16S rRNA–Based Analysis Reveals Differences in the Bacterial Community Present in Tissues of Choromytilus chorus (Mytilidae, Bivalvia) Grown in an Estuary and a Bay in Southern Chile

Round 1

Reviewer 1 Report

Reviewer comments: Manuscript ID: diversity-1160850

Type of manuscript: Article

Title: 16S rRNA–based analysis reveals differences in the bacterial community present in tissues of Choromytilus chorus (Mytilidae, Bivalvia) grown in an estuary and a Bay in southern Chile

Authors: Tamara Valenzuela, Joaquin Rilling, Giovanni Larama, Jacquelinne J.

This manuscript looks interesting, the investigation is well set up, rigorous and complete.

The work is an important contribution to improve the current knowledge of the autochthonous microbial population in mussels with a metagenomic approach, a sector still very little investigated.

The figures are well made and easy to understand. The bibliography is extensive and updated.

General consideration: it would be interesting in the future to verify the influence of seasonality on the composition of the microbial flora, in order to evaluate the stable presence of some bacterial groups over time.

I have no major revisions to propose, only minor revisions.

NB: In the draft I downloaded the numbered lines are not available and this makes the revision work a little more difficult!!.....anyway…

Pag. 1:…… Traditionally, it has been postulated by the local Nehuentue community that the mixture of seawater and river fresh-water gives C. chorus…….please change in the more complete sentence……….. Traditionally, it has been postulated by the local Nehuentue community that the brackishwater, a mixture of seawater and river fresh-water, gives C. chorus…

Pag. 1…. In contrast, studies have also revealed the occurrence of pathogenic bacteria in the micro-biota, not only pathogens for bivalve hosts (e.g., Vibrio, Nocardia and Roseovar-ius) but also for human consumers (e.g., Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus).

Two considerations:

The first, the species of pathogens is not specified for the bivalves, as it is specified for the zoonotic pathogens. Please specify: V. splendidus, V. aestuariuanus, N. asteroides…..

The second: perhaps it is not the case to put together V. cholerae, no halophile and that does not multiply outside the host (man), with the other vibrio that are halophilous and part of the native flora of marine bivalves. Therefore I would omit V. cholerae and would rather put only the others and possibly the halophilic NAG-vibrio.

Pag. 5… In this context, a recent report indicates that high proportions (97%) of many bacterial taxa across environments are culturable with known techniques [40]. Therefore, our results suggest that most of the bacteria in the sampled tissues of C. chorus could be culturable;

A bit risky this statement as it is based on a single publication. There are many other publications that claim the opposite. Compare for example the work of Rosenberg et al. 2014: The prokaryotes, firmicutes and tenericutes. Springer link. https://0-link-springer-com.brum.beds.ac.uk/referencework/10.1007%2F978-3-642-30120-9

References:

1-……..El estado mundial de la pesca y la acuicultura 2018……the comma is missing… El estado mundial de la pesca y la acuicultura, 2018

8- ………..Sci. World J. (6), 931–945……please change in …. Sci. World J. 6, 931–945

14- …….Barone, M., Palladino, G., & Candela, M. 2020. …… the comma is missing .. Barone, M., Palladino, G., & Candela, M., 2020

20- ………… Microb. Ecol. 64(2): 555–569.. ..please remove the brackets……… Microb. Ecol. 64(2), 555–569

27-………… Sci. Rep. 9:4950………….. please remove the brackets………… Sci. Rep. 9, 4950

42- Rao, K. V. S., Natarajan, K., Kumar, L. V., Manivel, V., Ahmad, J., Chatterji, A., Ansari, Z.A., 2004. Ex-tract from an Indian green mussel (Perna viridis) for differentiation and maturation of dendric cells. https://europepmc.org/article/pat/us2004009596 ....... please  complete...... Rao, K. V. S., Natarajan, K., Kumar, L. V., Manivel, V., Ahmad, J., Chatterji, A., Ansari, Z.A., 2004. Ex-tract from an Indian green mussel (Perna viridis) for differentiation and maturation of dendric cells. United States Patent Appl. Publ. within the TVPP US20030401622.  https://europepmc.org/article/pat/us2004009596

