Proximal and fatty acid analysis in Ostrea chilensis, Crassostrea gigas and Mytilus chilensis (Bivalvia: Mollusca) from southern Chile.

Oysters and blue mussels are important hydrobiological resources for aquaculture. In Chile, they are farming on the Chiloé island, where around 18% of the world's mussels are produced, however, their nutritional dynamics are largely unknown. For this reason, the objective of this study was to determine the proximal biochemical composition and the fatty acid profile in the Chilean oyster (Ostrea chilensis), the Pacific oyster (Crassostrea gigas) and the Chilean mussel (Mytilus chilensis), to perform an intra and interspecific comparison. Shellfish sampled in winter were characterized by a high protein content, followed by medium values for lipid content and a low carbohydrate content compared to similar species in Europe. Also, oysters and mussels were found to be rich in omega-3 long chain polyunsaturated fatty acid (n-3 LC-PUFA), so they can be considered excellent functional food option for a healthy human diet. Their high contribution of n-3 LC-PUFA ranged between 5.2-12.9 μg FA mg-1 dry weight with high n-3/n-6 ratios, which depends on both the species and the on-growing location. Both taxa can be considered a plausible option to promote a healthy diet of marine origin in future generations. Also, these results could benefit the projection and development of aquaculture of these mollusks.

Introduction

Proximal composition studies and fatty acid analysis make it possible to determine the nutritional value of any organism and provide relevant information to understand its metabolism and energy balance [1-3]. In bivalve mollusks, the nutritional value varies between species, including differences between males and females [4-9]. One of the relevant nutritional characteristics of bivalves is their production of omega-3 highly unsaturated fatty acids (n-3 HUFA) or long chain poly unsaturated fatty acids (n-3 LC-PUFA), especially they have high contents of eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) [10]. Both fatty acids are main components of phytoplanktonic diet of bivalves. The phytoplankton are the main producers of n-3 LC-PUFA in the marine food chain [11, 12]. [13] suggests that the EPA + DHA content of mussels, clams and oysters is usually higher than that of other bivalves. In the Magallana bilineata oyster (formerly Crassostrea madrasensis), polyunsaturated fatty acids (PUFAs) were estimated to be the highest total lipids, among which eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and acid linoleic stand out [14].

During the last decades, research has been focused on understanding the nutritional composition of bivalves of commercial interest, mainly because they are an important source of protein, lipids, carbohydrates, minerals and essential vitamins of great value for the human population [15-17]. However, most bivalves lack the ability to synthesize PUFA from saturated precursors [18]. Therefore, the levels of these components are determined by the food intake, or by the variability of the food they consume [19]. Other factors related to the proportion of fatty acids in mollusks have also been described, such as the physiological state of the organism, growth, the organism’s response to variations in the environment, and internal and external factors that affect a moment in the life cycle of each individual [2, 9, 20]. For example, investigations on marine mussels have described that the proximal composition is related to the gametogenic cycle and the availability and quality of the food [2, 21, 22]. Therefore, the cultivation area has a strong influence on the nutritional components that mollusks acquire [2, 16, 23].

Oysters and blue mussels are bivalve mollusks of aquaculture importance in Chile [24]. The Chilean oyster (Ostrea chilensis, Küster, 1844), the Pacific oyster (Crassostrea gigas, Thunberg, 1793) and the Chilean blue mussel (Mytilus chilensis, Hupé, 1854) inhabit the Southeast Pacific coast. Ostrea chilensis forms sea beds on rocky or muddy hard bottoms up to 8 m deep. It has a limited range of distribution in Chile between Chiloé Island and Guaitecas Islands [25] and also in the nearshore waters of New Zealand [26]. However, its cultivation has developed in the northern area of Chiloé Island (city of Ancud). Currently, harvesting about 400 tons per year and the demand for the product has increased during the last decade [24]. The Pacific Oyster or Crassostrea gigas is an exotic species introduced in Chile (Coquimbo Region) in 1978 for commercial purposes [27]. In Chile, its cultivation has been carried out mainly in the III and IV regions (26°30S to 29°54’S—North of Chile) to export seeds to the United States (https://fch.cl/noticia/la-ruta-de-las-semillas-de-ostra-de-cultimar/, last accesed 11/08/2020) for on-growing mainly in North of Chile (about 500 ton), while at The Lakes Region its harvesting is reduced to 11 tons at the island of Chiloé, Chile (e.g. Hueihue and Faro Corona, 42°52’S). On the other hand, the Chilean mussel (Mytilus chilensis), also known as ‘chorito’, is distributed in the Eastern Pacific Ocean from Puerto Saavedra (Chile) to Ushuaia (Argentina) [28]. In Chile, the annual landing reaches 379,096 tons, which explained 18% of world production in 2019 that are mainly exported to Asia and Europe [24, 29]. Chile is one of the main exporters of blue mussels in the world and all the production (99.9%) comes from The Lakes Region, mainly Chiloé island. Oysters and mussels have increased their production in Chile through the last decade, however, the variability of their nutritional components is largely unknown.

This study aimed to evaluate the proximal biochemical composition and the fatty acid profile of the Chilean oyster, Japanese oyster and Chilean mussel, in order to carry out an intra and interspecific comparison of commercial bivalves from aquaculture in southern Chile, to nutritionally characterize the food quality of these hydrobiological resources.

Material and methods

Sampling and determination of the proximal composition and fatty acid profile of mollusks

The bivalve samples used in this study were manipulated according to the criteria on the use of animals in research granted by the Bioethics Committee of the Universidad Austral de Chile.

The animals were collected on two occasions in the north of Chiloé Island. The first sample collection was performed in September 2017 (at the end of winter) when 60 individuals of Ostrea chilensis (Chilean oyster) were randomly extracted from the locality of Quempillén (41° 52′ 0″ S, 73° 49′ 60″ W), which were separated 3 groups of 20 individuals, each group was considered as a replicate. On the same occasion, 60 individuals of Ostrea chilensis were collected from Pullinque locality (41° 50’ 45" S, 73° 56’ 08" W) and 40 individuals of Cassostrea gigas (Pacific oyster) from Faro Corona locality (41° 47′ 06″ S, 73° 53′ 18″ W), each group of 20 individuals was considered as a replicate (Fig 1). Then, in August 2018 (late winter), 60 O. chilensis, 60 C. gigas and 60 M. chilensis specimens were randomly collected following the same type of sampling described above, all from Hueihue locality (41° 54’ 05" S, 73° 29’ 28" W), north of Chiloé Island (Fig 1). The environmental conditions of temperature and salinity for each zone were: Quempillén (14.7°C and 22.4 ppt), Pullinque (13.6°C and 30.6 ppt), Faro Corona (14.1°C and 29.1 ppt) and Hueihue (12.3°C and 31.8 ppt) [30]. The samples were transferred dry, with a transfer time of 2 hours to the Marine Invertebrate Hatchery of the Aquaculture Institute of the Universidad Austral de Chile (HIM-UACh), where the tissues were processed without shells. Each sample was stored in a refrigerated/frozen Ziploc bag with the meat of 20 individuals corresponding to replicates 1, 2 and 3 of each sampling. For each sample of 20 individuals the tissues were pooled and freeze dried in a Savant freeze dryer (-80°C), then they were ground to homogenize and stored at -41°C until the respective biochemical analysis. The total length of bivalves from each sample was individually measured with a caliper (± 0.1 mm) and weighed on a Sartorius analytical balance (± 0.01 g).

Fig 1

Map of the collection sites of oysters (Ostrea chilensis and Crassostrea gigas) and Chilean mussels (Mytilus chilensis) in the north of Chiloé Island, Chile.

The samples were dissected with the help of a scalpel. The relative weight index [31] based on the allometric equation between the whole wet weight vs the total length was calculated according to unpublished results, the bivalves in rearing conditions showed the allometric relationship of WW = 0.021*L2.16 for M. chilensis, WW = 0.0004*L2.77 for O. chilensis, and WW = 0.079*L1.48 for C. gigas, calculated from the same samplings of the study, where WW and L are the wet weight in g and total length in cm of the specimens. In this way, the relative weight index (Wr) for each species was calculated as Wr = WW / [0.021*L2.16], Wr = WW / [0.0004*L2.77], Wr = WW / [0.079*L1.48], respectively.

The proximal and fatty acid compositions of tissues were determined according to [32]. Carbon, hydrogen and nitrogen content were analyzed using a CHN analyzer (LECO CHN-900) with 1 mg sample weighed in a microbalance (± 0.001 mg; METTLER-TOLEDO XP2U). Crude protein was calculated based on the N x 6.25 and reported in percentage. The total nitrogen content of the sample was converted into a value called "crude protein" by multiplying it by 6.25, a constant based on the conventional assumption that any protein is composed of 16% nitrogen. Total lipid content was obtained gravimetrically, after extraction, through the method of [33]. Methylation and quantification of fatty acids were performed according to the method described by [34]. The fatty acid methyl esters (FAMEs) from the total lipids were analyzed in a gas chromatograph (Focus GC, Thermo Fisher Scientific Inc., Waltham, MA, USA) equipped with an autosampler. The separation was performed using hydrogen as a carrier gas in a capillary column RESTEC RT-2560 of 100 m length, 0.25 mm internal diameter and 0.2 mm phase, using a splitless injection program. The initial temperature of 140°C was maintained for 5 min before starting a ramp at a rate of 5°C min up to 240°C, held for 20 min. The detector was set at 260°C. Nonadecanoic acid (19:0) was used as an internal standard.

Ash content was obtained after calcination at 500°C for 6 h (Vulcan Muffle model A-550). The carbohydrates were calculated as the difference between 100% of the dry weight and the sum of protein, lipid, and ash. Values to calculate energy conversion were 23.7, 39.5 and 17.2 kJ g-1 for protein, lipid and carbohydrate, respectively [35].

The nutritional status indicators of bivalve meat were calculated in dry basis as:

Protein, lipid, carbohydrates and ash = per (g 100 g dry weight)-1.

Energy = (g protein g dry weight−1×23.7 kJ g−1) + (g lipid g dry weight−1×39.5 kJ g−1) + (g carbohydrate g dry weight-1x17.2 kJ g-1) = kJ g dry weight−1 x 1000 = MJ kg-1 dry weight.

Protein/energy ratio (P/E) = g protein per kg dry weight / MJ per kg dry weight.

Carbon/Nitrogen ratio (C/N) = [g carbon g dry weight-1/12 g] / [g nitrogen g dry weight-1/14 g].

