Glochidia Infection of Endemic Fishes from Lake Prespa, N. Macedonia

Round 1

Reviewer 1 Report

The authors described glochidia infection of two fish Prespa roach and Prespa nase from Lake Prespa, N. Macedonia. This is the first documented introduction of infection into this water area. Although it is a local introduction, it would be good to observe it from the standpoint of introducing a new pathogen into a particular area and shed as much light as possible on this introduction. My questions, comments and suggestions are also in this context.

Abstract suggestion:

Large freshwater mussels (Unionida) are very long-lived, have large bodies, and produce thousands to millions of larvae (glochidia) that usually must attach to host fish tissue to complete the life cycle. This is an obligate parasitic stage of mussel larvae. However, less than one in 1 million find a suitable host and survive. The degree of host specificity varies among unionid species from specialists that can successfully parasitize only one or a few closely related fish species to generalists that can complete development on a taxonomically broad range of fish species.

Freshwater mussels are among the most threatened groups of animals. This is due to habitat destruction, introduction of non-native species, and loss of host fish on which their larvae (glochidia) are obligate parasites. Glochidiosis harms fish by affecting their growth. On the other hand, freshwater mussels play an important role in freshwaters by improving water quality and constantly ridding the water of bacteria, algae, and pollutants, and they are also an indicator species of water quality.During our parasitological survey of fish from the Macedonian part of Lake Prespa in April 2022, many glochidia were found on the gills, skin and fins of two endemic fishes - Prespa roach (Rutilus prespensis) and Prespa nase (Chondrostoma prespense), in the range from tens to thousands on one host.

 

Add a map of the Macro and Micro Prespa, since these are new species and it is interesting to visualize the ecological relationship of the lakes to the environment.

Line 51-52. Freshwater mussels (order Unionoida) play an important role in lakes and rivers by constantly filtering water as they go, breathe, and feed.

Lines 52-54: They are filter feeders and improve water quality by filtering out bacteria, algae, organic matter and pollutants. In the process, they accumulate contaminants in their bodies but do not break them down.

Line 119: how much is any type of fish Prespa roach and Prespa nase, share in the total number 27? ie, sentence from lines 134-136, please move to material and methods.

 

Lines 158 and later, statements must be supported by literature.

Lines 173-175: how these new species were introduced?

Therefore, what are the possible measures to prevent these inputs into lake systems?

Author Response

Please see the attachment

Reviewer 2 Report

This is an interesting study investigating glochidia of swan mussel Anodonta cygnea infection on two endemic fishes - Prespa roach (Rutilus prespensis) and Prespa nase (Chondrostoma prespense) from Lake Prespa, N. Macedonia. The findings will contribute to enabling conservation of the freshwater mussels and endemic fishes. However, the presentation of the method and statistical analysis of the data need to be further improved. See the attached PDF with further comments.

Comments for author File: Comments.pdf

Author Response

Please see the attachment

Author Response File: Author Response.docx

Reviewer 3 Report

The paper under revision is dedicated to studying of the glochidia infection of two endemic fishes from the lake Prespa. This topic is high of importance as it expanded the known host range for glochidia of A. cygnea. It is also necessary information for conservation measures both fishes and mussels.

The manuscript leaves controversial impressions. Authors have a good field data to make a sound paper of a good quality about glochidia infestation of two endemic fishes. However, data, represented in the paper, are very brief and incomplete. The authors tried to accent on the impact of the glochidia infection on fishes, but do not have, or do not provide, appropriate data.

I would recommend rewriting the manuscript with main scope on analysis of infestation characteristics of two endemic fishes and discuss the results in the context of the global host diversity of A. cygnea.

Please keep in mind, that references are an important part of the scientific publication and the dogma of the scientific ethics.

I would also recommend proofreading the text by the person who fluent in English. I did not correct grammar and style, but it is definitely necessary.

I will look forward to the next round of revision of this manuscript, that could turn out into the nice paper.

Ilya V. Vikhrev

 

Additional comments regarding the text are below.

