Effects of Temperature and Salinity on the LMS (Lysosomal Membrane Stability) Biomarker in Clams Donax trunculus and Chamelea gallina

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Ms: Effect of temperature and salinity on the biomarker LMS (Lysosomal Membrane Stability) in clams Donax trunculus and Chamelea gallina.

In this study, the effects of temperature and salinity variations were evaluated on two mollusc species, namely Donax trunculus and Chamelea gallina. Molluscs were exposed under controlled laboratory conditions for 21 days. The animals were subjected to nine different combinations: 16 three temperature levels (12, 20 and 27.5 °C) and three salinity ranges (27-28, 32-33 and 37-38). During exposure, mollusc mortality was checked daily, whereas after exposure only one biomarker was measured in molluscs, namely lysosomal membrane stability in haemocytes.

Although I appreciated efforts made by the authors to assess effects of two important environmental parameters on molluscs, in my opinion results obtained are a bit informative. Consequently, the manuscript is not suitable for publication in its present form.

-        First of all, it is important to highlight that only one biomarker (LMS) was measured in molluscs. It is too little to be able to reach exhaustive conclusions on the effects of temperature and salinity on molluscs.

-        As well highlighted by the author themselves, “LMS in bivalve hemocytes is a core 84 biomarker of general stress of chemical pollution that has been widely recommended by different organizations such as the Barcelona Convention, International Council for Exploration of the Sea (ICES), and Oslo and Paris Conventions (OSPAR) and implemented in mussel monitoring programs of chemical pollution [33–35]”. Therefore, it is not clear why a contaminant toxicity biomarker was used, rather than other physiological biomarkers, in addition to the condition index.

-        Why were the hemolymph and the condition index evaluated only after 21 days and not at time intervals? Probably, after such a long period the animals have acclimatised to the experimental conditions, while an analysis at shorter times could have highlighted important responses of the molluscs.

-        As for statistical analysis, the authors used parametric (1-way ANOVA test) or Welch ANOVA (non-parametric test) when the assumption of homogeneity of variance was rejected to reveal differences between samples. I have serious doubts about this approach. For a more impressive evaluation of the results obtained, a multivariate data analysis approach should be used to assess the effects of the variables and their interaction.

-        Why didn't the authors measure other parameters of the seawater in the exposure tanks, such as pH and percent oxygen saturation?

-        It is unclear whether the animals were fed during the 21 days of exposure.

-        Considering the few biomarkers measured in this to evaluate the effects of temperature and salinity variations, I highlight that many sentences in the manuscript are speculative. See lines 309-311, 412-413, 422-424.

-        Lines 390-393: the authors have correctly summarized the important roles of molluscan haemocytes. So, I ask, why weren't other cellular parameters measured which would surely have helped the authors obtain more comprehensive results?

Comments on the Quality of English Language I suggest the authors have the English text checked by a native speaker,
because there are syntax errors in the manuscript.

 

Author Response

The authors would like to indicate to the Editor that they have considered all the reviewers' comments in the resubmission process. The authors thank the reviewers for all comments and suggestions that significantly contributed to improving the quality and clarity of the resubmitted manuscript.

Each reviewer comment is shaded in grey, and our clarifications appear directly below it. 

Reviewer #1:

1. First of all, it is important to highlight that only one biomarker (LMS) was measured in molluscs. It is too little to be able to reach exhaustive conclusions on the effects of temperature and salinity on molluscs.

The authors (we) thank the constructive comments of the reviewer. We would like to indicate that it was beyond the scope of this work to study the effects of temperature and salinity on different subcellular and physiological indicators in bivalves. As stated in the title of the manuscript, we investigated only the effects of temperature and salinity on the lysosomal membrane stability (LMS) in hemocytes of two marine clam species. The reasoning behind lays on the following facts

1.  hemocytes represent the primary line of internal defense for bivalves and a decreased functionality of them represent a direct key factor for survival.
2. LMS, in addition of being a robust biomarker of cytotoxicity (at the cellular-tissue level) it is also found to be a prognostic indicator for putative pathologies, and as such is an integrated pathophysiological indicator of health status and a proxy for possible effects at the population level (Moore et al., 2012) (indicated in the manuscript, lines 94-97).

Furthermore, the alterations in LMS can be used as a biomarker of organism health condition, and environmental assessment criteria has been established to assess properly the responses when using certain techniques, such as the Neutral Red Retention assay (the one used in our study).

1. We assessed our results of neutral red retention time (NRRT; min) against the background assessment criteria (BAC) and environmental assessment criteria (EAC) developed for the technique (Davies et al., 2012). NRRT shorter than the EAC level suggested the organism were severely stressed and probably exhibiting pathology. NRRT shorter than the BAC level but longer than the EAC level was considered to represent stressed but compensating organism (indicated in the resubmitted manuscript, lines 375-378).

Accordingly, we believe that results of LMS measured in living hemocytes of Donax trunculus and Chamelea gallina under different environmental conditions are robust enough to infer conclusions on the effects of temperature and salinity on these mollusk’s well-being.

