Vibrio parahaemolyticus becomes lethal to post-larvae shrimp via acquiring novel virulence factors

ABSTRACT Translucent post-larvae disease (TPD), caused by Vibrio parahaemolyticus (Vp TPD), has become an emerging shrimp disease, affecting more than 70%–80% of coastal shrimp nurseries in China in spring 2020. Here, we investigated the key virulence factors of Vp TPD by analyzing protein fragments, related genomic information, as well as experimental challenge tests. After investigating the toxic effects of purified protein fragments with different molecular weights (MWs) from Vp TPD, we found that only the protein fragments with MW >100 kDa showed similar lethality to live Vp TPD in the experimental challenge test using post-larvae shrimp. Meanwhile, similar histopathological changes exhibiting in the hepatopancreas and midgut of the diseased individuals were observed in the post-larvae shrimp challenged with either bacterial protein fragments (MW >100 kDa) or live Vp TPD. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry analyses, two novel proteins, Vibrio high virulent protein (VHVP)-1 and VHVP-2, were identified as the candidates of key virulence factors to cause TPD. Moreover, VHVP-1 and VHVP-2 were found to be encoded by two genes (vhvp-1 and vhvp-2) tandemly located on a 187,791-bp plasmid and were predicted to depend on the same promoter following a comparative genomic analysis. Further epidemiological investigation and challenge test indicated that the V. parahaemolyticus isolate carrying only the vhvp-1 gene and lacking vhvp-2 gene could not cause mortality of experimental Penaeus vannamei post-larvae. The mutant (Δvhvp-2) by deleting vhvp-2 gene could only cause 4.92% of accumulative mortality of post-larvae that is similar to the non-Vp TPD Vibrio strain. Additionally, the complemented strains, Δvhvp-2/pBT3-vhvp-2 and Δvhvp-2/pwtCas9-vhvp-2, showed similar virulence to the wild-type Vp TPD. The results demonstrated that V. parahaemolyticus becomes lethal to post-larval shrimp by acquiring the VHVP-2 virulence factor. This study sheds light on further investigations of the pathogenic mechanism of Vp TPD and the development of strategies for early diagnosis of TPD in shrimp hatcheries. IMPORTANCE As a severe emerging shrimp disease, TPD has heavily impacted the shrimp aquaculture industry and resulted in serious economic losses in China since spring 2020. This study aimed to identify the key virulent factors and related genes of the Vp TPD, for a better understanding of its pathogenicity of the novel highly lethal infectious pathogen, as well as its molecular epidemiological characteristics in China. The present study revealed that a novel protein, Vibrio high virulent protein-2 (MW >100 kDa), is responsible to the lethal virulence of V. parahaemolyticus to shrimp post-larvae. The results are essential for effectively diagnosing and monitoring novel pathogenic bacteria, like Vp TPD, in aquaculture shrimps and would be beneficial to the fisheries department in early warning of Vp TPD emergence and developing prevention strategies to reduce economic losses due to severe outbreaks of TPD. Elucidation of the key virulence genes and genomics of Vp TPD could also provide valuable information on the evolution and ecology of this emerging pathogen in aquaculture environments.


COVER LETTER.
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• Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process.Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript." Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me.If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail.Arrangements for payment must be made before your article is published.For a complete list of Publication Fees, including supplemental material costs, please visit our website.Q9: Fig. 2b, it is surprising that only a few major protein bands were detected in the >100 kDa fraction of the Vp lysate.This fraction contains a fourth band of around 100 kDa, which should be mentioned.The whole-cell lysate should be included as a control in this Figure or in Fig S1 .R9: From the electrophoretogram of denaturing SDS-PAGE of the bacterial lysate, there were only 3 proteins larger than 100 kDa, the fourth band in the electrophoretogram of SDS-PAGE was obviously less than 100 kDa in molecular weight.According to the instruction manual of the ultrafiltration membrane we used, the 100 kDa type ultrafiltration membrane can theoretically only intercept the protein that is obviously larger than 100 kDa in molecular weight, that is to say, the Vp TPD protein intercepted by ultrafiltration membrane of 100 kDa type is larger than 100 kDa.Therefore, there seems to be no more reason to analyze the fourth band of in the electrophoretogram of SDS-PAGE which is obviously smaller than 100 kDa.In addition, R10: Following the reviewer's suggestion, the Vhvp protein in the Figure 2c has been changed to Vp TPD _4-2-2 in the revised manuscript.Q11: Regarding protein identification by mass spectrometry.Based on their statement, they claim to have identified proteins by mass spectrometry prior to genomic analysis of the strain causing TPD.In this case, a protein with specific peptides can be determined, but it is not identical to the target protein.Should describe accurately.As they note that WP_269169668 and APX09935.1 are already in the GenBank, these proteins and the strains harboring them should be discussed more.