47-…… Wegner, K. M. 2016. The role of …. the comma is missing…… Wegner, K. M., 2016

52-……. & Planes, S. 2019. Microbiome …. the comma is missing……& Planes, S., 2019. Microbiome

54-……Kuske, C. R., & Smith, C. (2012). Analysis ….. please remove the brackets……… Kuske, C. R., & Smith, C., 2012. Analysis

60-……….. Furones, D., De Vico, G. 2013., the comma is missing…… Furones, D., De Vico, G.,2013.,

Author Response

Reviewer #1

This manuscript looks interesting, the investigation is well set up, rigorous and complete.

The work is an important contribution to improve the current knowledge of the autochthonous microbial population in mussels with a metagenomic approach, a sector still very little investigated.

The figures are well made and easy to understand. The bibliography is extensive and updated.

General consideration: it would be interesting in the future to verify the influence of seasonality on the composition of the microbial flora, in order to evaluate the stable presence of some bacterial groups over time.

I have no major revisions to propose, only minor revisions.

Answer: We appreciate the positive commentary made by Reviewer #1. The manuscript was carefully revised, and comments were addressed point-by-point below according to the Reviewer’s recommendations.

NB: In the draft I downloaded the numbered lines are not available and this makes the revision work a little more difficult!!.....anyway…

Answer: Sorry for this mistake. The original draft was uploaded with numbered lines, but Editorial changed to the format of Diversity journal where the numbers of lines were not included. In this revised version the numbers of lines were added for a better revision.

Pag. 1:…… Traditionally, it has been postulated by the local Nehuentue community that the mixture of seawater and river fresh-water gives C. chorus…….please change in the more complete sentence……….. Traditionally, it has been postulated by the local Nehuentue community that the brackishwater, a mixture of seawater and river fresh-water, gives C. chorus…

Answer: The sentence was modified as suggested by Reviewer (Page 1, lines 58-59).

Pag. 1…. In contrast, studies have also revealed the occurrence of pathogenic bacteria in the micro-biota, not only pathogens for bivalve hosts (e.g., Vibrio, Nocardia and Roseovar-ius) but also for human consumers (e.g., Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus).

Two considerations:

The first, the species of pathogens is not specified for the bivalves, as it is specified for the zoonotic pathogens. Please specify: V. splendidus, V. aestuariuanus, N. asteroides…..

Answer: The sentence was modified. I hope the changes solve the Reviewer’s concern (Page 1, lines 82-85).

The second: perhaps it is not the case to put together V. cholerae, no halophile and that does not multiply outside the host (man), with the other vibrio that are halophilous and part of the native flora of marine bivalves. Therefore I would omit V. cholerae and would rather put only the others and possibly the halophilic NAG-vibrio.

Answer: V. cholerae was deleted.

Pag. 5… In this context, a recent report indicates that high proportions (97%) of many bacterial taxa across environments are culturable with known techniques [40]. Therefore, our results suggest that most of the bacteria in the sampled tissues of C. chorus could be culturable;

A bit risky this statement as it is based on a single publication. There are many other publications that claim the opposite. Compare for example the work of Rosenberg et al. 2014: The prokaryotes, firmicutes and tenericutes. Springer link. https://0-link-springer-com.brum.beds.ac.uk/referencework/10.1007%2F978-3-642-30120-9

Answer: We thank to the reviewer for this commentary. To prevent an overinterpretation of our results, the sentence was deleted.