Yield = Meat wet weight x 100/ Total wet weight [36].

Statistical analysis

The data from each sampling year were treated independently. A one-way ANOVA was performed when the assumptions of normality and homoscedasticity (Levene’s test and White’s test, respectively) were fulfilled in the data corresponding to fatty acids and proximal composition values. If the data did not meet the statistical assumptions, a Kruskal-Wallis rank ANOVA was performed. In those cases, where significant differences were found, a Tukey or Dunn posterior test was carried out according to the analysis of variance test used. These significant differences are indicated with different letters (Tables 1 to 4).

Table 1

Proximal composition and condition indices of oysters and mussels from Hueihue (Chiloé Island, Chile).

Each value represents the average of three independent replicates ± standard error. The letters indicate significant differences between averages.

	Ostrea chilensis	Crassostrea gigas	Mytilus chilensis
	(Hueihue)	(Hueihue)	(Hueihue)
Proximal composition
Protein (% dry weight)	42.81 ± 0.24a	50.07 ± 0.88b	52.54 ± 0.29c
Lipid (% dry weight)	17.70 ± 1.11ab	19.54 ± 0.28b	14.50 ± 2.55a
Carbohydrate (% dry weight)	20.95 ± 1.15	11.50 ± 0.62	14.28 ± 4.42
Ash (% dry weight)	19.24 ± 1.08	18.77 ± 0.38	18.96 ± 4.81
Energy (MJ kg-1 meat)	20.75 ± 0.13	21.56 ± 0.14	20.63 ± 2.04
Condition indicators
C/N (atom C/ atom N)	7.41 ± 0.10	6.50 ± 0.10	5.58 ± 0.07
Protein/Energy (g protein/MJ)	20.67 ± 0.07a	23.20 ± 0.30ab	25.63 ± 1.56b
Yield (% total wet weight)	16.91 ± 0.33a	23.70 ± 0.52b	41.54 ± 0.68c
Wr (Relative weight index)	1.17 ± 0.05b	1.18 ± 0.04b	1.02 ± 0.02a

Table 4

Fatty acid content in oyster meat from Quempillén, Pullinque and Faro corona (Chiloé Island, Chile).

Each value represents the average of independent replicates ± standard error, except for C. gigas with two independent replicas ± standard error. The letters show significant differences between averages.

	O. chilensis	O. chilensis	C. gigas
	(Quempillén)	(Pullinque)	(Faro Corona)
Fatty acids (mg fatty acid g-1 dry weight of meat)
Myristic acid, 14:0	1.77 ± 0.15	2.14 ± 0.09	1.96 ± 0.22
Pentadecanoic acid, 15:0	0.39 ± 0.03a	0.51 ± 0.02b	0.47 ± 0.01ab
Palmitic acid, 16:0	14.12 ± 0.70	11.56 ± 1.48	16.50 ± 0.84
Heptadecanoic acid, 17:0	0.83 ± 0.09	0.95 ± 0.07	1.23 ± 0.06
Stearic acid, 18:0	4.13 ± 0.44	3.39 ± 0.41	3.23 ± 0.34
Oleic acid, 18:1n-9	3.11 ± 0.24	3.82 ± 1.07	3.74 ± 0.43
Linoleic acid, 18:2n-6	1.81 ± 0.21	2.40 ± 1.27	1.47 ± 0.20
Eicosenoic acid, 20:1n-9	2.99 ± 0.21b	2.57 ± 0.53b	1.62 ± 0.22a
Linolenic acid, 18:3n-3	3.58 ± 0.28	2.94 ± 0.33	2.76 ± 0.17
Eicosadienoic acid, 20:2n-6	0.16 ± 0.10	0.54 ± 0.48	n/a
Arachidonic acid, 20:4n-6 (ARA)	1.62 ± 0.15	1.51 ± 0.08	1.21 ± 0.03
Eicosapentaenoic acid, 20:5n-3 (EPA)	12.54 ± 1.81	8.51 ± 2.97	18.24 ± 0.86
Docosahexaenoic acid, 22:6n-3 (DHA)	8.70 ± 1.32ab	7.04 ± 2.01a	14.22 ± 0.33b
Other saturated fatty acids (SFA)	7.13 ± 0.73	6.19 ± 0.60	8.74 ± 0.34
Other monounsaturated fatty acids (MUFA)	0.64 ± 0.53	3.06 ± 2.63	1.32 ± 0.19
Other polyunsaturated fatty acids (PUFA)	3.89 ± 0.21	4.39 ± 0.50	3.07 ± 0.49
Total amount per group of fatty acids
SFA	21.96 ± 1.17	17.53 ± 2.39	23.84 ± 1.29
MUFA	8.34 ± 0.40	8.93 ± 0.47	8.60 ± 0.60
PUFA	28.40 ± 3.49	22.52 ± 4.84	37.97 ± 0.88
EPA + DHA	21.24 ± 3.13	15.55 ± 4.98	32.46 ± 1.19
Highly unsaturated fatty acids (HUFA)	21.76 ± 2.89	15.63 ± 5.06	32.52 ± 1.24
Ratios
n-3/n-6	8.17 ± 0.39ab	5.15 ± 1.77a	12.86 ± 0.98b
EPA/ARA	8.50 ± 0.49a	7.23 ± 1.37a	15.18 ± 1.07b
DHA/EPA	0.69 ± 0.01	0.87 ± 0.07	0.78 ± 0.02
16:1n-7/16:0	0.15 ± 0.008	0.18 ± 0.03	0.15 ± 0.001
SFA/PUFA	0.79 ± 0.05	0.81 ± 0.09	0.63 ± 0.05
MUFA/PUFA	0.30 ± 0.03	0.44 ± 0.11	0.23 ± 0.02
HUFA/PUFA	0.76 ± 0.01	0.66 ± 0.09	0.86 ± 0.01

To analyze the differences between profiles of fatty acids of the species at different locations, a multivariate analysis of principal components (PCA) was performed using a correlation matrix, to define new uncorrelated variables (components) established from linear combinations of the original variables (Fig 2).

Fig 2

Plot of scores on principal components from the fatty acids: (a) Chilean oyster (Ostrea chilensis), Pacific oyster (Crassostrea gigas) and Chilean mussel (Mytilus chilensis) from Hueihue (Chiloé Island, Chile); (b) Chilean oyster (Ostrea chilensis) and Pacific oyster (Crassostrea gigas) from Faro Corona, Pullinque and Quempillén (Chiloé Island, Chile) (for locations see Fig 1). The ovals around the three separate clusters of points are drawn to aid interpretation; they do not represent confidence intervals.

Results

Interspecific analysis

The three species analyzed (O. chilensis, C. gigas and M. chilensis) showed significant differences in protein content (H (2, n = 9) = 7.20; P = 0.027). The highest value was obtained by M. chilensis and the lowest by Chilean oysters (Table 1). Also, the lipid content showed significant differences in the mollusk tissue (F2,6 = 7.50; P = 0.02) (Table 1).

The C/N values did not present significant differences between the three bivalve mollusk species (mean = 6.5 ± 0.1). The relative weight index (Wr) showed a higher relative weight in oysters than mussels (13%). On the other hand, Chilean blue mussels registered the highest values in the protein/energy ratio (H (2, n = 9) = 6.54; P = 0.038). The yield also showed differences between species, being significantly higher in M. chilensis, and lower in oysters (Table 1).

The fatty acid analyzes showed that the mussels had significantly lower SFA values (between 31% and 69% less) and PUFA (between 19% and 70% less) (Table 2). They also had lower values in the following acids: 15:0 (up to 93% less), 16:0 (up to 64% less), 18: 3n-3 (up to 91% less) and 22:6n-3 (up to 68% less). On the other hand, Chilean oysters presented the highest values in 14:0, 16:0, 18:0, 18:3n-3 and 20:4n-6 acids among the species analyzed (Table 2).

Table 2

Fatty acid content of oysters and mussels from Hueihue (Chiloé Island, Chile).

Each value represents the mean of three independent replicates ± standard error. The letters indicate significant differences between averages.

	Ostrea chilensis	Crassostrea gigas	Mytilus chilensis
	Hueihue	Hueihue	Hueihue
Fatty acids (mg fatty acid g-1 dry weight of meat)
Myristic acid, 14:0	4.98 ± 0.37b	2.25 ± 0.72a	1.23 ± 0.30a
Pentadecanoic acid, 15:0	0.55 ± 0.04b	0.35 ± 0.12b	0.04 ± 0.04a
Palmitic acid, 16:0	16.08 ± 1.11b	13.23 ± 3.67ab	5.77 ± 1.36a
Heptadecanoic acid, 17:0	0.96 ± 0.07	0.65 ± 0.33	0.24 ± 0.04
Stearic acid, 18:0	4.64 ± 0.23b	2.44 ± 0.60a	1.29 ± 0.27a
Oleic acid, 18:1n-9	1.91 ± 0.11	2.33 ± 0.61	0.40 ± 0.08
Linoleic acid, 18:2n-6	1.28 ± 0.22	1.18 ± 0.29	0.42 ± 0.09
Eicoosenoic acid, 20:1n-11	1.10 ± 0.08	1.13 ± 0.36	0.35 ± 0.08
Linolenic acid, 18:3n-3	4.46 ± 0.39c	2.00 ± 0.58b	0.41 ± 0.09a
Eicosadienoic acid, 20:2n-6	0.22 ± 0.12	0.22 ± 0.09	0.18 ± 0.03
Arachidonic acid, 20:4n-6 (ARA)	1.99 ± 0.11b	0.75 ± 0.25a	0.57 ± 0.15a
Eicosapentaenoic acid, 20:5n-3 (EPA)	15.14 ± 1.50	12.90 ± 3.64	5.30 ± 1.65
Docosahexaenoic acid, 22:6n-3 (DHA)	9.05 ± 1.08b	8.89 ± 2.45b	2.88 ± 0.85a
Content of each group (mg g-1 meat)
SFA	27.78 ± 1.66b	19.29 ± 5.63ab	8.70 ± 1.98a
MUFA	5.01 ± 0.76	5.43 ± 1.76	3.49 ± 0.84
PUFA	32.31 ± 2.21b	26.22 ± 7.46ab	9.78 ± 2.79a
EPA + DHA	24,19 ± 2.58	21,79 ± 6.09	8.18 ± 2.50
Highly unsaturated fatty acids (HUFA)	24.27 ± 2.53	21.83 ± 6.07	8.21 ± 2.52
Ratios
n-3/n-6	7.84 ± 0.62	10.14 ± 0.73	7.13 ± 1.82
EPA/ARA	7.62 ± 0.57a	18.23 ± 1.88b	9.22 ± 2.09a
DHA/EPA	0.60 ± 0.01a	0.69 ± 0.01b	0.55 ± 0.01a
16:1n-7/16:0	0.15 ± 0.04a	0.12 ± 0.03a	0.46 ± 0.02b
SFA/PUFA	0.87 ± 0.09	0.73 ± 0.06	1.01 ± 0.27
MUFA/PUFA	0.15 ± 0.02	0.20 ± 0.02	0.42 ± 0.13
HUFA/PUFA	0.75 ± 0.02	0.83 ± 0.01	0.84 ± 0.04