R11:
According to the process of Vp TPD virulence gene identification in this study, we added adjunct word in front of the "virulence gene" to make the result description more accurate in the revised manuscript.We used "candidate virulence gene" in the analysis of SDS-PAGE and mass spectrometry, "putative virulence gene" in the analysis of comparative genome analysis, PCR epidemiological analysis, and "key virulence gene" in the analysis of knockout and compensation experiment.Further discussion of high homologous protein of putative virulence protein of Vp TPD has been added in the revised manuscript.Q12: Fig. 3a legend: should describe Vp TPD (left) and Vp1616 (right).The style of genome maps should be unified.R12: In order to better differentiate between the two strains, Vp TPD (left) and Vp 1616 (right), we have used different colors to express and display their genomic circular map.and we hope to keep the pictures of the two stains of Vp TPD (left) and Vp 1616 (right) in different colors in the manuscript for the convenience of readers to distinguish them.

Q13:
Are those genome sequences deposited in the public database?R13: The genome sequences has been deposited to NCBI, and the accession number has been included in the revised manuscript.

Q14:
Ln 155: "they depended on the same promoter in the plasmid".How did they claim this without any experimental data?This is also the case in the Abstract.

R14:
We have amended the sentence according to your suggestion and added "predict" to describe the actual situation of the possibility of sharing a predicted promoter of the virulence factor vhvp-1 and vhvp-2 in the genome in the revised manuscript.R15: It's been amended in the revised manuscript.Q16: Fig. 3d: why do they connect two protein sequences and display them as a single sequence?It is difficult to understand.The image quality is so bad.R16: The Fig. 3d has been changed in the revised manuscript according to your suggestions for better understanding of the two protein sequences.Q17: Fig. 4a, Is this vhvp-2?Do SpvB and TcdB_TN mean domains?Clarify.In addition, if images of amplicons are included in the diagram, at least match primer positions and amplicon widths.R17: The Fig. 4a has been amended for better understanding in the revised manuscript according to your suggestions.And the primer position and amplified fragment length has also been shown in details in the revised manuscript.Q18: Fig. 4b.Lane numbers are necessary in this case.

