Hemolymph Parameters Are a Useful Tool for Assessing Bivalve Health and Water Quality

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper “Hemolymph parameters are a useful tool for assessing bivalve health and water quality” collected the hemolymph of 15 commercially available species from wild populations at stations located in non-impacted and impacted water areas of the Sea of Japan. The results showed that hemolymph parameters are valid tools for assessing the health status of mollusks according to the environmental conditions of the water area they inhabit. Overall, the paper lacks scientific rigor and does not meet the requirements for journal acceptance. The specific issues with the article are as follows:

1. The article selected multiple locations as non-impacted areas and multiple locations as impacted areas to study the differences in various hemolymph parameters of different bivalves in the two types of regions. Beyond the existing literature on pollution, what criteria did the authors use to select multiple sampling points in these two regions? Was there a significant difference in the degree of pollution among the different sampling points? Did different types of pollution affect the experimental analysis results? It is suggested that the authors carefully consider and improve the rigor of the experimental design.

2. Since the impacted and non-impacted areas are different marine regions, were there differences other than pollution, such as water quality parameters? Were the differences in hemolymph parameters found in this study necessarily caused by pollution, or could they also be influenced by different water quality environments? The authors should carefully revise this section.

3. The authors collected the same species of bivalves from different marine regions for comparison. Did these bivalves from the two sources have similar growth and genetic backgrounds? Could the differences in hemolymph parameters found in this study be caused by physiological differences between different geographical populations? It is suggested that the authors optimize the sampling scheme.

4. The polluted areas in this article involved multiple types of pollution. Did hemolymph parameters respond to all these pollution stimuli, and what might be the mechanisms involved? It is suggested that the authors provide more relevant research background and selection basis in the introduction.

5. The reviewer considers the experimental design of the correlation analysis section to be unreasonable. Due to the overly random selection of sampling points and the limited data used for correlation analysis, the scientific value of the correlation analysis results is weak. It is suggested that the authors consider deleting this part of the study.

6. There are numerous language issues and several formatting problems in the article, such as "p<0.05" where the "p" should be italicized.

Comments on the Quality of English Language

    There are numerous language issues and several formatting problems in the article, such as "p<0.05" where the "p" should be italicized.

Author Response

Response to Reviewer 1

The Authors thank the Reviewer for carefully reading the manuscript and for constructive remarks. In the corrected version of the manuscript, we considered all the comments and suggestions. Those comments are all valuable and helpful for revising and improving our paper, as well as providing significant guidance to our research. We have added more detail and a better structure to explain our choice of water areas and make these explanations more understandable. The English writing has been checked and corrected by the Language Editing Service. We believe that the incorporation of the Reviewer's recommendations has significantly improved the manuscript’s quality. Please find a detailed point-by-point response to all comments below.

 

1. The article selected multiple locations as non-impacted areas and multiple locations as impacted areas to study the differences in various hemolymph parameters of different bivalves in the two types of regions. Beyond the existing literature on pollution, what criteria did the authors use to select multiple sampling points in these two regions? Was there a significant difference in the degree of pollution among the different sampling points? Did different types of pollution affect the experimental analysis results? It is suggested that the authors carefully consider and improve the rigor of the experimental design.

Answer: We thank the Reviewer for raising this issue. When choosing points for collecting material, the authors were guided mainly by the similarity of the geographical and hydrological conditions of the water areas, the occurrence of species, as well as the degree of pollution of the water areas. The degree of pollution of the water areas varied (see 2.2. Collection areas (this part was transferred from the Discussion)), among which the most polluted water area was Patrokl Bay and Vostok Cove, strongly polluted Sredneya Bay and Sportivnaya Gavan Bay, moderately polluted the water area near Cape Krasny. A detailed description of the degree and nature of water pollution is provided in the 2.2. Collection areas. Despite the similar geographical and hydrological conditions of impact water areas (see Table 1), some species of mollusks do not live in them. Other works have repeatedly shown the depletion of species diversity due to pollution, which, among other things, is one of the key indicators of the presence of a negative impact. Thus, the non-impacted water area (near the biological station “Vostok” in Vostok Bay), used as a reference, has a relative background pollution level for several environmental and chemical indicators, as a result of which most species of mollusks (12 out of 15) were collected precisely in this water area (except 3 species that have special hydrochemical requirements for water and soil type). Due to the gradient and quality of pollution of impacted water areas (described in detail in the 2.2. Collection areas), not all species of mollusks of interest to us were found in every water area, which determined the different species composition of samples from each water area. As is known, various species have different sensitivity and, accordingly, survival rates relative to several factors, which was also confirmed by our study.

