Transcriptome Analysis of Greenfin Horse-Faced Filefish (Thamnaconus septentrionalis) Gills in Response to Amyloodinium ocellatum (AO) Infection

Round 1

Reviewer 1 Report

 

In their manuscript, Yang et al. are studying the greenfin horse-faced filefish (Thamnaconus septentrionalis) in response to Amyloodinium ocellatum (AO) infection to understand the pathobiology caused by amyloodiniosis. Since the genome of filefish was only recently sequenced and assembled, studying the whole transcriptome in this fish species can provide new biological insights to the biology of filefish. In addition, better control measures or treatments against amyloodiniosis, would undobtedly be important e.g. for the aquaculture industry to prevent disease outbreaks. Here, the authors have used isolated gill samples from healthy and AO infected fish and performed RNA sequencing followed by gene ontology enrichment and pathway analyses. While the research question of the manuscript is highly relevant, there are major issues that would be important to assess prior publication.

 

Major comments:

-     - While it is well-justified to assess the pathobiological transcriptional response of AO infection by using the gills, other immunologically relevant tissues such as the spleen, liver or kidney would have provided important additional information on the host response against the parasite (similarly as in Syahputra et al., 2019; PMID: 31220151). Also, analyzing other parasite infested tissues such as skin could have revealed further insights. Consequently, the manuscript would be greatly improved by additional gene expression analyses performed from other tissues. Optimally RNA sequencing could be used. Alternatively, some of the genes introduced as differentially expressed in the gills, such as Leptin and Hepcidins, could be quantified from the liver using e.g. quantitative PCR. Also, basic flow cytometric analysis of blood cell populations could be analyzed from the blood, the kidney or the spleen to gain further immune cell-level information on the response (Langenau et al., 2003; PMID: 15123839 and Inoue et al., 2002; PMID: 12458744).

- Related to the previous comment, since the transcriptomic analysis has been made only after the symptoms of amyloodiniosis have developed, it is difficult to assess how the transcriptome changes as the disease progresses. In fact, this would be a highly important matter to be able to distinguish between early-stage host response against the parasite and the inevitable large-scale transcriptome modulation caused by a late-stage infection. To answer this question thoroughly, it would be necessary to set-up an experimental AO infection model, and to evaluate the response at different intervals post-infection (Xie et al., 2021; PMID: 33913520 and Oestmann  Lewis, 1995; PMID: 7483239). At minimum, as the temporality has not been studied, the authors should comment on the matter as a major limitation of the study.

- Figure 1 is missing micrographs from the skin and gills of healthy controls to make comparisons between infected and control groups possible. This is especially important for readers that are not familiar with the particular parasitic infection and its manifestations in the skin and the gills.

- In the results section 3.3. the authors describe only a few DEGs that are likely affecting the host response (e.g. Hepcidins). Although, these aforementioned example genes are later properly discussed, this section could describe several other DEG´s (over 2000 DEG´s identified in the paper). One possibility could be to include some of the genes shown in Figure 7 as example genes to the results section.

- The manuscript has a major focus on the pathway analyses and GO categories as well as on KEGG pathways. In fact, many of these have been mentioned in the Results sections 3.4. and 3.5. However, for this reviewer the relevance of the discovered pathways is unclear. In other words, the authors should discuss the significance of the identified pathways in the context of the manuscript. For example, what does it mean to have many genes enriched in Biological process – cellular process and why certain GO-classes show enrichment among both up- and down-regulated genes (Figure 5)? Or why certain KEGG pathways such as: Th17 cell differentiation, Malaria, Amoibiasis or Staphylococcus aureus infection are enriched (Shown in Figure 6B)? In addition, here the authors could also describe some of the specific genes that have been identified. For example, which are the genes enriched in AO infection that associate with Amoebiasis?

Minor comments:

-In row 18 and 183 the authors write that 624 new genes were detected. As the authors have not done genome sequencing it remains unclear for this reviewer that how this result was obtained. Consequently, the authors should clarify how they have used the RNA sequencing data to come to this conclusions.