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R18: Lane numbers has been added in the revised manuscript according to your suggestion.Q19: Fig. 5a.Indicate the dose and the number of samples.R19: the dose and the number of samples has been added in the section of "Materials and Methods" revised manuscript.Q20: Complementation should be included in Fig. 5a.R20: The complementary experiment has been done, and the results have been shown in the new Fig.5b in the revised manuscript according to your suggestion.Q21: Fig. 5b is unnecessary (or should be moved to supplemental figures).
R21：This sketch map could help readers to understand the content quickly and efficiently, and we have revised the Fig. 5b.and hope to keep it in the manuscript.Q22: Table 1.The targeted genes should be vhvp-2, not SpvB and TcdB?R22: The names of targeted genes have been corrected in the revised Table 1.Q23. Discussion can be reconsidered to be more informative for readers.R23: At your suggestion, the discussion has been revised, particularly to make it more informative for readers.Q24: Ln 221-228, a conventional method is just described.R24: We cited this conventional method to explain that the method used in this study is widely accepted and widely used in identifying the newly virulence proteins.Q25: Ln 234-252.Readers would want to know the discussion of the results rather than this introductory information.R25: Considering the very similar of the histopathological characteristics of Vp TPD infection in the P. vannamei are similar to those of acute hepatopancreaticne-crosis disease (AHPND).As the research progress and results of Vp AHPND may have important implications of enlightenment for the research of pathogenicity and diversity of Vp TPD , so we summarized the research process and results of the diversity of Vp AHPND pathogens here.Following your suggestion, the discussion of the results has also been added to the paragraph.Q26: Are VHVP-1 and VHVP-2 secreted?Have immersion experiments using purified proteins been examined?R26: VHVP-1 and VHVP-2 were not shown to be secreted as the supernatant after centrifugation of the bacterial solution did not show any virulent to the shrimp post larvae in our infection experiments.Thank you for submitting your manuscript to Microbiology Spectrum.Your manuscript has been re-reviewed by a previous reviewer and there are still many concerns with the revision, all of which need to be addressed.In addition, the reviewer noted in comment 1 that the complementation experiments need to have wild-type, deletion mutant and complemented strain analyzed together, which was not done in Fig. 5b.Both the reviewer and myself still have concerns about various grammatical errors and I suggest that you have it professionally proofread before resubmission.
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Reviewer comments:
Reviewer #1 (Comments for the Author): 1.The main criticism is regarding the infection experiment with the complemented strains, which is presented as Figure 5b in the revision.Figure 5a with WT and the deletion strain and Figure 5b with WT and complemented strains are clearly separated.This should be done including the deletion strain for comparison, otherwise it lacks legitimacy.
3. Ln 98-106.The purpose of this study is to investigate the virulence factors of Vp causing TPD and its epidemiology as stated by the authors.Nevertheless, this paragraph describing inactivation is confusing and wastes space for the reader.The reasons given in the rebuttal letter also do not seem logical and do not support the need for this section.There needs to be a reconsideration of this paragraph or a logical reason for its inclusion in this paper.4. Ln 112: "100% mortality was reached" to "mortality reached 100%" 5. Ln 116: "infected with <100 kDa proteins".Infecting proteins?7. Ln 142.A brief explanation should be provided here as to why VpTPD_4-2-2 (aconitate hydratase B) was excluded from subsequent analyses.
14. Ln 242-244: The claim that VHVP protein is not secreted must be taken with caution.Do these proteins possess a signal peptide?The culture conditions and data are not provided, so the details are unclear, but if these proteins were not detected in the culture supernatant under a single culture condition, this only indicates that they are not secreted under that specific condition.These should be described appropriately.15.Ln 258-261: "Interestingly ---, indicating that the strains carrying them should have been prevalent in the world for some time".It is unclear whether this claim is valid or just local, without information on the year and location of isolation of these strains.
16. Information about plasmid 2 (other than the presence of tra genes) is useful in considering the origin and evolution of the plasmid carrying this virulence gene and should be discussed further.S1, I would like to suggest that it would be better to separate it from Figure 4 and combine it with the summary of Table S1 results as an independent figure.

