Differential Study of Microbiota in the Gill and Intestine of Silver Carp (Hypophthalmichthys molitrix) from the Algae-Dominated and Hydrophyte-Dominated Areas of Taihu Lake, China
Round 1
Reviewer 1 Report
The manuscript submitted by Zhou et al., presents a study of microbiota of guts and gills of silver carp form Taihu Lake (China). The study included sampling of 7 fish and 3 water samples from two different zones, followed by tests to the diversity of microbial community. The analysis revealed significant differences in microbial community structures between gill and guts and between fish from the two habitats associated with tissue type and sampling site.
I consider this article as well prepared, but I have some comments and suggestions for the authors that may improve its quality.
Section 1:
· Line 56-63: Perhaps it is worthwhile to characterize both habitats (zones) in more detail either in the Introduction or in the Material and Methods.
Section 2:
- Line 96-97: Please, cite the source of the primers 515F/806R
Section 2.2: Material and methods: This section lacks data on the standard hydrological characteristics of both zones (depth, sediment type, suspended solids) from where the samples were taken (depth, surface water, how many meters from the shore, what distance are the sampling sites from each other, population density in the area, possible inflow of surface waters, watercourses; maybe a map of the lake with sampling point would be useful?)
Section 2.5: Statistical analyses:
· Line 120: Lack of the citation for the diversity indices
- Line 115, 118 and others, 127: GENG BANK?; PICRUSt, Graphpad Prism - please use the proper name and cite properly used software (according to MDPI guideline the name, version, corporation and location information for all software used should be clearly indicated)
Section 3:
- Line 131-135 and Supplementary Figure S1: Parameter range does not match the chart, incorrectly signed axis (twice body length), no legend on box plot (dots, whiskers meaning) ?
- Line 145, Figure 1: lack of “**” indicated P<0.01 on graphs
- Line 165, Figure 2: lack of interpretation of “**” visible on box plot B
- Section 3.3 Diversity analysis: Unclearly described results, e. g. in Figure 2 D-E we do not know whether the data refer to the sum of the results from both types of tissues or to one specific (gill or intestine)
- Line 176: The numbers and names of the axes in Fig. 3 are poorly visible (it would be good to enlarge the font)
Section 4:
· Line 205, Figure 4. Please, correct the description of the Figure 4; there are abbreviations ASI ASG HSI HSG in the graph and AI AG HI HG in the description
· Is the bacterial composition in the water samples typical of this lake? In the introduction, the articles on chlorophyll a and bacterioplankton (reference 24,25) in Lake Taihu were mentioned, and this was not used in the discussion, especially with regard to the gills, about which the lack of knowledge on the subject was emphasized in the Introduction (gap in the research - line 47)
Section 5:
· Line 344-345: The sentence “However, this study did not explore the associations of intestinal microbiota with host species, sex, age, and genotype” only emphasizes that the scope of the research work carried out was quite limited. Only 14 fish from of one species with undetermined sex were tested, so it is known that the association of individual features with the microbiota was not explored. I don't think you need to emphasize that.
· In my opinion the composition of bacteria in the water will determine the composition in the tissues, so in the conclusions it is rather worth considering, for example, that the seasonal changes in the bacterioplankton can be linked with fish microbiota, due to the large differences in water temperature between seasons. Unless the discussion shows that the bacterioplankton composition in the Taihu lake remains the same throughout the year…but I did not found it there.
In general, the obtained results are an interesting introduction to further research, I would suggest extending the scope of the research to other seasons of the year, maybe different sampling points, but the same size range of silver carp, and additionally fish from a different species, but e.g. predatory. A larger and more diversified amount of obtained data would then be valuable not only for local readers but also more globally.
Author Response
Dear reviewer,
On behalf of my co-authors, we appreciate you for the positive and constructive comments and suggestions on our manuscript entitled “Differential study of microbiota in the gill and intestine of silver carp (Hypophthalmichthys molitrix) from the algae-dominated and hydrophyte-dominated areas of Taihu Lake, China” (ID: fish-1907306). We have studied the reviewer’s comments carefully and revised our manuscript according to these comments. The revisions we made were highlighted with red font in the document named “revised manuscript”. Attached please find the revised version, which we would like to submit for your kind consideration.