Title

Swan Mussel Glochidia Infection of Some Two Endemic Fish Fishes from the Lake Prespa, N. Macedonia

Abstract

According to the “Instruction for Authors” of the journal, the abstract should provide information about background, methods, results, and conclusion. Please rewrite the abstract following the mentioned structure.

Keywords

I would recommend using different words in the title and keywords. It will help to gain more “visibility” fore searching algorithms in the internet.

Introduction

Since the main topic of the paper is how mussels infect fishes in the Lake Prespa, the introduction is better to start not with the story about the lake. Move the first paragraph (Lines: 34-48) in the end of the Introduction.

The significant portion of the Introduction (Lines: 51-95) provide overview of mussels’ ecosystem services and life cycle features, but with the only single reference, which is in the wrong style (Line 76). Please add the appropriate references.

It is also necessary to define the purpose of the work and main tasks that should be solved to achieve the purpose. Usually, this information is placed at the end of the section.

Materials and methods

L. 120: remove parasitologically. How many specimens of each species were investigated?

L. 122: Describe the methodology of the fish observation or provide the appropriate reference(s).

L. 129: Describe the methodology of the slide preparation or provide the appropriate reference(s).

L. 127: What kind of identification do you mean?

In the Results section you mention that A. cygnea specimens were sampled. Describe the purpose of the sampling and how it was performed.

Results and Discussion

Lines 133-136: Information about sample size and time of sampling must be moved to the Methods.

L. 140: Fig. 2 illustrates the shell, but not the data about glochidial load. Did authors identify the species definition of glochidia by its morphology?

L. 186: the reference not in the appropriate style.

L. 151: I do not see in the paper any evidence of the “clear harm” of glochidia load.

Please add the appropriate references from the line 158 and up to the end. I suppose, many facts you discussed are found in the literature.

 

The authors provided information about extensivity of infestation (how many fishes were infested) but did not about intensity (how many glochidia were found on each fish). Such kind of data is better to represent in a summary table with sample volumes and infestation values. It would be also interesting to see information how glochidia were distributed between different parts of the fish's body. The comparison between infestation of endemic fishes from the one side and common fishes from the other side would also contribute significantly to the paper.

Author Response

Please see the attachment

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

It's nice of you to have spent the time and energy to improve your manuscript according to the reviewers' suggestions. When there is will, nothing is difficult, and there is always a reason to improve the manuscript. So check your English again in the manuscript, like as suggestions:

 

Lines 206 – 209: ‘’There was a trend toward higher intensity of infection in the Prespa nase than in the Prespa roach. The mean (± S.E.) number of glochidia per infected fish was 40.0 ± 17.6 in the Prespa nase and 4.4 ± 0.9 in the Prespa roach, with only a marginally statistically significant difference ….’’

 

Lines 278 – 280: ‘’We expect to find out more data on the infestation rate and the effects of glochidia on the health status of the fish in the following studies. Already known host fishes of….’’

Author Response

Dear reviewer,

Thank you for your useful comments and suggestions. We give our best to provide the best answers to your comments within the 2 suggestions. We looking forward on acceptance of our last minor changes.

Kind regards,

Stojmir

Reviewer 2 Report

The revised manuscript has been greatly improved. I think it's ready for publication.

Author Response

Dear reviewer,

Thank you for your useful comments and suggestions. We made some minor changes within the text, in order to accept and the recommendations from the other reviewers. We looking forward on acceptance of our last minor changes.

Kind regards,

Stojmir

Reviewer 3 Report

Dear authors,

Unfortunately, I have to recommend to reject your paper. Since you did not provide the response to the reviewer's comments I do not know whether you agree or disagree with them and why. The information in the cover letter is very general. Moreover, I do not see that the manuscript has been improved. Most of the corrections looks formal. I do not attach the manuscript with my comments, because almost all of them remain the same as after the first revision, but one important thing I would like to mention here. The difference with the significancy value more or equal 0.05 (p ≥ 0.05) is not statistically significant.

