takagi et al. (2019) reported gonad size, color, texture and taste of the sea urchins. We changed the sentence “Takagi et al. [8] first improved the gonad taste of barren individuals to a more desirable level compared to that of individuals from an Eisenia kelp bed (fishing ground) by feeding them fresh cultivated Saccharina japonica during May–July” to “Takagi et al. [8] first improved the whole gonad quality (size, color, texture and taste) of barren individuals to a more desirable level compared to that of individuals from an Eisenia kelp bed (fishing ground) by feeding them fresh cultivated Saccharina japonica during May–July” on the lines 63–66, and added the sentence “Takagi et al. [8] conducted a sensory evaluation and measurements and analyses of gonad size, developmental stages, color, texture and free amino acid contents of sea urchins cultured and collected from the same algal beds by the same methods on the same days with the present study” on the lines 75–78 in Introduction.

7) L 280. I find the comparison between sea urchin and Benzaldehyde as almond, nutty and stone fruit to be perplexing – are these typical descriptors for urchin roe.

・Thank you for your comments. Since there are only four papers which conducted odor analyses of sea urchin gonads, it is difficult to judge whether the descriptions are typical for sea urchin gonads or not. “Almond” is also used as a description of sea urchin gonad odor in a past study which conducted a sensory evaluation (Phillips et al. 2009). Thus, we think using these descriptors are no problem.

8) L287 – Describing the same compound from two different sources as too different odours, could be due to a concentration affect, but I am also concerned about compound identification and panellist training – please include an explanation for this observation in the text.

・Referring to Question (2), the panelists of the sniffing analysis are well versed in sea urchin gonad quality and share common perceptions.

9) L294 – “reflect the sweet taste of their gonads??? Were the gonads tasted.

・As we mentioned on the lines 67–69 in Introduction, Takagi et al. (2019) evaluated the quality of the gonads of sea urchins collected in the same manner with CSU, ESU and BSU by a sensory test, and the gonads of CSU and ESU were evaluated sweeter than that of BSU. We suggested the sweet aromas could be affected by the strong sweetness of gonads of cultured sea urchins because flavor and odor could influence the taste of food.

10) Table 2 define ND – also does ND mean that it was not present? what was the detection limit. Why it <0.001 used in the Sato et al data – presumably this also means that it is ND.

・We apologize for making confusion. We did not refer to Sato et al. (2019) in Table 2. We provided the significant values in peak areas among groups analyzed by ANOVA in Table 2. We added the sentences “Significance of values in peak areas among groups analyzed by ANOVA are provided. ND indicate no detection” to the legend of Table 2. We apologize for no description in the previous MS.

11) I found the discussion in general to be a little limited, particularly with reference and published literature.

・Certainly, but there are only four paper which conducted odor analyses of sea urchin gonads (De Quirós et al. 2001; Niimi et al. 2010; Phillips et al. 2009; Sato et al. 2019), resulting in a limitation of references.

12) Why / how do the diets impact on aroma – what is in the diets – what is the mechanism that generates different compounds – do compounds differ or do the differences in odour reflect differences in concentration – is so what are trends in VOC.

・Referring to Question 11), there are only four studies analyzed odor of sea urchin gonads. Phillips et al. (2009) revealed that the odors of gonads of sea urchin collected from the North Island and the South Island of New Zealand varied but they did not mention about the reason. We used sea urchins cultured and fed fresh S. japonica kelp (CSU), and two types of wild sea urchins collected from a barren (BSU) and an Eisenia kelp bed (ESU). Past studies revealed that the difference in the habitat of sea urchins results in the variation of water temperature and the seaweed vegetation as sea urchin food, and these affect to the variation of body size, growth rate, and gonad development, color, texture and taste of sea urchins (e.g., Addis et al. 2015; Agatsuma et al. 2005; Ling et al. 2010; Takagi et al. 2017). In the present study, the culture and collection of sea urchins in the three groups were conducted in the same bay. Thus, the variation of gonad odor among the three groups could result from the food.

As you mentioned, we deleted the sentence “These results suggest that an extremely high content of ethylbenzene could result in an unpleasant odor of gonads” on the lines 286–287 because we do not have the confidence of effect of peak areas on the description.

We appreciate your valuable comments.

13) Did sampling only occur at a single timepoint? Sampling over time would have provided useful information

・Thank you for the comment. As we answered for Question (4), one of the purposes of the present study is to clarify the effect of S. japonica feeding to improve gonad flavor of sea urchins from a barren. In addition, the fishing season of M. nudus is between June and August (Kawamura 1993). Thus, sea urchin collection was conducted on 19 July 2016, at the end of the culture experiment.

14) L 330 – larger number of VOC with unpleasant odours …. Were detected …. which enhanced the pleasant aroma and richness of flavour, leading to high sensory evaluation. First of all what would more unpleasant odours improve taste? How was taste assessed? If this MS is part of a larger study – this neds to be made clearer in the MS and some data produced to support the statements.

