Cloning, Characterization and Functional Analysis of Caspase 8-like Gene in Apoptosis of Crassostrea hongkongensis Response to Hyper-Salinity Stress

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Author Response

Dear Editor and Reviewers,

Thank you very much for your comments and professional advice. These opinions help to improve the academic rigor of our article. Based on your suggestions and requests, we have made corrected modifications on the revised manuscript. We hope that our work can be improved again. The detailed corrections are listed below:

1．RNAi system should be specific to the target gene. In the current study, the transcripts of all apoptotic related genes were down regulated. This may indicate that RNAi system may work against apoptotic related genes as well. Thus, the author should add experiment to show that the used RNAi system is specific against caspase 8.

Response：Thank you for your reminder. Caspase 8 was located in upstream of the apoptotic pathway. Activation of caspase 8 subsequently triggered a downstream caspase cascade (Scatena et al., 2007), which activated downstream effector proteins and ultimately led to apoptosis (Lavrik et al., 2003). On the contrary, cells deficient in caspase-8 failed to trigger downstream genes’ expression and apoptosis (Jones & Gores, 1997; Kurokawa & Kornbluth, 2009). In our study, most detected genes were downstream genes of caspase 8. Therefore, we thought that the decrease of caspase 8 transcripts resulted in down-regulation of apoptotic related genes, but not because of the non-specificity of caspase 8 RNAi system.

2. Did the author try to detect expression level of caspase 8 or other apoptotic genes at the protein level using Western plot. They can use available commercial antibodies to detect expressed proteins. I believe that detection of protein will add solid results.

Response：The western blot experiment was proved unsuccessful in our previous experiment. The main reason was that it was difficult to get specific antibody of bivalve, because bivalve has no specific immunity. The commercial mammal caspase 8CASPASE8 antibody couldn’t specific recognize the caspase 8 of Crassostrea hongkongensis. Moreover, commercial antibody with tag was not suitable for in vivo experiments. Therefore, the western blot data was not provided in this study. In fact, most bivalve researchers had the same problems and had to provide data only on RNA levels instead of protein levels (Chen et al., 2011; Fang et al., 2023; Li et al., 2015; Zhu et al., 2020)

3. line 257, 3.5. The caspase 8 activity was increased by hyper-salinity stress, but was inhibited by ChCASP8-like silence. In Fig. 5 A, almost there is no inhibition. I can see only slight inhibition compared to negative controls. Actually, results of Fig 5 are confusing! Please make sure.

Response：Yes, the caspase 8 activity was only inhibited by 9.4% after ChCASP8-like silence. However, statistical analysis showed significant difference before and after ChCASP8-like silence. Therefore, the caspase 8 activity was considered to be reduced by ChCASP8-like silence.

In Fig 5B, the caspase 3 activity indeed was not significantly inhibited by ChCASP8-like silence. As known, caspase 8 could cleave its substrates caspase 3 and caspase 7, ultimately leading to apoptosis (Degterev et al., 2003). A plausible explanation was that the decrease of caspase 8 suppressed the caspase 7 activity more than caspase 3 activity. The hypothesis need further investigation.

4. The discussion section is poor. I recommend this section to be enhanced. The author should discuss add explanation for all observations in the result section.

For example, downregulation of caspase 8 transcript in adductor muscle upon hypersalinty stress! Also add explanation for Fig 5 B, why there is no caspase 3 activity after hypersalinty exposure?

Response：Thank you for your suggestion. We have revised and expanded the discussion section in the manuscript, trying to further explain the results.

5. Minor comments - I recommend modifying the title to be mor indicative Cloning, characterization and functional analysis of caspase 8-like gene in apoptosis of Crassostrea hongkongensis response to hyper-salinity Stress

Response：Thanks to your suggestion, we have changed the title to “Cloning, characterization and functional analysis of caspase 8-like gene in apoptosis of Crassostrea hongkongensis response to hyper-salinity stress” in the manuscript.

6.Please revise the language of manuscript thoroughly.

Response：Thanks for your suggestion. We have revised the language of manuscript thoroughly, trying to make it read more smoothly.