Principal component analyses explained 94.26% of all observed variance in the data. The first principal component (PC1) explained 78.12%, was negatively related to 16:0, 18:3n-3, 18:0, 14.0 and 20:4n-6 fatty acids. PC2 explained 16.14% of the variance of the data and was positively related to fatty acids 18:1n-9, 22:6n-3, 20:1n-11 and 20:5n-3 (Fig 2A). In graph PC1, the groups corresponding to the three species can be observed. Chilean blue mussels were separated from oysters due to their low C/N value and high protein/energy and yield values, while Pacific oysters were separated due to EPA/ARA and DHA/EPA values. The differentiating characteristics between the Chilean oyster and the other species were the high values of 18:3n-3, 20:4n-6, 20:5n-3 and 22:6n-3 (Fig 2A).

The EPA + DHA values were different between the three species. In this sense, the Chilean oyster was a better food to supply the daily requirement of EPA+DHA (250 mg EPA+DHA—suggested by Carboni et al. 2019). For 2018 samples, an amount of 174 g of Chilean mussel raw meat, or 65 g of Pacific oyster, or 59 g of Chilean oyster are needed to meet the recommended daily requirement (Fig 3).

Fig 3

Daily consumption of mg of EPA+DHA in 100 g of raw meat of Crassostrea gigas, Ostrea chilensis and Mytilus chilensis from Faro Corona, Quempillén, Pullinque and Hueihue (Chiloé Island, Chile) harvested in winter.

Analysis of oysters between locations

A higher protein content was estimated in Chilean flat oysters from Pullinque than in oysters from Quempillén. The Pacific oysters from Faro Corona had intermediate values (Table 3 - H (2, n = 8) = 5.55; P = 0.06). There were no significant differences in lipid and carbohydrate content between oyster species and localities (Table 3). The highest ash value was shown by the Pacific oyster and the lowest values were registered in the Chilean oysters (F2, 5 = 13.60; P = 0.009—Table 3).

Table 3

Proximal composition and condition index in oysters from Quempillén, Pullinque and Faro Corona (Chiloé Island, Chile).

Each value represents the average of three independent replicates ± standard error, except for C. gigas with two independent replicas ± standard error. The letters indicate significant differences between averages.

	O. chilensis	O. chilensis	C. gigas
	(Quempillén)	(Pullinque)	(Faro Corona)
Proximal composition
Protein (% dry weight)	47.05 ± 0.47a	53.39 ± 0.24b	51.22 ± 1.89ab
Lipid (% dry weight)	23.18 ± 0.30	22.36 ± 0.70	22.85 ± 0.65
Carbohydrate (% dry weight)	15.37 ± 1.20	11.65 ± 1.12	10.56 ± 2.57
Ash (% dry weight)	14.40 ± 0.46b	12.60 ± 0.31a	15.37 ± 0.04b
Energy (MJ kg-1 meat)	22.95 ± 0.05	23.49 ± 0.14	22.98 ± 0.26
Condition indicators
C/N (atom C/ atom N)	6.48 ± 0.11	6.16 ± 0.15	6.26 ± 0.21
Protein/Energy (g protein MJ-1)	20.50 ± 0.19a	22.73 ± 0.13b	22.29 ± 0.57b
Yield (% total wet weight)	17.16 ± 0.34b	15.07 ± 0.30a	15.27 ± 0.36a
Wr (Relative weight index)	0.93 ± 0.03a	1.20 ± 0.04b	0.88 ± 0.03a

The energy of Ostrea chilensis cultivated in Pullinque showed a significantly higher average value than the flat oysters from Quempillén (F2,5 = 5.06; P = 0.06). The protein/energy ratio was higher in the Chilean Pullinque oyster and in the Pacific oyster (F2,5 = 21.02; P = 0.004), while the C/N values did not present significant differences between the oysters and the sites (average = 6.3 ± 0.2—Table 3).

The fatty acid profile was similar in the two oyster species. However, there were significant differences in some fatty acids. For example, Chilean oysters had higher values of 20:1n-9 (F2,5 = 8.20; P = 0.03) and lower values of 22:6n-3 (F2,5 = 4.62; P = 0.07) than Crassostrea gigas. Also, the Chilean oysters from Pullinque showed higher values of 15:0 than the oysters from Quempillén (F2,3 = 9.88; P = 0.05—Table 4).

There were also significant differences in the relationships between fatty acids. In the n-3/n-6 relationship, the highest average value was recorded in Pacific oysters (F2,5 = 8.26; P = 0.03). The same occurred for EPA/ARA (F2,3 = 16.79; P = 0.02—Table 4).

The first two principal components explained 93.49% of the variability in the data. The PC1 and PC2 plots showed a slight separation between oyster species (Fig 2B). Chilean flat oysters from Pullinque and Quempillén formed two distinct but close groups. The Pullinque oysters occupied the lower left quadrant (with the exception of the point located in the upper right quadrant), and were characterized by lower values of EPA, DHA, 16:0, n-3/n-6 and yield. On the other hand, the Pacific oyster formed a distinct group in the lower right quadrant (Fig 2B).

Principal component 1 (PC1) correlated positively with 20:5n-3, 16:0, 22:6n-3 fatty acid, HUFA and EPA+DHA (Fig 2B).

For 2017 samples, an amount of 67 g of Chilean oyster from Quempillén sampling site or 92 g of Chilean oyster from Pullinque or 44 g of Pacific oyster are needed to achieve daily requirement (Fig 3).

Discussion

The concentration of omega-3 and omega-6 fatty acids are essential nutritional components for human health (i.e. prevent cardiovascular disease or depression–[37]). However, α-linolenic acid (18:3n-3, ALA) and linoleic acid (18:2n-6, LNA) cannot be synthesized within the human body, therefore they are essentials and must be obtained from the diet [38]. Besides, the main role of 18:3n-3 is to be precursor for biosynthesis of EPA and DHA, but its conversion to DHA is poor [39]. In this sense, WHO (World Health Organization) recommends including foods of marine origin because they have high concentrations of polyunsaturated fatty acids (PUFAs) [10], transforming them in functional foods, i.e., foods that provide beneficial physiological effects, not specifically nutritional, benefiting the health of consumers. Our results showed that both oysters and mussels can be considered a recommended option for a healthy human diet based on their high contribution of functional fatty acids n-3 LC-PUFA, specifically EPA and DHA [39]. The concentration of EPA ranged between 5.3 and 18.2 mg g-1 dry weight, for DHA the range was 2.88–14.22 mg g-1 dry weigh, with high n-3/n-6 ratios varying 5.2 and 12.9.

Oysters vs mussel

Oysters and mussels are filter feeder bivalves with similar feeding behaviors. However, our results demonstrated that the acquisition and synthesis of fatty acids are species-specific. The Chilean oyster (O. chilensis), showed a high content of EPA and DHA above the values registered in the Chilean mussels (see Table 2). The fatty acid profile of oysters resembles an omnivorous feeder [40, 41].

The meat yield of the mussels was on average 56% higher than that of the oysters. Perhaps it is for this reason that the mussels’ farming industry is successful in terms of meat yield. The meat yield values were similar to those reported previously by other authors (Mytilus sp–[42]; Ostrea sp–[43].

DHA/EPA values (<1) and 16:1n-7/16:0 (trophic marker) indicated that diatoms are an important food resource in all three species [43-45].

Stearic and oleic fatty acids are biomolecules of terrestrial origin; however, they have been found associated with aquaculture areas [46]. Stearic acid is normally found in particulate organic matter (POM) [44]. These particles come from terrigenous material washed away by the rains. In Chilean oysters we find the highest values of stearic acid. We can assume that this species of oyster is the one that consumes the most POM when compared to the other two bivalves.

Our results also showed that oleic, linoleic acids and DHA were more abundant in oysters than in mussels. These differences are likely to be associated with feeding strategies. Mussels ingest suspended particulate matter including inorganic material [47], while oysters have a diet that includes zooplankton, protozoa and/or dinoflagellates [44, 48, 49]. Crassostrea gigas and Ostrea chilensis had 8 and 14 times more pentadecanoic acid (15:0) than M. chilensis. The presence of this fatty acid is related to the intake of bacteria in the diet [49].

EPA+DHA values were different between the species studied. Presenting values higher than those reported for commercial species of bivalves from the northern hemisphere [50]. It has been reported that the recommended daily amount for human consumption should be 250 mg of EPA+DHA [51]. Therefore, 174 g of mussel meat (fresh), or 44 to 65 g of Pacific oyster, or 59 to 92 g of Chilean oyster should be consumed to meet the recommended daily requirement, considering the data obtained during the 2017 and 2018 sampling season. In this sense, the Chilean oyster is a good food to cover the daily requirement of EPA+DHA. Importantly, cooking techniques such as frying or boiling reduce the content of these essential fatty acids [52]. In addition, we found high values in the n-3/n-6 ratio for the oyster species analyzed (5.15 and 12.86). These values are much higher than the values reported for farmed and wild fish [53]. The higher this value, the healthier the diet, so oysters can be considered species of importance in human nutrition, opening a way to manage these nutrients through cultivation.

Western eating habits in recent times have drastically changed the nutritional content of food in developed countries, as a result of technological changes in the production and processing of high-calorie foods [54-56]. These have led to detrimental changes in nutrient metabolism leading to gene-diet interactions responsible for more obesity and systemic inflammation [57]. The role of n-3 PUFAs has been key in enhancing anti-inflammatory ability to control various chronic diseases, including atherosclerosis, coronary heart disease, diabetes, rheumatoid arthritis, depression, and cancer [58-60]. It has also been reported that n-3 and n-6 have a high potential for gastric cancer prevention and therapy [61, 62]. That is why the World Health Organization recommends including foods high in PUFA in daily consumption [63]. Under this context, the levels of n-3 LC-PUFA (mainly EPA and DHA) in oysters and mussels reported in this work may be beneficial for human health, and should be considered as a food alternative in the coming years.

