Seasonal Dynamics of Marine Bacterial Communities in Aquaculture Farms: The Case of the Northern Ionian Coastal Ecosystem (Mediterranean Sea)

Round 1

Reviewer 1 Report

This study investigates bacterial community profiles and seasonal patterns in the water column of coastal aquaculture systems. The diversity and composition of bacterial communities differed markedly between aquaculture systems and undeveloped areas. The subject of this study is well introduced and the manuscript is good and interesting. I had some comments below that I'd like to address to the authors.

1.       Line 35 to 38, “Marine environmental factors cause alterations in microbial communities that can herald changes in energy transfer pathways through the food web, biogeochemical cycles, and ecosystem biodiversity in general.” Please add relevant and recent references to this sentence.

2.       Please provide specific primer information and indicate what length range of sequences were intercepted for subsequent analysis.

3.       Whether the sequence numbers of all samples were resampled equal before calculating alpha diversity?

4.       The Simpson diversity index describes the probability that the number of individuals obtained from two consecutive samples from a community are of the same species, so the results of the Simpson index is the opposite of the alpha diversity, whereas the Inverse Simpson indexes and the alpha diversity are the same. So in Figure 1, why do the Simpson results and the Inverse Simpson results agree?

5.       Microbial community structure is influenced by a variety of environmental factors, and this study does not show data on the relevant environmental factors, but only emphasises that seasonal changes have a significant impact on microbial community structure, which is too broad and general. The results for NMDS and PCOA in Figure 2 are too messy, please indicate the sample groups by season with circles or other labels, and also show direct PERMANOVA results between different seasons, such as p-values calculated from Bray-Curtis and Jaccard distances.

Author Response

Reviewer #1: This study investigates bacterial community profiles and seasonal patterns in the water column of coastal aquaculture systems. The diversity and composition of bacterial communities differed markedly between aquaculture systems and undeveloped areas. The subject of this study is well introduced and the manuscript is good and interesting. I had some comments below that I'd like to address to the authors.

1. Line 35 to 38, “Marine environmental factors cause alterations in microbial communities that can herald changes in energy transfer pathways through the food web, biogeochemical cycles, and ecosystem biodiversity in general.” Please add relevant and recent references to this sentence.

References were added for this sentence, as suggested, in line 39 of the revised manuscript.

2. Please provide specific primer information and indicate what length range of sequences were intercepted for subsequent analysis.

The full sequences of the primers used and the amplicon size for the V3-V4 hypervariable regions of the 16S rRNA gene were added in the Materials and Methods section 2.3. “Library construction and sequencing”, lines 151-157.

3. Whether the sequence numbers of all samples were resampled equal before calculating alpha diversity?

In the initial alpha diversity analysis, sequence numbers were not rarefied. However, we have run the analysis after rarefying at the lowest sequence number of all samples, 14,512, and found no significant variations in alpha diversity index numbers or statistical significance of the results. Yet, in the revised manuscript, we replaced the plots in Figure 1 with those obtained after subsampling, as well as the results in Table S2 in the Supplementary Materials file, as suggested. Moreover, we added this step in the respective Materials and Methods section, lines 194-195.

4. The Simpson diversity index describes the probability that the number of individuals obtained from two consecutive samples from a community are of the same species, so the results of the Simpson index is the opposite of the alpha diversity, whereas the Inverse Simpson indexes and the alpha diversity are the same. So in Figure 1, why do the Simpson results and the Inverse Simpson results agree?

We thank the reviewer for the comment. The alpha diversity measures were calculated using the ‘estimate_richness’ function of the phyloseq package in R (https://www.rdocumentation.org/packages/phyloseq/versions/1.16.2/topics/estimate_richness). In the R Documentation it is stated that the internal functions are borrowed from the vegan package and specifically the function ‘diversity’ (https://www.rdocumentation.org/packages/vegan/versions/2.6-4/topics/diversity). Here, the Simpson index is defined as , or the probability of selecting two individuals from different species, with replacement. D is the sum of the squared proportions for each individual. The Inverse Simpson index is . Since these two indices are mathematically related, it is expected that their results would agree. Therefore, the Inverse Simpson index is not the opposite of the Simpson index, but rather a different measure of alpha diversity estimation, and calculated from the same data they will produce similar results.

5. Microbial community structure is influenced by a variety of environmental factors, and this study does not show data on the relevant environmental factors, but only emphasises that seasonal changes have a significant impact on microbial community structure, which is too broad and general. The results for NMDS and PCOA in Figure 2 are too messy, please indicate the sample groups by season with circles or other labels, and also show direct PERMANOVA results between different seasons, such as p-values calculated from Bray-Curtis and Jaccard distances.