However, after considering comment of the reviewer we found that the abstract was not properly addressed. Consequently, we have substantially modified the text in the abstract for a better understanding of the purpose of our study (see new abstract in the resubmitted version):

“Population of clams of Donax trunculus and Chamelea gallina have been declining significantly in recent decades, and environmental pollution and accelerated global warming have been proposed as contributing factors to this decline, in addition to overfishing. Lysosomal membrane stability (LMS) is sensitive indicator of health status of the organisms. In this study we investigate the LMS in these species after exposure for 21 days to nine combined conditions of water temperature (12, 20 and 27.5 °C) and salinity ranges (27-28, 32-33 y 37-38). LMS was assessed in living hemocytes by using the neutral red retention assay. Mortality and condition index of the organisms were evaluated as supporting parameters. The results indicated interspecies differences in the LMS under similar environmental conditions. Overall, LMS was found more sensitive to temperature than to salinity changes. Although both species can tolerate changes in either salinity or temperature seawater conditions, the tolerance range is narrower for D. trunculus, showing a significant cytotoxicity (NRRT <50 min) at temperature above 27.5 °C and salinities above 32, and 100% mortality at 27.5 °C and a low salinity range (27-28). This study is the first to assess the combined effect of temperature and salinity on the LMS in C. gallina and D. trunculus, and provide necessary information before using LMS as contaminant-related biomarker in field studies with these species”.

Moore, M. N.; Viarengo, A. G.; Somerfield, P. J.; Sforzini, S. Linking Lysosomal Biomarkers and Ecotoxicological Effects at Higher Biological Levels. Ecol. Biomark. Indic. Ecotoxicological Eff. 2012, 107–130.

Davies, I. M.; Gubbins, M.; Hylland, K.; Thain, J.; Maes, T.; Martínez-Gómez, C.; Giltrap, M.; Burgeot, T.; Wosniok, W.; Lang, T. 30 Technical Annex: Assessment Criteria for Biological Effects Measurements. Integr. Mar. Environ. Monit. Chem. Their Eff. 2012, 209.

 

2. As well highlighted by the author themselves, “LMS in bivalve hemocytes is a core biomarker of general stress of chemical pollution that has been widely recommended by different organizations such as the Barcelona Convention, International Council for Exploration of the Sea (ICES), and Oslo and Paris Conventions (OSPAR) and implemented in mussel monitoring programs of chemical pollution [33–35]”. Therefore, it is not clear why a contaminant toxicity biomarker was used, rather than other physiological biomarkers, in addition to the condition index.

We appreciate the comment of the reviewer on this topic because it has allowed us to improve the clarity on LMS as biomarker as well as the meaning and the value of our study in the resubmitted manuscript.

Keeping into account this comment, explanations given below and a new reference have been incorporated in the resubmitted version of the manuscript (see lines 88-92; 94-97; 97-101).

First of all, it must be underlined that LMS is an indicator of organism health status and it is also affected by non-contaminant factors (Moore N.M., 2012). Furthermore, LMS is a core biomarker of general stress of chemical pollution recommended/required by Regional Marine Conventions and expert groups in mussel monitoring programs of chemical pollution. This recognition is supported by an extensive knowledge on how LMS respond in mussels to extrinsic abiotic and intrinsic biotic factors, independently but also additionally to contaminants. However, the same cannot be said for the clam species we investigated.

Given the fact that a variety of factors, in addition to overfishing, such as environmental pollution has been proposed to explain the population significant declines of Donax trunculus and Chamelea gallina mollusks in the Mediterranean Region, interest and opportunities arise to use LMS as a general stress biomarker of chemical environmental pollution in these species (indicated in the manuscript, see lines 48-59). Several studies have shown that temperature and salinity are abiotic factors that affect lysosomal membrane stability and they may act as confounding factors when using LMS as a biomarker of environmental chemical pollution. Therefore, the use of LMS to monitor impact of chemical environmental pollution on these clam species should involve knowing the response of the LMS biomarker to the temperature and salinity variability to which these species might be subjected in their habitats, as they may act as confounding factors for the assessment of this biomarker.

Moore N.M.  2012. Background document: lysosomal stability as a global health status indicator in biomonitoring. Technical Annex 9. 68-70. In Davies, I. M. and Vethaak, A. D. 2012. Integrated marine environmental monitoring of chemicals and their effects. ICES Cooperative Research Report No. 315. 277 pp.

 

3. Why were the hemolymph and the condition index evaluated only after 21 days and not at time intervals? Probably, after such a long period the animals have acclimatised to the experimental conditions, while an analysis at shorter times could have highlighted important responses of the molluscs.

The choice of a 21-day sustained exposure period was made because we were looking for a time frame that reflected realistic environmental conditions of the different temperature-salinity regimes in our study area. In the southern sector of the Gulf of Valencia, temperatures of 27-28 ºC and 12-13 ºC persist for 21 days to one month (Historical data of the Valencia buoy, Puertos del Estado). Changes in salinity are associated with fluvial inputs, but mainly with groundwater flows that occur after heavy rainfall (Sospedra et al., 2018). These changes can be of more variable duration but often close to a month.

In order to better understand the reason for the chosen period, the following sentence has been added in the new version of the manuscript (lines 142-144): “Organisms were exposed for 21 days as this period reflects the time frame of sustained exposure to different salinity-temperature regimes in the southern sector of the Gulf of Valencia”.

Puertos del Estado. (s.f.). Boyas y mareógrafos. https://www.puertos.es/es-es/oceanografia/paginas/portus.aspx (accessed 2024-02-27).