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Response to Reviewer 1 Specific comments Query 1(Q1):
The main criticism is regarding the infection experiment with the complemented strains, which is presented as Figure 5b in the revision.Figure 5a with WT and the deletion strain and Figure 5b with WT and complemented strains are clearly separated.This should be done including the deletion strain for comparison, otherwise it lacks legitimacy.
Response 1 (R1): Thank you very much for your comments.The challenge tests of the wild-type, deletion mutant and complemented strains has been analyzed together and shown in the revised manuscript.Q2: I would like to recommend authors to check carefully before submitting as a professional.To give some examples, Ln 78, "hepatopancreatic ne-crosis"; Ln 191, "V.Owens" and "V.cambes"; Ln 229, remove the comma; Ln 239, remove "that of"; Ln 350, "famed"; Ln 465, "primes"; italicize gene names and Vibrio throughout.R2: The various grammatical errors have been amended carefully.Q3: Ln 98-106.The purpose of this study is to investigate the virulence factors of Vp causing TPD and its epidemiology as stated by the authors.Nevertheless, this paragraph describing inactivation is confusing and wastes space for the reader.The reasons given in the rebuttal letter also do not seem logical and do not support the need for this section.There needs to be a reconsideration of this paragraph or a logical reason for its inclusion in this paper.R3: Even if the virulence challenge assay is performed using the same amount of (total protein) of initial concentration of live Vp TPD , ultrasonically disrupted Vp TPD and different molecular weight potential virulence proteins of Vp TPD , live Vp TPD will continue to proliferate and increase its toxic effect by continuously producing virulence proteins.To eliminate the risk that ultrasonic disruption might not completely inactivate live Vp TPD , leading to the possibility that different molecular weights potential virulence proteins might carry residual active Vp TPD in subsequent experiments, we first tested the effect of thermal inactivation and ultrasonic disruption to inactivate Vp TPD .We have added the explanation in the revised manuscript.Q4: Ln 112: "100% mortality was reached" to "mortality reached 100%" R4: The text has been amended.Q5: Ln 116: "infected with <100 kDa proteins".Infecting proteins?R5: The text has been amended.Q6: Ln 123.Fig 1c, not 1d.Indicate how long the sample was infected with Vp TPD .R6: The error of figure number labeling has been amended and the time has been added in the revised manuscript.Q7: Ln 142.A brief explanation should be provided here as to why Vp TPD _4-2-2 (aconitate hydratase B) was excluded from subsequent analyses.R7: Both Vp TPD _4-2-1 and Vp TPD _4-2-2 were selected as the candidate virulence factors I and II of Vp TPD for further analysis, and Vp TPD _4-2-3 were excluded from subsequent analyses, as aconitate hydratase B is not a virulence protein according to previous reports.The brief explanation has been added in the revised manuscript.Q8: Ln 158.What are "deduced candidate virulence proteins I and II"? R8: The "deduced candidate virulence proteins I and II" has been revised to "deduced candidate virulence factors I and II", which firstly mentioned in the above paragraph in the revised manuscript.Q9: Ln 168: "depend on the same promoter" How did they predict that those genes depend on the same promoter?Rather, 'operon' is preferred in this case.R9: Here we used BPROM [1], which is a classic bacterial sigma70 promoter recognition program used in many publications, to predict the bacterial promoters.Reference: [1] V. Solovyev, A Salamov (2011) Automatic Annotation of Microbial Genomes and Metagenomic Sequences.In Metagenomics and its Applications in Agriculture, Biomedicine and Environmental Studies (Ed.R.W. Li), Nova Science Publishers, p. 61-78 Q10: Ln 202-204.The strain 20211213002-3 carrying vhvp-2 but lacking vhvp-1, appears without explanation.Given that the infection experiment with this strain shown in Fig. S5 is key to demonstrating the contribution of vhvp-2, but not vhvp-1, to virulence, a detailed explanation of how this strain was found should be provided.R10: The V. parahaemolyticus isolate 20211213002-3 was isolated as a common Vibrio in the epidemiological investigation as description in the M&M.The detailed information has been included in the context and the Supplemental Table1.Q11: Fig S5a: What is the PCR target region for?R11: The PCR primer set of Vp TPD -vhvp-1-F/R, targeting the conserved domain of TcdA, was designed to test the virulence gene of vhvp-1.And this description has been added in the revised figure caption of S5 Fig. Q12: Table S1.It is unclear which primer set is used to determine the plus/minus of Vp TPD .The results of PCR using vhvp-1F/R, vhvp-2F/R and vhvp-2F2/2R2 primers should