We thank the reviewer for the comments and suggestions. The revised manuscript now includes average seasonal measurements of environmental variables with standard deviations for the four sampling sites (Tables 1 and 2), namely temperature, salinity, chlorophyll a, and nitrogen compounds. However, data on environmental factors are now being considered for publication in another research paper, consequently, we have not provided additional analysis on the abovementioned measurements to avoid any conflict. Hence, we have incorporated a section for the measurement of those environmental parameters in 2.1. “Experimental design and sample collection” of the Materials and Methods section (lines 128-137) as well as Chapter 3.1. “Environmental variables characteristics” and Tables 1 and 2 in the Results section of the revised manuscript (lines 219-249). Regarding the beta diversity analysis, we attempted to indicate the seasons using circles in Figure 2 plots but they became harder to read, so instead, we changed the colors to indicate the different seasons, in order to make the plots more readable. Furthermore, we have added pairwise PERMANOVA results (Bray-Curtis and weighted UniFrac metrics) between seasons in Table S3 in the Supplementary Materials file and commented on these results in lines 288-289. Since data were provided by another researcher, his name was added among the names of the authors.

Author Response File: Author Response.docx

Reviewer 2 Report

 

The paper titled "Seasonal Dynamics of Marine Bacterial Communities in Aquaculture Farms: The Case of the Northern Ionian Coastal Ecosystem (Mediterranean Sea)" presents an investigation into the diversity and composition of bacterial communities in marine aquaculture farms in the northern Ionian Sea (Western Greece) compared to a control site unaffected by aquaculture activities. Overall, this study provides valuable insights into the seasonal dynamics of bacterial communities in Greek coastal aquaculture farms using amplicon metabarcoding analysis. It enhances our understanding of the impact of seasonality on the diversity and composition of marine bacterial communities in the context of coastal aquaculture systems.

 

However, I have a few minor revisions that I recommend before the manuscript can be accepted for publication. These revisions aim to improve the clarity and accuracy of the content:

 

1.     Do the authors have data on environmental factors for each month's samples, such as temperature, salinity, and nutrient concentrations? Including this information may help provide insights into the reasons for the seasonal changes in microbial diversity.

2.     In Chapter 2.1, it is difficult to correlate the sample names with the provided information. Could the authors provide a supplementary table that includes the location, depth, month, and season information for each sample?

3.     In Figure 2, could the authors consider plotting confident ellipses for each month or each season in the NMDS and PCoA plots? This would provide a clearer visual representation of the clustering patterns.

4.     In Figure 3, the authors present the top ten families in the bar plot. However, it is unclear why the total relative abundance of these ten families adds up to 100%. It should be lower than that. Please consider including an "other" category to represent the remaining families. The same comment applies to Figure S3 and S4.

5.     The top of Figure 5 appears to be cropped. Please provide the complete figure for better clarity.

6.     In Figure 5, could the authors explain the representation of the dots? Why are there multiple dots on a single row?

7.     The discussion of taxa is a bit confusing. On lines 251-252, the authors mention phyla, families, and genera, but in Figure S4, the results are presented at the class level. After describing the genus taxonomic level on lines 269-293, the authors revert back to the order level on line 294. Please ensure consistency in the taxonomic levels discussed.

8.     The authors present the average relative abundance of the genera for each season but do not provide information on significant changes or standard deviations. Consider including these details to support the findings.

9.     There seems to be a discrepancy in the taxonomic level discussed in the community composition (genus level) versus the differentially abundant taxa (family level) in Chapter 3.3. It is suggested that the authors polish the flow of the taxonomic results for better coherence.

10.   Is there a seasonal effect on fish farming? It would be valuable to see more discussion about bacterial community changes in relation to finfish aquaculture, as this would enhance the significance and depth of the paper.

11.   The discussion section would benefit from a clearer flow. For example, after mentioning the four interesting phyla on lines 424-432, the authors could discuss the interesting findings at the family and genus level within each phylum and their related ecological functions.

 

These revisions will help improve the clarity and organization of the manuscript and ensure its suitability for publication.

Author Response

Reviewer #2: The paper titled "Seasonal Dynamics of Marine Bacterial Communities in Aquaculture Farms: The Case of the Northern Ionian Coastal Ecosystem (Mediterranean Sea)" presents an investigation into the diversity and composition of bacterial communities in marine aquaculture farms in the northern Ionian Sea (Western Greece) compared to a control site unaffected by aquaculture activities. Overall, this study provides valuable insights into the seasonal dynamics of bacterial communities in Greek coastal aquaculture farms using amplicon metabarcoding analysis. It enhances our understanding of the impact of seasonality on the diversity and composition of marine bacterial communities in the context of coastal aquaculture systems.

However, I have a few minor revisions that I recommend before the manuscript can be accepted for publication. These revisions aim to improve the clarity and accuracy of the content:

1. Do the authors have data on environmental factors for each month's samples, such as temperature, salinity, and nutrient concentrations? Including this information may help provide insights into the reasons for the seasonal changes in microbial diversity.