Sospedra, J., Niencheski, L. F. H., Falco, S., Andrade, C. F., Attisano, K. K., & Rodilla, M. (2018). Identifying the main sources of silicate in coastal waters of the Southern Gulf of Valencia (Western Mediterranean Sea). Oceanologia, 60(1), 52-64.

 

4. As for statistical analysis, the authors used parametric (1-way ANOVA test) or Welch ANOVA (non-parametric test) when the assumption of homogeneity of variance was rejected to reveal differences between samples. I have serious doubts about this approach. For a more impressive evaluation of the results obtained, a multivariate data analysis approach should be used to assess the effects of the variables and their interaction.

We found this comment of the reviewer quite confusing. The suggestion of the reviewer to make a multivariate analysis considering all biomarker responses would be certainly appropriated, but unfortunately it is not possible in our study given that we only analyze a single dependent variable, the lysosomal membrane stability. Nevertheless, following the reviewer's recommendations, the statistical treatment of the data was modified. A Multifactor ANOVA procedure was used to determine whether or not there are significant differences between the means of NRRT, %LMS and %CI at the different levels of the factors (temperature and salinity) and whether or not there are interactions between the factors. Assumptions for using multifactor ANOVA include normal distribution of the data and homogeneity of variances.

In the first version of the manuscript, we could not perform this type of analysis because some of the data did not follow a normal distribution even though we had tried some simple transformations of the raw data to make the transformed data normal (i.e. squared values, square root, reciprocal of the data, logarithm of the data). 

Due to the reviewer's comments, we still try to transform the data, but this time we use BOX-COX transformation (lambda for the Box-Cox transformation was found using the Most Likely Estimate (MLE) approach). In this way, our transformed data could give rise to a normal distribution and thus use a two-way ANOVA.

In the new version of the manuscript, the Data analysis section was modified (lines 191-203), and the new results were incorporated in Tables 2 and 3; Figures 2, 4 and 6; and in the text in the results section.

 

5. Why didn't the authors measure other parameters of the seawater in the exposure tanks, such as pH and percent oxygen saturation?

The pH of seawater in the exposure tanks, not mentioned in the first version, has been included in the manuscript (lines 210-211). pH measurements were taken at the beginning and at the end of the experiment (after 21 days), obtaining values between 8 and 8.2. Daily measurements were not carried out because it was not considered a significant factor that could alter under the experimental conditions. This is because no contaminants were introduced into the aquaria and the pH of the seawater itself is very stable.

Conversely, dissolved oxygen was measured daily (as indicated in lines 138-140). Additionally, in lines 210-211, it is indicated that in all treatments, the percentage of saturation was 100%. Furthermore, dissolved oxygen values in mg/L for each treatment condition can be seen in Table 1 (in results). Dissolved oxygen levels were recorded in two ways: concentration in mg/L and percent oxygen saturation (% sat.). Following the reviewer's suggestion and in order to improve the understanding of the results, a new column has been added in Table 1 showing the percent oxygen saturation values.

 

6. It is unclear whether the animals were fed during the 21 days of exposure.

We would like to highlight that this aspect is elucidated in line 122 of the original manuscript, where it explicitly mentions the daily feeding regimen of clams with microalgae, specifically Isochrysis galbana.

 

7. Considering the few biomarkers measured in this to evaluate the effects of temperature and salinity variations, I highlight that many sentences in the manuscript are speculative. See lines 309-311, 412-413, 422-424.

Unfortunately, we disagree with this comment of the reviewer. The objective of our study was to specifically investigate the effect of temperature and salinity on the biomarker LMS (Lysosomal Membrane Stability) in clams Donax trunculus and Chamelea gallina but not to investigate the effect of temperature and salinity on a battery of sub-organismal biomarkers. This is clearly stated in the objectives and in the title itself of the manuscript.   Consequently, we consider that presentation of our results and conclusions are not speculative.

• 309-311: this study is the first to assess the combined effect of the environmental factors temperature and salinity on the stability of the lysosomal membrane in gallina and D. trunculus.

Authors find that sentence 309-311 is not speculative and it has been written on the bases of currently available in the scientific literature (see manuscript lines 331-339)

“Earlier works with C. gallina and other bivalve species (e.g. Crassostrea gigas, Ostrea edulis, Mytilus sp.) have found that LMS is sensitive to temperature fluctuations in both laboratory and field studies [50–52]. In contrast, few studies have evaluated the combined effects of both factors on LMS, in species such as Ostrea edulis, Ruditapes philippinarum and Anadara trapezia [53–55]. While it has been observed that phagocytic activity in C. gallina is differently altered depending on whether environmental parameter change individually or in combination [56], this study is the first to assess the combined effect of the environmental factors temperature and salinity on the stability of the lysosomal membrane in C. gallina and D. trunculus.”

• 412-413: This methodological adjustment would contribute to enriching the research and provide a more complete understanding of the environmental effects on the organisms studied.

