Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Please find attached document.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

N/A

Author Response

Response to Reviewer 1.

 

The manuscript by Sukhovskaya et al. is generally well written and offers an interesting perspective on an in vivo study involving LPS injection in fish to characterize the inflammatory response. In general, the manuscript is well written, but it can be improved in some areas, especially in the methodology and discussion section.

There are some critical pieces of information missing that are essential for reproducing experiments. I would highly recommend reading the manuscript again for grammar issues.

Answer: We sincerely appreciate your comprehensive evaluation of our manuscript and your positive assessment of our research significance and experimental design. Following your suggestions, we have carefully addressed several crucial and minor issues raised throughout the manuscript. We improved the grammar of the text with the assistance of MDPI team; ee believe that the language is now acceptable for the review process. Your feedback has greatly improved our manuscript.

Please see my comments below:

Section 2.2.

Please add the maintenance parameters for the fish (temperature of tank, size of tank, feed... etc). Also, where were the trout obtained? Males or females? How was the mortality recorded and were there any mortalities?

Reply: Thank you, the information you suggested (source of fish, sex, tank characteristics) has been added and is now in the text, please find out on lines 157-166, 185-186). The data on mortality was obtained daily and shown partial (10 per cent) fish mortality during acclimation period but not following treatment (injections) (lines 164-165). Feed trademark is now indicated (line 186), it was similar before and during the experimentation.

 

Line 26: These are not fish specific biomarkers. They are expressed in many other animals as well. Please change.

Reply: Thank you very much for pointing out this detail in our manuscript. Of course, we agree with your authoritative opinion, the words ‘fish-specific’ were deleted. It was rather used for the panel description.

 

Line 199:

Please italicize E. coli and check the manuscript for others (there are more cases) for example, in vivo should also beitalicized.

Reply: We sincerely thank the reviewer for pointing out these issues. All inconsistencies in italicization have been corrected.

 

Line 214:

Fish „were”euthanized.

Reply: Thank you, we have corrected this annoying grammar mistake.

 

Lines 222-223:

Please include the formula that you used to calculate this here not in another section.

Reply: We appreciate this valuable question. The order of Sections 2.4 and 2.5 has been reversed to ensure the formulae appears immediately after its first mention in the text.

 

Section 2.6:

How many fish were used to obtain serum? Was it a pooled sample?

Reply: According to your suggestion we substantially revised all the methodological information, the modified phrase is as follows: Serum from 12 fish per group (n=6 from each of the two replicate tanks) was analyzed, with each serum sample tested in three technical replicates.

 

Line274:

What is the reason for incubatingat this temperature? A trout would not experience this temperature in natural conditions

AND

Figure 4. A limitation of this assay should be discussed. This experiment could have been perform data lower temperature that is more representative of fish physiology.

Reply: Thank you very much for pointing out this detail in our manuscript. We completely agree with you regarding the crucial importance of temperature for trout physiology, including the immune response. The approach to temperature choice for the SBA assay varies—from lower temperatures typical for trout habitats to 37 °C, as prescribed by the standard protocol. We carried out the experiment in accordance with an established methodology, assuming that bacterial growth would be inhibited at lower temperatures. We also considered that all samples were treated under the same conditions, allowing for a valid internal comparison. Nevertheless, in our future studies, we will certainly perform the analysis at lower temperatures.

Line371:

The reis no need to indicate that it is not significant.

Reply: Though both LPS or PS administration regardless of the post-treatment time, 24 or 96 h, did not significantly affect SBA in fish, we decided to keep this evidence to emphasize the specificity of the model used including the experimental limitations, etc.

 

Figure7:

How many fields of view were used to obtain these results during microscopy? Please consider adding an image of the band neutrophils to the manuscript.

Reply: We thank the reviewer for their comment. The methodological detail regarding the number of fields of view has been added to the manuscript (Lines 327-328). Representative micrographs documenting cell morphology were reviewed as part of the technical analysis but are considered raw data not intended for publication.

 

 

 

 

 

General remarks:

The authors need to discuss the limitations of the study especially temperature.

Reply: Thank you for your useful comments. We recognize the limitations of our study; we suppose that the effects shown are associated with particular variables chosen for study, such as water temperature, LPS doses used, preconditioning, and even age and size of fish. According to the suggestion, all the variables leading to limitations have now been specified in detail both in the Methods and Conclusion parts.