 

2. Since the impacted and non-impacted areas are different marine regions, were there differences other than pollution, such as water quality parameters? Were the differences in hemolymph parameters found in this study necessarily caused by pollution, or could they also be influenced by different water quality environments? The authors should carefully revise this section.

Answer: Mollusks were caught from water areas with similar hydrolytic and geochemical regimes to exclude the influence of geographical factors since areas are located at almost the same latitude (Luchin et al., 2005; Danchenkov, 2023).

Literature data about the effect of various pollutants on hemolymph parameters (see Discussion) and pollutants detected in the studied water areas indicate that changes in the immunity parameters of the studied mollusks are caused precisely by the presence of pollutants in water areas that exceed the maximum permissible concentration (MPC) to varying degrees.

 

3. The authors collected the same species of bivalves from different marine regions for comparison. Did these bivalves from the two sources have similar growth and genetic backgrounds? Could the differences in hemolymph parameters found in this study be caused by physiological differences between different geographical populations? It is suggested that the authors optimize the sampling scheme.

Answer: In the experiment, sexually mature specimens of 4-7 years with average shell size were used (detailed information was added to the 2.1. Animals section of the Materials and Methods chapter (line 109) and Table 1).

As for the single genetic origin of the studied populations, the Authors find it difficult to answer this question since, unfortunately, there is limited data on the Far Eastern region, and it is mainly devoted to interspecific hybridization and introgression of genes.

 

4. The polluted areas in this article involved multiple types of pollution. Did hemolymph parameters respond to all these pollution stimuli, and what might be the mechanisms involved? It is suggested that the authors provide more relevant research background and selection basis in the introduction.

Answer: Each of the impacted water areas involved in the experiment includes several types of pollutants with different degrees of severity, but all exceed the maximum permissible concentration (see 2.2. Collection areas). The main polluting elements studied previously and having the most impact on living organisms are pesticides, phenols, petroleum products, diesel fuel and heavy metals which were also present in all impact water areas (above MPC) and described in detail in the 2.2. Collection areas (line 115). We did not undertake to give a definite answer to which of the pollutants played a primary role in modulating the immune system of mollusks but still tried to explain the effects we observed by comparing the results of our study with the work of other authors (see Discussion). In addition, each pollutant can act separately or synergistically, interacting with other pollutants, physical factors, and the organism itself (such as certain conditions, species, gender, age, etc.), depending on its dose and residence time in the environment. The development of indicators and biomarkers involves both field and controlled laboratory studies. It is important to examine the relationships between exposure to environmental pollutants and the effects they cause. This can be done by conducting controlled exposure experiments in the laboratory to confirm cause-and-effect relationships. Conversely, effects found in laboratory conditions should be studied in natural environments with less control over bioaccumulation and physiological status to gain a better understanding. Varied experimental approaches are necessary for comprehensive understanding. Currently, extensive data have been collected on assessing the parameters of the hemolymph of mollusks in laboratory conditions (see Discussion), while field studies are relatively limited. This work focuses on the conceptual significance and variability in the response of promising immunomarkers of bivalves in natural multifactorial conditions, which is especially important for aquaculture, as their farms are typically located in open water areas. In the future, after conducting a rapid assessment of the immunity of mollusks, and if deviations are detected, the user can find out the reason for this through further detailed studies of both the ecological state of the water area and the parasitic, histopathological, or physiological status of mollusks.

The Authors added a section (2.2. Collection areas) about sample collection areas in Materials and Methods to justify the choice of impact areas according to the Reviewer's suggestions.

 

5. The reviewer considers the experimental design of the correlation analysis section to be unreasonable. Due to the overly random selection of sampling points and the limited data used for correlation analysis, the scientific value of the correlation analysis results is weak. It is suggested that the authors consider deleting this part of the study.

Answer: Authors have reviewed the correlation analysis section and agree with the Reviewer that for this study, correlation analysis is not necessary. As a result, the parts of the manuscript containing correlation analysis were excluded.

