Molecular Cloning and Characterization of tlr1, tlr5s, tlr5m, and tlr14 Genes in the Swamp Eel, Monopterus albus
Round 1
Reviewer 1 Report
This is a sound paper, with a clear and easily interpreted presentation. I see no weaknesses in it, although I have to admit that my experience with toll-like receptors is limited to reading a few articles.
Author Response
Please see the attachment
Author Response File: 
Author Response.docx
Reviewer 2 Report
Comments fishes-1797000
Manuscript fishes-1797000 is an interesting work characterizing four toll like receptor genes (tlr1, tlr5s, tlr5m, and tlr14ˆ) swamp eel (Monoterus albus). Ms is well written and easy to follow. However, the current form of ms is not suitable yet for publication since many issues both major and minor found in the text. The revised ms could be considered after addressing those issues.
Title
- There is no molecular cloning activities explained in the ms, see comment on Materials and methods section. This gives a consequence to the current title!
Line 2-3 - it should be “Molecular cloning and characterization of tlr1, tlr5s, tlr5m, and tlr14 genes in swamp eel, Monopterus albus"
Abstract
16 - a) I would recommend the authors following the guidelines from Zebrafish regarding how to mention the gene names in the text, which can be found on https://zfin.org. Apply this for all genes mentioned in the ms. and b) (Monopterus albus) can be omitted. It has been mentioned on the title.
18 - “mRNA” can be omitted!
Highlights
35 - rewrite the sentence
38 - were involved
Introduction
70 - “(Monopterus albus)” can be omitted. Note that the Latin name is only included for the first appearance of species in the text. Apply this for all species mentioned in the ms.
75 - A. veronii
85 - provide
Materials and methods
- See the previous comment about the name of species on line 70!
- Detailed procedure for molecular cloning of genes is not found in the text!
88 - “and bacterial challenging” can be omitted!
90-1 - Did the authors evaluate other water quality parameters in addition to temperature during experiment! State this
91 - How were the fish fed? How many times a day? The amount of feed given? Feed specification (such as protein content). State this!
93-5 - these sentence could go to section 2.6
94-5 - a) Were the fish anesthetized before collecting tissue samples?, b) Where were the tissue samples kept before RNA extraction?
96-105 - this paragraph could go to section 2.8
101-3 - how many tanks/replications for each group? State this
106-7 - were the tissues sample placed in solution for RNA stabilization such as RNAlater for storage? State this
126-7 - Why did the authors use amino acid sequences for phylogenetic analysis instead of using cDNA sequences? State if there is any scientific reason!
133-4 - include the common name for each species mentioned!
138-40 - these sentences can be omitted since they have been mentioned previously!
141 - state the dye used for visualizing the RNA on agarose gel!
148-50 - modify the sentence!
159 - For gene expression analysis, gene expression data, typically lognormal distribution, should be presented as geometric mean instead of arithmetic mean!
165-6 - include the acc. Number of each gene in Table 1, and target size for each pair of primers!
168 - How did the authors evaluate the health status of fish in this study? Did the authors perform pathological test (including microbial and parasitological).
169 - 105 or 107 CFU/mL?
170-1 - double check the time points!
Results
- See the previous comment about the name of species on line 70!
177 - should be “The cDNA length
181 - What does pI stand for? State it!
232 - the unit (percentage, %) in Table 3 is missing!
279-81 - the Y axis should be written as “Relative gene expression”
282 - toll like receptor (TLR) genes
286-8 - these sentences could go to the Discussion section!
289-90 - this sentence can be omitted since it has been explained in the M&M section
290-303 - All these sentences could be combined as one paragraph. Normally, a paragraph consist of three and more sentences.
307 - were determined
Discussion
- See the previous comment about the name of species on line 70!
Conclusions
- Information regarding characteristic of tlr genes identified in this work should be included in the conclusions. See the aim of the study!
370-1 - this statement can be omitted!
Comments for author File: 
Comments.pdf
Author Response
Please see the attachment
Author Response File: Author Response.pdf
Reviewer 3 Report
The authors uses molecular cloning to identified and analyzed the sequence characteristics and homology of four TLR genes. Their results suggest that these genes may have a role in host immune defense. The manuscript has a potential for high impact to the readers of fishes.
Author Response
Please see the attachment
Round 2
Reviewer 2 Report
For reviewing the revised manuscript, the authors should include their response addressing each point of questions and pointing where revisions were made on the text
Author Response
Please see the attachment
Author Response File: Author Response.pdf
Round 3
Reviewer 2 Report
Comments fishes-1797000-v3
The authors have tried to improve manuscript fishes-1797000 by addressing the previous comments. At this moment, I only went through the response of authors based on the each question. Unfortunately, I found that most of the issues on the ms are not completely resolved, and even some of them raise new problems. I have therefore to say that the current form is not suitable to warrant publication in the journal.
80 - As previous comment, the authors did not include a procedure for molecular cloning as stated on the title. Later on, the authors suggested to see section of 2.8, that only explains the procedure for bacterial challenge experiment, molecular cloning. This needs to be clarified.
86-88 - rewrite the sentences!
91-3 - as suggested previously, a) information regarding tissue collection would be better to move to the section 2.6, and b) were the tissues samples kept in RNAlater before storage at deep freezing (-80 ℃)? State this!
111-2 - the authors did not answer the question regarding phylogenetic tree construction, please refer to the previous comment!
124 - Section 2.6 could be modified as “Tissue collections, total RNA extraction, and reverse transcription”
126 - “(SYBR Green I, Shanghai, China)” can be omitted!
127 - ……gel electrophoresis stained with SYBR Green I (Shanghai, China).
144-6 - it would be better to say “All data were evaluated for normality and homogeneity of variances before statistical analysis, and the gene expression data were expressed as geometric mean ± geometric standard deviation”
150 - the authors did not resolve the issues on Table 1 regarding the Acc. Number of genes and target size for each pair of primers. Please refer to the previous comment
152 - this section should be rewritten since duplication information exist between two paragraphs presented here. It seems that the authors only cut and paste the statement from section 2.1 without considering the existing information in the section 2.8.
153-4 - rewrite the sentence!
160 & 166 - double check the concentration of bacteria! Which one is correct? 105 or 107 CFU/mL?
209 - it would be better to state the units (%) next to the values rather than the species!
287 - are the main
Comments for author File: Comments.pdf
Author Response
Please see the attachment
Author Response File: Author Response.pdf
Round 4
Reviewer 2 Report
The quality of manuscript fishes-1797000 has been significantly improved by the author after revising 3-4 times. I only found two minor issues from the last previous comments as mentioned below. After addressing these issues, the revised ms could be acceptable for publication in fishes.
133 - were obtained
110-2 - then, state the authors’s explanation regarding construction of phylogenetic tree based on the amino acid sequence!
Author Response
Please see the attachment.
Author Response File: Author Response.docx
