Transcriptome Analysis of Immune Response against Streptococcus agalactiae Infection in the Nile Tilapia GIFT Strain
Round 1
Reviewer 1 Report
In the manuscript entitled "Transcriptome analysis of immune response against 2 Streptococcus agalactiae infection response in the Nile Tilapia 3 GIFT strain", Zhou et al., conducted a RNA-seq analysis regarding the fish gill damage infected with S. 33
agalactiae. The analysis is sound, the only thing that might need adjustment is probably the X-axis of the figure 4, from three digits to two or one digits.
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
This work contains a transcriptomic study of Nile tilapia gills upon Streptococcus agalactiae which results could give light in future preventive techniques against this pathogen that severely affects tilapia and causes high economic losses in Chinese aquaculture. The conceptualization and experimental design is appropriated, however, the manuscript should not be accepted in the present form. Concerns to be considered are described as follows:
- Introduction: the objectives described (lines 86-88) are not reflected in the abstract neither related with the results described in the abstract.
- Material and methods:
o Line 96: the meaning of BHI should be included.
o Lines 97-99: a clearer and more organized description of the groups would be appreciated.
- Results: In general, as in every transcriptomic assay, results are complex and large. If authors find a simpler way to describe them would be really appreciated as there are points that are not easy to follow (eg. Point 3.5.).
o Figure legend 1: A deeper explanation of venn graph would be appreciated to a better understanding of the results showed.
o Line 226: The top significant GO enrichment shown in Figure 3 is 18 not 10.
o Figure 7: red arrows are barely distinguished, please, try to use other symbol or colour.
- Discussion: major concerns are found in this part of the manuscript, that firstly, is poorly accurate in the definitions of immune system related concepts. To give only some examples:
o Line 352: pathways significantly changes could be for the better or for the worse so as it is described is unprecise.
o Lines 256-257: innate immune response is the main response in fish due to the less evolved adaptive immunity (fish poikilothermic condition, limited repertoire of antibodies and the slow proliferation and maturation of their lymphocytes) not because of being the first response as suggested by the authors.
o Lines 359-361: The defence against pathogens is initiated by several pathways and is a complex system of response. Whether authors focus on antibacterial responses (GBS), they should focus the discussion on that concrete pathways’ description. This sentence should be reformulated.
Or use inaccurate sentences. Eg:
o Line 461: Regarding: “They are released by various cells in the body”. Authors are encouraged to concrete sentences like this.
o Line 463: Regarding “Cytokines can be grouped by structure into different families and they are a group of cell signaling molecules produced and secreted by T-lymphocytes and mononuclear phagocytes participating in both the adaptive and innate immune pathways to initiate pathogen defense mechanisms”, authors are encouraged to focus the description to the families that are relevant for the manuscript. Moreover, the role of cytokines is far beyond the initiating the immune responses being the description also inaccurate.
Authors are encouraged to revise and rewrite the entire discussion focusing on the results obtained, avoiding large deffinitions and describing pathways/molecules and discussing results with greater accuracy.
- Format concerns:
o Please, all scientific names in italic letters.
o Names of the pathway in lower case letters.
o Line 33: Mechanisms of gill damage?
o English should be extensively revised.
Author Response
Dear reviewer,
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 3 Report
In this work, the transcriptomic expression of tilapia gills after infection with Streptococcus agalactiae is analyzed. Different times post-infection are analyzed (0, 6, 9, 15 and 18 hours) and a histopathology study is also carried out, which is an essay that provides great information when we talk about infection.
Abstract
The scientific name of S. agalactiae should be written in italics.
Introduction
References 11 and 12 are not correct, since from line 41 we are talking about S. agalactiae and these references correspond to S. iniae.
Line 82 refers to transcriptomes made in tilapia during infections, but references 27 and 28 have nothing to do with this topic.
Material and methods section
However, although it is specified that the fish are randomly divided into 9 groups of 50 fish per aquarium, it is not specified how many fish have been sampled for the RNA-Seq analysis nor how many fish have been fixed for histology. This is a crucial piece of information that must be specified, since it would allow us to know if the sample size is sufficient or not.
It is said that the water temperature was kept at 32 °C, why? The vast majority of studies done on tilapia infected with S. agalactiae are done between 26 and 28 °C.
Another meaningful test when describing the course of an infection is the survival test. Has a survival curve been made parallel to the collection of samples? Knowing the course of mortality is important to understand the reason for choosing the sampling points and to be able to relate the transcriptomic results with the course of the infection. If this test has not been carried out, I consider that it would be highly recommended to include a survival curve.
The threshold to determine the DEGs seems correct to me (FDR < 0.05 and FC ≥ 2), although it must be specified that the value 2 is an absolute value (│2│).
The Illumina validation data can also be improved. The use of the Pearson correlation coefficient between the values ​​obtained by Illumina and the values ​​obtained by Real-Time RT-PCR is a better way of validation.
Line 224 has two “that that” in a row.
Line 226 specifies that the top 10 significantly enriched GO terms in four comparisons are represented in figure 3, but the text at the bottom of figure 3 (line 231) says that the top 18 are represented.
Discussion
Line 386, Aeromonas salmonicida is not in italics.
Line 417 refers to a figure that is not mentioned, as well as line 420.
Line 471 does not follow the rules for bibliographic citations “(Wolf et al., 2020)”.
Conclusions
If at 9 hours post-infection the histopathology shows greater damage than at 18 hours, does that mean that the fish outgrow the disease? Can they recover? In that case, why is it important to study this disease if the fish improve? Or does this mean that the infection used in this study did not work? If so, all the results presented in the article correlate very little with what occurs in nature, in which we find high morbidity and mortality of tilapia infected with this pathogen.
I find it more and more necessary to use a survival curve to better explain the results.
Author Response
Dear reviewer,
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
I appreciate the efforts of the authors to improve the manuscript anwsering the concerns raised. However, some concerns are still unsolved. The main one is related to the discussion point, which despite being improved, when read as a whole it is not accurated and scarcely discusses results at some points (concerns proposed in the first review at this regard were only examples) so authors are encouraged to rewrite this point trying to focus only in the pathways that they want to discuss taking into account their results and giving concrete and focused deffinitions when needed.
Authors are also required to review english language with a native expert since there are many mistakes including basic english in all the manuscript.
Line 337: blue arrow instead bule arrow.
Line 387: Please, delete "relevant" as relevant lymphocytes provides inaccurate information about lymphocytes involved in that response.
Author Response
Dear reviewer,
Please see the attachment
Author Response File: Author Response.pdf
Reviewer 3 Report
You should specify how the survival test was done. A part from that, all ok.
Author Response
Dear reviewer,
Response: Thanks. We specified the survival test in line 106.