 

6. There are numerous language issues and several formatting problems in the article, such as "p<0.05" where the "p" should be italicized.

Answer: Authors modified the text of the manuscript according to the Reviewer’s remark.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The quality of English language of this paper is average.

Author Response

Response to Reviewer 2

The quality of English language of this paper is average.

There are a lot of papers published on hemocytes of bivalves (the authors missed a lot of relevant literature), so this work is not very innovative. Overall the manuscript is well designed and scientific writing is also good. The results have good reference value, and is worthy of publication. But I have observed that Introduction section needs more modifications and the methodology section is detailed but lacks clarity on the sampling process. It is not clearly mentioned how the sampling sites were selected and if there was any randomization in the selection of bivalves. Provide a clear description of the sampling strategy, including how sites were chosen and any measures taken to ensure representative sampling. I recommend a major revision, and it can be accepted for publication once the authors addressed my concerns. My detailed comments are given below.

Answer: The Authors are grateful to the Reviewer for their thoughtful comments and efforts towards improving our manuscript. The authors considered the Reviewer’s comments and suggestions and made all necessary corrections to the manuscript. The English writing has been checked and corrected by the Language Editing Service. We believe that the incorporation of the Reviewer's recommendations has significantly improved the manuscript’s quality.

The Authors acknowledge the Reviewer's point about the extensive research on hemocytes. However, our submitted work focuses on a comprehensive study of the mollusk immune system response to chronic environmental pollution in natural conditions. The study not only evaluates the cellular component of the hemolymph (hemocytes) but also the humoral component and their functional activity under normal conditions, as well as the impact of various levels of negative factors. Among the previous studies, fewer works assessed hemocyte parameters in mollusks from natural habitats or under aquaculture conditions, while most works focused on studying the parameters of hemocytes when simulating laboratory conditions, changing 2 to 3 factors, which is uncommon in nature, where as a rule, the organism is affected by a complex of factors that are also interconnected and influence each other (de la Ballina, 2022).

Our goal is to demonstrate that hemolymph parameters can effectively assess the physiological state of mollusks, similar to blood parameters in fish and humans; parameters are also sensitive to influence and can be used in aquaculture, which is especially in demand because of the active development of this industry. This method is non-lethal, relatively fast, applicable to large sample sizes, nearly automated, and does not require special skills compared to traditional histology.

The Authors agree with the Reviewer regarding the lack of description of the sampling methodology. Corresponding additions have been made to the Material and Methods (lines 109 and 115).

 

Introduction

Ln 40: The phrase "internal environment parameters" might be clearer as "internal environmental parameters" to specify the environment within the organism.

Answer: Authors modified the sentence according to the Reviewer’s remark.

 

The introduction references previous research on hemolymph parameters and bivalve health, it lacks sufficient context or discussion of these studies. The authors missed a lot of relevant references that can improve the quality of this manuscript (Hemocyte responses of the oyster Crassostrea hongkongensis exposed to diel-cycling hypoxia and salinity change; Differential in vivo hemocyte responses to nano titanium dioxide in mussels: Effects of particle size; Hemocyte responses of the thick shell mussel Mytilus coruscus exposed to nano-TiO2 and seawater acidification). Providing more background information and explaining the relevance of prior research findings would strengthen the introduction and support the rationale for the current study if possible.

The introduction briefly mentions the limitations of laboratory analysis and the need for field research; it does not adequately address potential challenges or limitations associated with using hemolymph parameters for assessing bivalve health. (see papers: Combined effects of seawater acidification and high temperature on hemocyte parameters in the thick shell mussel Mytilus coruscus; Effects of short-term hypoxia and seawater acidification on hemocyte responses of the mussel Mytilus coruscus; Immune toxicity of TiO2 under hypoxia in the green-lipped mussel Perna viridis based on flow cytometric analysis of hemocyte parameters;). Acknowledging and addressing potential uncertainties would add depth to the Introduction and demonstrate a comprehensive understanding of the research context.