Differences between populations of oysters

The two species of oysters (O. chilensis y C. gigas) had a high content of proteins, lipids, and carbohydrates in winter 2017, which is positive for the aquaculture industry due to the commercial importance of these oyster species for the development of aquaculture on Chiloé Island. On the other hand, fatty acids showed significant differences between species, for example, eicosadienoic acid (c20: 2n-6) was detected in all species except for the Pacific oysters from Faro Corona. The Chilean oysters from Pullinque showed high values of pentadecanoic acid (15:0) when compared to the oysters from Quempillén. 15:0 is a naturally rare, straight chain saturated fatty acid [64] and it has been described as a fatty acid that may decrease mother-to-child transmission of HIV through breastfeeding [65]. This fatty acid has not been previously reported for oysters but has been reported for mussels [66]. We do not understand very well what these differences between the localities are due to but we do know that 15:0 has been found within the seston. Furthermore, 15:0 is considered a bacterial marker [49, 67] and has been related to the growth of bivalves (e.g., clams). The positive association of saturated fatty acids with growth may indicate utilization of carbon derived from bacteria in the mollusk diet [66]. Bacteria have been considered an important food in the diet of oysters, both in the larval stage and in adulthood. For example, they have been reported to increase larval survival of Crassostrea gigas [68].

This study showed that the environment plays an important role in the fatty acid acquisition of the Chilean oyster. The concentration of PUFAs in the oysters was different between the oysters of the analyzed localities (Quempillén, Pullinque and Hueihue), even though the localities were close. Therefore, nutrient acquisition is a quantitative variable that can be used to identify commercial species with higher fatty acid acquisition. In this way, growers could have aquaculture productions with better nutritional characteristics [69]. However, it is necessary to understand the intra- and interspecific variability of the cultivated species, and our results contribute to the knowledge for these purposes.

One-hundred g of mussel or oyster meat provides a quarter of the daily amount of protein that an adult need. This same dose provides the recommended amount of vitamin B12. In addition, these mollusks provide essential trace elements such as Se, Fe and Zn [14, 43, 70, 71]. However, the nutritional profiles of these bivalves will also change seasonally, so it is necessary to expand the study not only to more nutrients relevant to human health but also to all seasons of the year, especially during harvest periods. In conclusion, we consider that the nutritional variability of marine food sources should continue to be investigated, especially that providing high production volumes. Further investigations are needed on the heritability of the nutritional attributes of mussels and oysters to improve those characteristics in the short term.

Conclusions

Shellfish sampled in winter were characterized by a high protein content, followed by medium values for lipid content and a low carbohydrate content compared to similar species in Europe. EPA + DHA values were different among the three species, with oysters showing the highest values in relation to mussels. It was also observed that the shellfish studied are rich in long-chain omega-3 polyunsaturated fatty acids (n-3 LC-PUFA), varying between 5.2–12.9 μg FA mg-1 dry weight with high n-3/n-6 ratios, depending on the species and the place of fattening. Keystone species can be considered as a sustainable option for a balanced and healthy human diet.

10 Dec 2021

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Reviewer #1: Partly

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

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Reviewer #1: The paper by A. Valenzuela et al. describes interesting data on the proximal composition and fatty acid content of three mollusc species from Chile, that can contribute to the literature already existing on bivalve molluscs.

The Authors also tried to assess differences existing between species or even between the same species growing in relatively close locations.

On the other hand, several aspects of the paper need to be improved, especially in terms of description of experimental procedures, statistical analysis and results.

A list of suggestions/comments that could help the Authors during the revision stage is thus provided below, following the order of appearance in the manuscript.

Materials and methods

The title of the first sub-section (Sampling) of this section should be integrated to clarify that the sub-section deals also with the determination of proximal composition and fatty acid profile of molluscs.

Lines 122-129: the use of the same symbol, WW, first for the calculated wet weight (lines 125-126) and then for the experimental wet weight (lines 128-129) can be misleading. A different subscript (i.e., calc and exp, according to the case) should be added to the WW symbol to avoid any misunderstanding. Wr would thus be expressed as WWexp/WWcalc.

Line 133. Details should be provided about the use of the N x 6.25 rule. This can not immediately understandable for all readers.

Line 137 – 138. Which detector was used after GC separations? I guess that it was a FID but this detail has to be provided.

Lines 146-150. The sentence was already reported at lines 131-135.

Lines 151-152. Is there any previous reference about this approach to estimate the carbohydrate content?

Lines 157, 158, and 160-162. The “g dry weight” expression should be put between parentheses and then the -1 exponent should be placed outside; as an alternative, the word “per” could be placed before “g dry weight”.

Lines 173-176. The type of data used as original variables for PCA must be cited.

Results

Lines 188, 190, 193 and 196: the meaning of the H and F symbols needs to be clarified. Since the Authors claimed that normality and homoscedasticity tests were performed on data, the results of those tests should also be described.

Line 213. Fig2 should be referred to as Fig2a.

Figure 2. I would strongly suggest adding also loading plots to the figure, to help the readers in understanding how the variables influenced the differences observed between samples in the score plot. It is very complicated to switch to the Supplementary Material to find this information using Figure S1.

Tables 1-4. It seems that the results of tests for the comparison between means were not reported for all data, as significant differences could be supposed to be present sometimes (based on numerical values) but superscripts indicating the outcome of those tests were missing, thus suggesting that no significant difference was observed. Is this correct?

I would suggest using a consistent number of significant figures to express standard deviations (for example two) and then round off mean and standard deviation values accordingly.

The number of replicates should be clearly indicated in the tables captions.

Line 309. I would suggest using the word “quadrant” or “semi-plane”, according to the case, instead of “hemisphere”.

Lines 306-311: it would be better to use the term “principal” instead of “main”, since PCA results are described. I would be very careful in writing that Chilean oysters from Pullinque and Quempillen made up different groups in the score plot. Indeed, a remarkable variability was observed for the samples collected in Pullinque and at least one of them was very close to the samples from Quempillen.

Figure S1. What is the meaning of the colour code (and then of the legend) adopted in the figure?

Figure 3. The figure does not show daily consumptions, as stated in the caption, but the amounts of the two fatty acids referred to 100 g of meat of each mollusc. Please clarify this issue. The number of replicates used to calculate error bars should be indicated for each set of data.

Lines 375. Why were just those two values reported for the n-3/n-6 ratio? If the Authors meant to indicate the entire interval observed for those ratios in all the analyzed species, then the minimum value would be 5.15, found for Chilean oysters from Pullinque. A clarification is needed.

Lines 412-413. This sentence seems to be conceptually linked to the previous one, yet it concerns animal genetic improvement, so how can it be related to the sentence in which the focus was on the locality? Please clarify.

Conclusions. I would suggest rephrasing this section, focusing on more specific aspects of the work, like the comparison between oysters and mussels in terms of key components and the subtle effects that the location seemed to have on the nutrient content of the considered shellfish.

Reviewer #2: Present study determined the proximal biochemical composition and the fatty acid profile in the Chilean oyster (Ostrea chilensis), the Pacific oyster (Crassostrea gigas) and the Chilean mussel (Mytilus chilensis), to perform an intra and interspecific comparison. To my understanding the interspecific comparison (O. chilensis, C. gigas and M. chilensis) was performed on samples that were all collected from Hueihue in 2018, and the intraspecific comparison was meant on O. chilensis that were sampled from two locations (Quempillen vs. Pullinque) in 2017. Additionally to the results of O. chilensis (2017) the authors present results of C. gigas (sampled from Faro Corona in 2017). However, this is irritating as C. gigas is a different species and the results do not fit to the “intraspecific comparison”.

Besides minor grammatical inaccuracies, the ms is well written and the results seem to be reliable supporting the conclusion that the investigated shellfish species can be a “sustainable option for a balanced and healthy human diet”. However, the finding that oysters and mussels are healthy food components in human diet is not new and the study will benefit from a clearer (hypothesis-driven) research question to more strongly emphasize its relevance to the scientific community. In addition, the ms can be improved by a more detailed description of the M&M part and an in-depth discussion.

Major comments:

1) The authors aim to “nutritionally characterize the food quality” of two oyster and one mussel species from aquaculture in Southern Chile. To improve the study, the results presented (content of lipids, protein etc. as well as fatty acid composition) need to be put in context with literature data and discussed appropriately; e.g. Do the results differ from bivalves obtained from European or US aquaculture facilities? If so, why? Investigations were conducted on bivalves collected during the winter season, and the data could be discussed/compared with data from bivalves collected during spring/summer.

2) Please highlight why the different locations were chosen for the intraspecific comparison. In what respect do they differ from each other? The intraspecific comparison aspect should be more clearly elaborated in the discussion. In its present form (under the heading “Differences between populations of oysters”) it is a bit confusing as the dataset of C. gigas is also discussed. Please comment on your decision to add the data of C. gigas (obtained in 2017) to the results part of the intraspecific comparison (line 243f).

3) With respect to the results in Table 1 showing highest proximal composition values in protein content (42 – 52% dry weight) and lower values in lipid (14.5 – 19.5%) and carbohydrates (11.5 to 20.9%), the statement “shellfish sampled in winter were characterized by high lipid content along with low protein and carbohydrates contents (line 36f, and 431f) is irritating. Please clarify.

Specific comments:

Line 26/27: Please clarify the definitions of equal contribution of the authors - ¶ vs. &.

Line 83: Change into “On the one hand…”

Line 107f: Sampling of animals: Please clarify the sentence "3 samples of 20 oysters" as it is 60 animals in total, isn't it? Please add more details about the environmental parameters at the time of collection (water temperature, salinity...) and transport of the animals (duration, transport on ice/water...). Were the animals kept in institutional aquarium systems prior to dissection? If yes, please add details on husbandry.

Line 121ff: Please comment on using the calculation of yield instead of condition index (soft body dry mass*100/shell dry mass) which is more common. In any case, data sets should be compared with literature data to increase the reliability of the results.

Line 122: Please add the underlying data for the calculation of the allometric relationship of WW. The cited reference (#30) is for Wr for fish. Is it also used for shellfish?