- Row 41: “AO salinities” is likely an incorrect way to describe the salinity range in which AO can survive. Please, check the correct description.

- Row 94: The methodology of the microscopic examination is not adequately described. Were the fish dead or alive during the process and if alive fish were used were they anesthetized during the examination?

- Row 100: Fish weight and length should be described separately for the infected and control groups.

- Row 107: What anesthetic system/agent was used?

- Row 113: How much RNA was used for RNA sequencing?

- Rows 114-115: Regarding the sentence “Samples 1-3 were each collected from one AO infected fish whereas samples 4-6 were each from one healthy fish”. For this reviewer this sentence gives the impression that only 1 infected and 1 control fish was used and 3 samples taken from both of these. Please, clarify the sentence.

- Row 165: In case the sentence “Corpses had missing eyes, mutilated fins and dull skin” is not related to the AO infection but rather caused by the cannibalism (that is common behavior), the necessity of this sentence is questionable.

- Histological analysis of gills and skin could be used to verify AO infection.

- Rows 169-170: An image of the agarose gel electrophoresis analysis of infected and control fish PCR products should be added to the supplementary to evaluate the specificity of detection.

- Figure 1: Scale bar(s) should be added to the micrographs.

- Figure 1: In the figure legend it is written “AO photomicrograph”. Since this image is not only about the parasite the legend should be modified.

- Rows 180 and 181: It is stated that the PCA results show high similarity within groups. However, it seems that while this is true for the infected samples, there is actually large variation between the controls. Please, justify the use of the sentence or modify accordingly.

- Figure 2: The sample labels for two of the infected samples are on top of each other and consequently difficult to read. Please, modify.

- Figure 3:

o   The volcano plot in A) should be larger to improve its readability.

o   At the same time, since the only information given in B) is gene numbers, there is not much information gained from this part. Consequently, DEG numbers could be written into the volcano plot.

o   Also, important example genes could be labelled into the volcano plot.

- Figure 4: The figure and the legend should be more informative. E.g. what does the scale bar represents?

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Letter to Authors
fishes-1884802-v1
Transcriptome analysis of greenfin horse-faced filefish (Thamnaconus septentrionalis) gills in response to Amyloodinium ocellatum (AO) infection
Li-Guo Yang, Yue Wang, Wen-Bing Xu, Bo Qin, Na Ying, Xue-Feng Song, Yan-Feng Yue, Xiao-Shan Wang, Bian-Bian Zhang, Yan-Qing Wu


220821


Dear authors,
Your MS is not well-written, though it is potentially interesting. Why greenfin horse-faced filefish? To present it clearly is not the current beginning of the introduction section. Placement of your study in an important research field will emphasize why filefish.
Method description is incomplete and unclear. This is equivalent to "control missing" that is a criterion of "major" of this journal. Result description is also unclear. Several genes first appear in the discussion section.
See below for detail. Words in braces indicate options. Bracketed words can be omitted.


L25 keywords
Thamnaconus septentrionalis; transcriptome analysis; Amyloodinium ocellatum; gill; infection -> 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: aquaculture, disease control, dinoflagellate, Thoracosphaeraceae, Tetraodontiformes, Monacanthidae, RNA-seq, pathway analysis, etc.

L28 introduction
Consider beginning with general statements about fish pathology to attract wider readership. General statements also will present placement of your MS in the research field.
Move the 3rd paragraph to the top is one point, though it is not the best. Some revision may be necessary to fit with this re-organization.

L59-70
Move to the top.

L59
Add a brief statement about importance of disease control (diagnosis or remedy) in aquaculture, stock enhancement seedling, aquarium, or else relevant to your material fish. One or two sentences are enough (<50 words). This will be a preparative statement for the next "Transcriptome sequencing is .. ".

L61
most previous studies focus on host response to virus or bacteria
Relevant reference should be cited. A review may be good, but Sudhagar et al (2018) reviewed vigorous fish transcriptome research activities on interaction both with viral, bacterial and eukaryotic pathogens.