It also discusses the use of hematological analysis in fish aquaculture, it may be overreliant on this comparison. Bivalves have distinct physiological characteristics, and while there may be parallels with fish, it's essential to justify why these comparisons are relevant and how they apply specifically to bivalves. (see: Effects of copper on hemocyte parameters in the estuarine oyster Crassostrea rivularis under low pH conditions; Combined effects of ZnO NPs and seawater acidification on the haemocyte parameters of thick shell mussel Mytilus coruscus; Immune parameter changes of hemocytes in green-lipped mussel Perna viridis exposure to hypoxia and hyposalinity; Immune responses to combined effect of hypoxia and high temperature in the green-lipped mussel Perna viridis).

Answer: The Introduction contains references to works devoted to valid methods for assessing the health of aquatic organisms (references 1-6). Even though mollusks are one of the key objects of fishing and aquaculture, formal non-lethal health assessment methods have been developed only for fish. Although the validity of assessing the physiological state of mollusks and their habitat based on hemolymph indicators has long been repeatedly demonstrated (references 7-13 in the Introduction). The focus of this study is not aimed at assessing the immune response of mollusks to discrete sources of pollution but at diagnosing the state of mollusks under conditions of chronic long-term pollution and modulating the immune system likely to contribute to their survival. Despite the similar geographical and hydrological conditions of the water areas, some of the species of mollusks do not live everywhere but inhabit water areas only with an "acceptable" level of pollution. Thus, the non-impacted water area (near the biological station “Vostok” in Vostok Bay), has a relative background level of pollution for several environmental and chemical indicators, as a result of which most species of mollusks (12 out of 15) were collected precisely in this water area (except 3 species that have special hydrochemical requirements for water and soil type). Due to the gradient and quality of pollution of impacted water areas (described in detail in the 2.2. Collection areas), not all species of mollusks of interest to us were found in every water area, which determined the different species composition of samples from each water area. As is known, various species have different sensitivity and, accordingly, survival rates relative to several factors, which was also confirmed by our study.

In this work, we consider the issues of conceptual significance and variability in the response of promising immunomarkers of bivalves in natural multifactorial conditions, which is especially important for aquaculture that locates their farms in open water areas. The Far Eastern District, and especially the Primorsky Territory, is one of the key port regions and the main supplier of seafood not only for Russia but for neighboring countries, so the need for the selection of indicators to assess the condition of both water areas and aquatic organisms is quite acute here.

The development of indicators and biomarkers involves both field and controlled laboratory studies. It is important to examine the relationships between exposure to environmental pollutants and the effects they cause. This can be done by conducting controlled exposure experiments in the laboratory to confirm cause-and-effect relationships. Conversely, effects found in laboratory conditions should be studied in natural environments with less control over bioaccumulation and physiological status and where interactions with multiple chemical compounds and biotic factors may occur. In addition, we must not forget that pulsed exposure in laboratory conditions, causing aberrations of physiology, especially in bivalves, can only be a short-term reaction or acclimatization without any consequences. A better understanding requires a variety of experimental approaches. Currently, extensive data has been accumulated on assessing the parameters of the hemolymph of mollusks in laboratory conditions (see Discussion), while there are comparatively fewer field studies.

Our study assessed the health status of mollusks at several levels of organization, affecting both humoral and cellular factors of the hemolymph, which in turn allows us to identify the most sensitive parameters for each species. Using a given area and resources, the researcher can select the appropriate species and set of biomarkers that are more likely to provide an effective evaluation of the state of the environment and mollusks.

The publications proposed by the Reviewer, are focused only on the experiments simulated in laboratory conditions and on hemocytes. Thus, the paper “Effects of copper on hemocyte parameters in the estuarine oyster Crassostrea rivularis under low pH conditions” addresses the study of the combined effects of Cu and low pH under strictly controlled laboratory conditions (pH 8.12, seawater temperature 24 - 25°C, salinity 22.0 ‰, microalgae Chlorella spp. diet) on the parameters of C. rivularis hemocytes (total hemocyte count, esterase activity, lysosomal content, hemocyte mortality, phagocytosis activity, and reactive oxygen species production), flow cytometry in vitro and in vivo. The work “Combined effects of ZnO NPs and seawater acidification on the hemocyte parameters of thick shell mussel Mytilus coruscus”  evaluates parameters of M. coruscus hemocytes under the combined effects of acidification and ZnO nanoparticles under strict laboratory conditions using flow cytometry. Other works “Immune parameter changes of hemocytes in the green-lipped mussel Perna viridis exposure to hypoxia and hyposalinity”, “Immune responses to the combined effect of hypoxia and high temperature in the green-lipped mussel Perna viridis”, “Effects of short-term hypoxia and seawater acidification on hemocyte responses of the mussel Mytilus coruscus" and "Immune toxicity of TiO2 under hypoxia in the green-lipped mussel Perna viridis based on flow cytometric analysis of hemocyte parameters", provides similar content, and devoted to the assessment of hemocyte parameters using flow cytometry after exposure to some agent or factor. Authors are grateful to one of the Authors of these works for valuable advice, and we will cite his work in similar works in the future.