 

Perhaps the authors can provide a rationale for using a human pathogen (Escherichia coli 055:B5). Why not a fish LPS?

Reply: To reproduce the model in trout we relied on available protocols, though the parameters recommended as well as inflammatory responses are highly variable. We acknowledge that an alternative pathogen could have been considered for this study. Nevertheless, our experimental design was based on published evidence demonstrating the use of Escherichia coli (055:B5) in fish objects, such as:

Corripio-Miyar Y., Bird S., Tsamopoulos K., Secombes C.J. Cloning and expression analysis of two pro-inflammatory cytokines, IL-1 beta and IL-8, in haddock (Melanogrammus aeglefinus) // Mol. Immunol. 2007, V. 44(6), P. 1361-1373. doi: 10.1016/j.molimm.2006.05.010

Liewes E.W., Van Dam R.H., Vos-Maas M.G., Bootsma R. Optimization and kinetics of in vitro stimulation of carp (Cyprinus carpio L.) leukocytes // Vet. Immunol. Immunopathol. 1982, V. 3(3), P. 325-343. doi: 10.1016/0165-2427(82)90006-x.

 

Once again, thank you for your insightful comments which provide invaluable guidance for our subsequent work.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Introduction

• Line 33:
  The phrase “LPS is poorly investigated in fish” is inaccurate. Suggest revising to: “LPS is poorly investigated in a variety of fish species” for accuracy. It is widely used in salmon.

• Line 38:
  The term “non-specific” immunity used here is unclear in context. Please clarify what “non-specific” refers to, e.g., innate immunity or broad-spectrum immune responses.

• Line 42:
  Add justification for the choice of sampling time points at 24 and 96 hours post-injection; explain why these intervals are biologically or experimentally relevant.

• Line 46:
  Suggest changing “does not involve” to “does not appear to involve” for scientific accuracy , as the study did not investigate earlier time points such as 6 hours.

• Line 122:
  The phrase “biochemical and molecular genetic hallmarks” is confusing. Clarify what is meant by “molecular genetic hallmarks,” or revise to “biochemical and molecular hallmarks.”

• General Language Issues:
  The introduction contains frequent language errors and awkward phrasing. Examples include:

  • “fastestgrowing” (should be “fastest-growing”)

  • “traditionally applied” (preferred “traditionally used”)
  • “PGE2. animals” (fragmented and unclear).
    the number 2 should be adjusted 

Methods

• “Water recycling system” (Line ~161):
  Clarify if this refers to a recirculating aquaculture system (RAS), as the terminology implies different systems.

• Light Regime (Lines 173–184):
  The reported “10 h/14 h (day/night)” photoperiod seems reversed; typically, the photoperiod is 14 h light/10 h dark. Please verify and correct.

• Line 191 (Experimental challenge):
  The manuscript does not specify the water temperature during the challenge exposures, nor does it state whether fish were fasted before injections.

• qPCR Primer Efficiency:
  Missing data on the amplification efficiency of each primer set, also the refrence genes M-value

• Serum Bactericidal Activity (SBA) Assay:

  • It is unclear whether individual flasks represent independent biological replicates or technical replicates.

  • Bacterial concentration used (CFU/mL) is not reported; this value is crucial for assay repeatability and comparison.

  • Number of technical and biological replicates per treatment should be clearly stated.

Discussion

• The discussion section requires improvement. Currently, it merely summarizes previous studies without critical evaluation or explanation of discrepancies. The authors should provide reasoning for any conflicting findings. Consider addressing:

  • Differences in LPS doses and fish species or strains.

  • Variability in biomarkers across studies.

  • Potential limitations or alternative interpretations.

Conclusion

• Both the doses used and the preconditioning may disqualify it as an inflammatory model, however, it provides a robust example of how management could affect the immune responses

General - please provide the diet used. 

Author Response

Response to Reviewer 2.

 

Comments and Suggestions for Authors

Thank you for your useful comments and suggestions on the language and scientific content of our manuscript. We have modified the manuscript accordingly, and detailed corrections are listed below point by point. Any changes made could be tracked in MS file as marked in red. Once again, thank you for your insightful comments.

