The Pathology and Splenic Transcriptome Profiling of Trionyx sinensis Challenged with Bacillus cereus

Round 1

Reviewer 1 Report

I found serious in methodology and experiment design. My comments are here

Lines 64-66, were T. sinensis free for specific pathogen free? If yes, how were they tested to be free from any infection before use them in the experiment.

It is not mentioned that how many turtles were used for the experimental infection?

Line 69, from where bacteria was isolated to be use in this study? What about its identification and authenticity? Which bacteria do you mean here? Is any name of it?

Line 72, why intraperitoneally approach used not bath infection? Bath treatment is a natural infection process in nature.

Line 74, I do not think if you store spleen directly in liquid nitrogen and will get the RIN value more that 8.0 for RNA-seq.

Line 99, RIN values of RNA samples are documented. Please provide the document.

Line 101, RNA concentration is missing.

Table 2: PCR product size of each tested gene is missing.

Raw data of RNA-seq have not been deposited to any gene bank e. g. NCBI.

This manuscript does not provide any accession number of generated sequencing data.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear Editor,

The manuscript entitled “The Pathology and Splenic Transcriptome Profiling of Trionyx sinensis Challenged with Bacillus cereus” by Jinbiao Jiao et al. presents a study of B. cereus effects on T. sinensis turtle with histopathology, PCR identification of virulence factors and transcriptomic analysis of differentially expressed genes of infected spleen. The authors also present results of GO and KEGG analysis.

Τhe manuscripts’ objects are interesting, it well written in a comprehensive way and the findings are interesting. Therefore, the manuscript could be accepted for publication after minor revisions:

1.      The key references [9] and [11] are in Chinese, therefore I cannot read it (and a great portion of Fishes readers I suppose). Please provide alternative references in English.

2.      The scale bars in figure 1 are not clear. Which are 50 and which are 100 μm?

3.      Figure 2 seems blurred and oversaturated. Please provide an image with better definition.

4.      Please provide a correct legend for figure 8, describing better each graph.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Letter to Authors
fishes-2114666-v1
The Pathology and Splenic Transcriptome Profiling of Trionyx sinensis Challenged with Bacillus cereus
Jinbiao Jiao, Jing Chen, Jiayun Yao, Yanlin Li, Xuemei Yuan, Lei Huang, Shengqi Su, Haiqi Zhang


230114


Dear authors,
Your potentially interesting MS needs at least a round of major revision. Introduction section is incomplete for presenting needs for your current study. Method description is unclear indicating a criterion of "major" (control missing) in the journal. Extensive English editing is also necessary.
See below for detail.


L14
sequencing 96 h -> sequencing at 96 h

L20
In summary, -> delete

L23 keywords
Bacillus cereus; Transcriptome- > replace
Avoid listing words which appear also in the title. Duplicate hits upon computer search do not make sense. Give words that do not appear in the title to draw attention from wider readership. Posting words that neither appear in the abstract is better, because even in full-text search/indexing robots may not weigh much on words deeper (posterior) in the text. Hint: opportunistic infection, resident bacteria, aquaculture, etc.

L25 introduction
Consider beginning with general statements. Beginning with your material animals clearly indicate this MS is good for softshell turtle geeks. There are few such geeks among readers. Beginning with general statements will not only attract wider readership and also present a placement of your research in a wider research field. In your case, disease control in aquaculture seems to be the inclusive research field. If so, give a top short paragraph (~100 words) telling importance/needs of disease control in aquaculture.

L28
But in recent years, -> In recent years, however,
Do not begin a sentence with "but".

L30-32
severe diseases. For example, there have been previous reports on Aeromonas 30 hydrophila [1], Edwardasiella tarda [2], Trionyx sinensis Hemorrhagic Syndrome Virus 31 (TSHSV) [3], and B. cereus [4]. -> revise
Do not make a simple reference list (A stated this, B analyzed that, C argued it, or alike). Simple reference lists bloat your MS, dilute your originality, and undersell your own research. Use noun phrases to make abstract contents of those references. In your case, particular pathogen names except for Bacillus are not essential.
 -> severe infectious diseases including Bacillus cereus [1-4].

L32
These have -> Outbreak of these pathogens has

L34
a Gram-positive, (does not make sense) -> a Gram-positive bacteria, which is
A what?

L37
factor -> factors

L38-40
species, for example, .. Apostichopus japonicus [10], -> [cultured] aquatic animal species [8-10],
Do not make a simple reference list. In your case, particular disease and animal names are not essential except for the softshell turtle.

L43
Indeed, (does not make sense) -> delete
Bacillus cereus -> B. cereus
See L32.
isolates were extracted from diseased T. sinensis in Taiwan, carrying cytK, HblC, NheA and entFM ? -> isolates, carrying cytK, HblC, NheA and entFM, were detected from diseased T. sinensis in Taiwan ?
Who carries these factors?