Reference:

Chen, J., Xiao, S., Deng, Y., Du, X., & Yu, Z. (2011). Cloning of a novel glutathione S-transferase 3 (GST3) gene and expressionanalysis in pearl oyster, Pinctada martensii. Fish Shellfish Immunol, 31(6), 823-830. https://doi.org/10.1016/j.fsi.2011.07.023

Degterev, A., Boyce, M., & Yuan, J. (2003). A decade of caspases. Oncogene, 22(53), 8543-8567. https://doi.org/10.1038/sj.onc.1207107

Fang, X., Liang, B., Liang, H., & Lu, J. (2023). The role of Bcl-2 family genes in the physiology and immune response of the Pinctada fucata martensii. Aquaculture, 574. https://doi.org/10.1016/j.aquaculture.2023.739636

Jones, B. A., & Gores, G. J. (1997). Physiology and pathophysiology of apoptosis in epithelial cells of the liver, pancreas, and intestine. Am J Physiol, 273(6), G1174-1188. https://doi.org/10.1152/ajpgi.1997.273.6.G1174

Kurokawa, M., & Kornbluth, S. (2009). Caspases and kinases in a death grip. Cell, 138(5), 838-854. https://doi.org/10.1016/j.cell.2009.08.021

Lavrik, I., Krueger, A., Schmitz, I., Baumann, S., Weyd, H., Krammer, P. H., & Kirchhoff, S. (2003). The active caspase-8 heterotetramer is formed at the CD95 DISC. Cell Death Differ, 10(1), 144-145. https://doi.org/10.1038/sj.cdd.4401156

Li, C., Qu, T., Huang, B., Ji, P., Huang, W., Que, H., Li, L., & Zhang, G. (2015). Cloning and characterization of a novel caspase-8-like gene in Crassostrea gigas. Fish Shellfish Immunol, 46(2), 486-492. https://doi.org/10.1016/j.fsi.2015.06.035

Scatena, R., Bottoni, P., Botta, G., Martorana, G. E., & Giardina, B. (2007). The role of mitochondria in pharmacotoxicology: a reevaluation of an old, newly emerging topic. Am J Physiol Cell Physiol, 293(1), C12-21. https://doi.org/10.1152/ajpcell.00314.2006

Zhu, Y., Pang, Y., & Li, Q. (2020). Molecular evolution of the tnfr gene family and expression profiles in response to pathogens in lamprey(Lethenteron reissneri). Fish Shellfish Immunol, 96, 336-349. https://doi.org/10.1016/j.fsi.2019.11.037

We are very grateful to the reviewers for their valuable feedback and to the editor for the opportunity to make improvements. Corrections have been made to the manuscript in the form of revisions. The revised manuscript, figures and tables have been submitted to the system.

Sincerely yours,

Feifei Yu

College of Fisheries, Guangdong Ocean University,

Zhanjiang, Guangdong 524088, China

 

Reviewer 2 Report

Comments and Suggestions for Authors

Specific comments

-    rows 58-61: a link between the stress due to salinity fluctuation and apoptosis could be useful

-     rows 89 and 174: the authors stated that “The specific primers (ChCASP8-like-outer-F and 89 ChCASP8-like-outer-R) were designed based on the partial sequence from the 90 transcriptome of C. hongkongensis”: more detailed explanation on the choice of primers should be included, also explaining why the identified cDNA is coding for a CASP8like protein

-     row 176: due to the used method, it can not be possible state that was isolated the full-length cDNA but only the “presumed” full-length

-     row 179: a more specific statement like this should be considered: “The bioinformatic analysis of cDNA sequence do not show the presence of signal peptide etc..”

-     row 198: the authors should explain the criteria with which have choice the species used in the phylogenetical analysis; for example, why Homo sapiens and Gallus gallus have been included and not Mus musculus, Saccharomyces, and others?

-     row 220: to be corrected to “in all analyzed tissues”

-     row 224: the authors should explain better that some tissues are “immune” tissues motivating their choice in the tissue expression analysis

    Figure 3: the use of lowercase and capital letters to indicate the differences among tissues is not clear and should be revised

-     rows 234 and following: in the experiment data on gene expressin with only CASP8-like silencing without hypersalinity stress would have been useful; the authors could insert a comment on this

-     Figure 4: the acronym NC in the legend should be precised

-     row 264: the statement seems to be a quite “strong”; the level of significance is evident for the caspase 8 activity after hypersalinity but is not the same with CASP8-like silencing; the statement should be corrected

-     Figure 6: numeric data are absent: one can simply count the percentage of TUNEL positive cells in the tissue section.