L63
parasite -> eukaryotic parasite (contrasting to "virus or bacteria" at L62)

L63-67
For example, .. (Salmo salar) [16]. -> delete
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 this case, particular species names except for AO and the file fish are not essential. I think citing Sudhagar et al (2018) is enough.

L68
, the parasite was only found in two fishes, European seabass and silver pomfret, belonging to different suborders -> were limited to a few fish species
Particular fish names are not essential.

L72,79
Irrelevant citation.

L73
species of fish caught -> fishery resource

L75,81,82,92,etc
the greenfin horse-faced filefish -> the filefish
I think an abbreviation is better than the wordy name from the second appearance and later.

L75
whole genome data has only been recently analyzed
Reference needed.

L90
The same batch -> A batch
naturally fertilized ? -> spontaneous spawning [in captivity] ?

L97
cultured in different tanks ?
Did you sampled and transferred the infected and uninfected fish to these tanks from a mass culture?
-> transferred to independent 100-L tanks supplied with filtered seawater ?

L98
Finally, ? -> X days later,
Raring duration should be presented.

L101
Fish were cultured in 1000-L tanks containing filtered seawater. (redundant) -> delete
See comment on L97.

L113
pooled ?
How did you prepare three samples from "pooled" RNA?

L117
paired-end mode
Length?

L130
The greenfin horse-faced filefish genome has been published [19]. (redundant) -> delete
See comment on L75.

L131
the reference genome was built -> the reference genome was built from a draft genome data [19]

L142
differentially expressed genes/transcripts -> differentially expressed genes/transcripts (DEGs)
See L149 and later.

L144
using R -> using R (http://www.rproject.org/)

L146
(http://www.rproject.org/) -> delete

L169
Moreover, (wordy) -> delete

L171 figure picture
Black lines are unnecessary. Delete the corresponding legend at L173-174.

L177
Do "raw reads" contain rRNA? If so, you should add a column to table 1 describing filtered clean reads. If not, "Raw Data" in the table body should be "Filtered Raw Data".

L181
high similarity within groups ?
Controls showed large variance on the plain.

L188 table 1 body
Raw Datas -> Raw Data

L189,202,220,239,243 figure pictures
Fonts in the figure pictures are too small to see. They should at least as large as the font used in the main text. For figure 7, use of larger fonts only for up- and down-regulated genes may be OK.

L201
Table S1 ? -> supplementary tables ?
fishes-1884802-supplementary.xlsx has multiple sheets, and it is unclear which is the table S1.

L202
Sub-figure B is unnecessary. Numbers may be presented in A in blue and red fonts at the upper left and right corners.

L220,243 figure 5 &7 pictures
Down-regulated genes should be presented in a blue color. Be consistent with preceding figures.

L248
green -> blue
See above.

L252
seabass, silver pomfret, and gilthead bream -> some fish

L254
first identified -> first identified in the filefish
Put your originality forward.

L255
differentially expressed genes -> DEGs

L257
marine fish-AO -> not only the filefish- but also marine fish-AO ?
See L69-70.

L261
Insert a brief summary of your PCR diagnosis.

L268-271
In the European sea bass .. were down-regulated [5]. -> revise
Do not make a simple reference list.
-> Transcriptome sequencing of some fish revealed several up- and down-regulated genes in which AO-infected fish exhibited more up-regulated genes than down-regulated [4,5].

L273-293
You should address first about these 15 genes/paralogs in the result section.

L273,275
Ccl21 and f11 are absent from your supplement.

L287
Hepcidin is an iron regulator that reduces iron required for pathogen growth (redundant) -> delete
See L293.

L287-290
and, in salmonids .. but not in nodular virus-infected tissues. -> (continue to L286) , but up-regulated in bacteria- or parasite-infected salmonids and sea bass [31,32].
Do not make a simple reference list.

L288
Furthermore, (wordy) -> delete

L290
What is Vibrio eel?