Below is our point-by-point response to the reviewers’ comments.
Comments and Suggestions for Authors
The manuscript submitted by Zhou et al. presents a study of microbiota of guts and gills of silver carp form Taihu Lake (China). The study included sampling of 7 fish and 3 water samples from two different zones, followed by tests to the diversity of the microbial community. The analysis revealed significant differences in microbial community structures between gill and guts and between fish from the two habitats associated with tissue type and sampling site.
I consider this article as well prepared, but I have some comments and suggestions for the authors that may improve its quality.
Section 1:
Line 56-63: Perhaps it is worthwhile to characterize both habitats (zones) in more detail either in the Introduction or in the Material and Methods.
Response: Thanks for the suggestion. We have added the details of both zones in the Material and Methods Section in the revised manuscript (Line 78-87).
Section 2:
Line 96-97: Please, cite the source of the primers 515F/806R
Response: Thanks for the suggestion. We have cited the source of the primers 515F/806R in the revised manuscript (Line 115).
Section 2.2: Material and methods: This section lacks data on the standard hydrological characteristics of both zones (depth, sediment type, suspended solids) from where the samples were taken (depth, surface water, how many meters from the shore, what distance are the sampling sites from each other, population density in the area, possible inflow of surface waters, watercourses; maybe a map of the lake with sampling point would be useful?)
Response: Thanks for the suggestion. We have added the hydrological characteristics of both zones in the revised manuscript (Line 78-87). In addition, we have drawn the map of Taihu Lake and sampling locations (Fig. S1) as the reviewer suggested.
Section 2.5: Statistical analyses:
Line 120: Lack of the citation for the diversity indices
Response: Thanks for the suggestion. We have added the citation for the diversity indices in the revised manuscript (Line 138-141).
Line 115, 118 and others, 127: GENG BANK?; PICRUSt, Graphpad Prism - please use the proper name and cite properly used software (according to MDPI guideline the name, version, corporation and location information for all software used should be clearly indicated)
Response: Thanks for the suggestion. We have modified it in the revised manuscript (Line 134, Line 137, Line 149) as the reviewer suggested.
Section 3:
Line 131-135 and Supplementary Figure S1: Parameter range does not match the chart, incorrectly signed axis (twice body length), no legend on box plot (dots, whiskers meaning) ?
Response: Thanks for the suggestion and sorry for our carelessness. The ordinate of the right drawing should be body weight. Each dot on the box plot represented one fish. We have corrected it in the supplementary data.
Line 145, Figure 1: lack of “**” indicated P<0.01 on graphs
Response: Thanks for the suggestion. “**” indicated P<0.01 in the caption of Figure 1 was redundant. We have deleted it in the revised manuscript.
Line 165, Figure 2: lack of interpretation of “**” visible on box plot B
Response: Thanks for the suggestion. “**” indicated P<0.01. We have added this interpretation in the caption of Figure 3 (Line 190-191).
Section 3.3 Diversity analysis: Unclearly described results, e. g. in Figure 2 D-E we do not know whether the data refer to the sum of the results from both types of tissues or to one specific (gill or intestine)
Response: Thanks for the suggestion and sorry for our unclear description. In fact, the intestine in Figure 3A-C represented a sum of intestine samples from two zones, and the gill represented a sum of gill samples from two zones. In Figure 3D-F, zone A represented a sum of gill and intestine samples from zone A, and zone H represented a sum of gill and intestine samples from zone H. We have added that information in the caption of Figure 3 in the revised manuscript (Line 185-190).
Line 176: The numbers and names of the axes in Fig. 3 are poorly visible (it would be good to enlarge the font)
Response: Thanks for the suggestion. We replaced the original Figure 3 with a high-visibility diagram in the revised manuscript.
Section 4:
Line 205, Figure 4. Please, correct the description of Figure 4; there are abbreviations ASI ASG HSI HSG in the graph and AI AG HI HG in the description
Response: Thanks for the suggestion. It has been corrected in Figure 4.