-     row 294: the choice to study caspase-8-like gene to investigate apoptosis as survival pathway should be argumented

-     row 314: to state “several tissues” instead of “all tissues” could be more correct

-     rows 337-339: the statement that  “the number of TUNEL-338 positive hemocytes were significantly increased after hyper-salinity stress, while 339 significantly decreased after silencing caspase8-like” is not supported by numerical data and should be revised

Author Response

Dear Editor and Reviewers,

Thank you very much for your comments and professional advice. These opinions help to improve the academic rigor of our article. Based on your suggestions and requests, we have made corrected modifications on the revised manuscript. We hope that our work can be improved again. The detailed corrections are listed below:

Reviewer 2:

1.rows 58-61: a link between the stress due to salinity fluctuation and apoptosis could be useful

Response：Thank you for your reminder. We have added the relationship between salinity stress and apoptosis in the third paragraph of the introduction section of the manuscript, as following. Salinity fluctuations were a key environmental factor that affects the reproduction, growth, development, and survival of aquatic animals. Changes in salinity created osmotic gradients between intra- and extracellular of aquatic species. If not compensated for, these changes may disrupt cell volume, impair protein function and ultimately lead to death (Abdelrahman et al., 2017). Therefore, shellfish living in estuaries must adjust their physiology to maintain homeostasis in the intracellular environment of their organisms during periods of fluctuating salinity (Burnett et al., 2007; Kültz, 2015). However, prolonged exposure to high salinity can result in a situation where the immunity of shellfish can decline rapidly, predisposing them to outbreaks of various diseases and mass mortality (Cheng et al., 2016; Lacoste et al., 2002). Apoptosis is a crucial process in oyster cells that effectively eliminates damaged, senescent, and infected cells without triggering inflammation (Xiao et al., 2018).

2. rows 89 and 174: the authors stated that “The specific primers (ChCASP8-like-outer-F and 89 ChCASP8-like-outer-R) were designed based on the partial sequence from the 90 transcriptome of C. hongkongensis”: more detailed explanation on the choice of primers should be included, also explaining why the identified cDNA is coding for a CASP8like protein

Response：Thanks to your suggestion. We have added a description of the primers at 2.3 of the Materials and Methods in the revised manuscript. Based on the ChCASP8-like partial sequences in the transcriptome data, specific primers were designed using Primer Premier 5.0 (https://www.premierbiosoft.com) (Table 1). The ChCASP8-like-outer-F and UMP primers were used to amply 3’ sequence of 667 bp, and ChCASP8-like-outer-R and UMP primers were used to amply 5’ sequence of 606 bp. The PCR products were detected using a gel imaging system (Bio-Rad, USA), and the target fragments were collected.

3.row 176: due to the used method, it can not be possible state that was isolated the full-length cDNA but only the “presumed” full-length

Response：After splicing “presumed” full-length using 3’ and 5’ regions sequence, the ChCASP8-like-test-F and ChCASP8-like-test-R primers were used to amplify and confirm the presumed full-length. The amplified fragment’s sequence was the same as splicing full-length sequence. Therefore, we thought the sequence was indeed the full-length, more than “presumed” full-length. The full-length cDNA was obtained by this method in other researches, which support our opinion (Fang et al., 2023; Li et al., 2015).

4. row 179: a more specific statement like this should be considered: “The bioinformatic analysis of cDNA sequence do not show the presence of signal peptide etc.”

Response：As suggested, we corrected the sentence as “The bioinformatics analysis of cDNA sequences did not reveal signal peptides or transmembrane domains.” in 3.1 of the Results section of the revised manuscript.

5. row 198: the authors should explain the criteria with which have choice the species used in the phylogenetical analysis; for example, why Homo sapiens and Gallus gallus have been included and not Mus musculus, Saccharomyces, and others?

Response：The species choose of phylogenetical analysis based on the species affinities and caspase 8 similarity. For example, most caspase 8 or caspase 8-like gene from bivalves with high simalirity were used in the phylogenetic tree. For alien species, only one species was usually chosen as representatives, such as Homo sapiens representing mammal, Gallus gallus representing bird. Moreover, we did not find the caspase 8 gene or caspase 8 gene in Saccharomyces.