be accurately described.R12: Both of the primer sets of Vp TPD -vhvp-1-F/R Vp TPD -vhvp-2-F/R have been used to test the potential virulence genes of the collected bacteria isolates, and the results have been added in the revised Table S1.We amended the Table S1 and added note to make this point clear in the revised manuscript.Q13: Ln 220: Δvhvp-2/pBT3-vhvp-2 and Δvhvp-2/pwtCas9-vhvp-2, instead of Δvhvp-2c+(P6-5) and Δvhvp-2c+(P3-2).Also, explain why the inducible vector was used here.R13: According to the reviewer's suggestion, we changed the names in original lines of Line 39, Line 224, Line 449, Line 470, Line 473, Line 717, Line 807, Line 809 in the revised manuscript For illustration of that the recovery of bacterial pathogenicity after complement has nothing to do with the complement methods, we conducted the complement experiment by using two different complement methods commonly used at present, including the methods based on both of non-inducible [2] and inducible [3] plasmids according to previous reports.References: [2] Zhang WW, Sun K, Cheng S, Sun L. Characterization of DegQVh, a serine protease and a protective immunogen from a pathogenic Vibrio harveyi strain.Appl Environ Microbiol.2008.74, 6254-6262.
[3] Liu X, Wang X, Sun B, Sun L. The Involvement of Thiamine Uptake in the Virulence of Edwardsiella piscicida.Pathogens.2022.11(4):464.Q14: Ln 242-244: The claim that VHVP protein is not secreted must be taken with caution.Do these proteins possess a signal peptide?The culture conditions and data are not provided, so the details are unclear, but if these proteins were not detected in the culture supernatant under a single culture condition, this only indicates that they are not secreted under that specific condition.These should be described appropriately.R14: The culture conditions have been shown in the section "Bacterial strains and growth conditions".Your comment is accurate and exact, and so we assed description of that the Vp TPD is not secreted under that specific condition in the revised discussion.Q15: Ln 258-261: "Interestingly ---, indicating that the strains carrying them should have been prevalent in the world for some time".It is unclear whether this claim is valid or just local, without information on the year and location of isolation of these strains.R15: The detailed information about the strains carrying the highly homologous protein gene has been added, and the context has been amended in the revised manuscript.Q16: Information about plasmid 2 (other than the presence of tra genes) is useful in considering the origin and evolution of the plasmid carrying this virulence gene and should be discussed further.R16: Thank you very much for your comments.The origin and evolution of plasmid 2 information is a very interesting issue.In consideration of that the primary concern in the present study is the key virulence factor, we did not discuss the origin and evolution of plasmid 2 in the discussion.The vhvp gene was identified in V. natriformis, V. Campbellii and V. alginolyticus, suggesting the high risk of horizontal transmission of the vhvp gene, so the presence of tra genes was also discussed in the manuscript.Q17: Ln 293-306.just repeating the results.R17: The paragraph has been compressed and refined in the revised manuscript according to your comment.Q18: Ln 307-329.Not just a list of past observations, I would like to see more discussion on the function of VHVP-2 based on the already-known information.R18: The context has been revised according to your comment.Q19: Ln 376.Is the Vp TPD strain used in this study identical to Vp-JS20200428004-2?It is not known from previous literature [Ref 3] and should be clearly indicated here and in Table 1.R19: Vp TPD strain used in this study is Vp-JS20200428004-2 and it was indicated here and in Table1.Q20: Ln 446.Accession numbers should be provided.R20: Accession numbers have been provided in the revised manuscript.Q21: Ln 471: "The resulting plasmid ---into the indicated Δvhvp-2+(3-2) strain by electroporation".This description is not appropriate.R21: The description has been changed to "The resulting plasmid pwtCas9-vhvp-2 was introduced into Δvhvp-2 strain by electroporation."Q22: Ln 502.Make the primer names consistent with those in Table 3. R22: The primer names have been revised to be consistent with those in Table 3. Q23: The legend for  S1, I would like to suggest that it would be better to separate it from Figure 4 and combine it with the summary of Table S1 results  Thank you for submitting your manuscript to Microbiology Spectrum.As you will see your paper is very close to acceptance.I have found some additional minor grammatical changes that I feel should be made.Please modify the manuscript along the lines I have recommended below.As these revisions are quite minor, I expect that you should be able to turn in the revised paper very quickly.