 

Introduction

Line 33:
The phrase “LPS is poorly investigated in fish” is inaccurate. Suggest revising to: “LPS is poorly investigated in a variety of fish species” for accuracy. It is widely used in salmon.

Reply: Thank you, we have included the recommended changes to the text.

 

Line 38:
The term “non-specific” immunity used here is unclear in context. Please clarify what “non-specific” refers to, e.g., innate immunity or broad-spectrum immune responses.

Reply: We fully agree with your opinion; ‘non-specific’ is addressed to immunity and to clarify it we revised the text as follows ‘innate immunity biomarkers, such as…’

 

Line 42:
Add justification for the choice of sampling time points at 24 and 96 hours post-injection; explain why these intervals are biologically or experimentally relevant.

Reply: In our study, which utilized a preconditioning model to investigate LPS effects, the rationale for the 24-hour sampling point was to capture the most pronounced inflammatory response following each LPS administration. The 72-hour interval between the initial and second injection was selected to allow the acute inflammatory response from the first treatment to resolve fully before applying the subsequent challenge.

Line 46:
Suggest changing “does not involve” to “does not appear to involve” for scientific accuracy, as the study did not investigate earlier time points such as 6 hours.

Reply: Thanks a lot; the text was corrected to be deliberately careful not to overstate our findings: ‘In contrast to warm-blooded animals, LPS challenge in trout did not appear to significantly elevate CRP levels or antioxidant enzyme activity’

 

Line 122:
The phrase “biochemical and molecular genetic hallmarks” is confusing. Clarify what is meant by “molecular genetic hallmarks,” or revise to “biochemical and molecular hallmarks.”

Reply: Thank you again for careful reading and the proposed way to change confusing collocation; corrected.

 

General Language Issues:
The introduction contains frequent language errors and awkward phrasing. Examples include:

“fastest growing” (should be “fastest-growing”)

Reply: Thank you for suggestion. We substantially improved clarity, grammar, and overall readability of the text and hope it now meets the scientific criteria.

 

“traditionally applied” (preferred “traditionally used”)

Reply: Thank you for your careful reading, it has been corrected as ‘traditionally used’

 

“PGE2. animals” (fragmented and unclear). the number 2 should be adjusted

Reply: the text was revised, and the collocation 'prostaglandin E2' was kept and PGE2 was decided to be excessive (please, find out on line 89)

 

Methods

“Water recycling system” (Line ~161): Clarify if this refers to a recirculating aquaculture system (RAS), as the terminology implies different systems.

Reply: Thanks for the clarification, corrected as ‘recirculating aquaculture system’ (lines 159, 169).

 

Light Regime (Lines 173–184):
The reported “10 h/14 h (day/night)” photoperiod seems reversed; typically, the photoperiod is 14 h light/10 h dark. Please verify and correct.

Reply: Deeply grateful to you for tracking the mistake; the data is now presented accordingly as 14 h/10 h (day/night) (line 180).

 

Line 191 (Experimental challenge):
The manuscript does not specify the water temperature during the challenge exposures, nor does it state whether fish were fasted before injections.

Reply: Thank you for your comment. The information you recommended is now included in the text. Please see lines 188-190 and 184-196.

 

qPCR Primer Efficiency:
Missing data on the amplification efficiency of each primer set, also the reference genes M-value

Reply: According to your suggestion, here we placed the data on the amplification efficiency of each primer set and reference genes M-values. We consider this data as technical and excessive to be added to the main text. We also refer that the source of primer sequences is the available publications (our previous works).

 

The amplification effectiveness and M-value of the reference genes

Gene name	Amplification efficiency, E %	Slope of curve, R
Elongation Factor	100.1	0.99
ß-actin	90.0	1.00
Interleukin 1ß	103.9	0.99
Interleukin 8	91.3	0.99

 

Ranking stability of housekeeping gene based on three widely using normalization algorithms

Method	Criteria	Actin	EF1
geNorm	M-value	0.974	0.989
NormFinder	Stability value (SV)	0.085	0.176
BestKeeper	SD	0.99	1.12
	CV	5.36	6.37
	r (coeff. of corr.)	0.987	0.990
	p-value	0.001	0.001

 

Serum Bactericidal Activity (SBA) Assay:

It is unclear whether individual flasks represent independent biological replicates or technical replicates.