L302
accelerated ? (no evidence) -> coincided with

L311-313
Cytokine-cytokine receptor .. was infected [4]. -> Many chemokine genes were altered when {fish, European sea bass or silver pomfret} was infected [4,5].
Do not make a simple reference list.

L324
in greenfin horse-faced filefish (redundant) -> delete

L349 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 seems actually occurred. It is the authors' responsibility that all references are properly cited.

L350,etc (many)
Make sure if book titles are in Italic title case. Check thoroughly.

L352,377,etc (many)
Make sure if paper titles are in lower case.

L354
Diseases -> Dis.
See L360.

L360
J -> J.
Make sure if abbreviated journal title words accompany a dot.

L362
Dis Aquat -> Dis. Aquat.
Make sure if abbreviated journal title words accompany a dot. Check thoroughly (L382,etc).

L369
artemia salina -> Artemia salina (in Italics)

L371
Emma J Fa jer-avila -> Fajer-avila, E. J.

The following item may be helpful for further discussion.

Sudhagar A, Kumar G, El-Matbouli M. 2018. Transcriptome analysis based on RNA-seq in understanding pathogenic mechanisms of diseases and the immune system of fish: A comprehensive review. Intl J Mol Sci 19:245.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

This article first shows the complete transcriptomic response of greenfin horse-faced filefish gills to infection with the ectoparasite Amyloodinium ocellatum.

 

The abstract contains very superfluous information such as the number of clean reads that they have obtained, which I think should not be in this section. Instead, it should have data that attracts the reader more and that was more relevant to the study.

Section 2.1 does not specify how many fish were sampled for survival control, nor does it specify at what time of infection samples are taken for transcriptomic analysis. Is it at the beginning of the infection or at the end? Can the post-infection day on which the samples are taken be specified? This would later help to better understand the entire transcriptomic response described, since it is known that the immune system does not act in the same way at the beginning of an infection or when it is already advanced.

Line 115 is misspelled, it has a ) symbol that it shouldn't.

The selection criteria for the so-called "representative SDRs" must be specified. And the name of the table does not correspond to the name of the file, there are many tabs in that Excel, it must be specified better.

In general, the article seems correct to me since it describes all the fields of transcriptomics.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Although Yang et al. have made important corrections to their manuscript, major issues related to the experimental design and execution of the work still remain. Additionally, the modified/revised parts of the manuscript would need language revision to match the otherwise fluent english.

Remaining issues are listed below:

- It seems that neither of the first two major concerns from round one has been addressed appropriately; 1. gills as the only analyzed tissue and 2. the temporality of amyloodiniosis. As already suggested previously, the authors could at minimum discuss the limitation of not knowing the exact time of disease onset at the time of sample collection. Also, it still remains unclear whether other tissues than gills were collected from the infected fish. In case other tissues were collected, additional analyzes such as qPCR analysis of hepcidin from the liver would be relatively straightforward to perform and would add value to the work.

- Since having proper controls is a prerequisite for any well-executed scientific experiment/work, the missing micrograph images of healthy controls is difficult to understand. However, since we are talking about healthy fish, using fish that have not been used in the RNA sequencing experiments could still be useful for comparisons. Of course, the fact that these fish were not contemporary to the infected fish would need to be mentioned e.g. in the figure legend.

- The relevance of many of the discovered GO-terms and KEGG-pathways still remains unclear. For example, why certain KEGG pathways
such as: Th17 cell differentiation, Malaria, Amoibiasis or Staphylococcus aureus infection are enriched in the particular infection (Shown in Figure 6B)? In other words, it would be necessary to discuss the results of the pathway analyses in more detail.

- It seems that the minor comment "Histological analysis of gills and skin could be used to verify AO infection." was understood incorrectly. Apologies for the possibly misleading comment that was initially meant to encourage the authors about the possibility of using histology in AO-identification. Also, in case this sentence would be used within the text it would need some rephraising and a literature reference to point out article(s) that have used histological examination of AO-infection (e.g. "Histological analysis of gills and skin have been previously used to verify AO infection", PMID: 35056010).