Authors find that sentence 412-413 can be improved for a better understanding but it lacks of a speculative character. On the contrary, in this sentence we recognize the limitation of our study and propose new avenues for future research on effects of water temperature on biomarker responses in these clam species. Anyhow, we have rephrased the paragraph in the resubmitted version of the manuscript to avoid any misunderstanding or speculative interpretation (see manuscript lines 440-445)

 “After conducting this study, certain limitations were identified that could be addressed in future research. Kruft Welton et al. [20] states that infaunal species, such as ours, may not be negatively affected by climate change stressors due to their ability to burrow as a strategy to avoid high temperatures. However, Monari et al (2007) found that the Chamelea gallina kept at 20 and 25 °C burrowed completely, whereas at 30 °C the clams progressively emerged from the sediment and then remained on the surface. Nonetheless, including a sand bed in the aquaria during the experimental period would provide results ecologically more relevant concerning the effects of water temperature on the LMS in these species with burying behaviour.”

• 422-424: Although both species can tolerate changes in either salinity or temperature sweater conditions, trunculus appears more sensitive than C. gallina to seawater temperature above 27.5 °C, showing a significant cytotoxicity (NRRT <50 min) at salinities above 32 and 100% mortality at low salinities (27-28).

Authors find that sentence 422-424 can be improved for a better understanding but it lacks of a speculative character as it has been written on the bases of the obtained results. (see new lines 455-458 of the manuscript).

“Although both species can tolerate changes in either salinity or temperature seawater conditions, the tolerance range is narrower for D. trunculus, showing a significant cytotoxicity (NRRT <50 min) at temperature above 27.5 °C and salinities above 32 and 100% mortality at 27.5ºC and low salinities (27-28)”.

 

8. Lines 390-393: the authors have correctly summarized the important roles of molluscan haemocytes. So, I ask, why weren't other cellular parameters measured which would surely have helped the authors obtain more comprehensive results.

As indicated above, the objective of our study was to specifically investigate the effect of temperature and salinity on the biomarker LMS (Lysosomal Membrane Stability) in clams Donax trunculus and Chamelea gallina, and it was beyond the scope of this work to study the effects of temperature and salinity on molluscan immune function.

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The manuscript has been revised by a native speaking person, and multiple grammatical mistakes (included those indicated below) have been fixed.

• Line 78: “suggest” instead “suggests”.
• Line 110: “of” instead “from” and “and” instead “from”.
• Line 111: “sample” instead “sampling”.
• Line 112: added “the”.
• Line 114: added “out” and, “on” instead “at”.
• Line 133: “held” instead “hold” and “for” instead “during”.
• Line 137: “monitored daily” instead “daily monitored”.
• Line 149: “from” instead “of”.
• Lines 261: added “the”.
• Line 276: “acute” instead “a cute”.
• Lines 278, 309, 311, 312, 314 and 317: added “the”.
• Line 336: “altered differently instead “differently altered”.
• Line 337: “parameter” instead “parameters”.
• Line 353: “patterns” instead “pattern”.
• Line 354: added “the”.
• Line 357: “show” instead “point”.
• Line 360: “signaling” instead “pointing”.
• Line 370: added “the”.
• Line 383: added “out”.
• Line 384: “since” instead “as”.
• Line 387: “at the” instead “with”.
• Line 391 and 393: added “the”.
• Line 402: “were showed” instead “they showed”.
• Line 403: “revealed extremely” instead “revelated very”.
• Line 411: “to” instead “with”.
• Line 423: added “,”.
• Line 448: “clarifying” instead “clarify”.
• Line 451: added “the”.
• Line 451: “similar” instead “certain”.
• Line 455-456: “seawater salinity or temperatures” instead “seawater conditions”.
• Line 459: “consolidating” instead “consolidates”.
• Line 460: “to” instead “with”.

 

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors, I enjoyed your paper and think some minor adjustments are necessary for its publication. My suggestions are listed below.

Line 26 (abstract): please inform that the values shown represent salinity ranges

Line 99 and on: here, I suggest the authors to inform the size ranges of the animals used (both species)

Lines 125-126: please inform the temperatures (low, medium, high), as shown in fig 1.

Lines 344-349: Longer NRRT times were also observed in brown mussel Perna perna (a subtropical-tropical coastal species that occurs mainly in South America and Africa) according to literature, with an optimum at 20ºC instead of 25ºC. Perhaps this information is useful to help in the interpretation of your results.

Lines 335-358: Literature shows that in higher temperatures, cell membranes (including lysosome membranes) become more fluidic and permeable, with implications to the ionic and osmotic balance in the hemocytes. This fact could be explored to explain the results obtained at 27/28ºC.

 

Author Response

The authors would like to indicate to the Editor that they have considered all the reviewers' comments in the resubmission process. The authors thank the reviewers for all comments and suggestions that significantly contributed to improving the quality and clarity of the resubmitted manuscript.

Each reviewer comment is shaded in grey, and our clarifications appear directly below it. 

Reviewer #2:

1. Line 26 (abstract): please inform that the values shown represent salinity ranges.

The abstract has been modified, so it is not necessary to include that some values represent salinity ranges.

 

2. Line 99 and on: here, I suggest the authors to inform the size ranges of the animals used (both species).

The size ranges of both species have been provided in line 115-117 of the new version.

 

3. Lines 125-126: please inform the temperatures (low, medium, high), as shown in fig 1.

We have updated lines 133-134 to include this information as requested.

 

4. Lines 344-349: Longer NRRT times were also observed in brown mussel Perna perna (a subtropical-tropical coastal species that occurs mainly in South America and Africa) according to literature, with an optimum at 20ºC instead of 25ºC. Perhaps this information is useful to help in the interpretation of your results.