 

The Introduction highlights the relevance of the Sea of Japan waters but does not delve into specific environmental challenges or factors unique to this region that may impact bivalve health. Providing more detail on the specific ecological conditions and stressors in this area would enhance the introduction's relevance and specificity.

Answer: In the Introduction, the authors focused on updating the examination of the condition of mollusks based on hemolymph parameters and field studies associated with the active development of aquaculture and its needs. Basic and detailed information about the state of the waters of the Peter the Great Bay of the Sea of Japan is provided in the 2.2. Collection areas. The authors chose not to include this information in the Introduction to avoid overloading it with less pressing issues for this area. However, the Authors made some additions to the Introduction regarding specific environmental conditions and stress factors (line 73), as per the Reviewer's recommendations.

 

Materials and Methods

Specify the sizes of bivalves.

Answer: Authors followed the reviewer’s recommendation and provided the information about shell length (Table 1).

 

Ln_88: "...water areas of the Sea of Japan (Table 1, Figure 1)": This sentence could be clearer if "water areas" were replaced with "marine environments" or "water bodies."

Answer: Authors changed the sentence according to the Reviewer’s suggestion.

 

Ln 109_110: "The rest of the hemolymph was centrifuged at 800 g for 12 min at 15 °C.": Consider using "were centrifuged" for agreement with "rest" as a plural noun.

Answer: Authors modified the sentence according to the Reviewer’s remark.

 

Ln 110_111: The supernatant was then frozen at –85°C for further humoral immunity activity.” It should be "for further analysis of humoral immunity activity" to convey the intended meaning clearly.

Answer: Authors changed the sentence according to the Reviewer’s comment.

 

Ln 115: Insert a space between "[18]" and "with" for clarity and consistency.

Answer: Authors apologize for the typo. It was fixed according to the Reviewer’s comment.

 

Ln 116_117: Consider specifying the duration or conditions of the incubation for completeness.

Answer: When writing the manuscript, the authors used the Instructions for Authors and provided links to previously published methods, as indicated in Instructions for Authors: “New methods and protocols should be described in detail while well-established methods can be briefly described and appropriately cited.” In the paragraph dedicated to the description of the assessment of hemolytic activity of plasma, to simplify the perception of information and not duplicate an already published method, a link is provided to a reference (line 206), which describes in detail the procedure for implementing this type of examination:

«Hemolytic activity was estimated according to a method described in detail by Grinchenko and colleagues [18]…» Grinchenko, A.; Sokolnikova, Y.; Korneiko, D.; Kumeiko, V. Dynamics of the Immune Response of the Horse Mussel Modiolus Kurilensis (Bernard, 1983) Following Challenge with Heat-Inactivated Bacteria. J Shellfish Res 2015, 34, 909–917, doi:10.2983/035.034.0321.

However, the Authors made a clarification in the part of the manuscript regarding the incubation conditions (Line 208).

 

Ln 122: Use "were" instead of "was" for subject-verb agreement with "type."

Ln 123: Correct it please “mollusk species”?

Ln 137: It should be "were incubated" for clarity and completeness.

Ln 146: Consider adding "were" before "washed" for parallel structure.

Answer: Authors changed the sentences according to the Reviewer’s suggestion.

 

Ln 150: A total of 100,000 events per sample were recorded.” This sentence could be clarified by specifying what types of events were recorded.

Answer: Debris was gated out by using forward scatter (FS)/side scatter (SS) data and red fluorescence of hemocyte nuclei associated with cell prestaining with 1 μg/ml propidium iodide. The corresponding clarification has been added to the text (line 244).

