Effects of Short-Term Salinity Stress on Ions, Free Amino Acids, Na⁺/K⁺-ATPase Activity, and Gill Histology in the Threatened Freshwater Shellfish Solenaia oleivora

Round 1

Reviewer 1 Report

Zhang et al…have analysed the osmotic response under high salinity stress conditions in a freshwater mollusc, observing an increase in total amino acid content in haemolymph and a drop in NKA activity in different tissues as well as alterations in gill histology.

In general, the manuscript is well structured, has a well defined objective with an experimental design adequate for the purposes of the study and the results are exposed efficiently.

However, the authors should clarify a number of doubts in the material and methods section:
1. Although in the text the authors indicate that the individuals were acclimatised in the laboratory for one week, the number of individuals at the beginning of the experiment (acclimatisation) is not indicated, nor how many are distributed for each treatment.

2. It seems that the inviduals in the control group came from the wild. Were they not acclimatised for 1 week? Please explain this section.

3. Explain the term "three duplications" (line 89). Would it not be better to indicate that it was done in triplicate?
4. In the legend of the graphs, n=6 is specified. This means, then, that the authors took two individuals from each treatment. The biochemical analyses were carried out individually or the tissues from each treatment were homogenised together.
5. Why the Duncan test was chosen instead of the Tukey test?
6. Finally, with the number of results obtained, I would like to point out the lack of a component variance analysis (PCA) as well as a redundancy analysis (RDA).

7. Review the citation of Kulac et al., 2013 in the text (line 201) and unify the citation form in the references section.

Author Response

Dear reviewer,

On behalf of my co-authors, we appreciate you for the positive and constructive comments and suggestions on our manuscript entitled “Effects of high salinity stress on ion, free amino acids, Na+/K+-ATPase activity, and gill histology in the threatened freshwater shellfish Solenaia oleivora” (ID: fishes-1986796). We have studied the reviewer’s comments carefully and revised our manuscript according to these comments. The revisons we made were highlighted with red in the document named “revised manuscript”.

Here below is our description of revision according to the reviewers’ comments.

Comments and Suggestions for Authors

Zhang et al…have analysed the osmotic response under high salinity stress conditions in a freshwater mollusc, observing an increase in total amino acid content in haemolymph and a drop in NKA activity in different tissues as well as alterations in gill histology.

In general, the manuscript is well structured, has a well defined objective with an experimental design adequate for the purposes of the study and the results are exposed efficiently.

However, the authors should clarify a number of doubts in the material and methods section:
1. Although in the text the authors indicate that the individuals were acclimatised in the laboratory for one week, the number of individuals at the beginning of the experiment (acclimatisation) is not indicated, nor how many are distributed for each treatment.

Response: Thanks for the meaningful suggestion and sorry for our unclear descriptions in the manuscript. After 1 week of acclimation, fifty-four S. oleivora were randomly divided into six groups (0 h, 3 h, 6 h, 12 h, 24 h, and 48 h) with 9 individuals and triplicate in each group. We have revised it in the manuscript (line 96-97).

2. It seems that the inviduals in the control group came from the wild. Were they not acclimatised for 1 week? Please explain this section.

Response: Thanks for the meaningful suggestion. The mussels used in this experiment were bred and cultured artificially in this institution, and all mussels including the control group were acclimatized for 1 week before the formal experiment. We have explained it in the manuscript (line 96-97).

3. Explain the term "three duplications" (line 89). Would it not be better to indicate that it was done in triplicate?

Response: Thanks for the meaningful suggestion. It should be in triplicate. We have revised it in the manuscript (line 97).

4. In the legend of the graphs, n=6 is specified. This means, then, that the authors took two individuals from each treatment. The biochemical analyses were carried out individually or the tissues from each treatment were homogenised together.

Response: Thanks for the meaningful suggestion. After each tissue was homogenized separately, homogenates of two tissues per replicate in the same group were mixed proportionally. There were three mixed tissue homogenates in each group for physiological and biochemical detection. We have explained it in the manuscript (line 117-119).

5. Why the Duncan test was chosen instead of the Tukey test?

Response: Thanks for the meaningful suggestion. Both Tukey and Duncan tests are effective methods for multiple comparisons in one-way ANOVA. Among them, Duncan analysis is suitable for the comparison of every two treatments in the agriculture and biology research. This study actually belongs to the field of agriculture and biology. Hence, Duncan test was chosen in this study.

6. Finally, with the number of results obtained, I would like to point out the lack of a component variance analysis (PCA) as well as a redundancy analysis (RDA).

Response: Thanks for the meaningful suggestion. We have added the PCA analysis in the manuscript (Abstract : line 25-28; Results: line 189-203: Discussion: line 286-298).