References:

1-……..El estado mundial de la pesca y la acuicultura 2018……the comma is missing… El estado mundial de la pesca y la acuicultura, 2018

8- ………..Sci. World J. (6), 931–945……please change in …. Sci. World J. 6, 931–94

14- …….Barone, M., Palladino, G., & Candela, M. 2020. …… the comma is missing .. Barone, M., Palladino, G., & Candela, M., 2020

20- ………… Microb. Ecol. 64(2): 555–569.. ..please remove the brackets……… Microb. Ecol. 64(2), 555–569

27-………… Sci. Rep. 9:4950………….. please remove the brackets………… Sci. Rep. 9, 4950

42- Rao, K. V. S., Natarajan, K., Kumar, L. V., Manivel, V., Ahmad, J., Chatterji, A., Ansari, Z.A., 2004. Ex-tract from an Indian green mussel (Perna viridis) for differentiation and maturation of dendric cells. https://europepmc.org/article/pat/us2004009596 ....... please  complete...... Rao, K. V. S., Natarajan, K., Kumar, L. V., Manivel, V., Ahmad, J., Chatterji, A., Ansari, Z.A., 2004. Ex-tract from an Indian green mussel (Perna viridis) for differentiation and maturation of dendric cells. United States Patent Appl. Publ. within the TVPP US20030401622.  https://europepmc.org/article/pat/us2004009596

47-…… Wegner, K. M. 2016. The role of …. the comma is missing…… Wegner, K. M., 2016

52-……. & Planes, S. 2019. Microbiome …. the comma is missing……& Planes, S., 2019. Microbiome

54-……Kuske, C. R., & Smith, C. (2012). Analysis ….. please remove the brackets……… Kuske, C. R., & Smith, C., 2012. Analysis

60-……….. Furones, D., De Vico, G. 2013., the comma is missing…… Furones, D., De Vico, G.,2013.,

Answer: The references were revised and corrected as requested by Reviewer.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript by Valenzuela and colleagues investigates the composition of the microbial community associated with the Chilean mussel CHoromytilus chorus in two different locations. This is a simple and straighforward study, which investigated a neglected topic and reports novel data for this economically relevant species. However, this study suffers from several limitations and the authors should try to better present those in the main text, i.e. expaining that reported data refers to a single sampling time. At the same time, the brief section which concers the phylogenetic placement of C. chorus samples was largely unsatisfying and should be entirely rewritten, with an appropriate phylogenetic analysis. Please find attached my detailed comments below.

-is the water salinity range tolerance of C. chorus known? Does this species occurr in the same geographical areas of other mussel species, such as Mytilus chilensis?

-while talking about the role of pathogenic bacteria, the authors could cite some out of the several papers that have linked shifts in microbiota composition to bivalve mass mortality events (see Clerissi et al. 2020, Milan et al. 2018, Lasa et al. 2019, and others).

Section 2.1. I think it would be crucial here to provide some information about sampling dates and associated environemntal parameters, as bivalve-associated microbial communities can undergo significant modifications throughtou the year along with seasonal variations in water temeprature, food availability and others.

Section 2.2. Please provide some additional information about the samples. What was the size of the specimens sampled? Was their sex determined?

-Section 2 would benefit from the addition of a clear explanation about the number of samples which have been actually analyzed, including as a key information the number of biological replicates.

Figure 2 is currently not informative at all. First, the tree is represented as a circular cladogram (i.e. no information about branch length is reported, and the note about the interpretation of the bar in teh text was clearly wrong). Second, no elements to evaluate the robustness of phylogenetic reconstruction have been provided. Please use an outgroup to root the tree (e.g. the sequence of a non-mytilid pteriomorph bivalve), explain in the materials and methods section the details of the phylogenetic inference analysis (e.g. the molecular model of evolution that was used and how it was selected, the number of bootstrap replciates, etc.) and display the tree as a rooted phylohgram, with bootstrap support values shown close to each node.

Sample nomenclature is somewhat misleading. Given the presence of 9 samples, one would expect them to be named S1-S9, but three different samples named “S4” are reported in the tree.

Figure 5B should be accompanied by an appropriate legend. The reader can only guess what the colors stand for.

Section 4. First line: this sentence is unclear, as it does not explain whether 99% refers to % of aligned sequence, or to sequence identity %. These sequence data should be discussed in a much more appropriate way, by referencing a properly constructed phylogenetic tree and verifying the well-supported monophyletic placement (as expected) of all C. chorus sequences.

The quote from reference [40] exaggerates the contents of the referenced paper, which in no way states that 97% of bacteria are culturable with known techniques. This should be definitely toned down, as the ovwerwhelming consensus of scientific literature agrees about the impossibility to cultivatate more than half of marine bacteria with known techniques.