 

The locations of sampling sites are mentioned, details on why these specific sites were chosen and how they represent non-impacted and impacted areas are lacking. Providing justification for site selection and describing the criteria used to classify them as non-impacted or impacted would enhance the methodology.

Answer: In the Materials and Methods on the line 130 it is stated that “…the impacted water areas do not meet the sanitary and epidemiological requirements for the levels of phenols, petroleum products, and pathogenic bacteria (such as enterococci), nor for the concentrations of oxygen, phosphorus, and heavy metals, as well as several other parameters...” A more detailed description of the ecological and microbiological status of water areas is given in the 2.2. Collection areas (this part was transferred from the Discussion). The Authors also clarified the Materials and Methods (line 119) “The collection of certain species of mollusks from water areas was based on the similar geographical and hydrological conditions of the water areas and the occurrence of a particular species.”

 

Ln 101_103 The timing of sample collection (September-October 2023) is mentioned, but there is no discussion on why these months were chosen or whether seasonal variations in environmental factors were considered. Providing justification for the chosen sampling period and discussing potential seasonal effects would improve the methodology's section.

Answer: The period for sample collection was selected upon reaching the period of sexual inertia for each species. The Authors have made clarifications to the text of the manuscript (line 110).

 

There is no mention of quality control checks performed during the study.

Answer: It is not clear to the Authors what the Reviewer meant under quality control. If the Reviewer meant quality control of personnel, then the authors of this article are highly qualified specialists in this field of research (PhD, bachelor's degree, master's student, or postgraduate). If the Reviewer meant quality control of the methods used in the work, then the authors tested and adapted the described methods over 10 years in previously published works (Grinchenko et al., 2015-2021; Sokolnikova et al., 2014-2024; Kumeiko et al., 2018-2021), including patent (RU2014150448). If the Reviewer meant control samples, then the Authors used hemolymph parameters of mollusks from non-impacted water areas as a control. All protocols for collecting, processing, and samples storage are also described in detail in the relevant sections of the Materials and Methods. The reactions were performed in triplicate, as indicated in the description of each of the methods. The equipment of the A.V. Zhirmunsky National Scientific Center of Marine Biology and the Far Eastern Federal University undergoes mandatory periodic verification in accordance with the established metrological requirements of each device. All analyses and data collection were carried out using automatic devices and instruments.

 

Results

Ln 161: The sentence should be like this ….hemolytic activity (HL) of plasma were identified in T. boucardi.

Conclusion

Ln 545: Add coma (,) after M. gigas.

Answer: Authors changed the sentence according to the Reviewer’s remarks.

 

Ln 556_560: The sentence should be like this “In the future, the assessment of hemolymph parameters combined with molecular analysis, reflecting the health status of aquatic organisms, will ensure greater efficiency in their cultivation. This, in turn, will lead to an increase in their quality of life, improvement in physiological state, increased survival, and higher bioproductivity."

Answer: Authors modified the sentence into two, according Reviewer’s suggestion.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript diversity-3033275 by the authors Grinchenko et al. entitled “Hemolymph parameters are a useful tool for assessing bivalve health and water quality" is a MS with valuable results of haemolymph analyses of commercial bivalves that are worth studying and falls within the scope of MDPI Diversity Journal - Special Issue: Biodiversity as Tools to Assess Impacts on Coastal Ecosystems.

 

Bivalves are a widespread group of invertebrates of high ecological and economic importance and a key to the development, functioning and sustainability of coastal environments (goods and services of marine bivalves). Coastal areas are their most important habitats and growing areas, which are under increasing pressure from various anthropogenic activities. In this work, the authors analysed 11 haemolymph parameters of 15 commercially available species from wild populations at stations in non-impacted and impacted water areas of the Sea of Japan.

 

The most pronounced and reliable changes were observed in the number of hemolymph cells, the percentage of hemocyte types, phagocytic activity, reactive oxygen species and protein concentration. The species most indicative of pollution were M. chinensis, M. yessoensis, M. gigas and C. grayanus, in which most of the 11 hemolymph parameters examined changed significantly.

 

I agree with the authors that there is a lack of large-scale monitoring studies at regional and global levels to assess the health status of commercial shellfish. Therefore, efforts to establish reference ranges for selected cellular and humoral parameters of bivalve haemolymph from aquatic areas with different ecological conditions and pollution are welcome.