We thank the reviewer for the comments and suggestions. The revised manuscript now includes average seasonal measurements of environmental variables with standard deviations for the four sampling sites (Tables 1 and 2), namely temperature, salinity, chlorophyll a, and nitrogen compounds. Since the specific data on environmental factors are now being considered for publication in another research paper, we have not provided additional analysis on the abovementioned measurements to avoid any conflict. Hence, we have incorporated a chapter for the measurement of those environmental parameters in 2.1. “Experimental design and sample collection” of the Materials and Methods section (lines 128-137) as well as Chapter 3.1. “Environmental variables characteristics” and Tables 1 and 2 in the Results section of the revised manuscript (lines 219-249). Finally, we have included information of these results in the discussion section (e.g., lines 474-479) to provide further insights into the factors impacting seasonality of bacterial populations. Since data were provided by another researcher his name was added among the names of the authors.

2. In Chapter 2.1, it is difficult to correlate the sample names with the provided information. Could the authors provide a supplementary table that includes the location, depth, month, and season information for each sample?

In the revised manuscript, we have included detailed information such as the location within the farm, month, and season of each collected sample in Supplementary Material Table S1. The depth information is not included in the table because, as we mention in the Materials and Methods section of the manuscript, lines 108-117, for each sample analyzed in the present study, two samples were collected from two different depths in each collection point, then they were mixed and 1L of the mixture was used for 16S metabarcoding analysis. In lines 112-117, we have included the middle and bottom depths in meters from the surface for each sampling site (Lorida, Skaloma, Bastia, and Control).

3. In Figure 2, could the authors consider plotting confident ellipses for each month or each season in the NMDS and PCoA plots? This would provide a clearer visual representation of the clustering patterns.

We attempted to indicate the seasons using circles in Figure 2 plots but they became harder to read, so instead, we have changed the colors in the beta diversity plots to indicate the different seasons, in order to make the plots more readable.

4. In Figure 3, the authors present the top ten families in the bar plot. However, it is unclear why the total relative abundance of these ten families adds up to 100%. It should be lower than that. Please consider including an "other" category to represent the remaining families. The same comment applies to Figure S3 and S4.

Thank you for the suggestion. In the revised manuscript we added an “Other” category in all barplots, which comprises all the remaining taxa not shown individually, as suggested. In addition, we chose to present the top 20 families, to give the reader more information on the abundance of bacterial families. We have also revised Figures S3 and S4 in the Supplementary Materials file accordingly.

5. The top of Figure 5 appears to be cropped. Please provide the complete figure for better clarity.

We have provided the complete plot in Figure 5 in the revised manuscript.

6. In Figure 5, could the authors explain the representation of the dots? Why are there multiple dots on a single row?

We have given a more detailed description of the dots in the legend of Figure 5 in the revised manuscript (lines 443-446). Specifically, each dot in the plots represents a differentially abundant ASV with adjusted p-value < 0.01 and absolute log₂(fold-change)>= 2. Since the differentially abundant ASVs are presented at the family taxonomic level (y-axis), multiple dots in the same row depict ASVs that belong to the same family.

7. The discussion of taxa is a bit confusing. On lines 251-252, the authors mention phyla, families, and genera, but in Figure S4, the results are presented at the class level. After describing the genus taxonomic level on lines 269-293, the authors revert back to the order level on line 294. Please ensure consistency in the taxonomic levels discussed.

We have made the appropriate changes in line 302 and the description of the genera in the revised manuscript (lines 322-375) in order to improve the clarity and consistency of the results we present, taking also into consideration the suggestions of comment 9.

8. The authors present the average relative abundance of the genera for each season but do not provide information on significant changes or standard deviations. Consider including these details to support the findings.

We have added the standard deviations throughout the manuscript wherever an average value is mentioned (e.g., lines 322-375).

9. There seems to be a discrepancy in the taxonomic level discussed in the community composition (genus level) versus the differentially abundant taxa (family level) in Chapter 3.3. It is suggested that the authors polish the flow of the taxonomic results for better coherence.

We revised the Results section's description of the relative abundance composition (lines 322-375) to achieve a clearer flow, as suggested, taking into account the description of the differential abundance analysis at the family taxonomic level in Chapter 3.4 “Seasonal patterns of differentially abundant taxa in the marine aquaculture systems” of the Results section.

10. Is there a seasonal effect on fish farming? It would be valuable to see more discussion about bacterial community changes in relation to finfish aquaculture, as this would enhance the significance and depth of the paper.

We thank the reviewer for the comment. We have included information on seasonal changes of environmental parameters in the aquaculture and control sites in the discussion section. Specifically, we have revised the text in lines 465-479 and 496-503 to provide more insight into the seasonal variations in relation to the bacterial community, as suggested.

11. The discussion section would benefit from a clearer flow. For example, after mentioning the four interesting phyla on lines 424-432, the authors could discuss the interesting findings at the family and genus level within each phylum and their related ecological functions.

We have made several changes in the discussion section to achieve a better flow. Moreover, we have revised the text in lines 514-528 and 573-579 for better clarity and discussed specific genera in the most prevalent phyla that were identified in our analysis, as suggested.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

No further comments