The claim “our results suggest that most of the bacteria in the sampled tissues of C. chorus could be culturable” is unsubstantiated and should be removed.

The comparison with freshwater mussels seems inappropriate, as freshwater mussels are not Mytiloida and not even Pteriomorphia, but rather belong to Palaeoheterodonta.

The discussion shoud make it clear that the analyses presented in this study represent a snapshot of the microbial composition associated with C. chorus tissues at a single seasonal samplign point, and therefore they should not be considered as reliable indicators of the microbial community associated with the species in other geographical locations and in other seasons. To the very least, the authors should report the sampling period and broadly discuss the associated ecological parameters (temperature, freshwater input, food availability, etc.)

Author Response

Reviewer #2

The manuscript by Valenzuela and colleagues investigates the composition of the microbial community associated with the Chilean mussel CHoromytilus chorus in two different locations. This is a simple and straighforward study, which investigated a neglected topic and reports novel data for this economically relevant species. However, this study suffers from several limitations and the authors should try to better present those in the main text, i.e. expaining that reported data refers to a single sampling time. At the same time, the brief section which concers the phylogenetic placement of C. chorus samples was largely unsatisfying and should be entirely rewritten, with an appropriate phylogenetic analysis. Please find attached my detailed comments below.

Answer: We appreciate the commentaries made by Reviewer #2. The manuscript was carefully revised, and comments were addressed point-by-point below according to the Reviewer’s recommendations.

-is the water salinity range tolerance of C. chorus known? Does this species occurr in the same geographical areas of other mussel species, such as Mytilus chilensis?

Answer: We understand the reviewer’s concern and we are agreed on the relevance (or influence) of the environment on Mytilus and their microbiota. However, this study represents our first approach to elucidate the C. chorus microbiota (data derived from an undergraduate thesis) and the study in this stage did not consider the measurements of environmental parameters. We appreciate the reviewer’s comment, and the salinity and other environmental parameters will be considered in further studies.

In relation to the salinity tolerance of C. chorus in water, this tolerance ranges from 24 ppt to 30 ppt (37.8 mS/cm a 46.2 mS/cm) according to Navarro (1998).

Navarro, J. M. (1988). The effects of salinity on the physiological ecology of Choromytilus chorus (Molina, 1782) (Bivalvia: Mytilidae). Journal of Experimental Marine Biology and Ecology, 122(1), 19–33. doi:10.1016/0022-0981(88)90209-2

Different species of mytilus can coexist in the same geographical area, particular in natural bank. This statement is mentioned in the text (Page 3, line 143-144).

-while talking about the role of pathogenic bacteria, the authors could cite some out of the several papers that have linked shifts in microbiota composition to bivalve mass mortality events (see Clerissi et al. 2020, Milan et al. 2018, Lasa et al. 2019, and others).

Answer: A new sentence and their references was added in the introduction as requested by reviewer (Pag. 1, lines 85-87).

Section 2.1. I think it would be crucial here to provide some information about sampling dates and associated environemntal parameters, as bivalve-associated microbial communities can undergo significant modifications throughtou the year along with seasonal variations in water temperature, food availability and others.

Answer: Information on conductivity and temperature was added in text as requested (Page 2, line 113-114)

Section 2.2. Please provide some additional information about the samples. What was the size of the specimens sampled? Was their sex determined?

Answer: The size of sampled specimens was added in text (Page 2, line 125). In addition, a picture showing the size of specimens was added in the Fig. 1. However, the sex was not determined in this study. We apologies for this.

-Section 2 would benefit from the addition of a clear explanation about the number of samples which have been actually analyzed, including as a key information the number of biological replicates.

Answer: The method section was revised, information on number of samples was added in text (Page 2, line 124; Page 3, line 135, 144, 167 and lines 170-173), and the experimental design was added in the Fig. 1. We hope the changes satisfy the reviewer’s commentary.