We appreciate the reviewer's comment providing us with examples of other species that also show longer NRRT at temperatures of 20ºC. Despite conducting an exhaustive search on Perna perna and NRRT, we were unable to find the work where what the reviewer mentioned is cited. However, it helped us find examples of other species where it is noted that temperatures close to 20ºC have been found to be optimal for maintaining hemocyte lysosomal stability, with membrane stability decreased both when increasing and decreasing the temperature (Yu et al., 2009, Zhao et al., 2011). This information was added in lines 388-390.

Yu, J. H., Song, J. H., Choi, M. C., & Park, S. W. (2009). Effects of water temperature change on immune function in surf clams, Mactra veneriformis (Bivalvia: Mactridae). Journal of invertebrate pathology, 102(1), 30-35.

Zhao, C.; Li, X.; Luo, S.; Chang, Y. Assessments of Lysosomal Membrane Responses to Stresses with Neutral Red Retention Assay and Its Potential Application in the Improvement of Bivalve Aquaculture. Afr. J. Biotechnol. 2011, 10 (64), 13968–13973

 

5. Lines 335-358: Literature shows that in higher temperatures, cell membranes (including lysosome membranes) become more fluidic and permeable, with implications to the ionic and osmotic balance in the hemocytes. This fact could be explored to explain the results obtained at 27/28ºC.

It is true that lipid membranes can be more or less fluid and permeable depending on temperatures, which has implications for the ionic and osmotic balance of hemocytes. However, since no variables have been measured in relation to the phenomenon, and considering there are adaptation mechanisms such as changes in lipid composition and changes in membrane proteins that will be synthesized in response to temperature and/or salinity changes, we believe it is prudent not to engage in speculations that we would have no way to support. We will take into account the reviewer's suggestion and in the future, we will incorporate transcriptomic studies associated with our experiments that would shed light on this issue.

Pernet, F., Tremblay, R., Comeau, L., & Guderley, H. (2007). Temperature adaptation in two bivalve species from different thermal habitats: energetics and remodelling of membrane lipids. Journal of Experimental Biology, 210(17), 2999-3014.

Rais, A., Miller, N., & Stillman, J. H. (2010). No evidence for homeoviscous adaptation in intertidal snails: analysis of membrane fluidity during thermal acclimation, thermal acclimatization, and across thermal microhabitats. Marine biology, 157, 2407-2414.

Los, D. A., & Murata, N. (2004). Membrane fluidity and its roles in the perception of environmental signals. Biochimica et Biophysica Acta (BBA)-Biomembranes, 1666(1-2), 142-157.

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The manuscript has been revised by a native speaking person, and multiple grammatical mistakes (included those indicated below) have been fixed.

• Line 78: “suggest” instead “suggests”.
• Line 110: “of” instead “from” and “and” instead “from”.
• Line 111: “sample” instead “sampling”.
• Line 112: added “the”.
• Line 114: added “out” and, “on” instead “at”.
• Line 133: “held” instead “hold” and “for” instead “during”.
• Line 137: “monitored daily” instead “daily monitored”.
• Line 149: “from” instead “of”.
• Lines 261: added “the”.
• Line 276: “acute” instead “a cute”.
• Lines 278, 309, 311, 312, 314 and 317: added “the”.
• Line 336: “altered differently instead “differently altered”.
• Line 337: “parameter” instead “parameters”.
• Line 353: “patterns” instead “pattern”.
• Line 354: added “the”.
• Line 357: “show” instead “point”.
• Line 360: “signaling” instead “pointing”.
• Line 370: added “the”.
• Line 383: added “out”.
• Line 384: “since” instead “as”.
• Line 387: “at the” instead “with”.
• Line 391 and 393: added “the”.
• Line 402: “were showed” instead “they showed”.
• Line 403: “revealed extremely” instead “revelated very”.
• Line 411: “to” instead “with”.
• Line 423: added “,”.
• Line 448: “clarifying” instead “clarify”.
• Line 451: added “the”.
• Line 451: “similar” instead “certain”.
• Line 455-456: “seawater salinity or temperatures” instead “seawater conditions”.
• Line 459: “consolidating” instead “consolidates”.
• Line 460: “to” instead “with”.

 

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript “Effect of temperature and salinity on the biomarker LMS (Lysosomal Membrane Stability) in clams Donax trunculus and Chamelea gallina” studied the cellular stress caused by the action of combined temperature and salinity conditions in the marine clams C. gallina and D. trunculus. This investigation intends to demonstrate the interspecies differences in the background response of lysosomal membrane stability biomarkers under certain environmental conditions. Given the importance of the effect of climate changes on aquatic organisms, these results have a relevant interest in the potential use of LMS as a biomarker in these species. This research contributes to consolidating the finding that climate changes can affect hemocyte functionality, which is directly related to bivalve immune defense capabilities.

I consider this manuscript suitable for publication following my specific comments.

 

Specific comments:

 

M&M

Line 130 – 131 - The authors should include how they mesured temperature, salinity and dissolved oxygen.

 

Results

 

The authors should add some pictures of Lysosomal Membrane stained with neutral red.

 Line 228 – Replace “that” with “than”.

Table 3- The authors should explain why certain results were not included in the table.

 

Line 239 – Please remove “of temperature and salinity”

Author Response

The authors would like to indicate to the Editor that they have considered all the reviewers' comments in the resubmission process. The authors thank the reviewers for all comments and suggestions that significantly contributed to improving the quality and clarity of the resubmitted manuscript.