Line 133: Please clarify how samples were treated prior to analysis to ensure homogeneity, as parameters were determined in whole animal extracts.

Line 133: Unclear, please add more information how crude protein was calculated.

Line 146f: Repetition, see line 131f.

Line 161: Please provide more details concerning the equation parameters “12g” and “15g”.

Line 163: Consider rephrasing “performance”. If it is a common term in aquaculture, please provide a citation for the calculation.

Line 165f: Statistics: Missing info of p level.

Line 191: Revise sentence “higher compared to …”. Please check the whole ms regarding this aspect. Furthermore, using the mean values of lipid content (Table 1) the values (9 to 25%) do not fit. Please clarify.

Line 202: Table 1 and all other Tables: Please add experimental number. Energy is expressed as MJ per kg dry meat, isn’t it? Please specify that “different” letters indicate differences.

Line 206: see comments to line 191.

Line 317f: What about the EDA+DHA concentration of nearly 600mg/100g ww meat determined in C. gigas samples collected in 2017?

Line 368f: This is a repetition, see line 317f.

Line 375: Please clarify: 7.13 “y” 12.86.

Line 376: Please add more details/information. The reader wonders, so what?

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Reviewer #1: No

Reviewer #2: No

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20 Jan 2022

REPLIES TO REVIEWERS

PONE-D-21-28284

Proximal and fatty acid analysis in Ostrea chilensis, Crassostrea gigas and Mytilus chilensis (Bivalvia: Mollusca) from Southern Chile.

Dear Academic Editor

Below you can find the corrections made in the manuscript according to the suggestions made by you and reviewers #1 and 2:

ACADEMIC EDITOR

A.E: Both reviewers find merit in your study, but raise concerns. I agree that you should provide more details describing sampling conditions (abiotic factors, water depth) and reproductive status of the animals, as this has a strong impact on fatty acid profiles. Also, the method and results section need to describe in more detail, how many replicate animals were used for each analysis and statistical tests. The manuscript also would benefit from being proof-read by a native speaker.

Reply: we took these considerations into account and they were included in the manuscript in each of the sections.

A.E: Please include the following items when submitting your revised manuscript:

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Reply: The three files mentioned in this paragraph were created and submitted separately.

A.E: If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

Reply: Without changes

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Reply: all the requirements requested by PLOS ONE were considered.

2. In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the field site access and, if no permits were required, a brief statement explaining why.

Reply: Additional permits are not required for field work. All locations are freely accessible and the collection of samples for research is allowed.

3. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service.

Reply: the manuscript was reviewed by a native speaker (Michael Note) before being resubmitted.

4. Thank you for stating the following financial disclosure: “Dr. Jorge Toro received the following funding: Fondef ID19I10214 and ID16I10018”

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Reply: Dr. Jorge Toro worked on the design of the study, the decision to publish, and the preparation of the manuscript. An amended statement of the funder's role will be included in the cover letter.

5. Thank you for stating the following in the Acknowledgments Section of your manuscript:

“Fondef ID19I10214 and ID16I10018 financed this study. To Justo García Campos from the town of Hueihue and Juan Pablo Fuentes Cid from Quempillén, for their collaboration in the installation of the experiments.

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

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Fondef ID19I10214 and ID16I10018”

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Reply: the codes of the projects responsible for the financing were removed from the acknowledgments section. Codes were placed on the application and an amended statement of project funding was included in the cover letter.

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Reply: The data from this research can be found in the tables and figures presented in the manuscript and in the supplementary material, and are freely available to the journal and those who work in bivalve mollusc nutrition research.

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Reply: the ethics statement was included in the methods section of the manuscript, including the full name of the ethics committee that approved the study.

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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: Yes

REPLY: the manuscript was made based on a robust and solid research work, with data that support the conclusions. All considerations were taken to carry out a rigorous experimental design, with adequate controls, replicates and sample sizes.

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

REPLY: The treatment of the data was carried out in an adequate and rigorous manner according to the nature of the data.

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

REPLY: The data is available to the journal and for universal use by those working in science, especially in marine bivalve nutrition.

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

REPLY: the manuscript is written in standard English, proofread and proofread by native speaker Michael Note.

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: The paper by A. Valenzuela et al. describes interesting data on the proximal composition and fatty acid content of three mollusc species from Chile, that can contribute to the literature already existing on bivalve molluscs.

The Authors also tried to assess differences existing between species or even between the same species growing in relatively close locations.

On the other hand, several aspects of the paper need to be improved, especially in terms of description of experimental procedures, statistical analysis and results.

A list of suggestions/comments that could help the Authors during the revision stage is thus provided below, following the order of appearance in the manuscript.

Materials and methods

The title of the first sub-section (Sampling) of this section should be integrated to clarify that the sub-section deals also with the determination of proximal composition and fatty acid profile of molluscs.

Reply: Done

Lines 122-129: the use of the same symbol, WW, first for the calculated wet weight (lines 125-126) and then for the experimental wet weight (lines 128-129) can be misleading. A different subscript (i.e., calc and exp, according to the case) should be added to the WW symbol to avoid any misunderstanding. Wr would thus be expressed as WWexp/WWcalc.

Reply: The WW values cited between lines 122-129 are any WW obtained experimentally to apply the index Wr, that allows to determine if a WW sampled in an individual of species A relates to the expected WLR for species A with a values inferior, similar or superior to 1; ie, there are no calculated WW values, only experimental values in all cases

Line 133. Details should be provided about the use of the N x 6.25 rule. This can not immediately understandable for all readers.

Reply: In nutrition, the total nitrogen content of the sample is converted into a value called "crude protein" by multiplying it by 6.25, a constant based on the conventional assumption that any protein is composed of 16% nitrogen.

Line 137 – 138. Which detector was used after GC separations? I guess that it was a FID but this detail has to be provided.

Reply: Flame ionization detector (FID) Thermo Scientific was used

Lines 146-150. The sentence was already reported at lines 131-135.

Reply: corrected

Lines 151-152. Is there any previous reference about this approach to estimate the carbohydrate content?

Reply: the methods of substraction of total carbohydrate from proximal composition as cited by Metzger LE and Nielsen SS, 2019. Chapter 3 Nutrition Labelling. In: Food Analysis. Fifth edition, SS Nielsen (ed), Food Science Text Series, Springer, Cham, Switzerland, pp 35-44

Lines 157, 158, and 160-162. The “g dry weight” expression should be put between parentheses and then the -1 exponent should be placed outside; as an alternative, the word “per” could be placed before “g dry weight”.

Reply: done

Lines 173-176. The type of data used as original variables for PCA must be cited.

Reply: The data used for PCA analysis were the values of each replicate for: every fatty acid, every relationship between fatty acids, every performance indicator, and every proximal content. Finally, only those variables most correlated with the first two components were selected for final PCA analysis.

Results

Lines 188, 190, 193 and 196: the meaning of the H and F symbols needs to be clarified. Since the Authors claimed that normality and homoscedasticity tests were performed om data, the results of those tests should also be described.

Reply: done

Line 213. Fig2 should be referred to as Fig2a.

Reply: done

Figure 2. I would strongly suggest adding also loading plots to the figure, to help the readers in understanding how the variables influenced the differences observed between samples in the score plot. It is very complicated to switch to the Supplementary Material to find this information using Figure S1.

Reply: Figure 2a and b was improved and replaced with data from the supplementary material. Therefore, the supplementary material was eliminated, and its data were incorporated along with the variables in Figure 2, for a better understanding of the results.

Tables 1-4. It seems that the results of tests for the comparison between means were not reported for all data, as significant differences could be supposed to be present sometimes (based on numerical values) but superscripts indicating the outcome of those tests were missing, thus suggesting that no significant difference was observed. Is this correct?

I would suggest using a consistent number of significant figures to express standard deviations (for example two) and then round off mean and standard deviation values accordingly.

The number of replicates should be clearly indicated in the tables captions.

Reply: done.

Line 309. I would suggest using the word “quadrant” or “semi-plane”, according to the case, instead of “hemisphere”.

Reply: done.

Lines 306-311: it would be better to use the term “principal” instead of “main”, since PCA results are described. I would be very careful in writing that Chilean oysters from Pullinque and Quempillen made up different groups in the score plot. Indeed, a remarkable variability was observed for the samples collected in Pullinque and at least one of them was very close to the samples from Quempillen.

Reply: done.

Figure S1. What is the meaning of the colour code (and then of the legend) adopted in the figure?

Reply: In blue (active) it refers to the active variables that influence the principal components and in red (suppl) to the supplementary variables that have no influence on the analysis of the principal components, but do help to interpret the results. However, this graph was corrected and the supplementary material was removed, Figure 2 and Figure S1 were merged for a better understanding of the results.

Figure 3. The figure does not show daily consumptions, as stated in the caption, but the amounts of the two fatty acids referred to 100 g of meat of each mollusc. Please clarify this issue. The number of replicates used to calculate error bars should be indicated for each set of data.

Reply: Figure 3. Potential intake of EPA + DHA in a 100 g edible portion from Crassostrea gigas, Ostrea chilensis and Mytilus chilensis.

Lines 375. Why were just those two values reported for the n-3/n-6 ratio? If the Authors meant to indicate the entire interval observed for those ratios in all the analyzed species, then the minimum value would be 5.15, found for Chilean oysters from Pullinque. A clarification is needed.

Reply: corrected

Lines 412-413. This sentence seems to be conceptually linked to the previous one, yet it concerns animal genetic improvement, so how can it be related to the sentence in which the focus was on the locality? Please clarify.

Reply: corrected

Conclusions. I would suggest rephrasing this section, focusing on more specific aspects of the work, like the comparison between oysters and mussels in terms of key components and the subtle effects that the location seemed to have on the nutrient content of the considered shellfish.

Reply: Reworded this section as suggested by reviewer.

Reviewer #2: Present study determined the proximal biochemical composition and the fatty acid profile in the Chilean oyster (Ostrea chilensis), the Pacific oyster (Crassostrea gigas) and the Chilean mussel (Mytilus chilensis), to perform an intra and interspecific comparison. To my understanding the interspecific comparison (O. chilensis, C. gigas and M. chilensis) was performed on samples that were all collected from Hueihue in 2018, and the intraspecific comparison was meant on O. chilensis that were sampled from two locations (Quempillen vs. Pullinque) in 2017. Additionally, to the results of O. chilensis (2017) the authors present results of C. gigas (sampled from Faro Corona in 2017). However, this is irritating as C. gigas is a different species and the results do not fit to the “intraspecific comparison”.

