Description of Life Cycle Stages of Fish Parasite Cymothoa pulchrum (Isopoda: Cymothoidae), with DNA Barcode Linked to Morphological Details
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThis paper discussed the life stages of Cymothoa pulchrum and its genetic sequence. The information is needed and valuable for future research.
Some of the images are blurry and should be replaced or deleted.
There is some repetition and unnecessary information in the methods section that needs to be addressed. Some clarity is needed in the descriptions too.
The different life stages are clearly distinguished and drawings are provided of each. It would be helpful to know how many of each life stage were observed.
More small edits are provided in the pdf.
Comments for author File: 
Comments.pdf
Author Response
Prof. Maria Angeles Esteban
Editor-in-Chief
Fishes
Dear Editor:
We wish to re-submit the manuscript titled “Description of life cycle stages of fish parasite Cymothoa pulchrum (Isopoda: Cymothoidae), with DNA barcode linked to morphological details” The manuscript No. is 3530247.
We thank you and the reviewer for your thoughtful suggestions and insights. The manuscript has benefited from these insightful suggestions. We look forward to working with you and the reviewers to move this manuscript closer to publication in Fishes.
The manuscript has been rechecked and the necessary changes have been made in accordance with the reviewer’ suggestions. The responses to all comments have been prepared and attached herewith below.
Thank you for your consideration. I look forward to hearing from you.
Sincerely,
Hiroki Fujita
R1
This paper discussed the life stages of Cymothoa pulchrum and its genetic sequence. The information is needed and valuable for future research.
Some of the images are blurry and should be replaced or deleted.
There is some repetition and unnecessary information in the methods section that needs to be addressed. Some clarity is needed in the descriptions too.
The different life stages are clearly distinguished and drawings are provided of each. It would be helpful to know how many of each life stage were observed.
More small edits are provided in the pdf.
Response: Thank you for your helpful comments and suggestions. We responded to each of the points raised as follows.
Comments in PDF
L34: The
Response: I don't think articles should be added to scientific names.
L76: Under the tongue?
Response: Fish don't have tongue, they just have basihyal. I added an explanation.
L81: A lot of this information is repeated in the discussion. A more condensed version needs to be provided here to avoid repetition. Just numbers of hosts and how long they lived or something similar.
Response: We have removed repetition in the discussion.
L95: This reference is not in the reference list
Response: Agreed and revised as your suggestion.
L104: This seems like results and should be lower down
Response: Agreed and revised as your suggestion.
Figure 3: Some of these images are very blurry. Can you maybe get better images? Otherwise remove this plate as it is not helpful at this point.
Response: A high quality version was inserted.
L211: What about the mouthparts?
Response: The information was omitted to match the information listed in this taxonomy.
L221: What is a pleopod gill?
Response: It was not appropriate and has been removed.
L404: Is a pearl yellow?
Response: We think this is pearl yellow.
L427: I assume this was manca II?
Response: If pre-manca is called manca I, manca is called manca II. We added the explanation in the first mention (L56). The figure of pereopod 7 has also been added.
L429: In what way is it underdeveloped?
Response: We added the explanation in the first mention (L402).
L459: How many observations of each stage were made?
Response: Individuals observed are listed in Material examined.
L500; Are these all accurate identifications? Would you move some of the life stages into different life stages?
Response: I confirmed the life cycle stages described in each paper.
L569: Looking at this paper I see the original name was also Aulopus japonicus and this is most likely a taxonomic misapplied name problem (where the "new" species name was not correct at the time).
Response: Although I was unable to find any taxonomic history that would explain why the two species were confused, I changed the expression to a milder one.
R2
I have reviewed the manuscript titled “Description of life cycle stages of the fish parasite Cymothoa pulchrum (Isopoda: Cymothoidae), with DNA barcode linked to morphological details.” The manuscript is well-written and provides valuable taxonomic information on this parasitic isopod species. The illustrations are of high quality, and the study utilizes two relevant molecular markers for species identification. This work contributes to a better understanding of the biodiversity of isopod parasites in fish. Below, I provide some minor comments for consideration.
Response: Thank you for your helpful comments and suggestions. We responded to each of the points raised as follows.
