Study on the Microflora Structure in a Litopenaeus vannamei–Sinonovacula constricta Tandem-Culture Model Based on High-Throughput Sequencing under Different Culture Densities
Round 1
Reviewer 1 Report
This study describes an interesting tandem culture approach to cultivation of a shrimp and a shellfish. It focuses on the microbiota associated with the two species.The Tables and figures describes the results well. I would have liked to see a schematic figure explaining the experimental upset, though.
My main objection to the presentation is that large parts of section 3.1.2 appears to be non-formatted. The same applies to several sections of the Discussion. This must be changed.
See "comments to authors". The English is OK, the formatting is not.
Author Response
Dear Reviewer 1,
Thank you very much for giving us an opportunity to revise our manuscript. We appreciate for your constructive comments and suggestions on our manuscript entitled “Study on the microflora structure in Litopenaeus vannamei-Sinonovacula constricta tandem culture model based on high-throughput sequencing under different culture density”. We have carefully considered your comments and made revision, and we have modified it according to your requirements in the corresponding places. We appreciated your careful reading and efforts for our manuscript.
Responds to comments:
Comment 1: My main objection to the presentation is that large parts of section 3.1.2 appears to be non-formatted. The same applies to several sections of the Discussion. This must be changed.
Response: Many words containing “uncultured_bacterium_f_” appear in the article, because these bacteria can only be identified to the genus, and cannot be accurate to the species. This naming method is determined by the company that we send samples to. We refer to some articles (The successional trajectory of bacterial and fungal communities in soil are fabricated by yaks' excrement contamination in plateau, China. DOI: 10.3389/fmicb.2022.1016852; A polysaccharide from Fagopyrum esculentum Moench bee pollen alleviates microbiota dysbiosis to improve intestinal barrier function in antibiotic-treated mice, DOI: 10.1039/d0fo01948h), and they are also written in this way. Because these words are too long to be segmented, there will be some blank spaces in the format. We have now left-aligned the corresponding parts to make them look more standardized than before. Hope to get your understanding, thank you!
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for Authors
: As aquaculture using microorganisms has recently become active, research on microbial communities is considered a valuable study. Overall, the study stands out for its interesting themes and detailed investigation. During the normal breeding process, the microbial community produces significantly different results depending on the introduced nutrients, and the perspective of viewing the results also changes. It is thought that it is necessary to write information on water, feed, and food, and if this information is supplemented, I think I will be able to understand the results of this study as a reviewer and review what needs to be supplemented in the review.
I send a message of support for the efforts of the authors.
Introduction
Page 2, Line 75
: Typo correction is required (Vannabei). - Pacific white shrimp.
Materials and methods
Page 3, Line 103
: Typo correction is required (Vannabei). There are also typos in the Results and Discussion section.
Page 3, Line 110
: Information about the weight of shrimp and clam needs to be filled out.
Page 3, Line 119
: Diet and diet are major factors influencing the microbial community, so more detailed information about diet and feeding is needed.
It is necessary to write basic information about whether natural seawater or artificial seawater was used, and if natural seawater was used, which area water was used. Since the distribution of microorganisms in water in each region is different, it is necessary to prepare basic information for future reference.
Page 4, Line 123
: Typo correction is required (micromicrococcus).
Result
: The Lactiobacillus results in table 5 are significantly different for different densities. This result seems to change the perspective of seeing the results depending on the shrimp feed composition table. Please fill out the feed composition table.
Discussion
: It seems appropriate to abbreviate terms such as uncultured_bacterium in manuscripts and
Comments for author File: Comments.pdf
Typo correction is required.
Author Response
Dear Reviewer 2,
Thank you very much for giving us an opportunity to revise our manuscript. We appreciate for your constructive comments and suggestions on our manuscript entitled “Study on the microflora structure in Litopenaeus vannamei-Sinonovacula constricta tandem culture model based on high-throughput sequencing under different culture density”. We have carefully considered your comments and made revision, which is marked in yellow color in the latest manuscript. We appreciated your careful reading and efforts for our manuscript.
Responds to comments:
Comment 1: Page 2, Line 75: Typo correction is required (Vannabei). - Pacific white shrimp.
Page 3, Line 103: Typo correction is required (Vannabei). There are also typos in the Results and Discussion section.
Response: We have modified the place you mentioned, and now it has been replaced by Pacific white shrimp. Thank you very much for your advice. (Line 77 and 106 in revised manuscript)
Comment 2: Page 3, Line 110: Information about the weight of shrimp and clam needs to be filled out.