Abstract section: Principal component analysis (PCA) showed that the first two principal components (PC1 and PC2) cumulatively explained 77.6 % of the total variation. The NKA activity was positively associated with PC1, while ion concentration and most FAA were negatively associated with PC1.

Results section: 3.5. PCA analysis

Regarding PCA (Figure 5), the first two principal components (PC1 and PC2) cumulatively explained 77.6 % of the total variation. The NKA activities in the four tissues were positively associated with PC1. Ion concentration and most FAA were negatively associated with PC1. Furthermore, all salinity stress groups were clearly separated to control group. The distance between control group and 4 salinity stress groups (6 h, 12 h, 24 h, and 48 h) was greater than that between control group and 3 h group. The 0 h and 3 h groups were close to the NKA, and other groups were closed to FFA and inorganic ion.

Discussion section: The PCA analysis showed that NKA activity was positively associated with PC1, and ion concentration and most FAA were negatively associated with PC1, suggesting again NKA activity that NKA, inorganic ions, and FAA played an important role in the osmoregulation of S. oleivora. The distance between the control group and the 4 salinity stress groups (6 h, 12 h, 24 h, and 48 h) was greater than that between the control group and the 3 h group. This result indicated that with the extension of stress time, the difference between the salinity stress group and the control group was greater. Furthermore, the 0 h and 3 h groups were close to the NKA, and other groups were close to FFA and inorganic ions. This finding implied that at the initial stage (0-3 h) of salinity stress, NKA was mainly involved in the osmoregulation of S. oleivora, but with the extension of stress time, inorganic ions and FAA were mainly responsible for the osmoregulation. In general, the osmoregulation mechanism of S. oleivora in a hypertonic environment is multidimensional.

7. Review the citation of Kulac et al., 2013 in the text (line 201) and unify the citation form in the references section.

Response: Thanks for the meaningful suggestion. We have revised it in the manuscript (line 233).

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript describes the osmotic responses of a freshwater shellfish, Solenaia oleivora following short-term exposure to salinity stress. Results short that S. oleivora are  osmoconformers, and the inorganic ions, FAA, NKA, and changes in gill structures have important roles in the osmoregulation process of these organisms.

The manuscript is straightforward and provides significant information in the biology of this species of shellfish that would lead towards conservation efforts. I have some comments/suggestions, which I hope the authors will take into consideration when they revise the manuscript.

1. In the Introduction, the authors should provide a strong justification how this study is relevant in the conservation efforts of this species considering that they have mentioned in the title that this is a threatened species.

2. In line 89, authors wrote, " three duplications in each group". What do they mean by this? It is in triplicate. Please change the term that is being used in the manuscript.

3. Authors used a salinity of  2.2‰, and they have stressed that this is a high salinity level. Is this level of salinity considered as high? Kindly qualify the use of high salinity in your manuscript.

4. Shellfish were sampled at different time intervals up to 48 hrs. What is the basis of using 48hrs? Authors should explain and contextualize whether this is considered as short-term or long-term exposure.

5. Where is the data on survival? This has to be given in support of how the shellfish respond to salinity changes.

6. Authors had a sample size of 6 shellfish in all their assays. However, in their groups they mentioned each group had 3 duplicates (triplicate). How many animals were there in each replicate? If the authors used 6 shellfish, how did the authors collect the 6 animals? Kindly elucidate. In my opinion, 6 animals per group considering that there were 3 replicates in each group is few. It would have been better to increase the sample size, for example, to 3 per replicate or for a total of 9 animals per group.

7. Please provide better resolution of Fig 4, especially on the structures of the tissues. It is better to provide photos from at least 3 shellfish at each time point and the structures have to be clearer and bigger so that the readers could immediately see the differences.

I hope the authors will take these suggestions into consideration when they revise the manuscript.

Author Response

Dear reviewer,

On behalf of my co-authors, we appreciate you for the positive and constructive comments and suggestions on our manuscript entitled “Effects of high salinity stress on ion, free amino acids, Na+/K+-ATPase activity, and gill histology in the threatened freshwater shellfish Solenaia oleivora” (ID: fishes-1986796). We have studied the reviewer’s comments carefully and revised our manuscript according to these comments. The revisons we made were highlighted with red in the document named “revised manuscript”.

Here below is our description of revision according to the reviewers’ comments.

Comments and Suggestions for Authors

The manuscript describes the osmotic responses of a freshwater shellfish, Solenaia oleivora following short-term exposure to salinity stress. Results short that S. oleivora are  osmoconformers, and the inorganic ions, FAA, NKA, and changes in gill structures have important roles in the osmoregulation process of these organisms.

The manuscript is straightforward and provides significant information in the biology of this species of shellfish that would lead towards conservation efforts. I have some comments/suggestions, which I hope the authors will take into consideration when they revise the manuscript.

1. In the Introduction, the authors should provide a strong justification how this study is relevant in the conservation efforts of this species considering that they have mentioned in the title that this is a threatened species.