Introduction
Line 48. Do all males transition to females, or do some remain male throughout their entire lives?
Response: The exact details are not known, so we will avoid mentioning them.
Line 58. The presence of C. pulchrum in Tetraodontiformes is mentioned twice in consecutive lines. Please reword to avoid repetition.
Response: Agreed and revised as your suggestion.
Methods
Lines 146 – 150. In this paragraph, it would be helpful to specify how many other species were included in the phylogenetic trees. You could also mention that the GenBank accession numbers are provided in the respective section below. If deemed useful, the authors may consider providing a table with relevant information on these species, such as their locality, hosts, and references if previously published.
Response: Agreed and revised as your suggestion.
R3
- lines 125-126. The amount of samples: only 3 parasites (each of them is different ) is too small to get DNA information.
Response: These populations are considered to be sufficient for DNA barcoding.
- line 196. If the female was damaged what kind of precise morphological information could be achieved?
Response: As written in the material examibed, that individual was not used for detailed morphological observations.
- lines 514-521. The information in this paragraph is not convincing enough. It consist only of suppositions. I suggest that the authors study it more carefully before including it in the article or exclude it from the article.
Response: The relevant discussion has been deleted.
R4
The manuscript provides a comprehensive morphological description of Cymothoa pulchrum across its life stages, supported by molecular phylogenetic analysis using COI and 16S rRNA sequences. This research contributes significantly to cymothoid taxonomy by addressing the lack of morphological data beyond adult stages. However, substantial revisions are needed to improve data presentation, methodological justification, and phylogenetic interpretation.
Response: Thank you for your helpful comments and suggestions. We responded to each of the points raised as follows.
The genetic division of C. pulchrum into two groups in COI and 16S rRNA trees raises critical taxonomic concerns, yet the manuscript does not assess whether this divergence indicates cryptic speciation. It remains unclear whether genetic distance analyses, such as pairwise K2P distances, were conducted to evaluate intra- and interspecific variation or whether additional nuclear markers (e.g., 18S rRNA, ITS) were used to validate species delimitation.
Response: We added analysis of perawise intra- and inter specific genetic distances.
Furthermore, the relationship between C. pulchrum and C. eremita is ambiguous, with overlapping COI clades suggesting potential historical misidentifications in GenBank, necessitating further morphological verification of voucher specimens.
Response: MK430019 - MK430026 are sequences obtained at the same time as morphological descriptions by taxonomists, so the risk of misidentification has probably been eliminated. Since there are sequences of eremita that have been reliably identified as species, it is possible to discuss and debate them (L565-587).
The manuscript also inconsistently states that C. pulchrum is specific to Tetraodontiformes while acknowledging its presence in Carangiformes and Acanthuriformes, raising questions about the criteria for determining host specificity.
Response: The claim that it is specific to Tetraodontiformes is that of Martin et al., and we are citing it. The wording has been changed (L570).
Additionally, the proposed life cycle hypothesis lacks direct experimental evidence; stable isotope or gut content analyses would strengthen claims that mancae and juveniles obtain nutrients from their hosts while attached externally. If these concerns cannot be fully addressed, they should be acknowledged as study limitations.
Response: The purpose of this is to present it as a future research topic, so I revised the text.
The writing also lacks clarity and conciseness, with redundant morphological descriptions in the Results and Discussion sections that hinder readability. Summarizing these details in a comparative table would improve organization and enhance academic rigor.
Response: This method of description is a common taxonomic method and should not be changed. If a comparison is needed in the future, the delta file provided as S1 can be used.
Reviewer 2 Report
Comments and Suggestions for AuthorsI have reviewed the manuscript titled “Description of life cycle stages of the fish parasite Cymothoa pulchrum (Isopoda: Cymothoidae), with DNA barcode linked to morphological details.” The manuscript is well-written and provides valuable taxonomic information on this parasitic isopod species. The illustrations are of high quality, and the study utilizes two relevant molecular markers for species identification. This work contributes to a better understanding of the biodiversity of isopod parasites in fish. Below, I provide some minor comments for consideration.
Introduction
Line 48. Do all males transition to females, or do some remain male throughout their entire lives?
Line 58. The presence of C. pulchrum in Tetraodontiformes is mentioned twice in consecutive lines. Please reword to avoid repetition.