Response: We have supplemented the initial weights of shrimp and Sinonovacula constricta. (Line 110-111 in revised manuscript)
Comment 3: Page 3, Line 119: Diet and diet are major factors influencing the microbial community, so more detailed information about diet and feeding is needed. It is necessary to write basic information about whether natural seawater or artificial seawater was used, and if natural seawater was used, which area water was used. Since the distribution of microorganisms in water in each region is different, it is necessary to prepare basic information for future reference.
Response: We have added information on shrimp feed and seawater, thank you very much for your suggestions. (Line 127-128 and 136-138 in revised manuscript)
Comment 4: Page 4, Line 123: Typo correction is required (micromicrococcus).
Response: We are very sorry that we have made a spelling mistake, it has now been corrected to “micrococcus”. (Line 131 in revised manuscript)
Comment 5: The Lactiobacillus results in table 5 are significantly different for different densities. This result seems to change the perspective of seeing the results depending on the shrimp feed composition table. Please fill out the feed composition table.
Response: The feed we used during the experimental period was a commercial compound feed, and we have listed the specific four components. (Line 127-128 in revised manuscript)
Comment 6: It seems appropriate to abbreviate terms such as uncultured_bacterium in manuscripts and tables.
Response: We refer to some articles (The successional trajectory of bacterial and fungal communities in soil are fabricated by yaks' excrement contamination in plateau, China. DOI: 10.3389/fmicb.2022.1016852; A polysaccharide from Fagopyrum esculentum Moench bee pollen alleviates microbiota dysbiosis to improve intestinal barrier function in antibiotic-treated mice, DOI: 10.1039/d0fo01948h), and they are also written in this way. Because these words are too long to be segmented, there will be some blank spaces in the format. Thank you very much for understanding this way of writing!
Author Response File: Author Response.pdf
Reviewer 3 Report
This is an interesting manuscript looking at the diversity of bacterial communities of two different species, i.e. marine shrimp and clams, in a co-culture situation. Overall, the manuscript has good preliminary information, but a lot of pertinent information necessary for publication is missing. There is a lack of consistency of terms used throughout the manuscript, numerous run-on sentences that could be better divided into multiple sentences with the same meaning, and a number of misspelled words. The authors need to describe how the shrimp and clams were killed, and include an ethics statement about animal use. Even though Table 1 shows how many animals were in each system, there is no mention of how many animals were samples from each system and if the samples were individual or pooled. The manuscript could also be reduced in length by moving a lot of the redundant information (i.e. Tables 2-9) to a Supplemental Material section. In addition, there are a significant number of English edits (in the edited pdf of the manuscript) that will need to be corrected before accepting this manuscript for publication.
Significant issues that the authors should correct or clarify in a revision:
Lines 10, 23 – the intestinal contents were not “measured”, replace with “evaluated”.
Lines 10-11 – common names of the organisms should be mention in the Abstract.
Line 20 - What does under-lined phrase mean?
Line 22 - "national standards" of who or what country?
Line 96 - Replace “breeding” with "culture" tank as this study is not about breeding.
Line 109 - This figure legend is not descriptive enough for 4 images.
Line 120 - Replace with "Shrimp were fed (Yuchai shrimp compound, location of source).." Authors also need to include if diet was pelleted or crumbles, and the composition of the diet, i.e. protein, CHO, fats, etc.
Line 123 - What was original concentration of algae?
Line 185 – OTU needs to be defined (i.e. operational taxonomic unit) in manuscript.
Lines 296-303 – Tables 2 through 9 should be placed into a supplemental material section.
Line 304 – Four images of Figure 7 are too small.
Line 500 - The darkening of the biomedia does not indicate that more microorganisms were present, only that more detritus (i.e. uneaten food and feces) were present in the system due to the increased number of animals and increased feeding which is getting caught in biomedia.
Line 503 - Increased survival does not mean the animals are healthier. Delete this statement.
Lime 510 - This is over-stepping the data, there is no data to suggest that shrimp or clams were healthier. Survival does not equate with healthier animals.
Line 528 - Add "presumably" since there is no data to confirm the bioturbation, only that the biomedia was darker.
Line 533 - Need to describe the "situation".
Comments for author File: Comments.pdf
There is a lack of consistency of terms used throughout the manuscript, numerous run-on sentences that could be better divided into multiple sentences with the same meaning, and a number of misspelled words. There is not ethics statement about animal use. In addition, there are a significant number of English edits (in the edited pdf of the manuscript) that will need to be corrected before accepting this manuscript for publication.
Author Response
Dear Reviewer 3,
Thank you very much for giving us an opportunity to revise our manuscript. We appreciate for your constructive comments and suggestions on our manuscript entitled “Study on the microflora structure in Litopenaeus vannamei-Sinonovacula constricta tandem culture model based on high-throughput sequencing under different culture density”. We have carefully considered your comments and made revision, which is marked in red color in the latest manuscript. We appreciated your careful reading and efforts for our manuscript.