Figure 2 is currently not informative at all. First, the tree is represented as a circular cladogram (i.e. no information about branch length is reported, and the note about the interpretation of the bar in teh text was clearly wrong). Second, no elements to evaluate the robustness of phylogenetic reconstruction have been provided. Please use an outgroup to root the tree (e.g. the sequence of a non-mytilid pteriomorph bivalve), explain in the materials and methods section the details of the phylogenetic inference analysis (e.g. the molecular model of evolution that was used and how it was selected, the number of bootstrap replciates, etc.) and display the tree as a rooted phylohgram, with bootstrap support values shown close to each node.

Answer: Sorry for this misunderstanding, the objective of tree was to confirm that specimens Choromytilus chorus species were collected to prevent in the study the collection of other mytilus species. We did not pretend to establish a phylogenetic relationship among the sampled specimens and/or the populations from two different geographical locations. However, attending to the reviewer’s comments, (1) the sentences were modified in text (Page 3, lines 144-145), (2) “Phylogeny” term was deleted and the titles of sections 2.4 (Page 3, line 142) and 3.1 (Page 4, line 240) were changed to “Collection of Choromytilus chorus”, and (3) the tree (Fig. 2) is now presented as rooted phylogram indicating values of bootstrap (%) in each branch as requested by reviewer.

Sample nomenclature is somewhat misleading. Given the presence of 9 samples, one would expect them to be named S1-S9, but three different samples named “S4” are reported in the tree.

Answer: The tree was newly built and now it is presented with 20 sequences from DNA samples. Only longest high-quality sequences (20 from 27 sequences) were used in this analysis as explained in text (Page 3, line 167).

Figure 5B should be accompanied by an appropriate legend. The reader can only guess what the colors stand for.

Answer: We thank to the reviewer for this commentary. The legend was revised (Page 12, lines 332-335) and Fig. 5B was modified to a better understanding.

Section 4. First line: this sentence is unclear, as it does not explain whether 99% refers to % of aligned sequence, or to sequence identity %. These sequence data should be discussed in a much more appropriate way, by referencing a properly constructed phylogenetic tree and verifying the well-supported monophyletic placement (as expected) of all C. chorus sequences.

Answer: As mentioned above, this section was modified to prevent misunderstanding. We hope the changes are according to the reviewer’s comments.

The quote from reference [40] exaggerates the contents of the referenced paper, which in no way states that 97% of bacteria are culturable with known techniques. This should be definitely toned down, as the ovwerwhelming consensus of scientific literature agrees about the impossibility to cultivatate more than half of marine bacteria with known techniques.

Answer: We are agreed with this commentary and the sentence was removed.

The claim “our results suggest that most of the bacteria in the sampled tissues of C. chorus could be culturable” is unsubstantiated and should be removed.

Answer: We are agreed with this commentary and the sentence was removed.

The comparison with freshwater mussels seems inappropriate, as freshwater mussels are not Mytiloida and not even Pteriomorphia, but rather belong to Palaeoheterodonta.

Answer: We are agreed with this commentary and the sentence was removed.

The discussion shoud make it clear that the analyses presented in this study represent a snapshot of the microbial composition associated with C. chorus tissues at a single seasonal samplign point, and therefore they should not be considered as reliable indicators of the microbial community associated with the species in other geographical locations and in other seasons. To the very least, the authors should report the sampling period and broadly discuss the associated ecological parameters (temperature, freshwater input, food availability, etc.).

Answer: We thank to the reviewer for this commentary. A new paragraph was added to indicate the limitations highlighted by the reviewer (Page 16, lines 502-510). As mentioned above, this is our first approach to elucidate the C. chorus microbiota and we expect include in further studies the influence of environmental parameters and spaciotemporal variability on microbiota.

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript entitled “16S rRNA–based analysis reveals differences in the bacterial community present in tissues of Choromytilus chorus (Mytilidae, Bivalvia) grown in an estuary and a Bay in southern Chile” by Valenzuela et al. focused on the composition and predicted functions of the bacterial community in tissues of Choromytilus chorus specimens grown in an estuary and a bay were studied. It is interesting but found some minor corrections.