 

Comments

- Moderate – Extensive editing and improvement of the English language is required!

- Instead of organism e.g. “fish condition” line 46, 60. use the term “health” or “health status”

- Methods should be described in more detail. For example, there is missing information on hemolymph sample types (line 104), incubation conditions (line 117), etc. In addition, the number of specimens per location and number of technical repetitions is missing for all described analyses. This information should be provided for all investigated species. 

- References are only sparingly provided at the end of the paragraphs. Also, some sections of materials and methods are missing references. This should be corrected as it is difficult to check the manuscript content and referred statements. - Figures are indicated jointly, and this reduces the amount of space they would otherwise take. However, they should be placed after the text that describes them.

- It is rather not clear the selection of confidence intervals instead of using standard deviation/standard errors. 

- The within population variability is often commented, but it is not described in the material and methods section. How was it evaluated? It should be noted that the population variance is not equal to standard deviation or standard error and it is calculated differently. 

 

Line 20

-Instead, sensitive write “indicative species of pollution”

 

Line 22

-> “Health status cards”

 

Line 66

“Field researches have more advantages.” – it is a too general statement! Similar sentences should be revised throughout the manuscript. 

 

Line 81

-> Accepted names of species Spisula sachalinensis and Azumapecten farreri should be provided. Authors are invited to consult databases such as WoRMS. 

 

Line 106

-> “precooled (+4C)”

 

Line 107

-> Units should be written as indicated in the Journal’s guidelines (for ex., mL instead of ml).

 

 

Line 113-131

Hemolytic activity, hemagglutination reaction and Protein conc. were measured in plasma? Please give more details. Usually for protein concentration Bradford assay and BSA standards are used.

 

Line 243

- Figure 4C should be corrected as the confidence interval is not fully visible (y-axis values should be increased).

 

Line 292

-> “…bioindicators of the quality of marine environment.” 

 

Line 306

 

- Reference 25, Newton and Cope (2006:) analysed biomarker responses of Unionid mussels not Mytilids to environmental contamination.

Comments on the Quality of English Language

Moderate – Extensive editing and improvement of the English language is required!

Author Response

Response to Reviewer 3

The Authors are grateful to the Reviewer for the careful and insightful review of our manuscript and kind words. The authors considered the Reviewer’s comments and suggestions and made all necessary corrections to the manuscript. The English writing has been checked and corrected by the Language Editing Service. We believe that the incorporation of the Reviewer's recommendations has significantly improved the manuscript’s quality.

 

Comments

- Methods should be described in more detail. For example, there is missing information on hemolymph sample types (line 104), incubation conditions (line 117), etc. In addition, the number of specimens per location and number of technical repetitions is missing for all described analyses. This information should be provided for all investigated species. 

Answer: The Authors agree with the Reviewer's comment about the lack of details in the Materials and Methods and have made the appropriate changes.

However, the Authors, unfortunately, did not understand what the Reviewer meant by “information on hemolymph sample types (line 104).”

Regarding “missing, incubation conditions (line 117)…”, while writing the manuscript, the Authors used Instructions for Authors and provided links to previously published methods, as indicated in Instructions for Authors: “New methods and protocols should be described in detail while well -established methods can be briefly described and appropriately cited.” In the paragraph dedicated to the description of the assessment of hemolytic activity of plasma, to simplify the perception of information and not duplicate an already published method, a link is provided to a reference (line 206), which describes in detail the procedure for implementing this type of examination:

«Hemolytic activity was estimated according to a method described in detail by Grinchenko and colleagues [18]…» Grinchenko, A.; Sokolnikova, Y.; Korneiko, D.; Kumeiko, V. Dynamics of the Immune Response of the Horse Mussel Modiolus Kurilensis (Bernard, 1983) Following Challenge with Heat-Inactivated Bacteria. J Shellfish Res 2015, 34, 909–917, doi:10.2983/035.034.0321.

However, the Authors clarified information regarding the incubation conditions (line 208).

The Authors agree with the Reviewer regarding “the number of specimens per location” and have made the appropriate changes (line 111).

As for the “number of technical repetitions is missing for all described analyses,” the reactions were performed in triplicate, as indicated in the description of each of the methods that required this (for example, the line 226 or corresponding link with a description of the methodology (line 235)

 

- References are only sparingly provided at the end of the paragraphs. Also, some sections of materials and methods are missing references. This should be corrected as it is difficult to check the manuscript content and referred statements.