L45
And -> delete
Do not begin a sentence with "and".
a strain SYJ15 -> a B. cereus strain SYJ15
Make sure what strain.

L46
Insert a sentence telling needs for further characterization of the particular B. cereus strain isolated previously [11]. Without this insertion, detection of virulence factors from this strain (2.3 & 3.2) does not make sense.
Whereas little .. Therefore, it is of great significance -> Because little is currently known about how T. sinensis responds to B. cereus isolated previously [11], it is of great significance

L50-54
immune gene identification .. in Eriocheir japonica sinensis 53 [16]. (simple reference list) -> immune gene identification in many aquatic animals under bacterial challenge [13-15].
Digestive enzyme genes are out of scope.

L54
Relatively, (doe not make sense) -> delete

L65
Ten turtles were randomized into two groups ?
Does each group consisted of ten turtles (20 in total)?

L74-78
Your tissue sampling scheme is unclear.

L74
random -> randomly chosen

L77
hpi -> hpi (hours post injection)
spleen?
Was this spleen from the same individual turtle given in the previous sub-section?
.. were immediately extracted ?
How many replicates?

L80
staining (repeated word) -> delete

L94 table 1 body
Make the column of primer sequence wider to avoid line overflow. When the current line width is too narrow for make space, consult with the assigned assistant editor for overhanging into the left margin.

L109
The sequencing data .. in a FASTQ format. -> delete

L111
low-quality reads (< Q20) ?
Average? Containing a stretch of nucleotides?

L112
Reference and version information are needed for Trinity.

L120
References and version information are needed for the three softwares.
software -> softwares

L132
Reference and version information are needed for KOBAS.

L138
Supplier's information for Primer Premier is needed.

L145
Reference is needed for 2-ΔΔCT method.

L150-153
by the Student's t-test, and .. by GraphPad Prism version 8 software. (back-and-forth) -> by Student's t-test using SPSS version 25 (supplier?). A significance threshold was set at p < 0.05. Graph assembly was visualized by GraphPad Prism version 8 software (supplier?).

L157
in the tissues -> in the tissues (Figure 1)

L167 figure picture
Fonts used to indicate scale lengths are too small to see. They should be at least equal to those in the main text. Scale bars themselves are also too narrow. Make them broader.

L168
What do "CK" and "GD" mean?

L169
Where is a note for normal liver (a)?

L182
ces and cytK -> cytK and ces
Order as table 1 and Figure2.

L183
didn't match the length of the target product -> didn't match (> 500 bp) the length of the target product (428 bp) (reader-friendly)

L189
After being sequenced on an Illumina HiSeq 4000 platform, (redundant) -> delete

L195,270
And -> delete
Do not begin a sentence with "and".

L196-197
Detailed length .. described in Table 4. -> delete
Wording like "X is shown in figure/table Y" imposes killing readers' times to read such an information deficient sentence telling only that there is a figure/table. You should present an outline or a perspective drawn from the figure/table and cite it in parentheses at the end. In this case you may cite it at the end of previous sentence in parentheses.

L199-203
Change format following that of L205-207.

L214
Homologous species -> Homologous species (top-hit species upon database search)
was shown in Figure 3, it -> delete
Figure 3 may be cited at the and in parentheses.

L215
high -> highly

L243,254,287,291 figure pictures
Fonts in pictures are too small. They should be equal to or larger than those in the main text.

L246,261 tables 6 & 7 bodies
Make the description column wider to avoid line overflow. When the current line width is too narrow for make space, consult with the assigned assistant editor for overhanging into the left margin. Capitalize the head of lines in Table 7.

L248
analyze -> explore

L249
MA plot -> an MA plot

L253
partial information is listed in Table 7 -> (Table 7)

L267
The top ten .. are listed in Figure 6. (does not make sense) -> delete
See L268-275 for how many.
You may cite this figure at the end of previous sentence.

L280
The top 30 .. shown in Figure 7a. (does not make sense) -> delete
See L281-286 for how many.
You may cite this figure at the end of previous sentence.

L295
, larger bubbles indicate more genes; -> delete

L300
The fold change .. in Figure 8. -> delete
You may cite this figure at the end of previous sentence.

L310
But -> However,

L329
in the infected parrots -> delete

L332-333
A study on Galleria ..t for virulence effects [28]. -> delete

L333
Insert an abstract of your result on bceT, cytK and ces.
"absence of commonly found bceT, cytK and ces" ?