Is the bacterial composition in the water samples typical of this lake? In the introduction, the articles on chlorophyll a and bacterioplankton (reference 24,25) in Lake Taihu were mentioned, and this was not used in the discussion, especially with regard to the gills, about which the lack of knowledge on the subject was emphasized in the Introduction (gap in the research - line 47)
Response: Thanks for the suggestion. The bacterial composition in the water samples is not typical of this lake. In this study, the predominated bacterial phyla in water were also Actinobacteria, Proteobacteria, Cyanobacteria, and Bacteroidetes at both sampling sites. A previous study also found that the dominant bacteria in Taihu Lake were the above four phyla, but the abundance of these microbiotas varied in different habitats. Similarly, in other lakes, such as East Lake, Hulun Lake, Shahu Lake, and Neisha Lake, the main microorganisms in water bodies are also Actinobacteria, Proteobacteria, Cyanobacteria, and Bacteroidetes. It was worth noting that the changes of some specific microbiota in the water environment and fish were consistent. For instance, the abundance of Cyanobacteria and Microcystis_PCC-7914 in water from zone A was high than from zone H, as well as in fish, both in gills and gut. The abundance of Acinetobacter and Cyanobium_PCC_6307 in water in water from zone H was high than from zone H, and so did in fish gills and guts. Moreover, water from zone H had a higher abundance of Proteobacteria than from zone H, and a similar result was found in fish gills. Firmicutes showed a higher abundance in both fish guts and water from zone H than those from zone A. These data indicated the impacts of microbiota in water on fish-associated microbiota.
Section 5:
Line 344-345: The sentence “However, this study did not explore the associations of intestinal microbiota with host species, sex, age, and genotype” only emphasizes that the scope of the research work carried out was quite limited. Only 14 fish from of one species with undetermined sex were tested, so it is known that the association of individual features with the microbiota was not explored. I don't think you need to emphasize that.
Response: Thanks for the suggestion. This sentence has been removed.
In my opinion the composition of bacteria in the water will determine the composition in the tissues, so in the conclusions it is rather worth considering, for example, that the seasonal changes in the bacterioplankton can be linked with fish microbiota, due to the large differences in water temperature between seasons. Unless the discussion shows that the bacterioplankton composition in the Taihu lake remains the same throughout the year…but I did not found it there.
Response: Thanks for the suggestion. As the reviewer mentioned, the composition of bacteria in the water will determine the composition in the tissues. We are sorry we didn't show that in our discussion and conclusions. In the revised manuscript, we have supplemented the relevant results on bacteria composition in water, and have illustrated its impacts on fish related-microbiota in revised manuscript (Line 217-221, Line 371-390, Line 392-407).
In general, the obtained results are an interesting introduction to further research, I would suggest extending the scope of the research to other seasons of the year, maybe different sampling points, but the same size range of silver carp, and additionally fish from a different species, but e.g. predatory. A larger and more diversified amount of obtained data would then be valuable not only for local readers but also more globally.
Response: Thanks for the meaningful and constructive suggestion. This study only investigated the differences in gill and intestinal microbiota of a filtering-feeding fish species silver carp in different sampling sites of Taihu Lake. Our findings indicated that the microbial communities of silver carp in Taihu Lake were different with different tissues and habitats. As suggested by the reviewer, based on this study, we will expand the scope to different seasons, different fish species, more sampling sites, or more other variables in the future, to accumulate more valuable data.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The work is interesting and instructive for scientists, it provides new knowledge. However, before the paper is accepted for publication, it needs to be refined - the experimental part should be better explained. Comments are given in the paper.
Comments for author File: 
Comments.pdf
Author Response
Dear reviewer,
On behalf of my co-authors, we appreciate you for the positive and constructive comments and suggestions on our manuscript entitled “Differential study of microbiota in the gill and intestine of silver carp (Hypophthalmichthys molitrix) from the algae-dominated and hydrophyte-dominated areas of Taihu Lake, China” (ID: fish-1907306). We have studied the reviewer’s comments carefully and revised our manuscript according to these comments. The revisions we made were highlighted with red font in the document named “revised manuscript”. Attached please find the revised version, which we would like to submit for your kind consideration.
Below is our point-by-point response to the reviewers’ comments.
Comments and Suggestions for Authors
The work is interesting and instructive for scientists, it provides new knowledge. However, before the paper is accepted for publication, it needs to be refined - the experimental part should be better explained. Comments are given in the paper.