Reply: According to your recommendation we have specified the description of a procedure as follows: 'Into three replicate sterile flasks containing 200 mL of meat-peptone broth (MPB), a lyophilized E. coli culture was inoculated using a sterile microbiological loop. The flasks were incubated at 37 °C...' and then 'Every 24 h, the optical density at 605 nm in three aliquots from each of three flasks was measured against the blank medium...'

 

Bacterial concentration used (CFU/mL) is not reported; this value is crucial for assay repeatability and comparison.

Reply: Thank you for the addition. The authors agree that the bacterial concentration measured in CFU/mL is important to assess repeatability and accuracy of measurements; however, we assumed that it is likely not essential for group comparisons, as the same bacterial concentration (of the same stock) was used for all compared groups. We will certainly take this suggestion into account in future studies.

 

Number of technical and biological replicates per treatment should be clearly stated.

Reply: Many thanks for your suggestion. We have revised it; accordingly, it has been corrected as 'Serum of six individuals of each of two replicated tanks per group (n=12 per group) was examined for SBA in three technical replicates each'.

 

Discussion

The discussion section requires improvement. Currently, it merely summarizes previous studies without critical evaluation or explanation of discrepancies. The authors should provide reasoning for any conflicting findings. Consider addressing:

• Differences in LPS doses and fish species or strains.
• Variability in biomarkers across studies.
• Potential limitations or alternative interpretations.

Reply: We revised the Discussion to follow your suggestions. For comparisons we refer to the relevant data on other LPS doses, other fish species from available sources. We also emphasized similar and specific responses. According to available protocols, LPS used at highly variable doses and the results differ from study to study; some references and possible explanation have now been included to MS. We also explain variability in biomarkers across the studies. We clearly indicate some limitations of our experiment and tried to interpret the differences in the effects observed.

 

Conclusion

Both the doses used and the preconditioning may disqualify it as an inflammatory model, however, it provides a robust example of how management could affect immune responses.

Reply: We totally agree with your reputable opinion that we reproduced a model in the particular limitations due to temperature, doses, and preconditioning used. According to the suggestion, all the variables have now been specified in detail both in the Methods and Conclusion parts.

 

General - please provide the diet used. 

Reply: We thank the reviewer for their suggestion. We have now added the feeding schedule and diet details to the manuscript as follows: 'Following the four-week acclimation period, the fish were fed twice daily (10:00 and 19:00) with a commercial trout feed (AquaRex, Russia; 2.5 mm granule size), identical to the diet used at the source farm.'

We decided not to include the detailed feed composition as this study was not a feeding trial, and both the control and experimental fish received the same diet, ensuring that feed was not a variable in the experiment. The feed coefficient of 2.3, which was set according to the manufacturer's recommendations and adjusted for the water temperature, can be found in the manuscript (Lines 185-189).

 

Once again, thank you for your insightful comments which provide invaluable guidance for our subsequent work.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The MS identify specific biomarkers of inflammation reliable in assessing health and immune state of reared fish. My suggestions as follow:

1. In qRT-PCR assay, the ef1α and ß-actin were both selected as reference gene, how the result of gene transcripts was calculated from ΔΔCt including normalizing which reference gene? The criterion of choosing reference gene should be declared.
2. The reason of using 300 and 600 ug LPS for challenge should be
3. Line 34 and 56， the word of “Oncorhynchus mykiss” should be italic.
4. Line 138, “Tlr4” or “TLR4”?
5. Line 248, “splenocytes” or “spleen”
6. Line 272, the word of “E. coli” should be italic.
7. Line 336, what’s the mean of IQR?
8. In table 2, the difference was shown with letters, while in figure 3, the difference was present with asterisk, please uniform the statement.

Author Response

Response to Reviewer 3.

 

Comments and Suggestions for Authors

The MS identify specific biomarkers of inflammation reliable in assessing health and immune state of reared fish.

We sincerely thank the reviewer for their valuable comments. All issues have been carefully addressed: typographical and language errors corrected, scientific content refined, and the discussion strengthened.

 

My suggestions as follow: In qRT-PCR assay, the ef1α and ß-actin were both selected as reference gene, how the result of gene transcripts was calculated from ΔΔCt including normalizing which reference gene?