Besides minor grammatical inaccuracies, the ms is well written and the results seem to be reliable supporting the conclusion that the investigated shellfish species can be a “sustainable option for a balanced and healthy human diet”. However, the finding that oysters and mussels are healthy food components in human diet is not new and the study will benefit from a clearer (hypothesis-driven) research question to more strongly emphasize its relevance to the scientific community. In addition, the ms can be improved by a more detailed description of the M&M part and an in-depth discussion.

Major comments:

1) The authors aim to “nutritionally characterize the food quality” of two oyster and one mussel species from aquaculture in Southern Chile. To improve the study, the results presented (content of lipids, protein etc. as well as fatty acid composition) need to be put in context with literature data and discussed appropriately; e.g. Do the results differ from bivalves obtained from European or US aquaculture facilities? If so, why? Investigations were conducted on bivalves collected during the winter season, and the data could be discussed/compared with data from bivalves collected during spring/summer.

Reply: This comment has been considered in the discussion to deepen the comparison between the three species of bivalves.

2) Please highlight why the different locations were chosen for the intraspecific comparison. In what respect do they differ from each other? The intraspecific comparison aspect should be more clearly elaborated in the discussion. In its present form (under the heading “Differences between populations of oysters”) it is a bit confusing as the dataset of C. gigas is also discussed. Please comment on your decision to add the data of C. gigas (obtained in 2017) to the results part of the intraspecific comparison (line 243f).

Reply: The locations chosen in this study for the comparison between oyster species were selected because Quempillén and Pullinque correspond to two areas of natural banks of O. chilensis on Chiloé Island. The town of Quempillén is an estuarine zone with high salinity fluctuations. Instead, the town of Pullinque is a bay in the Gulf of Quetalmahue with depths of 7 to 8 meters. The species C. gigas was added because it is the second most important commercially important oyster species in Chile and because it also has natural banks on Chiloé Island.

3) With respect to the results in Table 1 showing highest proximal composition values in protein content (42 – 52% dry weight) and lower values in lipid (14.5 – 19.5%) and carbohydrates (11.5 to 20.9%), the statement “shellfish sampled in winter were characterized by high lipid content along with low protein and carbohydrates contents (line 36f, and 431f) is irritating. Please clarify.

Reply: The paragraphs mentioned in lines 36f and 431f were corrected.

Specific comments:

Line 26/27: Please clarify the definitions of equal contribution of the authors - ¶ vs. &.

Reply: the authors' equal contribution to:

¶ = all of these authors contributed to study design, data collection and analysis, decision to publish, or manuscript preparation.

&= all these authors contributed to the experimental and field work.

Line 83: Change into “On the one hand…”

Reply: done.

Line 107f: Sampling of animals: Please clarify the sentence "3 samples of 20 oysters" as it is 60 animals in total, isn't it? Please add more details about the environmental parameters at the time of collection (water temperature, salinity...) and transport of the animals (duration, transport on ice/water...). Were the animals kept in institutional aquarium systems prior to dissection? If yes, please add details on husbandry.

Reply: For each locality, 60 sampled specimens were collected from cultivation areas, where 3 samples (replicates) of 20 individuals were randomly made, except for C. gigas, which were fewer specimens (40 individuals). Then, in the laboratory, the tissues without the shell were worked, each sample was stored in a refrigerated/frozen ziplox bag with the meat of 20 individuals corresponding to replica 1, 2 and 3 of each sampling.

Line 121ff: Please comment on using the calculation of yield instead of condition index (soft body dry mass*100/shell dry mass) which is more common. In any case, data sets should be compared with literature data to increase the reliability of the results.

Reply: The samples were processed in the field, where the wet weight was directly measured. In our laboratory, the samples were lyophilized to be considered for proximal content and fatty acid content. The work of Sing & Ransangan (2019) measure yield according to wet weight.

Sing, O. F., & Ransangan, J. (2019). Effect of physicochemical parameters and phytoplankton composition on growth performance of green mussel (Perna viridis) in Ambong Bay and Marudu Bay, Sabah, Malaysia. Journal of Fisheries and Environment, 43(1), 50-68.

Line 122: Please add the underlying data for the calculation of the allometric relationship of WW. The cited reference (#30) is for Wr for fish. Is it also used for shellfish?

Reply: Now the underlying data suggested is:

The relative weight index [30] is based on the length -weight relationships (LWRs). The total wet weight in grams (WW), and shell length in milimeters (L) obtained from each individual sampled during the study were used. The wet weights were fit using a power regression as a function of shell lenght to obtain WW = aLb , where a is the intercept (condition factor) and b is the slope (relative growth) following Sousa et al (2020 a, b). The relationships were WW = 0.021*L2.16 for M. chilensis with 60 individuals (R2= 0.48), WW = 0.0004*L2.77 for O. chilensis with 180 individuals (R2= 0.51), and WW = 0.079*L1.48 for C. gigas with 100 individuals (R2= 0.67).

The use of WLRs are commonly used for shellfish see Sousa et al 2020a, b. The WLRs are used to compare different ecological, geographical, reproductive, etc., conditions for some particular species, and the comparisons is about the exponent of WLRs.

So the Wr is only a ratio WW/WLR to show if the animals are more slim or fat respect to an average condition represented by WLR of the species in different geographical areas, or growth conditions, or etc, Therefore it is applicable to shellfish, the same as WLRs

Line 133: Please clarify how samples were treated prior to analysis to ensure homogeneity, as parameters were determined in whole animal extracts.

Reply: For each sample of 20 individuals the tissues were pooled and freeze dried in a Savant freeze dryer (-80°C), then they were ground to homogenize and saved at -41°C until the respective biochemical analysis.

Line 133: Unclear, please add more information how crude protein was calculated.

Reply: In nutrition, the total nitrogen content of the sample is converted into a value called "crude protein" by multiplying it by 6.25, a constant based on the conventional assumption that any protein is composed of 16% nitrogen.

Line 146f: Repetition, see line 131f.

Reply: corrected

Line 161: Please provide more details concerning the equation parameters “12g” and “15g”.

Reply: correspond to the molecular weights of carbon and nitrogen. The nitrogen value was corrected to 14.

Line 163: Consider rephrasing “performance”. If it is a common term in aquaculture, please provide a citation for the calculation.

Reply: CI commercial = wet meat weight x 100 /whole wet weight (live) = Meat wet weight x 100/ Total wet weight following Hickman and Illingworth (1980)

Line 165f: Statistics: Missing info of p level.

Reply: p< 0.05

Line 191: Revise sentence “higher compared to …”. Please check the whole ms regarding this aspect. Furthermore, using the mean values of lipid content (Table 1) the values (9 to 25%) do not fit. Please clarify.

Reply: corrected

Line 202: Table 1 and all other Tables: Please add experimental number. Energy is expressed as MJ per kg dry meat, isn’t it? Please specify that “different” letters indicate differences.

Reply: done

Line 206: see comments to line 191.

Reply: done

Line 317f: What about the EDA+DHA concentration of nearly 600mg/100g ww meat determined in C. gigas samples collected in 2017?

Reply: For 2017 samples, an amount of 67 g of Chilean oyster from Quempillén sampling site or 92 g of Chilean oyster from Pullinque or 44 g of Pacific oyster are needed to achieve daily requirement (Fig.3).

Line 368f: This is a repetition, see line 317f.

Reply: corrected

Line 375: Please clarify: 7.13 “y” 12.86.

Reply: corrected.

Line 376: Please add more details/information. The reader wonders, so what?

Reply: Reworded this paragraph to be more consistent.

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5 Apr 2022

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Reviewers' comments:

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Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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28 Apr 2022

REPLIES TO REVIEWER

PONE-D-21-28284

Proximal and fatty acid analysis in Ostrea chilensis, Crassostrea gigas and Mytilus chilensis (Bivalvia: Mollusca) from Southern Chile.

Dear Academic Editor

Below you can find the corrections made in the manuscript according to the suggestions made by the reviewer # 2:

REVIEWER #2 COMMENTS:

Line 26/27: Please clarify the definitions of equal contribution of the authors - ¶ vs. &.

Reply: the authors' equal contribution to:

¶ = all of these authors contributed to study design, data collection and analysis, decision to publish, or manuscript preparation.

&= all these authors contributed to the experimental and field work.

�  Thank you, but this information should also be in the paper, shouldn't it?

Reply: We appreciate your clarification. Your suggestion will be included in the manuscript.

Line 83: Change into “On the one hand…”

Reply: done.

�  hm,, the authors revised the sentence but not as suggested. That's fine but the reply "done" is irritating.

Reply: We regret the incorrect use of the word "done". We will take this into account in future corrections.

Line 107f: Sampling of animals: Please clarify the sentence "3 samples of 20 oysters" as it is 60 animals in total, isn't it? Please add more details about the environmental parameters at the time of collection (water temperature, salinity...) and transport of the animals (duration, transport on ice/water...). Were the animals kept in institutional aquarium systems prior to dissection? If yes, please add details on husbandry.

Reply: For each locality, 60 sampled specimens were collected from cultivation areas, where 3 samples (replicates) of 20 individuals were randomly made, except for C. gigas, which were fewer specimens (40 individuals). Then, in the laboratory, the tissues without the shell were worked, each sample was stored in a refrigerated/frozen ziplox bag with the meat of 20 individuals corresponding to replica 1, 2 and 3 of each sampling.

�  Please add more details….” This comment wasn't addressed, neither in the reply nor in the revised ms.

Reply: We improved the paragraph, including the required information in the manuscript. We appreciate your helpful suggestions.

Line 121ff: Please comment on using the calculation of yield instead of condition index (soft body dry mass*100/shell dry mass) which is more common. In any case, data sets should be compared with literature data to increase the reliability of the results.

Reply: Samples were processed in the field, where 20 individuals were randomly selected for each sample, measured and weighed wet, shells were immediately removed and the soft bodies of the 20 individuals were grouped in a properly labeled bag. The bags were stored cold for transport to the laboratory. In the laboratory, the samples were frozen, freeze-dried, macerated and grouped by bag, kept frozen until analysis for proximal and fatty acid content.

The index was calculated according to Hickmann and Illingworth (1980).

These authors compared different condition index formulas that are weight/weight ratios and that reflect changes in body proportions in bivalve populations.