Responds to comments:
Comment 1: Lines 10, 23 - the intestinal contents were not “measured”, replace with “evaluated”.
Response: Thank you for your kind suggestion, we have corrected the place you mentioned. (Line 11,24 in revised manuscript)
Comment 2: Lines 10,11 - Common names of the organisms should be mention in the Abstract.
Response: Thank you for your kind suggestion, we have added the common name of the species in the corresponding place. (Line 11,12 in revised manuscript)
Comment 3: Line 20 - What does under-lined phrase mean?
Response: Many words containing “uncultured_bacterium_f_” appear in the article, because these bacteria can only be identified to the genus, and cannot be accurate to the species. This naming method is determined by the company that we send samples to. We refer to some articles (The successional trajectory of bacterial and fungal communities in soil are fabricated by yaks' excrement contamination in plateau, China. DOI: 10.3389/fmicb.2022.1016852; A polysaccharide from Fagopyrum esculentum Moench bee pollen alleviates microbiota dysbiosis to improve intestinal barrier function in antibiotic-treated mice, DOI: 10.1039/d0fo01948h), and they are also written in this way. Hope to get your understanding, thank you!
Comment 4: Line 22 - “national standards” of who or what country?
Response: In this study, we used the national standards of China, and we apologize for leaving this information out earlier. (Line 24 in revised manuscript)
Comment 5: Line 96 - Replace “breeding” with “culture” tank as this study is not about breeding.
Response: Thank you for your kind suggestion, we have corrected the place you mentioned. (Line 99 in revised manuscript)
Comment 6: Line 109 - This figure legend is not descriptive enough for 4 images.
Response: After your reminding, we realized this, and now we have supplemented the legend. (Line 113-116 in revised manuscript)
Comment 7: Line 120 - Replace with “Shrimp were fed (Yuchai shrimp compound, location of source)..” Authors also need to include if diet was pelleted or crumbles, and the composition of the diet, i.e. protein, CHO, fats, etc.
Response: We have corrected it according to your requirements, and added four main compositions of feed. (Line 127,128 in revised manuscript)
Comment 8: Line 123 - What was original concentration of algae?
Response: The type of algae is Nannochloropsis, and the original concentration of algae cells is 7×105 ind/mL. (Line 131,132 in revised manuscript)
Comment 9: Line 185 - OTU needs to be defined (i.e. operational taxonomic unit) in manuscript.
Response: Thank you very much for your suggestion. We have supplemented the definition of OTU in the corresponding places. (Line 201-204 in revised manuscript)
Comment 10: Lines 296-303 - Tables 2 through 9 should be placed into a supplemental material section.
Response: Thank you very much for your suggestion. We have moved these tables to supplementary materials.
Comment 11: Line 304 - Four images of Figure 7 are too small.
Response: Thank you very much for your suggestion. Now we have re-created the Figure 7 and adjusted the definition. (Line 315 in revised manuscript)
Comment 12: Line 500 - The darkening of the biomedia does not indicate that more microorganisms were present, only that more detritus (i.e. uneaten food and feces) were present in the system due to the increased number of animals and increased feeding which is getting caught in biomedia.
Response: We strongly agree with your point and have now amended the corresponding section. (Line 503-505 in revised manuscript)
Comment 13: Line 503 - Increased survival does not mean the animals are healthier. Delete this statement.
Response: We have deleted it according to your request. (Line 507 in revised manuscript)
Comment 14: Line 510 - This is over-stepping the data, there is no data to suggest that shrimp or clams were healthier. Survival does not equate with healthier animals.
Response: Thank you very much for your suggestion. We realized that we really exaggerated the data, and now we have deleted this part. (Line 513 in revised manuscript)
Comment 15: Line 528 - Add “presumably” since there is no data to confirm the bioturbation, only that the biomedia was darker.
Response: Thank you very much for your suggestion. We did make some mistakes in using words, and now we have corrected them. (Line 531 in revised manuscript)
Comment 16: Line 533 - Need to describe the “situation”.
Response: According to your suggestion, we have made some supplements in the legend part. (Line 536,537 in revised manuscript)
Thank you again for giving us a lot of useful suggestions and helping us correct many grammatical mistakes!
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
I have some issues with the nomenclature/identifiction of various bacterial strain, with a series of underlines. However, it is done consistently in the manuscript, and I thus can accept it.
Author Response
Dear Reviewer 1,
Thank you very much for giving us an opportunity to revise our manuscript. We appreciate for your constructive comments and suggestions on our manuscript entitled “Study on the microflora structure in Litopenaeus vannamei-Sinonovacula constricta tandem culture model based on high-throughput sequencing under different culture densities”. We appreciated your careful reading and efforts for our manuscript.