Methods
Lines 146 – 150. In this paragraph, it would be helpful to specify how many other species were included in the phylogenetic trees. You could also mention that the GenBank accession numbers are provided in the respective section below. If deemed useful, the authors may consider providing a table with relevant information on these species, such as their locality, hosts, and references if previously published.
Author Response
Prof. Maria Angeles Esteban
Editor-in-Chief
Fishes
Dear Editor:
We wish to re-submit the manuscript titled “Description of life cycle stages of fish parasite Cymothoa pulchrum (Isopoda: Cymothoidae), with DNA barcode linked to morphological details” The manuscript No. is 3530247.
We thank you and the reviewer for your thoughtful suggestions and insights. The manuscript has benefited from these insightful suggestions. We look forward to working with you and the reviewers to move this manuscript closer to publication in Fishes.
The manuscript has been rechecked and the necessary changes have been made in accordance with the reviewer’ suggestions. The responses to all comments have been prepared and attached herewith below.
Thank you for your consideration. I look forward to hearing from you.
Sincerely,
Hiroki Fujita
R1
This paper discussed the life stages of Cymothoa pulchrum and its genetic sequence. The information is needed and valuable for future research.
Some of the images are blurry and should be replaced or deleted.
There is some repetition and unnecessary information in the methods section that needs to be addressed. Some clarity is needed in the descriptions too.
The different life stages are clearly distinguished and drawings are provided of each. It would be helpful to know how many of each life stage were observed.
More small edits are provided in the pdf.
Response: Thank you for your helpful comments and suggestions. We responded to each of the points raised as follows.
Comments in PDF
L34: The
Response: I don't think articles should be added to scientific names.
L76: Under the tongue?
Response: Fish don't have tongue, they just have basihyal. I added an explanation.
L81: A lot of this information is repeated in the discussion. A more condensed version needs to be provided here to avoid repetition. Just numbers of hosts and how long they lived or something similar.
Response: We have removed repetition in the discussion.
L95: This reference is not in the reference list
Response: Agreed and revised as your suggestion.
L104: This seems like results and should be lower down
Response: Agreed and revised as your suggestion.
Figure 3: Some of these images are very blurry. Can you maybe get better images? Otherwise remove this plate as it is not helpful at this point.
Response: A high quality version was inserted.
L211: What about the mouthparts?
Response: The information was omitted to match the information listed in this taxonomy.
L221: What is a pleopod gill?
Response: It was not appropriate and has been removed.
L404: Is a pearl yellow?
Response: We think this is pearl yellow.
L427: I assume this was manca II?
Response: If pre-manca is called manca I, manca is called manca II. We added the explanation in the first mention (L56). The figure of pereopod 7 has also been added.
L429: In what way is it underdeveloped?
Response: We added the explanation in the first mention (L402).
L459: How many observations of each stage were made?
Response: Individuals observed are listed in Material examined.
L500; Are these all accurate identifications? Would you move some of the life stages into different life stages?
Response: I confirmed the life cycle stages described in each paper.
L569: Looking at this paper I see the original name was also Aulopus japonicus and this is most likely a taxonomic misapplied name problem (where the "new" species name was not correct at the time).
Response: Although I was unable to find any taxonomic history that would explain why the two species were confused, I changed the expression to a milder one.
R2
I have reviewed the manuscript titled “Description of life cycle stages of the fish parasite Cymothoa pulchrum (Isopoda: Cymothoidae), with DNA barcode linked to morphological details.” The manuscript is well-written and provides valuable taxonomic information on this parasitic isopod species. The illustrations are of high quality, and the study utilizes two relevant molecular markers for species identification. This work contributes to a better understanding of the biodiversity of isopod parasites in fish. Below, I provide some minor comments for consideration.
Response: Thank you for your helpful comments and suggestions. We responded to each of the points raised as follows.
Introduction
Line 48. Do all males transition to females, or do some remain male throughout their entire lives?
Response: The exact details are not known, so we will avoid mentioning them.
Line 58. The presence of C. pulchrum in Tetraodontiformes is mentioned twice in consecutive lines. Please reword to avoid repetition.
Response: Agreed and revised as your suggestion.