CIwet = [wet meat volumen/(whole volumen - Shell volumen)] * 100

CIvolume = [dry meat wt / (whole volumen - shell volumen)] * 100

CIweight = [dry meat wt/(whole wt - shell wt)] * 100

CIcommercial = [Meat wet wt / Total wet weight] * 100

They found that CIcommercial correlated 92.7% with CIwet, 90.4% with CIvolume and 90.9% with CIweight.

According to Hickmann and Illingworth (1980), although CIweight is the most advisable for reliable comparisons, it is possible to standardize the measurement of wet weight in an index such as CIcommercial to minimize errors by always adopting the same weighing routines, which is what was applied in this study.

Line 133: Please clarify how samples were treated prior to analysis to ensure homogeneity, as parameters were determined in whole animal extracts.

Reply: For each sample of 20 individuals the tissues were pooled and freeze dried in a Savant freeze dryer (-80°C), then they were ground to homogenize and stored at -41°C until the respective biochemical analysis.

�  This is an important information that the tissue of 20 individuals was pooled prior to analyses and must be given in the ms.

Reply: Thank you for the clarification. This information has been included in the manuscript.

Line 133: Unclear, please add more information how crude protein was calculated.

Reply: In nutrition, the total nitrogen content of the sample was converted into a value called "crude protein" by multiplying it by 6.25, a constant based on the conventional assumption that any protein is composed of 16% nitrogen.

�  Again, this is an information that should be given in the ms.

Reply: Thank you for the clarification. This information has also been included in the manuscript.

Line 146f: Repetition, see line 131f.

Reply: corrected

�  Sorry, the repetition is still in the revised version.

Reply: This repetition has been removed. Thanks for the clarification

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30 May 2022

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2 Jun 2022

Dear Academic Editor

The points suggested by reviewer 2 in the second revision and also the rest of the original points raised by reviewer 1 and 2 in the first revision were fully addressed.

I appreciate your support and understanding.

Below you can find the corrections made in the manuscript according to the suggestions made in the first (reviewer 1 and 2) and second revision (reviewer 2).

REPLIES TO REVIEWER

PONE-D-21-28284

Proximal and fatty acid analysis in Ostrea chilensis, Crassostrea gigas and Mytilus chilensis (Bivalvia: Mollusca) from Southern Chile.

Second revision R2 (May 2022)

REVIEWER #2 COMMENTS:

Line 26/27: Please clarify the definitions of equal contribution of the authors - ¶ vs. &.

Reply: the authors' equal contribution to:

¶ = all of these authors contributed to study design, data collection and analysis, decision to publish, or manuscript preparation.

&= all these authors contributed to the experimental and field work.

�  Thank you, but this information should also be in the paper, shouldn't it?

Reply: We appreciate your clarification. Your suggestion will be included in the manuscript.

Line 83: Change into “On the one hand…”

Reply: done.

�  hm,, the authors revised the sentence but not as suggested. That's fine but the reply "done" is irritating.

Reply: We regret the incorrect use of the word "done". We will take this into account in future corrections.

Line 107f: Sampling of animals: Please clarify the sentence "3 samples of 20 oysters" as it is 60 animals in total, isn't it? Please add more details about the environmental parameters at the time of collection (water temperature, salinity...) and transport of the animals (duration, transport on ice/water...). Were the animals kept in institutional aquarium systems prior to dissection? If yes, please add details on husbandry.

Reply: For each locality, 60 sampled specimens were collected from cultivation areas, where 3 samples (replicates) of 20 individuals were randomly made, except for C. gigas, which were fewer specimens (40 individuals). Then, in the laboratory, the tissues without the shell were worked, each sample was stored in a refrigerated/frozen ziplox bag with the meat of 20 individuals corresponding to replica 1, 2 and 3 of each sampling.

�  Please add more details….” This comment wasn't addressed, neither in the reply nor in the revised ms.

Reply: We improved the paragraph, including the required information in the manuscript. We appreciate your helpful suggestions.

Line 121ff: Please comment on using the calculation of yield instead of condition index (soft body dry mass*100/shell dry mass) which is more common. In any case, data sets should be compared with literature data to increase the reliability of the results.

Reply: Samples were processed in the field, where 20 individuals were randomly selected for each sample, measured and weighed wet, shells were immediately removed and the soft bodies of the 20 individuals were grouped in a properly labeled bag. The bags were stored cold for transport to the laboratory. In the laboratory, the samples were frozen, freeze-dried, macerated and grouped by bag, kept frozen until analysis for proximal and fatty acid content.

The index was calculated according to Hickmann and Illingworth (1980).

These authors compared different condition index formulas that are weight/weight ratios and that reflect changes in body proportions in bivalve populations.

CIwet = [wet meat volumen/(whole volumen - Shell volumen)] * 100

CIvolume = [dry meat wt / (whole volumen - shell volumen)] * 100

CIweight = [dry meat wt/(whole wt - shell wt)] * 100

CIcommercial = [Meat wet wt / Total wet weight] * 100

They found that CIcommercial correlated 92.7% with CIwet, 90.4% with CIvolume and 90.9% with CIweight.

According to Hickmann and Illingworth (1980), although CIweight is the most advisable for reliable comparisons, it is possible to standardize the measurement of wet weight in an index such as CIcommercial to minimize errors by always adopting the same weighing routines, which is what was applied in this study.

Line 133: Please clarify how samples were treated prior to analysis to ensure homogeneity, as parameters were determined in whole animal extracts.

Reply: For each sample of 20 individuals the tissues were pooled and freeze dried in a Savant freeze dryer (-80°C), then they were ground to homogenize and stored at -41°C until the respective biochemical analysis.

�  This is an important information that the tissue of 20 individuals was pooled prior to analyses and must be given in the ms.

Reply: Thank you for the clarification. This information has been included in the manuscript.

Line 133: Unclear, please add more information how crude protein was calculated.

Reply: In nutrition, the total nitrogen content of the sample was converted into a value called "crude protein" by multiplying it by 6.25, a constant based on the conventional assumption that any protein is composed of 16% nitrogen.

�  Again, this is an information that should be given in the ms.

Reply: Thank you for the clarification. This information has also been included in the manuscript.

Line 146f: Repetition, see line 131f.

Reply: corrected

�  Sorry, the repetition is still in the revised version.

Reply: This repetition has been removed. Thanks for the clarification

First revision, R1 (January 2022)

REVIEWERS #1 and #2 COMMENTS:

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: The paper by A. Valenzuela et al. describes interesting data on the proximal composition and fatty acid content of three mollusc species from Chile, that can contribute to the literature already existing on bivalve molluscs.

The Authors also tried to assess differences existing between species or even between the same species growing in relatively close locations.

On the other hand, several aspects of the paper need to be improved, especially in terms of description of experimental procedures, statistical analysis and results.

A list of suggestions/comments that could help the Authors during the revision stage is thus provided below, following the order of appearance in the manuscript.

Materials and methods

The title of the first sub-section (Sampling) of this section should be integrated to clarify that the sub-section deals also with the determination of proximal composition and fatty acid profile of molluscs.

Reply: Done

Lines 122-129: the use of the same symbol, WW, first for the calculated wet weight (lines 125-126) and then for the experimental wet weight (lines 128-129) can be misleading. A different subscript (i.e., calc and exp, according to the case) should be added to the WW symbol to avoid any misunderstanding. Wr would thus be expressed as WWexp/WWcalc.

Reply: The WW values cited between lines 122-129 are any WW obtained experimentally to apply the index Wr, that allows to determine if a WW sampled in an individual of species A relates to the expected WLR for species A with a values inferior, similar or superior to 1; ie, there are no calculated WW values, only experimental values in all cases

Line 133. Details should be provided about the use of the N x 6.25 rule. This can not immediately understandable for all readers.

Reply: In nutrition, the total nitrogen content of the sample is converted into a value called "crude protein" by multiplying it by 6.25, a constant based on the conventional assumption that any protein is composed of 16% nitrogen.

Line 137 – 138. Which detector was used after GC separations? I guess that it was a FID but this detail has to be provided.

Reply: Flame ionization detector (FID) Thermo Scientific was used

Lines 146-150. The sentence was already reported at lines 131-135.

Reply: corrected

Lines 151-152. Is there any previous reference about this approach to estimate the carbohydrate content?

Reply: the methods of substraction of total carbohydrate from proximal composition as cited by Metzger LE and Nielsen SS, 2019. Chapter 3 Nutrition Labelling. In: Food Analysis. Fifth edition, SS Nielsen (ed), Food Science Text Series, Springer, Cham, Switzerland, pp 35-44

Lines 157, 158, and 160-162. The “g dry weight” expression should be put between parentheses and then the -1 exponent should be placed outside; as an alternative, the word “per” could be placed before “g dry weight”.

Reply: done

Lines 173-176. The type of data used as original variables for PCA must be cited.

Reply: The data used for PCA analysis were the values of each replicate for: every fatty acid, every relationship between fatty acids, every performance indicator, and every proximal content. Finally, only those variables most correlated with the first two components were selected for final PCA analysis.

Results

Lines 188, 190, 193 and 196: the meaning of the H and F symbols needs to be clarified. Since the Authors claimed that normality and homoscedasticity tests were performed om data, the results of those tests should also be described.

Reply: done

Line 213. Fig2 should be referred to as Fig2a.

Reply: done

Figure 2. I would strongly suggest adding also loading plots to the figure, to help the readers in understanding how the variables influenced the differences observed between samples in the score plot. It is very complicated to switch to the Supplementary Material to find this information using Figure S1.

Reply: Figure 2a and b was improved and replaced with data from the supplementary material. Therefore, the supplementary material was eliminated, and its data were incorporated along with the variables in Figure 2, for a better understanding of the results.

Tables 1-4. It seems that the results of tests for the comparison between means were not reported for all data, as significant differences could be supposed to be present sometimes (based on numerical values) but superscripts indicating the outcome of those tests were missing, thus suggesting that no significant difference was observed. Is this correct?

I would suggest using a consistent number of significant figures to express standard deviations (for example two) and then round off mean and standard deviation values accordingly.

The number of replicates should be clearly indicated in the tables captions.

Reply: done.

Line 309. I would suggest using the word “quadrant” or “semi-plane”, according to the case, instead of “hemisphere”.

Reply: done.

Lines 306-311: it would be better to use the term “principal” instead of “main”, since PCA results are described. I would be very careful in writing that Chilean oysters from Pullinque and Quempillen made up different groups in the score plot. Indeed, a remarkable variability was observed for the samples collected in Pullinque and at least one of them was very close to the samples from Quempillen.