6. row 220: to be corrected to “in all analyzed tissues”

Response：As suggested, in 3.3 of the results section of the revised manuscript, we have corrected “in all tissues” to “in all analyzed tissues.”

7.row 224: the authors should explain better that some tissues are “immune” tissues motivating their choice in the tissue expression analysis

Response：Thanks to your careful review. ChCASP8-like transcripts were significantly up-regulated in immune tissues such as gills, hepatopancreas and hemocytes after 48 h of hyper-salinity stress. We have made changes and additions to section 3.3 in the Results section of the revised manuscript.

8.Figure 3: the use of lowercase and capital letters to indicate the differences among tissues is not clear and should be revised

Response：Thanks to your suggestion. In Figure 3, uppercase letters represent variations in caspase 8-like gene expression after salt stress. Lowercase letters indicated the expression differences of caspase 8-like gene before salt stress. Different letters meant significant differences, while identical letters indicated nonsignificant difference.

 

9. rows 234 and following: in the experiment data on gene expression with only CASP8-like silencing without hypersalinity stress would have been useful; the authors could insert a comment on this.

Response：We are sorry we haven’t detect the gene expression with only CASP8-like silencing without hyper-salinity stress. The main reason was that our aim was to analyze the capase8-like genes in apoptosis after salinity stimulation of C. hongkongensis. So we thought the sample under salinity stress was more suitable as the control. We speculated that CASP8-like silencing without hyper-salinity stress would significantly affect the gene expression, because the apoptosis was not activated.

10. Figure 4: the acronym NC in the legend should be precised

Response：The initials “NC” in Figure 4 referred to the negative control group. It has been labeled in the legend in revised manuscript.

11. row 264: the statement seems to be a quite “strong”; the level of significance is evident for the caspase 8 activity after hypersalinity but is not the same with CASP8-like silencing; the statement should be corrected

Response：Thank you for the reminder. The sentence has been rewritten as “The data showed that caspase 8 activity was significantly activated by hyper-salinity stress, but was slightly reduced by ChCASP8-like silencing, consistent with apoptotic genes expression analysis” in section 3.5 of the revised manuscript results.

12. Figure 6: numeric data are absent: one can simply count the percentage of TUNEL positive cells in the tissue section.

Response：As suggested, after hyper-salinity stress, approximately 87.5% of the hemocytes showed DNA breaks in the positive cells. After silencing ChCASP8-like, fewer DNA breaks were observed in hemocytes, even under hyper-salinity stress, in approximately 36.4% of the positive cells. This percentage of positive cells has been added to the revised manuscript results at 3.6.

13. row 294: the choice to study caspase-8-like gene to investigate apoptosis as survival pathway should be argumented

Response：Thanks to your suggestion. Caspase 8 genes are members of the caspase family, which act as initiators in apoptosis by activating downstream effector proteins that promote apoptosis. The caspase 8 associated with Fas-associated protein with death domain (FADD) to form the death-inducing signaling complex (DISC), undergoed self-cleavage and activated downstream caspase proteins to trigger apoptosis.

14. row 314: to state “several tissues” instead of “all tissues” could be more correct

Response：As suggested, in the fourth paragraph of the revised manuscript discussion, we have replaced “ all tissues” with “several tissues”.

15. rows 337-339: the statement that “the number of TUNEL-338 positive hemocytes were significantly increased after hyper-salinity stress, while 339 significantly decreased after silencing caspase8-like” is not supported by numerical data and should be revised

Response：Thanks to your suggestion. As suggested, after hyper-salinity stress, approximately 87.5% of the hemocytes showed DNA breaks in the positive cells. After silencing ChCASP8-like, fewer DNA breaks were observed in hemocytes, even under hyper-salinity stress, in approximately 36.4% of the positive cells. This percentage of positive cells has been added to the revised manuscript results at 3.6.