・As we answered for Question (9), Takagi et al. (2019) conducted a sensory evaluation on the sea urchins. We added the sentence “Takagi et al. [8] conducted a sensory evaluation and measurements and analyses of gonad size, gonad color, texture and free amino acid contents of sea urchins cultured and collected from the same algal bed by the same methods on the same day with the present study” on the lines 75–78 in Introduction.

Following your suggestion, we changed the sentence “From the gonads of cultured M. nudus, a larger number of odor-active VOCs with unpleasant odors, a green scent, and sweet aromas were detected than from those of wild sea urchins, which enhanced the pleasant aroma and richness of flavor, leading to high sensory evaluation [8]” to “In conclusion, from the gonads of cultured M. nudus, a larger number of odor-active VOCs with a green scent and sweet aromas were detected than from those of wild sea urchins, which would enhance the pleasant aroma and richness of flavor, leading to high sensory evaluation [8]” on the lines 326–329 in Discussion.

Thank you very much.

15) The MS seemed to lack a conclusion

・Certainly. We changed the sentence “From the gonads of cultured M. nudus, a larger number of odor-active VOCs with unpleasant odors, a green scent, and sweet aromas were detected than from those of wild sea urchins, which enhanced the pleasant aroma and richness of flavor, leading to high sensory evaluation [8]” to “In conclusion, from the gonads of cultured M. nudus, a larger number of odor-active VOCs with a green scent and sweet aromas were detected than from those of wild sea urchins, which would enhance the pleasant aroma and richness of flavor, leading to high sensory evaluation [8]” on the lines 326–329 in Discussion.

Thank you for your valuable, insightful comments.

References

Addis P, Moccia D, Secci M (2015) Mar. Ecol. 36: 178–184

Agatsuma Y, Sato M, Taniguchi K (2005) Aquaculture 249: 449–458

de Alencar DB, Diniz JC, Rocha SAS, Pires-Cavalcante KMS, Freitas JO, Nagano CS, Sampaio AH, Saker-Sampaio S (2017) J. Appl. Phycol. 29: 1571–1576

De Quirós ARB, López-Hernández J, González-Castro MJ, de la Cruz-García C, Simal-Lozano J. (2001) Eur. Food Res. Technol. 212: 643–647

Kawamura (1993) Uni Zouyoushoku to Kakou Ryutsu. Sapporo, Japan: Hokkai Suisan Shinbun Company. 253 pp (in Japanese)

Lehtinen J, Veijanen A (2011) Water Air Soil Pollut. 218: 185–196

Ling SD, Ibbott S, Sanderson (2010) J. Exp. Mar. Biol. Ecol. 395: 135–146

Niimi J, Leus M, Silcock P, Hamid N, Bremer P. (2010) Food. Chem. 121: 601–607.

Phillips K, Bremer P, Silcock P, Hamid N, Delahunty C, Barker M et al. (2009) Aquaculture 288: 205–215.

Phillips K, Niimi J, Hamid N, Silcock P, Delahunty C, Barker M, et al. (2010) LWT-Food Sci. Technol. 2010; 43: 202–213.

Sato Y, Takagi S, Inomata E, Agatsuma Y. (2019) Food and Nutrition Sciences 10: 860–875.

Takagi S, Murata Y, Inomata E, Aoki MN, Agatsuma Y (2019) J. Appl. Phycol. 31: 4037–4048.Zhu X, Gao Y, Chen Z, Su Q (2009) Chromatographia 69: 735–742.

Other changes

We changed the sentences “The data below the detection limits were inputted as 0.0001” to “The values of not detected compounds were inputted as 0.0001.” on the lines 162–163 in Materials and Methods.

We changed the sentence “Benzaldehyde has pleasant almond, nutty and stony fruit aromas of peach (Prunus persica)” to “Benzaldehyde has pleasant almond, nutty and stony fruit like aromas of peach (Prunus persica)” on the lines 277–278 in Discussion.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (49KB, docx)

Decision Letter 1

Filippo Giarratana

30 Mar 2020

Odor-active compounds from the gonads of Mesocentrotus nudus sea urchins fed Saccharina japonica kelp

PONE-D-20-02508R1

Dear Dr. Yukio Agatsuma,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Filippo Giarratana

Academic Editor

PLOS ONE

Acceptance letter

Filippo Giarratana

2 Apr 2020

PONE-D-20-02508R1

Odor-active compounds from the gonads of Mesocentrotus nudus sea urchins fed Saccharina japonica kelp

Dear Dr. Agatsuma:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Filippo Giarratana

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Table. Raw data of chromatogram and peak area of volatile organic compounds from gonads of Mesocentrotus nudus of each group.

    (XLSX)

    Click here for additional data file. (651.7KB, xlsx)
    Attachment

    Submitted filename: Response to Reviewers.docx

    Click here for additional data file. (49KB, docx)

    Data Availability Statement

    All relevant data are within the paper and its Supporting Information files.

    Articles from PLoS ONE are provided here courtesy of PLOS

    RESOURCES