Each reviewer comment is shaded in grey, and our clarifications appear directly below it. 

Reviewer #3:

1. Line 130 – 131 - The authors should include how they measured temperature, salinity and dissolved oxygen.

In lines 138 – 140 of the revised manuscript, we have addressed how temperature, salinity, and dissolved oxygen were measured.

 

2. The authors should add some pictures of Lysosomal Membrane stained with neutral red.

The pictures have been added (Figure 3 of the submitted manuscript).

 

3. Line 228 – Replace “that” with “than”.

The change has been made.

 

4. Table 3- The authors should explain why certain results were not included in the table.

In Table 3 (referring to the condition index), it was not possible to obtain the reference values at low temperature (12 ºC) for both species due to logistical problem. In addition, in the same table, for D. trunculus the CI values are not included, in the treatment at high temperature and salinity 27-28, due to the 100% mortality obtained during the exposure in that treatment.

In the table 3 (lines 266-267), the following has been added: “–“ no data and “X” indicates that there were no organisms due to mortality. 

 

5. Line 239 – Please remove “of temperature and salinity”.

This part of the sentence has been removed.

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The manuscript has been revised by a native speaking person, and multiple grammatical mistakes (included those indicated below) have been fixed.

• Line 78: “suggest” instead “suggests”.
• Line 110: “of” instead “from” and “and” instead “from”.
• Line 111: “sample” instead “sampling”.
• Line 112: added “the”.
• Line 114: added “out” and, “on” instead “at”.
• Line 133: “held” instead “hold” and “for” instead “during”.
• Line 137: “monitored daily” instead “daily monitored”.
• Line 149: “from” instead “of”.
• Lines 261: added “the”.
• Line 276: “acute” instead “a cute”.
• Lines 278, 309, 311, 312, 314 and 317: added “the”.
• Line 336: “altered differently instead “differently altered”.
• Line 337: “parameter” instead “parameters”.
• Line 353: “patterns” instead “pattern”.
• Line 354: added “the”.
• Line 357: “show” instead “point”.
• Line 360: “signaling” instead “pointing”.
• Line 370: added “the”.
• Line 383: added “out”.
• Line 384: “since” instead “as”.
• Line 387: “at the” instead “with”.
• Line 391 and 393: added “the”.
• Line 402: “were showed” instead “they showed”.
• Line 403: “revealed extremely” instead “revelated very”.
• Line 411: “to” instead “with”.
• Line 423: added “,”.
• Line 448: “clarifying” instead “clarify”.
• Line 451: added “the”.
• Line 451: “similar” instead “certain”.
• Line 455-456: “seawater salinity or temperatures” instead “seawater conditions”.
• Line 459: “consolidating” instead “consolidates”.
• Line 460: “to” instead “with”.

 

Reviewer 4 Report

Comments and Suggestions for Authors

The paper presents strong composition; however, it is not suitable for publication due to several issues that significantly impact the experiment and the authors' interpretations:

1. Sampling Periods: The use of three sampling periods (March, May, and June) raises concerns as environmental conditions fluctuate, potentially influencing the health state of the species and confounding the results. Moreover, the assignment of individuals to different temperature categories based on the month of sampling could introduce bias, especially considering the reproductive state of May and June individuals.

2. Dissolved Oxygen Control: The lack of controlled dissolved oxygen levels, particularly in compensating for temperature effects, complicates the interpretation of results. Without adequate control, it becomes statistically challenging to discern the influence of temperature on the response variables.

3. Statistical Analysis: The use of one-way ANOVA is inappropriate for a two-way crossed factorial design where salinity varies across three levels for each temperature level. Consequently, the incorrect statistical test invalidates subsequent post-hoc analyses, necessitating a reassessment using the appropriate methodology.

4. Experimental Design and Data Reporting: Discrepancies in individual sizes among treatment groups, as evident from Table 3, raise concerns about the experimental design's integrity. Additionally, the presentation of mean size and standard error (SE) should adhere to normal distribution assumptions, and information on the initial and final size distribution within each tank is necessary for comprehensive analysis.

5. Replication and Tank Variation: The absence of tank replication and insufficient tank numbers for each group (ideally 3-5 tanks per group) introduces the potential for uncontrolled and unmeasured factors influencing treatment group differences. This lack of replication undermines the reliability and validity of the experimental findings.

In summary, addressing these issues is crucial for ensuring the validity and credibility of the study's conclusions. Consideration should be given to revising the experimental design, statistical analysis, and data reporting before reconsidering publication.

Comments on the Quality of English Language

There are many paragraphs composed by one or two sentences. Paragraphs should be composed with an opening sentence supported by examples and clarifications and a conclusion. However, I find the manuscript well written

Author Response

The authors would like to indicate to the Editor that they have considered all the reviewers' comments in the resubmission process. The authors thank the reviewers for all comments and suggestions that significantly contributed to improving the quality and clarity of the resubmitted manuscript.

Each reviewer comment is shaded in grey, and our clarifications appear directly below it. 

Reviewer #4:

1. Sampling Periods:The use of three sampling periods (March, May, and June) raises concerns as environmental conditions fluctuate, potentially influencing the health state of the species and confounding the results. Moreover, the assignment of individuals to different temperature categories based on the month of sampling could introduce bias, especially considering the reproductive state of May and June individuals.