Line 136-142 In the experimental part, you did not explain how you determined these parameters. Add to the experimental section the subtitle Analytical Analysis and explain all these analyses.
Response: Thanks for the suggestion. We have added the details of the physicochemical analysis in the revised manuscript (Line 98-107, Line 149-150).
Line 189-191 Please explain why? Have other studies obtained similar results? It is necessary to write which and what kind of bacteria can cause problems for fish and whether they can be transferred to the human body through the food chain.
Response: Thanks for the suggestion. In this study, we found that in comparison to the gills, guts had higher abundances of LD29, Legionella, and Phreatobacter, while had a lower abundance of Pseudomonas (Fig. 5B). The possible explanation for the difference may be that the gut of fish may have a certain filtering effect on the environment or gill microorganisms. Among these four genera, only Pseudomonas is reported as the potentially pathogenic bacteria of fish. Additionally, a previous study revealed that the abundance of Pseudomonas in the skin of flag cichlid was higher than that in the gut, which was similar to this study. LD29, Legionella, and Phreatobacter are bacterial genera that mainly exist in various aquatic ecosystems and are rarely found in the tissues of fish or other aquatic animals. So far, no studies have shown that these bacteria can be transmitted through the food chain to humans. We have added this content in the revised manuscript (Line 306-314).
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
There are still some minor additions to be made:
Line 80-86: quote where the data on the reservoir was taken, depth, type of sediment, etc., unless they come from the authors, then describe it in the Material and methods.
Line 138 QIIME 2 software, quote properly (Caporaso et al. 2010).
J Gregory Caporaso, Justin Kuczynski, Jesse Stombaugh, Kyle Bittinger, Frederic D Bushman, Elizabeth K Costello, Noah Fierer, Antonio Gonzalez Pena, Julia K Goodrich, Jeffrey I Gordon, Gavin A Huttley, Scott T Kelley, Dan Knights, Jeremy E Koenig, Ruth E Ley, Catherine A Lozupone, Daniel McDonald, Brian D Muegge, Meg Pirrung, Jens Reeder, Joel R Sevinsky, Peter J Turnbaugh, William A Walters, Jeremy Widmann, Tanya Yatsunenko, Jesse Zaneveld and Rob Knight; Nature Methods, 2010; doi:10.1038/nmeth.f.303
Author Response
Dear reviewer,
On behalf of my co-authors, we would like to thank the reviewer again for their valuable comments our manuscript entitled “Differential study of microbiota in the gill and intestine of silver carp (Hypophthalmichthys molitrix) from the algae-dominated and hydrophyte-dominated areas of Taihu Lake, China” (ID: fish-1907306). We have studied the reviewer’s comments carefully and revised our manuscript according to these comments.
Below is our point-by-point response to the reviewers’ comments.
Comments and Suggestions for Authors
There are still some minor additions to be made:
Line 80-86: quote where the data on the reservoir was taken, depth, type of sediment, etc., unless they come from the authors, then describe it in the Material and methods.
Response: Thanks for the suggestion. We have cited the source of the these data in the revised manuscript.
Line 138 QIIME 2 software, quote properly (Caporaso et al. 2010).
J Gregory Caporaso, Justin Kuczynski, Jesse Stombaugh, Kyle Bittinger, Frederic D Bushman, Elizabeth K Costello, Noah Fierer, Antonio Gonzalez Pena, Julia K Goodrich, Jeffrey I Gordon, Gavin A Huttley, Scott T Kelley, Dan Knights, Jeremy E Koenig, Ruth E Ley, Catherine A Lozupone, Daniel McDonald, Brian D Muegge, Meg Pirrung, Jens Reeder, Joel R Sevinsky, Peter J Turnbaugh, William A Walters, Jeremy Widmann, Tanya Yatsunenko, Jesse Zaneveld and Rob Knight; Nature Methods, 2010; doi:10.1038/nmeth.f.303.
Response: Thanks for the suggestion. It has been corrected as the reviewer suggested.
Author Response File: Author Response.docx
Reviewer 2 Report
The paper can be accepted in this form.
Author Response
Dear reviewer,
We would like to thank the reviewer again for the time to review our manuscript and for your recognition of our manuscript.