L334-336
for example, .. cytK-1 and cytK-2 in 335 oyster farming areas [30], and even bceT of a strain from dairy products [31]. (simple reference list) -> [4,25,29-31]

L338
species differences ?
Which species?
-> specificity in host-pathogen affinity

L345
interleukin, complement, chemokine, tumor necrosis factor, interferon, nuclear factor, immunoglobulin, antimicrobial peptides -> move to L253

L352-354
This was similar .. exposed to LPS or poly I: C (simple reference list) -> delete

L356
IL-8, also referred -> new paragraph

L358
Neutrophils migrate to .. further eliminate invading pathogens (simple reference list) -> delete

L361
Previous studies -> A previous study

L362
A. hydrophila -> bacterial

L372
Indeed, (verbose) -> delete

L374
What is "this"?

L377
activated, the detailed (unclear connection) -> activated. The detailed (break sentence here)

L383-385
In Monopterus albus, ..ated with A. hydrophila infection, which was resistant to pathogen infection in a classical way (simple reference list) -> delete
You may cite the reference at the previous sentence.

L386
In Oplegnathus fasciatus, .. after E. tarda infection (simple reference list) -> delete
You may cite the reference at L383.

L390
Taking into .. grass carp [53,54] (back-and-forth simple reference list) -> delete

L392
Our results -> Our results, however, ?

L403
enrichment -> enriched

L404
derived -> infected

L405-409
Phagosome was .. in Penaeus monodon [59]. (simple reference list) -> delete
You may cite the reference at the previous sentence.

L414
debris[62] -> debris [62] (insert a white space)

L410-433
Insert an abstract of your result on immune-related pathways at an appropriate position.

L439
up-regulated and down-regulated -> up- and down-regulated

L455
RNA-seq results (FastQ files) should be deposited in a read archive such as SRA.
L459 references
Check the reference list carefully again from the beginning. Reference lists are frequently hotbeds of errors. You might add, omit or swap citation in the main text on the way internal revision. Numbering of the references might then shift. If so, readers think you are making irrelevant citation. It is the authors' responsibility that all references are properly cited.

L465,532,etc
Make sure if paper titles are in lower case. Check thoroughly.

L486
nigrum -> in Italics

L492
& nbsp; -> delete

Following items may be helpful for further discussion.

Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A. 2011. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 29:644-652. doi: 10.1038/nbt.1883

Li B, Dewey CN. 2011. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinf 12:323. doi: 10.1186/1471-2105-12-323

Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CT method. Methods 25:402-408.

Mao X, Cai T, Olyarchuk JG, Wei L. 2005. Automated genome annotation and pathway identification using the KEGG Orthology (KO) as a controlled vocabulary. Bioinformatics 21:3787-3793 doi: 10.1093/bioinformatics/bti430

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Authors improved the manuscript as per the suggestions. However, they should discuss splenic transcriptome porofiling of Trionyx sinensis with other fish species, rainbow trout spleen profiles https://www.nature.com/articles/s41598-018-31982-6

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Letter to Authors
fishes-2114666-v2
The Pathology and Splenic Transcriptome Profiling of Trionyx sinensis Challenged with Bacillus cereus
Jinbiao Jiao, Jing Chen, Jiayun Yao, Yanlin Li, Xuemei Yuan, Lei Huang, Shengqi Su, Haiqi Zhang


230126


Dear authors,
This revised MS needs a round of minor revision. Several issues have been newly introduced in the revised statements. Extensive English editing is still necessary. See below for detail.


L49-50
According to .. high mortality [14]. -> delete
It may be OK to retain this nonsensical sentence when particular virulence factors have one-by-one relations with particular symptoms of the disease.
Instead, insert a short sentence telling isolation of XS0724, or otherwise, readers will not understand why XS0724 is important.

L74-76
B. cereus strain XS0724 .. in our laboratory [14]. -> delete
See above and next.

L76
B. cereus strain XS0724 -> B. cereus strain XS0724 isolated previously [14]

L81
hour post-infection (grammar) -> hours post-infection

L91
microscopy[19] -> microscopy [19] (insert a white space)

L95
NEB Biolab, England -> {NEB, New England Biolabs}, Ipswich, MA, USA

L100
Bio-Rad, USA -> Bio-Rad, Hercules, CA, USA

L103
Gel Red -> GelRed (Biotium, Fremont, CA, USA)

L107
Takara, Japan -> Takara, Shiga, Japan

L109
Thermo Scientific, USA -> Thermo Fisher Scientific, Waltham, MA, USA

L110
Agilent Technologies, USA -> Agilent Technologies, Santa Clara, CA, USA

L112
Invitrogen, USA -> Invitrogen, Waltham, MA, USA

L117
Illumina, USA -> Illumina, San Diego, CA, USA

L385
researches (uncountable) -> {studies, reports}

Author Response

Please see the attachment.

Author Response File: Author Response.pdf