Ilya V. Vikhrev

Author Response

Dear reviewer,

Thank you for your useful comments and suggestions. We'd like to apologies for the misunderstandings, as we give our best to incorporate all the three reviewers suggestions and recommendations without losing our main aims and we missed out to provide you point by point explanation during the previous review round.

Please find below our responses to your comments regarding your most important recommendation during the second review: "... but one important thing I would like to mention here. The difference with the significancy value more or equal 0.05 (p ≥ 0.05) is not statistically significant. " - Within our results we did not state that the difference was statistically significant, but statistically marginally significant. P-value gives the probability of erroneously discarding the null hypothesis. Usually the criterion p < 0.05 is set as the sign of statistical significance. As well, frequently, values 0.05<p<0.10 are regarded as statistically marginally significant. Thus, our interpretation is that p = 0.083 is statistically marginally significant.

Hopefully with this second round we will satisfy your expectations.

Kind regards,

Stojmir

Round 3

Reviewer 3 Report

Dear Stojmir,

Thank you for the response and clarification.

I see that the manuscript has been improved and some important concerns have been corrected.

Regarding the statistical significance. Ok, I agree that the statement of “marginally significancy” is not a methodological error. However, it is a big discussion about how to represent and interpreting results which are close to significance threshold:

https://www.tandfonline.com/doi/full/10.1080/00031305.2018.1518788

https://www.medrxiv.org/content/10.1101/2021.03.01.21252701v1

https://www.psychologicalscience.org/observer/how-marginal-are-marginally-significant-p-values

https://www.reddit.com/r/AskStatistics/comments/9lp3cj/whats_the_correct_way_to_interpret_marginally/

In your case I do not think that using “marginally significant” has more sense than “insignificant”, but it is on the Editor’s decision. What is arbitrary necessary is the table showing how many glochidia per fish, and even better per fish’s body part, have been recorded. Such table will help to make you study reproducible.

One of the main and valuable findings of you research is that you expanded range of host fishes of A. cygnea and introduced two new host to the list. You should mention it in the Abstract and write somewhere in the beginning of the Results and Discussion.

L. 264: Is there only A. cygnea inhabits the lake or other naiades also?  If several Unionid taxa present in the lake, did you find their glochidia?

L. 272: Did you identify glochidia by morphology? Briefly describe characters that allowed you to identify glochidia as belonged to A. cygnea

L. 276: Did you investigate correlation between the fish’s size/weight and glochidial load?

 

Figure 4: fish names in the figure should be in italic

Author Response

Dear Reviewer,

Thank you for your feedback. Unfortunately at this point we cannot provide a genuine table with the infestation rate of how many glochidia per fish/or per body part have been found. This was the novel finding for us (never met before) and at that moment and overload with lab work that was focused on other fish parasites we overlooked this details. For sure for future research we'll keep this in mind.

To respond on your recommendations:

We add the first records of infestation with A. cyanea glochidia on this endemic species within the abstract and in the first paragraph in the result and discussion section.

1. 264: Is there only A. cygnea inhabits the lake or other naiades also?  If several Unionid taxa present in the lake, did you find their glochidia?

- Up to day the only recorded naiad species into the  Lake Prespa is A. cygnea.

1. 272: Did you identify glochidia by morphology? Briefly describe characters that allowed you to identify glochidia as belonged to A. cygnea

- As the only recorded species is A. cygnea was easy to determine that this glochidia belonged to this species. The species was confirmed during my STSM visit over COST project - Conservation of Freshwater Mussels Pan-European Approach (CA18239) with Dr. Jouni Taskinen in Finland (Department of Biological and Environmental Science, University of Jyväskylä).

1. 276: Did you investigate correlation between the fish’s size/weight and glochidial load?

 - As above mentioned we didn't had a chance to investigate this correlation during the lab. work.

- we corrected the names adding font italic in Figure 4.

Kind regards,

Stojmir

Author Response File: Author Response.docx