- Running the agarose gel electrophoresis again would definitely add value to the manuscript and should be performed.

- Adding the scale bars does not necessarily require a specific computer software. One easy solution would be to take an extra image from e.g. Burker chamber using the same microscope and magnification, and since the lenght of each line in the chamber is fixed, this information can in principle be transferred to any image.

 

 

 

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Letter to Authors
fishes-1884802-v2
Transcriptome analysis of greenfin horse-faced filefish (Thamnaconus septentrionalis) gills in response to Amyloodinium ocellatum (AO) infection
Li-Guo Yang, Yue Wang, Wen-Bing Xu, Bo Qin, Na Ying, Xue-Feng Song, Yan-Feng Yue, Xiao-Shan Wang, Bian-Bian Zhang, Yan-Qing Wu


220827


Dear authors,
Your v2 MS needs still a round of substantial revision. Extensive English editing is still necessary especially regarding revised parts. See below for detail.


L60
Disease control critical (grammar) -> Disease control is critical

L63
previous studies focus on -> previous studies focused on

L64
eukaryotic pathogens ?
If this is the case, entire this sentence does not make sense.

L65
Do not make a simple reference list.

L91-93
Three living fish .. killed for gill collection. (redundant) -> delete
See L99 and L108.

L100
healthy fish (uninfected) -> healthy fish (uninfected) in a separate tank ?
Make sure again what is described in L97.

206
see supplementary tables (redundant) -> delete

L214
control1 -> control 1 (insert a white space)
infected1 -> infected 1

L215
presents (grammar) -> was presented under

L227,246,250 figure pictures (Figs. 5-7)
Fonts in the figure pictures are still too small to see. They should at least as large as the font used in the main text. For figure 7, use of larger fonts only for up- and down-regulated genes may be OK.

L343 references
Check the reference list carefully again from the beginning.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 3

Reviewer 1 Report

While the authors have answered some of the remaining concerns, issues regarding their work still exist. Consequently, I cannot support the publication of the work in its current form.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Letter to Authors
fishes-1884802-v3
Transcriptome analysis of greenfin horse-faced filefish (Thamnaconus septentrionalis) gills in response to Amyloodinium ocellatum (AO) infection
Li-Guo Yang, Yue Wang, Wen-Bing Xu, Bo Qin, Na Ying, Xue-Feng Song, Yan-Feng Yue, Xiao-Shan Wang, Bian-Bian Zhang, Yan-Qing Wu


220831


Dear authors,
Your v3 MS needs still a round of substantial revision. Extensive English editing is still necessary especially regarding revised parts. See below for detail.


L64
viral and bacterial -> viral and bacterial pathogens

L65
the parasite (what parasite?) -> eukaryotic parasites (in general)
Make contrast with viral and bacterial pathogens.

L65-70
the interaction between Cryptocaryon irritans .. at the mucosal level. -> delete
Do not make a simple reference list. References may be cited at the end of previous sentence, or you may omit them. AO infection can be an "example" (for example ..).

L101
Due to the limitation .. sampling and testing. (grammar) -> Due to uncertainty on the exact time of disease onset, the time-point of sample collection was 2 days after infection being noticed.

L252 figure 7 picture
Fonts in the figure pictures are still too small to see. They should at least as large as the font used in the main text. Use of larger fonts only for up- and down-regulated genes may be OK.

L326
Second, -> delete

L327
Molecular -> molecular

L331
Malaria, Amoebiasis (English) -> malaria, amoebiasis
Staphylococcus aureus (Latin) -> in Italics

L332
infection are enriched -> infection pathways are enriched

L333
Amoebiasis -> amoebiasis

L334
Staphylococcus aureus -> in Italics
infection are essential -> infection pathways are essential
Infectious diseases -> response to infectious diseases
diseases [41], and the enrichment -> diseases [41]. The enrichment
Is this reference relevant?

L335
What is "basis"?

Author Response

Please see the attachment.

Author Response File: Author Response.docx