Minor corrections to be considered:

• In the Abstract, However, …unknown, correct and rewrite this sentence.
• In this study,..studied, substitute the word studied with other, as it seems repeated. (Line number not given for the manuscript).
• Introduction lacks global information about mussels.
• However,..thus far. .. Please rewrite this sentence.
• ..functions of the bacterial community present in the digestive gland and intestinal tissues of chorus grown in an estuary and a Bay in southern Chile. .. correct it grammatically.,
• What does a Bay indicate?
• Materials and methods - After collection, the specimens were placed in a poly- styrene box and immediately transported on ice to the Applied Microbial Ecology Laboratory from La Frontera University, where they were processed. … Rewrite this sentence to be clear for the readers.
• Figure 1: magnified part of the map (as 2 maps of Chile were present in the figure) has to be shown more particularly
• Both tissues are commonly used as model tissues in environmental and biological studies in bivalves The samples were placed in sterile propylene 2–mL tubes and stored at –20°C until molecular analysis. … correct it to - propylene tubes (2 mL)
• 6. PCR conditions were run with an enzyme activation … rewrite
• Most of the times in the materials and methods section authors used the word “were” where, are, and was can be used.. correct the writeup grammatically
• Results: qPCR did not show significant differences (P≤0.05) in samples of digestive glands between the two locations… sentence may start with “The qPCR”
• Same grammatical errors as mentioned in previous sections, language editing service is suggested for this manuscript.
• In section 3.3, results showed the high and low abundant taxa. Can you please write in case if you know the functions of these taxa and the role of these taxa might be the reason for the variability in the ratio of availability?
• Please write more observations about the differences in the bacterial communities using Figures 4 and 5.
• Discussion: Our results are difficult to discuss because population studies on Mytilus in Chilean coasts are scarce; however, our study contributes a number of sequences from two specific unstudied geographical areas, authenticating the distribution of chorus along with Chile with novel data as recommended … Modify and rewrite this sentence.

Hence the overall recommendation is to address the minor suggestions in the manuscript with moderate revisions that meet the required suggestions mentioned above.

Author Response

Reviewer #3

The manuscript entitled “16S rRNA–based analysis reveals differences in the bacterial community present in tissues of Choromytilus chorus (Mytilidae, Bivalvia) grown in an estuary and a Bay in southern Chile” by Valenzuela et al. focused on the composition and predicted functions of the bacterial community in tissues of Choromytilus chorus specimens grown in an estuary and a bay were studied. It is interesting but found some minor corrections.

Answer: We appreciate the positive commentary made by Reviewer #3. The manuscript was carefully revised, and comments were addressed point-by-point below according to the Reviewer’s recommendations.

Minor corrections to be considered:

In the Abstract, However, …unknown, correct and rewrite this sentence.

Answer: “However” was deleted and sentence accordingly revised (Page 0, line 20).

In this study,..studied, substitute the word studied with other, as it seems repeated. (Line number not given for the manuscript).

Answer: The sentence was accordingly modified. “Studied” was replaced by “investigated” (Page 0, line 23). The original draft was uploaded with numbered lines, but Editorial changed to the format of Diversity journal. In this revised version the number of lines was added for a better revision.

Introduction lacks global information about mussels.

Answer: A sentence on marine bivalve production was added in the Introductory. We hope this change solve the Reviewer’s concern (Pages 0-1, lines 43-45).

However,..thus far. .. Please rewrite this sentence.

Answer: The sentence was revised (Page 1, lines 68-70).

..functions of the bacterial community present in the digestive gland and intestinal tissues of chorus grown in an estuary and a Bay in southern Chile. .. correct it grammatically.,

What does a Bay indicate?

Answer: The sentence was modified and “Bay” was deleted (Page 2, line 104-107).

Materials and methods - After collection, the specimens were placed in a poly- styrene box and immediately transported on ice to the Applied Microbial Ecology Laboratory from La Frontera University, where they were processed. … Rewrite this sentence to be clear for the readers.

Answer: The sentence was revised and modified (Page 2, line 116-118).