Specific comments
Line 29: Remove the word that.The sentence should read...post larvae shrimp challenged with either... Line 37: deleating should be changed to deleting Line 39: complement should be changed to complemented Line 42: the word shed should be changed to sheds Line 54: change word "of" to the word "to".The sentence should read.....and would be beneficial to the fisheries department... Lines 100-106.The section within these lines does not make sense (at least to me).Is it possible to simply delete this section and start with line 106?This could now read as: "We first tested the effects of thermal inactivation and ultrasonic disruption to inactivate.....If this doesn't work for you, please clarify this section to be more clear.
When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues raised by the reviewers as file type "Response to Reviewers," not in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only".Please use this link to submit your revised manuscript.Detailed instructions on submitting your revised paper are below.

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Editor, Microbiology Spectrum Reviewer comments:

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according to the reviewer's suggestion, the whole-cell lysate has been included as a control in Fig S1 in the revised manuscript.Q10: Figure 2c, to be shown as VpTPD_4-2-2 instead of Vhvp protein.
Zhang Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences Qingdao China Re: Spectrum00492-23R1 (Vibrio parahaemolyticus becomes lethal to shrimp post-larvae for acquiring novel virulence factors) Dear Dr. Qingli Zhang:
11. Fig S5a: What is the PCR target region for?12. Table

17 .
Ln 293-306.just repeating the results.18. Ln 307-329.Not just a list of past observations, I would like to see more discussion on the function of VHVP-2 based on the already-known information.19.Ln 376.Is the VpTPD strain used in this study identical to Vp-JS20200428004-2?It is not known from previous literature [Ref 3] and should be clearly indicated here and in Table 1 .

20 .
figure.25.Ln 811: "at the same pathogen dose".Indicate dose.26.Fig 1a, 2a and 5c: It is rather a waste of space for the reader to illustrate such common methods, which may be excluded or moved to supplement.27.Fig 3a.If the authors used different colors for 'the convenience of readers to distinguish them' as in their response letter, the presented style makes unnecessary confusion for the reader.The label VpTPD or Vp1616 is sufficient to distinguish them with a consistent style.Furthermore, VpTPD should be in panel a, and Vp1616 should be in panel b.28.Fig 3a and b.It would be better if the displayed size of chromosomes and plasmids reflect their actual size.29.Fig 3d.Indicate as VHVP-1 and VHVP-2 here, not GE005140 and GE005139 in the legend and RF+1 superfamily in the figure.Also, the quality of the figure is still poor.Should be reconstructed using the protein sequence (size indicates bp) and made clear to the reader.30.Fig 4c: Since Figure 4c is related to TableS1, I would like to suggest that it would be better to separate it from Figure4and combine it with the summary of TableS1results as an independent figure.
• Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred

Fig 1 .
The infection dose should be also indicated in figure legends.R23: The infection dose has been also indicated in figure legends.Q24: Ln 800: "Schematic structure of ---gene".This description needs to be changed to properly describe the content of the figure.R24: The description has been changed according to your suggestion.Q25: Ln 811: "at the same pathogen dose".Indicate dose.R25: The dose has been indicated in the revised title caption.Q26: Fig 1a, 2a and 5c: It is rather a waste of space for the reader to illustrate such common methods, which may be excluded or moved to supplement.R26: For many potential readers, especially the shrimp farmers, the illustration of the methods would be very helpful for easier and better understanding of the present study.Q27: Fig 3a.If the authors used different colors for 'the convenience of readers to distinguish them' as in their response letter, the presented style makes unnecessary confusion for the reader.The label Vp TPD or Vp 1616 is sufficient to distinguish them with a consistent style.Furthermore,VpTPD should be in panel a, and Vp 1616 should be in panel b.R27: The genomic chromosome figures of Vp TPD and Vp 1616 have been re-labelled according to your suggestions.Q28: Fig 3a and b.It would be better if the displayed size of chromosomes and plasmids reflect their actual size.R28: The size of chromosomes and plasmids has been clearly labelled in the figures.The authors have attempted to adjust the size of figures of chromosomes and plasmids to reflect their actual size, and the layout of the figures is extremely incongruous.Q29: Fig 3d.Indicate as VHVP-1 and VHVP-2 here, not GE005140 and GE005139 in the legend and RF+1 superfamily in the figure.Also, the quality of the figure is still poor.Should be reconstructed using the protein sequence (size indicates bp) and made clear to the reader.R29: The Fig3d has been redrawn.The vhvp-1 and vhvp-2 have been highlighted according to your suggestions, and GE005140/GE005139 have been added in parentheses as an explanation to show the corresponding virulence genes in the Vp TPD genome.Q30: Fig 4c: Since Figure 4c is related to Table as an independent figure.R30: Fig. 4c has been redrawn.And the new Fig.4c includes the map and the summary of Table S1 results as you suggested.Zhang Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences Qingdao China Re: Spectrum00492-23R2 (Vibrio parahaemolyticus becomes lethal to post-larvae shrimp via acquiring novel virulence factors) Dear Dr. Qingli Zhang: