Effect of Salinity on the Gut Microbiome of Pike Fry (Esox lucius)
Round 1
Reviewer 1 Report
This paper deals with the possible effects of salinity on the gut microbiome of the pike fry. I think the methods, results and Discussion sections need improving:
- How many replicates per treatment were done?
- 30 fish were used in microbiological analysis but then only 20 were sequenced?
- (LINES 134-147) I did not understand how the authors went from having 1,533,836 merged reads, from which 213,390 reads were removed in quality filtering, but ended up with only 228,667 reads. Please explain or correct.
- Permanova detects differences between groups, but pairwise comparisons were never significant between control and 7%. salinity. However pairwise comparisons between 3%. group and both control and 7%. treatments were different. This in my view indicates that intermediate salinities had a higher effect on beta diversity than 7%, and i cannot see how this fits the authors conclusion.
- The authors state that "The pairwise test on the unweighted UniFrac data showed that the guts of fish in 3‰ salinity were habituated by significantly more species than those of other groups (see Table 1 OTU index).". If alpha diversity metrics were not significantly different between groups, what do the authors mean by this? PERMANOVA is not testing differences in the number of OTUS right?
- In conclusions the authors state: "This observation suggests that these phyla play valuable roles in the health and digestion of pike." I don't understand how this can be a conclusion of this study, as there was no testing of the hypothesis that either of these groups can contribute to health (or fitness). On the contrary, since there was no differences in these taxa abundances but mortalities were quite different between groups I would say they do not contribute to host health.
- Again another far fetched conclusion linked thier findings to host health: "Despite these fact, our results suggest that Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria, which dominate in the fish gut may play important roles in the health and digestion of pike at an early life stage."
- The authors state" Alphaproteobacteria predominated in 7‰ salinity, whereas in 3‰ and control tanks, Betaproteobacteria were more abundant". However in individuals plots one can see extremely high inter-individual variation and based on those plots I think these results are being mostly driven by only 2 individuals in 7%. group and one individual in the 0%. group.
- in their conclusion the author describe non-significant differences between the abundance of certain taxa, i think this is misleading
Author Response
Please see the attachment.
Author Response File: 
Author Response.docx
Reviewer 2 Report
It is an interesting and well written paper with the appropriate experimental design. In the text, writing in the first person plural should be avoided and should be corrected.
Author Response
Please see the attachment.
Author Response File: Author Response.docx
Reviewer 3 Report
This study deals with microbiota profiles of pike fry rearing in different NaCl concentration. The study attempts to provide an important contribution to aquaculture technologies.
While the discussion of the results presented is sound and presented well, I have concerns about the experimental design and pike fry condition which the authors utilized. I found it a little difficult to decide what to make of this work.
Major Point
- The condition of pike fry in 7‰ should be analyzed. The mortality of 7‰ is 37%.
Is the fry in stable condition? Are there any possibility the fry of 7‰ going to die?
Otherwise, the data of 7‰ should be removed.
I recommend confirming the plasma osmolality of fry. Authors need to certify the fry condition by using any scientific methods.
- Please refer to the gut environment and/or the osmolality of intestinal fluid. Please provide the osmolality of intestinal fluid.
- Please mention about the microbiota of the ambient tank water. As increase in NaCl concentration, the profile of water microbiota change. Fry might drink tank water for osmoregulation.
- The amount of artemia should be changed as the number of fry in tanks. Usually the amount of artemia is decided by the fry body mass in tanks.
- Authors conducted STAMP. Please provide any information about taxonomic or metabolic profiles.
Others
Material and Methods
Lines 93-94: How many replications are carried out?
Lines 100-101: Ethics statement is required because fry was scarified.
Line119: Add the place of company
Line 121: The title should be “16S rRNA”
Line 130: Add the company information of Nextera
Line 153: SRA numbers are appropriate for accession number rather than bioproject number.
Lines 166 and more: Use italic for P value.
Figure 1: The variation of rearing experiments should be exhibited.
Table S1: The first row in phylum should be Proteobacteria.
Figure 4: gammaproteobacterial is missed.
The legends are difficult to follow. Similar colors appear many times.
Please put the legends by order as the bar graph.
Line 515: please correct “e1-e1”.
Author Response
Please see the attachment.
Author Response File: Author Response.docx
Round 2
Reviewer 3 Report
This second version of the paper has been improved.While I appreciate the effort of the revised manuscript, I still have concerns. The paper should be published but only after revision.
MAJOR
Response 4: The amount of Artemia was calculated based on fish living in the tanks at current day. This information was add in line 99: “The fish were fed with commercial Artemia Premium Cysts three times per day in the amount depending on fish number in the tank.”.
Lines 319-322, is it true that the lowest density of fry could easily to catch the lowest density of Artemia? In addition to this, is there any possibility of growth hormone affection in 7‰?
Response 5: Taxonomic profile with the mean percent abundance of each classified taxa in each group can be found in supplementary material – S1 Table.
Did not authors attempt to analyze the metabolic profiles using STAMP? The paper would be greatly improved adding by a metabolic profile analysis. Only proportional changes of microbiota is a routine report.
MINOR
Response 7: In our country an ethic statement or any approval is not required in this type of research. An ethical statement is required in different cases such as the injection of external substances and not in the case of scarifying animals without protection. This statement can be found in line 103-104: ‘An ethics statement is not required for this type of research. No specific permissions were required for the described studies. The studies did not involve endangered or protected species.’ All experimental procedures were conducted in accordance with polish law.
I recommend adding the sentence “All experimental procedures were conducted in accordance with polish law.”
Response 8: Done. Line 122: ‘To quantify DNA, a Qubit 2•0 Fluorometer (Invitrogen, USA) was used.’
Please uniform the style. The name of city has been inserted in lines 131 and 133. Moreover, remove “San Diego, USA” in line 133.
Response 11: Done. Sentence was changed in line 157 to : ‘Sequencing data were exported ….. under the accession no. PRJNA578832.’
Providing only bioproject number is not recommended. The GenBank, WGS or SRA accession numbers are preferable. (cf. https://www.ncbi.nlm.nih.gov/bioproject/docs/faq/#should-i-cite-bioproject-accessi FAQ :Should I cite BioProject accession numbers in my manuscript?)
Point 13: Figure 1: The variation of rearing experiments should be exhibited.
Authors conducted in two replicates. The variation between two replicates should be exhibited.
Author Response
Please see the attachment.
Author Response File: Author Response.docx