Figure 1: magnified part of the map (as 2 maps of Chile were present in the figure) has to be shown more particularly

Both tissues are commonly used as model tissues in environmental and biological studies in bivalves The samples were placed in sterile propylene 2–mL tubes and stored at –20°C until molecular analysis. … correct it to - propylene tubes (2 mL)

Answer: The sentence was modified (Page 2, line 131).

6. PCR conditions were run with an enzyme activation … rewrite

Answer: The sentence was modified (Page 4, line 198).

Most of the times in the materials and methods section authors used the word “were” where, are, and was can be used.. correct the writeup grammatically

Answer: Before submission, the whole manuscript was subjected to the Professional English Editing service by “American Journal Experts (AJE; https://www.aje.com)”, which has a wide experience in the scientific article writing. However, additionally, the current version was revised and edited by native speaker, Sr. Andrew Ogram from Department of Soil and Water Sciences, Institute of Food and Agricultural Science (IFAS), University of Florida, USA.

Results: qPCR did not show significant differences (P≤0.05) in samples of digestive glands between the two locations… sentence may start with “The qPCR”

Answer: The sentence was modified (Page 5, line 257).

Same grammatical errors as mentioned in previous sections, language editing service is suggested for this manuscript.

Answer: As mentioned above, in addition to editing by AJE, the current version was additionally revised and edited by native speaker, Sr. Andrew Ogram from Department of Soil and Water Sciences, Institute of Food and Agricultural Science (IFAS), University of Florida, USA.

In section 3.3, results showed the high and low abundant taxa. Can you please write in case if you know the functions of these taxa and the role of these taxa might be the reason for the variability in the ratio of availability?

Answer: The functionality of taxa found is discussed in Discussion section; however, sentences was added in Result and discussion sections describing the diversity and functionality of low abundant taxa (Page 13, lines 353-355; Page 16, lines 477-479 and 492-494). We hope these changes solve the reviewer’s concern.

In relation to the reason for the variability is not possible with the current data and we want not to fall in overinterpretation of our results. We also appreciate this comment, which will be considered in further investigations on mussel microbiota.

Please write more observations about the differences in the bacterial communities using Figures 4 and 5.

Answer: The paragraph was revised and a better description of differences in bacterial communities was added (Page 10, lines 318-324).

Discussion: Our results are difficult to discuss because population studies on Mytilus in Chilean coasts are scarce; however, our study contributes a number of sequences from two specific unstudied geographical areas, authenticating the distribution of chorus along with Chile with novel data as recommended … Modify and rewrite this sentence.

Answer: The sentence was revised and divided (Page 14; lines 366-370).

Hence the overall recommendation is to address the minor suggestions in the manuscript with moderate revisions that meet the required suggestions mentioned above.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

I would like to thank the authors for the efforts made in the improvement of their manuscript, which is now in my opinion nearly ready for publication. I feel like all the major points have been appropropriately discussed and solved, and I am personally very open to these kind of exploratory studies in non-model bivalves.
I just have a single comment. Based on the satisfying explanation provided bt the authors, I believe that the inclusion of Figure 2 in the text is not necessary. The authors may simply say that the verification of amplicon sequences fully confirmed the correct species identification of all the samples collected and analyzed.

Author Response

Reviewer #2

I would like to thank the authors for the efforts made in the improvement of their manuscript, which is now in my opinion nearly ready for publication. I feel like all the major points have been appropropriately discussed and solved, and I am personally very open to these kind of exploratory studies in non-model bivalves.

I just have a single comment. Based on the satisfying explanation provided bt the authors, I believe that the inclusion of Figure 2 in the text is not necessary. The authors may simply say that the verification of amplicon sequences fully confirmed the correct species identification of all the samples collected and analyzed.

Answer: We thank for the positive commentaries made by Reviewer #2. In relation to last comment, we understand the concern of the reviewer; however, as other reviewers (# 1 and #3) did not comment about the pertinence of Fig. 2 in the text, we decided not deleted it from the article, but transfer as Supplementary Material (end of Article). I hope this change satisfy the Reviewer #2. In the section of results, the sentence was also modified according to the reviewer suggestion (Page 5, lines 299-301)

Author Response File: Author Response.docx