As the reviewer says, it is true that sampling at different periods could have introduced a bias due to spawning, but it was decided to sample the mollusks at different periods when the difference between the natural temperature in the field and the test temperature in the laboratory was minimal (lines 110-113 submitted manuscript). Consequently, it was assumed that they were almost naturally acclimatized to the different field temperatures, and the period during which individuals were kept in the laboratory aquariums for the experiments was limited (a procedure similar to that adopted by Ansell et al., 1980).

In addition, the possible effects of spawning on NRRT results has been addressed considerably in the discussion (lines 412-426).

Ansell, A. D.; Barnett, P. R. O.; Bodoy, A.; Massé, H. Upper Temperature Tolerances of Some European Molluscs II. Donax Vittatus, D. Semistriatus and D. Trunculus. Mar. Biol. 1980, 58 (1), 41–46. https://doi.org/10.1007/BF00386878.

 

2. Dissolved Oxygen Control:The lack of controlled dissolved oxygen levels, particularly in compensating for temperature effects, complicates the interpretation of results. Without adequate control, it becomes statistically challenging to discern the influence of temperature on the response variables.

As stated by the reviewer, it is well known that an increase in temperature causes a reduction of oxygen solubility. In our study, we conducted daily measurements of dissolved oxygen levels, as outlined in lines 136-138. The dissolved oxygen values (in mg/L) for each treatment condition are presented in Table 1, revealing a decline in dissolved oxygen levels as temperature increased, ranging from 9 to approximately 7 mg/L. However, it is essential to emphasize that despite this decrease, the oxygen saturation in all nine treatments consistently remained above 100%, as depicted in lines 210-211 of the manuscript. Thus, it is evident that dissolved oxygen conditions were adequately controlled (> 7 mg/L and 100% saturation), and any observed effects cannot be attributed to changes in this variable (as elaborated in the discussion, lines 361-365).

Following the reviewer's suggestion and to facilitate comprehension of the results, we have incorporated a new column in Table 1 displaying the percent oxygen saturation values.

 

3. Statistical Analysis:The use of one-way ANOVA is inappropriate for a two-way crossed factorial design where salinity varies across three levels for each temperature level. Consequently, the incorrect statistical test invalidates subsequent post-hoc analyses, necessitating a reassessment using the appropriate methodology.

Following the reviewer's recommendations, the statistical treatment of the data was modified. A Multifactor ANOVA procedure was used to determine whether or not there are significant differences between the means of NRRT, %LMS and %CI at the different levels of the factors (temperature and salinity) and whether or not there are interactions between the factors. Assumptions for using multifactor ANOVA include normal distribution of the data and homogeneity of variances.

In the first version of the manuscript, we could not perform this type of analysis because some of the data did not follow a normal distribution even though we had tried some simple transformations of the raw data to make the transformed data normal (i.e. squared values, square root, reciprocal of the data, logarithm of the data). 

Due to the reviewer's comments, we still try to transform the data, but this time we use BOX-COX transformation (lambda for the Box-Cox transformation was found using the Most Likely Estimate (MLE) approach). In this way, our transformed data could give rise to a normal distribution and thus use a two-way ANOVA.

In the new version of the manuscript, the Data analysis section was modified (lines 191-204), and the new results were incorporated in Tables 2 and 3; Figures 2, 4 and 6; and in the text in the results section.

 

4. Experimental Design and Data Reporting: Discrepancies in individual sizes among treatment groups, as evident from Table 3, raise concerns about the experimental design's integrity. Additionally, the presentation of mean size and standard error (SE) should adhere to normal distribution assumptions, and information on the initial and final size distribution within each tank is necessary for comprehensive analysis.

Regarding the reviewer’s comment, the size range of the organisms in the three samplings period (March, May and June), by species, has been incorporated in lines 115-117 of the new manuscript.

Regarding the sizes of the organisms used in the analysis of lysosomal membrane stability (LMS), in the new version of the manuscript they have been indicated on line 168-170 (line 129 in the previous version of the manuscript). “The size of the organisms used to analyze lysosomal membrane stability ranged from 13.9 to 23.9 mm (in March) and from 20.8 to 31.44 mm (in May and June) for C. gallina and from 16.1 to 28.5 mm for D. trunculus”.

For the condition index (CI) analysis, we chose to standardize the sizes of the organisms used due to the disparities in size between the three samplings, as previously noted in the previous manuscript version (now lines 177-180 of the new manuscript). In this way, it was possible to compare the health status of the organisms after 21 days of exposure to each temperature treatment (at the three salinity ranges) with the health status (reference CI) of the organisms sampled in the three periods (March, May and June).

 

5. Replication and Tank Variation:The absence of tank replication and insufficient tank numbers for each group (ideally 3-5 tanks per group) introduces the potential for uncontrolled and unmeasured factors influencing treatment group differences. This lack of replication undermines the reliability and validity of the experimental findings.

We agree with the reviewer that absence of tank replication in the sampling design used in our work limits the robustness of the experimental findings but still we find that they are reliable and valuable enough to be published.

