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:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

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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1. When submitting your revision, we need you to address these additional requirements. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

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”

Please state what role the funders took in the study. If the funders had no role, please state: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.""

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

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.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

“Dr. Jorge Toro received the following funding:

Fondef ID19I10214 and ID16I10018”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

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.

6. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

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.

7. Please include your full ethics statement in the ‘Methods’ section of your manuscript file. In your statement, please include the full name of the IRB or ethics committee who approved or waived your study, as well as whether or not you obtained informed written or verbal consent. If consent was waived for your study, please include this information in your statement as well.

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.

8. We note that Figure 1 in your submission contain [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

Respuesta: Figure 1 of the manuscript corresponds to a map made by the main author of this manuscript, using the ArcGIS Program.

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.

Attachments

Attachment

Submitted filename: Response to Reviewers.doc

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

Attachments

Attachment

Submitted filename: Response to Reviewers.doc

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.

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