Effect of Astragalus membranaceus on Transcriptome and Survival of Hybrid Yellow Catfish (Pseudobagrus vachellii ♂ × Tachysurus fulvidraco ♀) in Response to Aeromonas hydrophila Challenge
Round 1
Reviewer 1 Report
A. membranaceus can effectively enhance disease resistance, and reduce mortality
hybrid yellow catfish (Pseudobagrus vachellii 3×Tachysurus fulvidraco) in response to Aeromonas hydrophila challenge.
The materials and methods section needs to be upgraded, especially the immune stimulatory procedure, the amount of stimulant used?
The Results section also needs upgrading, especially the challenge experiment. Please include a figure of mortality of fish day by day or relative percentage survival.
The English language needs amendment: The authors are urged to contact an English speaking person.
The manuscript contains several incomplete sentences and som grammatical errors.
Author Response
Question1: The materials and methods section needs to be upgraded, especially the immune stimulatory procedure, the amount of stimulant used?
Answer: Hybrid yellow catfish were fed with A. membranaceus two times at the ratio of 4% for 28 days. Hybrid yellow catfish were challenged intraperitoneally by injecting 0.2 mL live A. hydrophila containing 2 × 105 cells at LD50 dosage on day 28.
Question2: The Results section also needs upgrading, especially the challenge experiment. Please include a figure of mortality of fish day by day or relative percentage survival.
Answer: The mortalities was 0%, 3.33%, 6.67%, 10%, 10%, 10%, 10% from the first day to the seventh day in A. membranaceus group, and in the control group, the mortality was 20%, 46.7%, 56.7%, 63.3%, 66.7%, 70%, and 70% from the first day to the seventh day. The time to reach highest mortality was on fourth and sixth day in A. membranaceus group and the control group respectively (Figure 6).
Question3: The English language needs amendment: The authors are urged to contact an English speaking person. The manuscript contains several incomplete sentences and some grammatical errors.
Answer: The manuscript has been revised by an English speaking person.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
In this manuscript, the authors describe effect of A. membranaceus onto gene expression in the hybrid yellow catfish in reponse to A. hydrophila. Such studies are important for aquacultures, however in my opinion this manuscript needs major revision since some fundamental information is missing.
Major part of the article is based on qRTPCR analysis of only 7 genes, whereas RNA-seq should be major part of manuscript. In my opinion authors need to add more information about DEGs obtained from RNA-seq analysis. First of all I recommended to add Table S1 - table with list of DEGs obtained through sequencing, with their log expression values and annotations. Then these result should be described in results and discussion sections.
Other things: line 37: is there some newer publication ?
Material and methods: RNA was obtained from one fish ? it's need to be clarified. Same about sequencing : only spleen was sequenced? More information should be added to methods.
RIN values missing
Why Authors choose mapping to reference genome instead of de novo assembly ?
Degs annotation: how enrichment analyses have been done ? there is no information in methods but figures suggest enrichment analyses. How degs have been annotated ?
How genes used for qrtpcr validation have been selected?
lines 112-116: Interval like in line 110 would be better - these details are in table 2
Figure 1 and figure 2 present same information - please delete one figure. Further, lines 123-124 should be moved into figure description , and I see blue dots not green and grey dots are even not presented on figure 2.
Line 125: On the figure we can see only that there were more down- than up-regulated genes. To check magnitude change levels some stats should be done e.g. wilcoxon test, and I recommend to do this.
Line 129-133: it is hard to read
Discussion parts should be rewritten after adding more information from RNA-seq.
Correlation between qrtpcr and rna-seq should be done.
Some minor things: on figures full pathway name should be provided. gene names- low italics; full gene names should be provided. Spaces and paragraph numbers should be verified.
Moderate editing of English language required
Author Response
Reviewer #2:
Question1: Major part of the article is based on qRTPCR analysis of only 7 genes, whereas RNA-seq should be major part of manuscript. In my opinion authors need to add more information about DEGs obtained from RNA-seq analysis. First of all I recommended to add Table S1 - table with list of DEGs obtained through sequencing, with their log expression values and annotations. Then these result should be described in results and discussion sections.
Answer: DEGs obtained through sequencing, with their log expression values and annotations was in Table S1. And some DEGs were described in results and discussion.
Question2: Other things: line 37: is there some newer publication ?
Answer: In line 37, the original cited article was replaced by an article published in 2020.
Question3: Material and methods: RNA was obtained from one fish ? it's need to be clarified. Same about sequencing : only spleen was sequenced? More information should be added to methods.
Answer: In each group, three fish from different tanks were prepared for RNA extraction. Total RNA of spleen, intestine and liver from one hybrid yellow catfish was extracted by Trizol reagent (Sangon, China). Only spleen RNA was used for library construction and RNA sequencing.