Reply: done.

Figure S1. What is the meaning of the colour code (and then of the legend) adopted in the figure?

Reply: In blue (active) it refers to the active variables that influence the principal components and in red (suppl) to the supplementary variables that have no influence on the analysis of the principal components, but do help to interpret the results. However, this graph was corrected and the supplementary material was removed, Figure 2 and Figure S1 were merged for a better understanding of the results.

Figure 3. The figure does not show daily consumptions, as stated in the caption, but the amounts of the two fatty acids referred to 100 g of meat of each mollusc. Please clarify this issue. The number of replicates used to calculate error bars should be indicated for each set of data.

Reply: Figure 3. Potential intake of EPA + DHA in a 100 g edible portion from Crassostrea gigas, Ostrea chilensis and Mytilus chilensis.

Lines 375. Why were just those two values reported for the n-3/n-6 ratio? If the Authors meant to indicate the entire interval observed for those ratios in all the analyzed species, then the minimum value would be 5.15, found for Chilean oysters from Pullinque. A clarification is needed.

Reply: corrected

Lines 412-413. This sentence seems to be conceptually linked to the previous one, yet it concerns animal genetic improvement, so how can it be related to the sentence in which the focus was on the locality? Please clarify.

Reply: corrected

Conclusions. I would suggest rephrasing this section, focusing on more specific aspects of the work, like the comparison between oysters and mussels in terms of key components and the subtle effects that the location seemed to have on the nutrient content of the considered shellfish.

Reply: Reworded this section as suggested by reviewer.

Reviewer #2: Present study determined the proximal biochemical composition and the fatty acid profile in the Chilean oyster (Ostrea chilensis), the Pacific oyster (Crassostrea gigas) and the Chilean mussel (Mytilus chilensis), to perform an intra and interspecific comparison. To my understanding the interspecific comparison (O. chilensis, C. gigas and M. chilensis) was performed on samples that were all collected from Hueihue in 2018, and the intraspecific comparison was meant on O. chilensis that were sampled from two locations (Quempillen vs. Pullinque) in 2017. Additionally, to the results of O. chilensis (2017) the authors present results of C. gigas (sampled from Faro Corona in 2017). However, this is irritating as C. gigas is a different species and the results do not fit to the “intraspecific comparison”.

Besides minor grammatical inaccuracies, the ms is well written and the results seem to be reliable supporting the conclusion that the investigated shellfish species can be a “sustainable option for a balanced and healthy human diet”. However, the finding that oysters and mussels are healthy food components in human diet is not new and the study will benefit from a clearer (hypothesis-driven) research question to more strongly emphasize its relevance to the scientific community. In addition, the ms can be improved by a more detailed description of the M&M part and an in-depth discussion.

Major comments:

1) The authors aim to “nutritionally characterize the food quality” of two oyster and one mussel species from aquaculture in Southern Chile. To improve the study, the results presented (content of lipids, protein etc. as well as fatty acid composition) need to be put in context with literature data and discussed appropriately; e.g. Do the results differ from bivalves obtained from European or US aquaculture facilities? If so, why? Investigations were conducted on bivalves collected during the winter season, and the data could be discussed/compared with data from bivalves collected during spring/summer.

Reply: This comment has been considered in the discussion to deepen the comparison between the three species of bivalves.

2) Please highlight why the different locations were chosen for the intraspecific comparison. In what respect do they differ from each other? The intraspecific comparison aspect should be more clearly elaborated in the discussion. In its present form (under the heading “Differences between populations of oysters”) it is a bit confusing as the dataset of C. gigas is also discussed. Please comment on your decision to add the data of C. gigas (obtained in 2017) to the results part of the intraspecific comparison (line 243f).

Reply: The locations chosen in this study for the comparison between oyster species were selected because Quempillén and Pullinque correspond to two areas of natural banks of O. chilensis on Chiloé Island. The town of Quempillén is an estuarine zone with high salinity fluctuations. Instead, the town of Pullinque is a bay in the Gulf of Quetalmahue with depths of 7 to 8 meters. The species C. gigas was added because it is the second most important commercially important oyster species in Chile and because it also has natural banks on Chiloé Island.

3) With respect to the results in Table 1 showing highest proximal composition values in protein content (42 – 52% dry weight) and lower values in lipid (14.5 – 19.5%) and carbohydrates (11.5 to 20.9%), the statement “shellfish sampled in winter were characterized by high lipid content along with low protein and carbohydrates contents (line 36f, and 431f) is irritating. Please clarify.

Reply: The paragraphs mentioned in lines 36f and 431f were corrected.

Specific comments:

Line 26/27: Please clarify the definitions of equal contribution of the authors - ¶ vs. &.

Reply: the authors' equal contribution to:

¶ = all of these authors contributed to study design, data collection and analysis, decision to publish, or manuscript preparation.

&= all these authors contributed to the experimental and field work.

Line 83: Change into “On the one hand…”

Reply: done.

Line 107f: Sampling of animals: Please clarify the sentence "3 samples of 20 oysters" as it is 60 animals in total, isn't it? Please add more details about the environmental parameters at the time of collection (water temperature, salinity...) and transport of the animals (duration, transport on ice/water...). Were the animals kept in institutional aquarium systems prior to dissection? If yes, please add details on husbandry.

Reply: For each locality, 60 sampled specimens were collected from cultivation areas, where 3 samples (replicates) of 20 individuals were randomly made, except for C. gigas, which were fewer specimens (40 individuals). Then, in the laboratory, the tissues without the shell were worked, each sample was stored in a refrigerated/frozen ziplox bag with the meat of 20 individuals corresponding to replica 1, 2 and 3 of each sampling.

Line 121ff: Please comment on using the calculation of yield instead of condition index (soft body dry mass*100/shell dry mass) which is more common. In any case, data sets should be compared with literature data to increase the reliability of the results.

Reply: The samples were processed in the field, where the wet weight was directly measured. In our laboratory, the samples were lyophilized to be considered for proximal content and fatty acid content. The work of Sing & Ransangan (2019) measure yield according to wet weight.

Sing, O. F., & Ransangan, J. (2019). Effect of physicochemical parameters and phytoplankton composition on growth performance of green mussel (Perna viridis) in Ambong Bay and Marudu Bay, Sabah, Malaysia. Journal of Fisheries and Environment, 43(1), 50-68.

Line 122: Please add the underlying data for the calculation of the allometric relationship of WW. The cited reference (#30) is for Wr for fish. Is it also used for shellfish?

Reply: Now the underlying data suggested is:

The relative weight index [30] is based on the length -weight relationships (LWRs). The total wet weight in grams (WW), and shell length in milimeters (L) obtained from each individual sampled during the study were used. The wet weights were fit using a power regression as a function of shell lenght to obtain WW = aLb , where a is the intercept (condition factor) and b is the slope (relative growth) following Sousa et al (2020 a, b). The relationships were WW = 0.021*L2.16 for M. chilensis with 60 individuals (R2= 0.48), WW = 0.0004*L2.77 for O. chilensis with 180 individuals (R2= 0.51), and WW = 0.079*L1.48 for C. gigas with 100 individuals (R2= 0.67).

The use of WLRs are commonly used for shellfish see Sousa et al 2020a, b. The WLRs are used to compare different ecological, geographical, reproductive, etc., conditions for some particular species, and the comparisons is about the exponent of WLRs.

So the Wr is only a ratio WW/WLR to show if the animals are more slim or fat respect to an average condition represented by WLR of the species in different geographical areas, or growth conditions, or etc, Therefore it is applicable to shellfish, the same as WLRs

Line 133: Please clarify how samples were treated prior to analysis to ensure homogeneity, as parameters were determined in whole animal extracts.

Reply: For each sample of 20 individuals the tissues were pooled and freeze dried in a Savant freeze dryer (-80°C), then they were ground to homogenize and saved at -41°C until the respective biochemical analysis.

Line 133: Unclear, please add more information how crude protein was calculated.

Reply: In nutrition, the total nitrogen content of the sample is converted into a value called "crude protein" by multiplying it by 6.25, a constant based on the conventional assumption that any protein is composed of 16% nitrogen.

Line 146f: Repetition, see line 131f.

Reply: corrected

Line 161: Please provide more details concerning the equation parameters “12g” and “15g”.

Reply: correspond to the molecular weights of carbon and nitrogen. The nitrogen value was corrected to 14.

Line 163: Consider rephrasing “performance”. If it is a common term in aquaculture, please provide a citation for the calculation.

Reply: CI commercial = wet meat weight x 100 /whole wet weight (live) = Meat wet weight x 100/ Total wet weight following Hickman and Illingworth (1980)

Line 165f: Statistics: Missing info of p level.

Reply: p< 0.05

Line 191: Revise sentence “higher compared to …”. Please check the whole ms regarding this aspect. Furthermore, using the mean values of lipid content (Table 1) the values (9 to 25%) do not fit. Please clarify.

Reply: corrected

Line 202: Table 1 and all other Tables: Please add experimental number. Energy is expressed as MJ per kg dry meat, isn’t it? Please specify that “different” letters indicate differences.

Reply: done

Line 206: see comments to line 191.

Reply: done

Line 317f: What about the EDA+DHA concentration of nearly 600mg/100g ww meat determined in C. gigas samples collected in 2017?

Reply: For 2017 samples, an amount of 67 g of Chilean oyster from Quempillén sampling site or 92 g of Chilean oyster from Pullinque or 44 g of Pacific oyster are needed to achieve daily requirement (Fig.3).

Line 368f: This is a repetition, see line 317f.

Reply: corrected

Line 375: Please clarify: 7.13 “y” 12.86.

Reply: corrected.

Line 376: Please add more details/information. The reader wonders, so what?

Reply: Reworded this paragraph to be more consistent.

Submitted filename: Response to Reviewers.doc

Click here for additional data file.

21 Jun 2022

Proximal and fatty acid analysis in Ostrea chilensis, Crassostrea gigas and Mytilus chilensis (Bivalvia: Mollusca) from Southern Chile.

PONE-D-21-28284R3

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23 Jun 2022

PONE-D-21-28284R3

Proximal and fatty acid analysis in Ostrea chilensis, Crassostrea gigas and Mytilus chilensis (Bivalvia: Mollusca) from Southern Chile

Dear Dr. Valenzuela:

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