Reference:

Abdelrahman, H., ElHady, M., Alcivar-Warren, A., Allen, S., Al-Tobasei, R., Bao, L., Beck, B., Blackburn, H., Bosworth, B., Buchanan, J., Chappell, J., Daniels, W., Dong, S., Dunham, R., Durland, E., Elaswad, A., Gomez-Chiarri, M., Gosh, K., Guo, X., . . . Zhou, T. (2017). Aquaculture genomics, genetics and breeding in the United States: current status, challenges, and priorities for future research. BMC Genomics, 18(1), 191. https://doi.org/10.1186/s12864-017-3557-1

Burnett, K. G., Bain, L. J., Baldwin, W. S., Callard, G. V., Cohen, S., Di Giulio, R. T., Evans, D. H., Gómez-Chiarri, M., Hahn, M. E., Hoover, C. A., Karchner, S. I., Katoh, F., Maclatchy, D. L., Marshall, W. S., Meyer, J. N., Nacci, D. E., Oleksiak, M. F., Rees, B. B., Singer, T. D., . . . Crawford, D. L. (2007). Fundulus as the premier teleost model in environmental biology: opportunities for new insights using genomics. Comp Biochem Physiol Part D Genomics Proteomics, 2(4), 257-286. https://doi.org/10.1016/j.cbd.2007.09.001

Cheng, B. S., Chang, A. L., Deck, A., & Ferner, M. C. (2016). Atmospheric rivers and the mass mortality of wild oysters: insight into an extreme future? Proc Biol Sci, 283(1844). https://doi.org/10.1098/rspb.2016.1462

Fang, X., Liang, B., Liang, H., & Lu, J. (2023). The role of Bcl-2 family genes in the physiology and immune response of the Pinctada fucata martensii. Aquaculture, 574. https://doi.org/10.1016/j.aquaculture.2023.739636

Kültz, D. (2015). Physiological mechanisms used by fish to cope with salinity stress. J Exp Biol, 218(Pt 12), 1907-1914. https://doi.org/10.1242/jeb.118695

Lacoste, A., Malham, S. K., Gélébart, F., Cueff, A., & Poulet, S. A. (2002). Stress-induced immune changes in the oyster Crassostrea gigas. Developmental and Comparative Immunology, 26(1), 1-9. https://doi.org/10.1016/s0145-305x(01)00067-2

Li, C., Qu, T., Huang, B., Ji, P., Huang, W., Que, H., Li, L., & Zhang, G. (2015). Cloning and characterization of a novel caspase-8-like gene in Crassostrea gigas. Fish Shellfish Immunol, 46(2), 486-492. https://doi.org/10.1016/j.fsi.2015.06.035

Xiao, S., Wong, N. K., Li, J., & et al. (2018). Analysis of in situ Transcriptomes Reveals Divergent Adaptive Response to Hyper- and Hypo-Salinity in the Hong Kong Oyster, Crassostrea hongkongensis. Front Physiol, 9, 1491. https://doi.org/10.3389/fphys.2018.01491

We are very grateful to the reviewers for their valuable feedback and to the editor for the opportunity to make improvements. Corrections have been made to the manuscript in the form of revisions. The revised manuscript, figures and tables have been submitted to the system.

Sincerely yours,

Feifei Yu

College of Fisheries, Guangdong Ocean University,

Zhanjiang, Guangdong 524088, China

 

Reviewer 3 Report

Comments and Suggestions for Authors

The article by Lin et al., details a study investigating acute (48 h) apoptotic related effects of the oyster, C. hongkongensis, associated with a transfer to hypersaline conditions.  The authors cloned and performed qPCR analysis of the caspase 8-like gene to examine tissue expression profiles and changes in response to salinity.  Additional apoptosis related genes were also similarly analyzed.  Caspase activity was also assessed as was a histological analysis using the TUNEL assay to evaluate apoptosis.  The authors also implemented gene silencing for ChCASP8-like using RNAi.

General Comments:

Overall, this is an interesting study that will aid in furthering our understanding of the effects of acute hypersalinity exposure on the economically important oyster species, C. hongkongensis.  My main concerns are that some of the key details related to the experimental design and methodology are either missing or unclear.  Moreover, the Discussion is largely a review of the results and little context and comparison with other relevant studies is provided. 

Specific Comments:

Line 15:  suggest omitting second use of “two”

Line 39:  what is meant by “mitochondrial cells”?

Lines 42-43:  please revise “downstream caspases protein kinases”; I assume the authors left out an “and”

Line 57:  “estuarine” is an adjective; I assume the authors meant “estuaries”

Line 58:  suggest “euryhaline” rather than “eurysalinity”

Lines 72-73:  please include SEM for these mean values

Line 35:  is 3 days sufficient time for acclimation of oysters prior to testing?

Line 82:  how much RNA was used as template?  What type of primers were used – poly-T, random hexamers?