Question4: RIN values missing
Answer: RIN values was added in Table 2. RIN values of all specimens were tested, which ranged from 9.2 to 9.9 and qualified for library construction.
Question5: Why Authors choose mapping to reference genome instead of de novo assembly ?
Answer: Because yellow catfish genome (NCBI_ASM372403v1) has been published in NCBI database, we can use it as reference genome.
Question6: DEGs annotation: how enrichment analyses have been done ? there is no information in methods but figures suggest enrichment analyses. How DEGs have been annotated ?
Answer: Significantly DEGs were selected by using |log2foldchange| ≥ 1 and q value < 0.05 as the threshold. DESeq (2012) R package was used to analysis DEGs. All DEGs were mapped to GO database and KEGG database.
Question7: How genes used for qrtpcr validation have been selected?
Answer: Seven genes which related immune, apoptosis, antigen processing and presentation from RNA sequencing results were selected to analysis qRTPCR.
Question8: lines 112-116: Interval like in line 110 would be better - these details are in table 2
Answer: In table 2, line intervals were set as single space.
Question9: Figure 1 and figure 2 present same information - please delete one figure.
Answer: Figure 1 was deleted.
Question10: Further, lines 123-124 should be moved into figure description , and I see blue dots not green and grey dots are even not presented on figure 2.
Answer: lines 123-124 have been moved into figure description. Grey dots were deleted by me last time, and they are recovered and presented on figure 1.
Question11: To check magnitude change levels some stats should be done e.g. wilcoxon test, and I recommend to do this.
Answer: In order to check magnitude change levels, DEGs between A. membranaceus group and control group were selected by |log2foldchange| ≥ 1 and q value < 0.05. Altogether 396 DEGs were identified between control group and A. membranaceus group, containing 263 up-regulated and 133 down-regulated DEGs (Figure 1).
Question12: Line 129-133: it is hard to read
Answer: In line 129-133 have been rewritten.
Original line 129-133:
“challenged with lipopolysaccharide, some genes related to immune and transcription were indentified in yellow catfish[27]. when challenged with poly I:C, 307 genes were up-regulated and 215 genes down-regulated in yellow catfish[13]. Researchers found 5527 DEGs in catfish when infected Edwardsiellaictaluri[28]. The Japanese flounder showed1037 DEGs in gills at 48 h after infected with E. ictaluri [29].”
Revised as:
“In the head kidney of yellow catfish challenged with lipopolysaccharides, immune- and transcription-related genes, cytoskeleton-related genes, apoptosis, and cell cycle-related genes were identified [38]. When challenged with poly I:C, 307 genes were upregulated, and 215 genes were downregulated in yellow catfish[21]. A previous study found that 5,527 DEGs are present in yellow catfish infected with Edwardsiella ictaluri. Of these, 2,265 are upregulated and 3,262 are downregulated [39]. When the Japanese flounder (Paralichthys olivaceus) was challenged with E. tarda for different stress durations, 456 and 1037 DEGs were identified in the gills at 8 hours and 48 hours after injection, respectively [ [40].”
Question13: Discussion parts should be rewritten after adding more information from RNA-seq.
Answer: More information and discussion about RNA-seq has been added in the manuscript.
Question14: Correlation between qrtpcr and rna-seq should be done.
Answer: The correlation analysis between RNA-Seq data and qRTPCR results of the seven DEGS in spleen was applied through cor() in the R environment. R2 was 0.86 , 0.95, 0.90, 0.93, 0.80, 0.86, 0.36 in Aatk, DAPK2, IL6ST, Bax, Cd63, Inppl1b, and Sh2d1a. All the seven DEGs in spleen have the same line correlation between qPCR and RNA-seq. Correlation analysis showed that qRTPCR and RNA-seq have significant tendency (P<0.05) in Aatk , DAPK2, IL6ST, Bax, Cd63, and Inppl1b (Figure 5) .
Question15: Some minor things: on figures full pathway name should be provided. gene names- low italics; full gene names should be provided. Spaces and paragraph numbers should be verified.
Answer: Full pathway name has been provided on figure3 and figure 4. Gene names have been changed to low italics in the article. Spaces and paragraph numbers were verified when the article revised.
Question16: Comments on the Quality of English Language, Moderate editing of English language required
Answer: The manuscript has been revised by an English speaking person.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
The manuscript is amended according to the suggestions of the reviewer. It may be accepted for publication.
Reviewer 2 Report
Many thanks to Authors for improving manuscript.
Some minor thing: Some editors errors and all gene names should be in low italics