The underlying reasons about the absence of tank replication were related from one hand with the number of samples to be analyzed at the same time and, from the other hand, with the volume capacity of the environmental chambers where tanks (aquariums) were placed.  In our work, experimental units used for each treatment were all located inside insulation chambers (closed room) which significantly minimized the potential effects of ambient factors such as luminosity, temperature, ambient humidity, etc. that could affect our experimental conditions. Furthermore, dissolved oxygen was forced to be ~100% saturation at all experimental conditions by continuously air pumping.

Concerning the number of samples to be analyzed, we have to underline that NRR assay is a “in vivo” technique (monoculture cell samples to be analyzed cannot be frozen or stored for further analysis) that last 4 hours. Personnel trained in the use of this technique are capable of analyzing 8 individual samples at the same time (Martínez-Gómez, personal communication). To solve this logistic reason, many authors replicate the tanks (usually 3 replicates) but reduce the number of organisms to be analyzed from each replicated tank (usually 4 organisms/replicate for each treatment) (Couglan et al., 2009; Rafiq, A. et al., 2023; Martínez-Gómez et al., 2023). However, some other authors using NRR assay in experimental studies have performed sampling designs similar to ours (Monari et al., 2007; Matozzo et al., 2009). While recognizing the limitations of our work, we want to emphasize that in most of experimental studies using NRR assay, the total number of individual organisms analyzed per treatment hardly are higher than 12 and to compensate for the lack of experimental replication, in our study we analyzed 16 individuals per treatment.

 

Coughlan, B. M., Moroney, G. A., Van Pelt, F. N. A. M., O’Brien, N. M., Davenport, J., & O’Halloran, J. (2009). The effects of salinity on the Manila clam (Ruditapes philippinarum) using the neutral red retention assay with adapted physiological saline solutions. Marine Pollution Bulletin, 58(11), 1680-1684

Martínez-Gómez C, Llorca M, Oporto T, Rapuano S, García Pimentel MM, Farré M. “Quantification of polyethylene in mussel hemolymph and its limited additive effect on immune function induced by bezafibrate”. Proceedings of the 3rd International Conference on Microplastic Pollution in the Mediterranean Sea. 2023. Ed. MC Cocca et al. Springer Waters. ISBN 978-3-031-34454-1; ISBN 978-3-031-34455-8 (eBook) https://link.springer.com/book/10.1007/978-3-031-34455-8

Matozzo, V., Monari, M., Foschi, J., Cattani, O., Serrazanetti, G. P., & Marin, M. G. (2009). First evidence of altered immune responses and resistance to air exposure in the clam Chamelea gallina exposed to benzo (a) pyrene. Archives of environmental contamination and toxicology, 56, 479-488.

Monari, M., Matozzo, V., Foschi, J., Cattani, O., Serrazanetti, G. P., & Marin, M. G. (2007). Effects of high temperatures on functional responses of haemocytes in the clam Chamelea gallina. Fish & Shellfish Immunology, 22(1-2), 98-114.

Rafiq, A., Capolupo, M., Addesse, G., Valbonesi, P., & Fabbri, E. (2023). Antidepressants and their metabolites primarily affect lysosomal functions in the marine mussel, Mytilus galloprovincialis. Science of the Total Environment, 903, 166078.

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The manuscript has been revised by a native speaking person, and multiple grammatical mistakes (included those indicated below) have been fixed.

• Line 78: “suggest” instead “suggests”.
• Line 110: “of” instead “from” and “and” instead “from”.
• Line 111: “sample” instead “sampling”.
• Line 112: added “the”.
• Line 114: added “out” and, “on” instead “at”.
• Line 133: “held” instead “hold” and “for” instead “during”.
• Line 137: “monitored daily” instead “daily monitored”.
• Line 149: “from” instead “of”.
• Lines 261: added “the”.
• Line 276: “acute” instead “a cute”.
• Lines 278, 309, 311, 312, 314 and 317: added “the”.
• Line 336: “altered differently instead “differently altered”.
• Line 337: “parameter” instead “parameters”.
• Line 353: “patterns” instead “pattern”.
• Line 354: added “the”.
• Line 357: “show” instead “point”.
• Line 360: “signaling” instead “pointing”.
• Line 370: added “the”.
• Line 383: added “out”.
• Line 384: “since” instead “as”.
• Line 387: “at the” instead “with”.
• Line 391 and 393: added “the”.
• Line 402: “were showed” instead “they showed”.
• Line 403: “revealed extremely” instead “revelated very”.
• Line 411: “to” instead “with”.
• Line 423: added “,”.
• Line 448: “clarifying” instead “clarify”.
• Line 451: added “the”.
• Line 451: “similar” instead “certain”.
• Line 455-456: “seawater salinity or temperatures” instead “seawater conditions”.
• Line 459: “consolidating” instead “consolidates”.
• Line 460: “to” instead “with”.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have carefully revised their manuscript according to the reviewers’ comments. Therefore, the manuscript can be accepted for publication in its current form.

Reviewer 4 Report

Comments and Suggestions for Authors

I genuinely appreciate your thorough explanation of your motivations and the edits you've made to enhance the manuscript. Despite these efforts, I remain concerned that the experimental design may not yield sufficiently robust results. Regarding the handling of non-normal data with covariates, I would recommend considering a PERMANCOVA analysis. Finally, I'd like to underscore that sound statistical practices and experimental design are the bedrock of credible research studies. I acknowledge the economic and logistical challenges inherent in conducting experiments, but these, and the fact that others have done worse, should never serve as justification for conducting subpar studies.