Table 1:  suggest including a column for amplicon sizes for those entries that produce a PCR product

Lines 92-93:  please clarify the purpose of the test PCR – what nucleotides are being linked?

Line 108-112:  replication in this section is not clear as described; please revise.  first, there are two tanks described, but later the experiment is described as being performed in triplicate.

Lines 113-119:  please clarify replication for all tissue samples – only hemolymph is clearly described

Lines 130-131:  again sampling and replicates are not clearly defined here

Line 137:  “contains” seems like an inappropriate word here

Line 140:  this method assumes reaction efficiencies near 100% - did the authors confirm reaction efficiencies for all primer pairs. also, were amplicons sequence verified and was a melt analysis performed after each experiment to confirm specificity?  Were negative controls included?  Was beta-actin confirmed to not change with experimental treatment?

Section 2.7:  why was Caspase 3 investigated?  this is not explained until the Discussion, which is too late

Line 170:  an ANOVA is not performed to determine mean values – please revise.

Figure 4:  technically, gene expression refers to protein synthesis, so I suggest that “mRNA” be added after “Relative” on y axis.  What tissue is this?  Please include in figure caption.

Line 292:  suggest instead “threat of fluctuating salinity.”

Line 310: suggest starting sentence with “This” rather than “The”

Line 333:  TUNEL should be spelled out on first use, not here late in the Discussion

 

Comments on the Quality of English Language

The manuscript is reasonably well organized, but suffers a bit from poor grammar times.  I have pointed out some of the errors, but there are many more, so I suggest the authors have a native English speaker review if possible. 

Author Response

Dear Editor and Reviewers,

Thank you very much for your comments and professional advice. These opinions help to improve the academic rigor of our article. Based on your suggestions and requests, we have made corrected modifications on the revised manuscript. We hope that our work can be improved again. The detailed corrections are listed below:

1.Line 15: suggest omitting second use of “two”

Response: Thank you for your reminder. We have removed the second “two” in the abstract of the revised manuscript.

2.Line 39:what is meant by “mitochondrial cells”?

Response: We apologize for our carelessness. Due to my carelessness in adding the extra word “cell”, we have corrected the first paragraph of the preface of the revised manuscript.

3. Lines 42-43: please revise “downstream caspases protein kinases”; I assume the authors left out an “and”

Response: In the first paragraph of the introduction to the revised manuscript, we have revised “downstream caspases protein kinases” as “downstream caspases proteins”.

4.Line 57: “estuarine” is an adjective; I assume the authors meant “estuaries”

Response: Thanks to your reminder. The “estuarine” has been changed to “estuaries” in the third paragraph of the introduction to the revised manuscript.

5.Line 58:  suggest “euryhaline” rather than “eurysalinity”

Response: We apologize for our carelessness. The “eurysalinity” has been changed to “euryhaline” in the third paragraph of the introduction to the revised manuscript.

6.Lines 72-73: please include SEM for these mean values

Response: Thank you for your reminder. We added SEM images of these averages to section 2.1 of the revised manuscript in the Materials and Methods.

7.Line 75: is 3 days sufficient time for acclimation of oysters prior to testing?

Response: We thought 3 days is sufficient for oysters to acclimatize to their new environment. Our opinion was supported by previous studies (Ertl et al., 2019; JO et al., 2008; Xie et al., 2022), which showed that the oyster adaptation period typically fell within the range of 3-7 days.

8.Line 82: how much RNA was used as template? What type of primers were used – poly-T, random hexamers?

Response: Thank you for your reminder. 0.1 μg of RNA was used as a template and cDNA was synthesized using 1 μL of 0.1 μg/μL of random primers. The description was added into the revised manuscript.

9.Table 1: suggest including a column for amplicon sizes for those entries that produce a PCR product

Response: As suggested, we have added a column for PCR products size in Table 1.

10.Lines 92-93: please clarify the purpose of the test PCR – what nucleotides are being linked?

Response: The test PCR was performed to validate the spliced cDNA sequence. In section 2.3 of the revised manuscript Materials and Methods, we have revised it to “The full-length cDNA sequence of ChCASP8-like was validated by conducting a test-PCR using primers”.

 

11.Line 108-112:  replication in this section is not clear as described; please revise.  first, there are two tanks described, but later the experiment is described as being performed in triplicate.