Response: Thanks for the meaningful suggestion. Our results elucidated the osmotic response and osmoregulatory mechanisms of S. oleivora under hypertonic conditions. These findings indicated that S. oleivora has a system to cope with hypertonic stress, which can adjust its osmotic pressure to a certain extent to adapt to the change of environmental salinity. This study made up the gap in osmotic pressure of S. oleivora, accumulated data for the study of environmental adaptability of S. oleivora, and provided a theoretical basis for its habitat protection and artificial breeding. We have revised it in the manuscript (line 79-86).

2. In line 89, authors wrote, " three duplications in each group". What do they mean by this? It is in triplicate. Please change the term that is being used in the manuscript.

Response: Thanks for the meaningful suggestion. We have revised it in the manuscript (line 97).

3. Authors used a salinity of 2‰, and they have stressed that this is a high salinity level. Is this level of salinity considered as high? Kindly qualify the use of high salinity in your manuscript.

Response: Thanks for the meaningful suggestion. The high salinity in this study is a relative concept. High salinity only refers to a higher salinity than the control group. In order to improve the legibility of the manuscript, we did not use the description of high salinity in the revised manuscript.

4. Shellfish were sampled at different time intervals up to 48 hrs. What is the basis of using 48hrs? Authors should explain and contextualize whether this is considered as short-term or long-term exposure.

Response: Thanks for the meaningful suggestion. On the one hand, we referred to the study of Cai et al. [1], who reported that within 48 h, the osmotic pressure of Anadara broughtonii increased first and then tended to be stable under the stress of high salinity (35‰ and 40‰) stress. On the other hand, our previous study showed that the antioxidant, immune, transcriptome and respiratory metabolism indexes of Solenaia oleivora changed significantly after salinity stress for 48 h [2-3]. The purpose of this study was to investigate the osmotic response of S. oleivora under short-term salinity stress. Hence, the experiment time of this study was set as 48 h. The experiment duration of this study only lasted up to 48 h, which was a short-term exposure experiment. We have statemented this in the title of the revised manuscript.

References

[1] Cai, X., Zhang, X., Tian, L.,Zhang, P. Effect of salinity stress on hemolymph osmolality and gill Na+/K+-ATPase activity of juvenile ark shell (Anadara broughtonii). South China Fisheries Science, 2015. 11(2): 12-19.

[2] Zhang, T., Yao, J., Xu, D., Ma, X., Jin, W., Lv, G., Gu, R., Wen, H.,Zhou, Y. Gill physiological and transcriptomic response of the threatened freshwater mussel Solenaia oleivora to salinity shift. Comp Biochem Physiol Part D Genomics Proteomics, 2021. 40: 100913.

[3] Lv, G., Zhang, T., Ding, T., Xu, D., Zhan, M., Gu, R., Xu, P., Ma, X., Jin, W., Dong, X., Wen, H. Effect of salinity on survival, respiration, feeding metabolism and energy budget of juvenile freshwater mussel Solenaia oleivora. Journal of Dalian Ocean University, 2022, 5: 802-808.

5. Where is the data on survival? This has to be given in support of how the shellfish respond to salinity changes.

Response: Thanks for the meaningful suggestion. The data on survival of S. oleivora is shown in the figure below. The data on survival of S. oleivora was included in our previous published paper and we have cited this result in the introduction of manuscript (line 71-73).

6. Authors had a sample size of 6 shellfish in all their assays. However, in their groups they mentioned each group had 3 duplicates (triplicate). How many animals were there in each replicate? If the authors used 6 shellfish, how did the authors collect the 6 animals? Kindly elucidate. In my opinion, 6 animals per group considering that there were 3 replicates in each group is few. It would have been better to increase the sample size, for example, to 3 per replicate or for a total of 9 animals per group.

Response: Thanks for the meaningful suggestion and sorry for our unclear descriptions in the manuscript. S. oleivora were randomly divided into six groups (0 h, 3 h, 6 h, 12 h, 24 h, and 48 h) with 9 individuals and triplicate in each group. The gills, hepatopancreas, adductor muscle, axe foot, and hemolymph of S. oleivora (2 individuals from each replicate and 6 individuals in each group) were collected for the detection of hemolymph osmotic pressure, ion concentration, enzyme activity and free amino acids. The gill tissue of the remaining 3 individuals in each group was collected for histological examination. We have revised it in the manuscript (line 102-105).

7. Please provide better resolution of Fig 4, especially on the structures of the tissues. It is better to provide photos from at least 3 shellfish at each time point and the structures have to be clearer and bigger so that the readers could immediately see the differences.

Response: Thanks for the meaningful suggestion. We have provided photos of three shellfish in each group in Figure S1. In addition, we have replaced the original photos in Figure 4 with  clearer and bigger photos.

Author Response File: Author Response.docx