Answer: Authors changed references position in the manuscript according Reviewer’s suggestion.

 

- Figures are indicated jointly, and this reduces the amount of space they would otherwise take. However, they should be placed after the text that describes them.

Answer: All Figures are placed according to generally accepted rules: immediately after the first paragraph in which the Figure is mentioned. Thus, Figure 1 is on line 125, and its first mention is on line 119. Figure 2 is located on line 264, and is first mentioned on line 255. Figure 3 is on line 299, after its first mention in a sentence on line 290. And Figure 4 is mentioned on line 333 and is located on line 324. Therefore, after the first mention and placement of the figure, there are usually no more than 9 lines of the paragraph.

 

- It is rather not clear the selection of confidence intervals instead of using standard deviation/standard errors. 

Answer: There is currently no certain, generally accepted way to display sampling variability. In the case of the mean, this can be the standard deviation, the error of mean, or the confidence interval. At the same time, for formal analysis, we used nonparametric Mann-Whitney analysis, but for simplicity of data presentation, we still used the average, and not the median and ranges. At the same time, the overlap of confidence intervals is indicative in the case of parametric statistics (t-test, for example) and, thus, additionally verifies the differences identified by the nonparametric Mann-Whitney test.

 

- The within population variability is often commented, but it is not described in the material and methods section. How was it evaluated? It should be noted that the population variance is not equal to standard deviation or standard error and it is calculated differently. 

Answer: The authors, using the phrase “population variability,” meant the variability of the obtained values of a particular indicator within the sample. To eliminate inaccuracies, Authors changed this phrase in the manuscript.

 

Line 66

“Field researches have more advantages.” – it is a too general statement! Similar sentences should be revised throughout the manuscript. 

Answer: Authors changed the statement according Reviewer’s suggestion.

 

Line 107

-> Units should be written as indicated in the Journal’s guidelines (for ex., mL instead of ml).

Answer: Authors corrected all units according Reviewer’s suggestion.

 

Line 113-131

Hemolytic activity, hemagglutination reaction and Protein conc. were measured in plasma? Please give more details. Usually for protein concentration Bradford assay and BSA standards are used.

Answer: Humoral parameters (hemolytic activity, hemagglutination reaction, and protein concentration) were measured in plasma, the corresponding clarifications were included in the Materials and Methods (line 201).

The Bradford method is by far one of the most accurate methods for measuring protein concentration. However, measuring protein absorbance at 280 nm (A280) is also often used (Kielkopf et al., 2020; Reinmuth-Selzle et al., 2022; Goldring, 2012; Porterfield et al., 2010), and this is one of the oldest and simplest methods (Warburg and Christian, 1942; Layne, 1957), and does not require incubating the sample with exogenous chromophores. This method also uses a BSA solution as a standard. We chose the UV method due to its speed and efficiency, which fully meets our needs.

 

Line 243

- Figure 4C should be corrected as the confidence interval is not fully visible (y-axis values should be increased).

Answer: Authors changed Figure 4C according to the Reviewer’s suggestion.

 

Line 20

-Instead, sensitive write “indicative species of pollution”

Line 22

-> “Health status cards”

- Instead of organism e.g. “fish condition” line 46, 60. use the term “health” or “health status”

Line 106

-> “precooled (+4C)”

Line 292

-> “…bioindicators of the quality of marine environment.” 

Answer: Authors changed the sentences according to the Reviewer’s comment.

 

Line 81

-> Accepted names of species Spisula sachalinensis and Azumapecten farreri should be provided. Authors are invited to consult databases such as WoRMS. 

Line 306

- Reference 25, Newton and Cope (2006:) analysed biomarker responses of Unionid mussels not Mytilids to environmental contamination.

Answer: Authors are grateful to the Reviewer for the valuable remarks. Corresponding changes have been made.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

This paper can be accepted in present form.

Comments on the Quality of English Language

This paper can be accepted in present form.

Reviewer 2 Report

Comments and Suggestions for Authors

There are a lot of papers published on hemocytes of bivalves, so this work is not very innovative.

Comments on the Quality of English Language

English language is ok