Reply: Thank you for your question. Our rationale was that gene expression data can be presented as a ΔΔCt, which involves normalizing the Ct values for the target gene relative to the reference gene in each sample (ΔCt), and then comparing the delta Ct values of the experimental samples with the delta Ct values of the control samples. Data can also be presented separately for the control (ΔCt contr) and experimental samples (ΔCt exp). We calculated the relative expression level of a target gene using the second formula to the power of ΔCt, where ΔCt is the difference between the threshold cycle of the reference gene and the threshold cycle of the target gene (DOI: 10.1006/meth.2001.1262; DOI: 10.1016/j.aquatox.2017.07.009).

Now in the MS: The relative transcript levels of il1ß and il8 were calculated using the ΔΔCt method [32], with normalization to the geometric mean of two reference genes, elongation factor-1 al-pha (ef1α) and ß-actin (actb).

 

The criterion of choosing reference gene should be declared.

Reply: The main criteria for selecting reference genes were stable expression, universality, and lack of variability. When selecting reference genes, we also relied on the already published protocols to optimize methodology for Oncorhynchus mykiss. According to our study, both genes, ef1α and ß-actin, could be used as reference due to their stable and predictable expression (as you can see in tables below), and the way to increase reliability of a target gene expression is in using both reference genes.

 

The amplification effectiveness and M-value of the reference genes

Gene name	Amplification efficiency, E %	Slope of curve, R
Elongation Factor	100.1	0.99
ß-actin	90.0	1.00
Interleukin 1ß	103.9	0.99
Interleukin 8	91.3	0.99

 

Ranking stability of housekeeping genes based on three widely used normalization algorithms

Method	Criteria	Actin	EF1
geNorm	M-value	0.974	0.989
NormFinder	Stability value (SV)	0.085	0.176
BestKeeper	SD	0.99	1.12
	CV	5.36	6.37
	r (coeff. of corr.)	0.987	0.990
	p-value	0.001	0.001

 

The reason of using 300 and 600 ug LPS for challenge should be

Reply: LPS dosages recommended to induce inflammation in trout are quite varied according to the published reports (references 29 to 31, Jeon et al., 2014; Rathinam et al., 2019; Huang et al., 2025). Our rationale in determining the appropriate LPS concentrations for preconditioning and stress-inducing was that desired doses should be below and above the tolerance limit of trout to LPS. In a preliminary survey with ranging LPS dosages (the results are included in the manuscript, point 2.4) we found reliable inflammatory responses, including leukocyte profile and intestinal edema to 500 μg/ml LPS. Based on these findings, we selected the concentrations for the main experiment; for the shock dose we used a slightly higher concentration (600 μg/mL) than in the preliminary survey to reduce the risk of fish mortality, while for preconditioning we chose a concentration half that of the main dose (300 μg/mL).

 

Line 34 and 56, the word of “Oncorhynchus mykiss” should be italic

Reply: Thank you very much; it has been searched and corrected throughout the text.

 

Line 138, “Tlr4” or “TLR4”?

Reply: Thanks for your question. TLR4 means a protein in human/mammals, and Tlr4 – its orthologue in fish.

 

Line 248, “splenocytes” or “spleen”

Reply: Many thanks for your suggestion. We have revised it accordingly.

 

Line 272, the word of “E. coli” should be italic.

Reply: Thank you for the clarification; it has been corrected throughout the text.

 

Line 336, what’s the mean of IQR?

Reply: Thank you for reading carefully. The data are presented as median ± 1/2 IQR; the meaning was revised in MS.

The Interquartile Range (IQR) is a measure of variability that shows the range of the middle 50% of values. It is calculated as the difference between the third quartile (Q3) and the first quartile (Q1). This is a way to show the variability, or spread, of values.

 

In table 2, the difference was shown with letters, while in figure 3, the difference was present with asterisk, please uniform the statement.

Reply: We would like to clarify the reason for using different symbols to indicate significant differences in the table and figures. In Table 2, different letters indicate the presence of significant differences between comparison groups (in pair-wise comparison) at similar p ≤ 0.05 (as stated in the sub-table description). The meaning of the asterisks in Figures 3 to 7 is that they denote different levels of significance (p-value); the lines on the figures indicate group pairs which differ substantially.

 

Once again, we sincerely thank the reviewers for their valuable time and insightful comments.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I am satisfied with the changes made by the authors. The current manuscript has been greatly improved and the findings will expand our knowledge on fish immunology.