Response: We are sorry we confused you. The two tanks were used for hyper-salinity seawater (40‰) and natural seawater (20‰) treatment. But in every treatment group, 5 individuals were collected for subsequent gene expression analysis. We have added the description into the revised manuscript.

 

12.Lines 113-119: please clarify replication for all tissue samples – only hemolymph is clearly described

Response: Thank you for your reminder. In section 2.4 of the revised manuscript Materials and Methods, we have clarified the replication of all tissue samples by adding the description “After 0 and 48 h of salt stress, adductor, mantle, gill, hepatopancreas and gonad of 5 individuals were collected for subsequent gene expression analysis.”

 

13.Lines 130-131: again sampling and replicates are not clearly defined here

Response: Thank you for your reminder. In the second paragraph of section 2.5 of the revised manuscript Materials and Methods, the hemocytes from 3 individuals were mixed as a sample, and tissues 5 parallel samples were collected for TUNEL assay. The gill, hepatopancreas, mantle, adductor muscle and gonads was collected from each experimental individual for gene expression analysis and caspase activity analysis. 5 parallel samples were collected. We have added the description into the revised manuscript.

 

14.Line 137: “contains” seems like an inappropriate word here

Response: As suggested, we have replaced “contains” with “conditions” in section 2.6 of the revised manuscript Materials and Methods.

15.Line 140: this method assumes reaction efficiencies near 100% - did the authors confirm reaction efficiencies for all primer pairs. also, were amplicons sequence verified and was a melt analysis performed after each experiment to confirm specificity?  Were negative controls included?  Was beta-actin confirmed to not change with experimental treatment?

Response: Yes. Before qRT-PCR experiments, we validated the specificity of all primers used. The melt analysis was performed after each experiment to confirm the specificity. The transcript level of β-actin was dectected as internal control. The relative expression level of each target gene was calculated by specific gene/ β-actin.

16.Section 2.7: why was Caspase 3 investigated?  this is not explained until the Discussion, which is too late

Response: Caspase-3 is an effective cysteine protease primarily responsible for cleaving structural and regulatory proteins in the nucleus and cytoplasm to either activate or inactivate them. It cleaves a wide variety of intracellular substrates, including major structural elements of the cytoplasm and nucleus, components of the DNA repair machinery, and several protein kinases, ultimately leading to apoptosis (Kurokawa & Kornbluth, 2009; Xu et al., 2016). We have added the description into the revised manuscript.

17.Line 170: an ANOVA is not performed to determine mean values – please revise.

Response: Thank you for your reminder. In section 2.9 of the revised manuscript Materials and Methods, we have made a change to “A one-way analysis of variance (ANOVA) was performed to determine the significant difference in different samples by SPSS (Chicago, USA)”.

18.Figure 4: technically, gene expression refers to protein synthesis, so I suggest that “mRNA” be added after “Relative” on y axis.  What tissue is this?  Please include in figure caption.

Response: As suggested, we have added “mRNA” to the y-axis in Fig. 4 and in the figure caption indicated the gill tissue used in the manuscript.

19.Line 292:  suggest instead “threat of fluctuating salinity.”

Response: As suggested, in the first paragraph of the discussion of the revised manuscript, we have changed “threaten of salinity-fluctuating” to “threat of fluctuating salinity”.

20.Line 310: suggest starting sentence with “This” rather than “The”

Response: As suggested, in the third paragraph of the discussion of the revised manuscript, we have changed“The”to “This”.

21.Line 333: TUNEL should be spelled out on first use, not here late in the Discussion

Response: We have added the full name of “TUNEL” at the first time it appeared in Introduction part.

22.Comments on the Quality of English Language

The manuscript is reasonably well organized, but suffers a bit from poor grammar times.  I have pointed out some of the errors, but there are many more, so I suggest the authors have a native English speaker review if possible. 

Response: Thanks to your suggestion. We have tried our best to modify the manuscript and make it more fluent.

Reference:

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We are very grateful to the reviewers for their valuable feedback and to the editor for the opportunity to make improvements. Corrections have been made to the manuscript in the form of revisions. The revised manuscript, figures and tables have been submitted to the system.

Sincerely yours,

Feifei Yu

College of Fisheries, Guangdong Ocean University,

Zhanjiang, Guangdong 524088, China

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors responded to all my comments and inquiries and the manuscript can be accepted for publication.