Responds to comments:
Comment 1: I have some issues with the nomenclature/identifiction of various bacterial strain, with a series of underlines. However, it is done consistently in the manuscript, and I thus can accept it.
Response: Thank you very much for your understanding. We look up some articles and find that there are many ways to deal with similar situations. For example, some will write it as “Pseudobacteriovoracaceae family” (Gut interkingdom predator-prey interactions are key determinants of shrimp health, https://doi.org/10.1016/j.aquaculture.2021.737304), some will write it as “norank_f_” (Polygonatum sibiricum polysaccharides improve cognitive function in D-galactose-induced aging mice by regulating the microbiota-gut-brain axis, https://doi.org/10.1016/j.jff.2023.105476), and some articles will write it as “uncultured_bacterium_f_” like us (Comparative study on the microbiota of colostrum and nipple skin from lactating mothers separated from their newborn at birth in China, https://doi.org/10.3389/fmicb.2022.932495). Once again, we would like to express our thanks to you for your help in our article!
Author Response File: Author Response.pdf
Reviewer 3 Report
The authors have done an excellent job of revising this manuscript based on reviewer comments, see attached pdf of manuscript (“fishes-2380306 - revised marked, reviewer edits”) for a few additional minor edits. There are still two issues the journal editor and authors should discuss.
1. The authors need to describe how the shrimp and clams were humanely killed, and include an ethics statement about animal use. Most journals today require this information for publication.
2. The authors have responded to the question of why there was some unusual underling of bacteria by stating that “Many words containing “uncultured_bacterium_f_” appear in the article, because these bacteria can only be identified to the genus, and cannot be accurate to the species. This naming method is determined by the company that we send samples to.” However, this is not standard scientific nomenclature, and suggest that all bacteria identified this way be changed to “Genus sp.”. Unfortunately, it looks like most of the bacteria identified this way are only identified to the Family level and not genus level.
Comments for author File: Comments.pdf
Revision corrected most of English problems
Author Response
Dear Reviewer 3,
Thank you very much for giving us an opportunity to revise our manuscript. We appreciate for your constructive comments and suggestions on our manuscript entitled “Study on the microflora structure in Litopenaeus vannamei-Sinonovacula constricta tandem culture model based on high-throughput sequencing under different culture densities”. We have carefully considered your comments and made revision, which is marked in yellow color in the latest manuscript. We appreciated your careful reading and efforts for our manuscript.
Responds to comments:
Comment 1: The authors need to describe how the shrimp and clams were humanely killed, and include an ethics statement about animal use. Most journals today require this information for publication.
Response: Thanks for your valuable advice. We have added an ethical statement about the use of animals in the corresponding paragraphs, and described the sampling of cultured organisms. All experimental procedures conformed to the Standard Operating Procedures (SOPs) of the Guide for Use of Experimental Animals of Ningbo University. The studies involving animals were reviewed and approved by the Ningbo University Laboratory Animal Center under permit number no. SYXK (ZHE2008-0110). (Line 151-154, 163-167 in revised manuscript)
Comment 2: The authors have responded to the question of why there was some unusual underling of bacteria by stating that “Many words containing “uncultured_bacterium_f_” appear in the article, because these bacteria can only be identified to the genus, and cannot be accurate to the species. This naming method is determined by the company that we send samples to.” However, this is not standard scientific nomenclature, and suggest that all bacteria identified this way be changed to “Genus sp.”. Unfortunately, it looks like most of the bacteria identified this way are only identified to the Family level and not genus level.
Response: We are very sorry that we made a mistake in the first reply. Usually, we can only identify the genus by sequencing the V3-V4 region. After confirmation, we found that the bacteria containing “uncultured_bacterium_f_” were only identified to the family. We look up some articles and find that there are many ways to deal with similar situations. For example, some will write it as “Pseudobacteriovoracaceae family” (Gut interkingdom predator-prey interactions are key determinants of shrimp health, https://doi.org/10.1016/j.aquaculture.2021.737304), some will write it as “norank_f_” (Polygonatum sibiricum polysaccharides improve cognitive function in D-galactose-induced aging mice by regulating the microbiota-gut-brain axis, https://doi.org/10.1016/j.jff.2023.105476), and some articles will write it as “uncultured_bacterium_f_” like us (Comparative study on the microbiota of colostrum and nipple skin from lactating mothers separated from their newborn at birth in China, https://doi.org/10.3389/fmicb.2022.932495). Reviewer 1 has raised the same question as you, and he thinks he can accept this writing since the whole text is unified. We hope to get your understanding.
Once again, we would like to express our thanks to you for your help in our article!
Author Response File: Author Response.pdf