Methods
Lines 146 – 150. In this paragraph, it would be helpful to specify how many other species were included in the phylogenetic trees. You could also mention that the GenBank accession numbers are provided in the respective section below. If deemed useful, the authors may consider providing a table with relevant information on these species, such as their locality, hosts, and references if previously published.
Response: Agreed and revised as your suggestion.
R3
- lines 125-126. The amount of samples: only 3 parasites (each of them is different ) is too small to get DNA information.
Response: These populations are considered to be sufficient for DNA barcoding.
- line 196. If the female was damaged what kind of precise morphological information could be achieved?
Response: As written in the material examibed, that individual was not used for detailed morphological observations.
- lines 514-521. The information in this paragraph is not convincing enough. It consist only of suppositions. I suggest that the authors study it more carefully before including it in the article or exclude it from the article.
Response: The relevant discussion has been deleted.
R4
The manuscript provides a comprehensive morphological description of Cymothoa pulchrum across its life stages, supported by molecular phylogenetic analysis using COI and 16S rRNA sequences. This research contributes significantly to cymothoid taxonomy by addressing the lack of morphological data beyond adult stages. However, substantial revisions are needed to improve data presentation, methodological justification, and phylogenetic interpretation.
Response: Thank you for your helpful comments and suggestions. We responded to each of the points raised as follows.
The genetic division of C. pulchrum into two groups in COI and 16S rRNA trees raises critical taxonomic concerns, yet the manuscript does not assess whether this divergence indicates cryptic speciation. It remains unclear whether genetic distance analyses, such as pairwise K2P distances, were conducted to evaluate intra- and interspecific variation or whether additional nuclear markers (e.g., 18S rRNA, ITS) were used to validate species delimitation.
Response: We added analysis of perawise intra- and inter specific genetic distances.
Furthermore, the relationship between C. pulchrum and C. eremita is ambiguous, with overlapping COI clades suggesting potential historical misidentifications in GenBank, necessitating further morphological verification of voucher specimens.
Response: MK430019 - MK430026 are sequences obtained at the same time as morphological descriptions by taxonomists, so the risk of misidentification has probably been eliminated. Since there are sequences of eremita that have been reliably identified as species, it is possible to discuss and debate them (L565-587).
The manuscript also inconsistently states that C. pulchrum is specific to Tetraodontiformes while acknowledging its presence in Carangiformes and Acanthuriformes, raising questions about the criteria for determining host specificity.
Response: The claim that it is specific to Tetraodontiformes is that of Martin et al., and we are citing it. The wording has been changed (L570).
Additionally, the proposed life cycle hypothesis lacks direct experimental evidence; stable isotope or gut content analyses would strengthen claims that mancae and juveniles obtain nutrients from their hosts while attached externally. If these concerns cannot be fully addressed, they should be acknowledged as study limitations.
Response: The purpose of this is to present it as a future research topic, so I revised the text.
The writing also lacks clarity and conciseness, with redundant morphological descriptions in the Results and Discussion sections that hinder readability. Summarizing these details in a comparative table would improve organization and enhance academic rigor.
Response: This method of description is a common taxonomic method and should not be changed. If a comparison is needed in the future, the delta file provided as S1 can be used.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe topic of this article is very interesting and the authors have done very through work describing different stages of life cycle of Cymothoa pulchrum. They received new data which will be useful for the specialists in fish parasitology.
Nevertheless, I have some questions and comments.
- lines 125-126. The amount of samples: only 3 parasites (each of them is different ) is too small to get DNA information.
- line 196. If the female was damaged what kind of precise morphological information could be achieved?
- lines 514-521. The information in this paragraph is not convincing enough. It consist only of suppositions. I suggest that the authors study it more carefully before including it in the article or exclude it from the article.
Author Response
Prof. Maria Angeles Esteban
Editor-in-Chief
Fishes
Dear Editor:
We wish to re-submit the manuscript titled “Description of life cycle stages of fish parasite Cymothoa pulchrum (Isopoda: Cymothoidae), with DNA barcode linked to morphological details” The manuscript No. is 3530247.
We thank you and the reviewer for your thoughtful suggestions and insights. The manuscript has benefited from these insightful suggestions. We look forward to working with you and the reviewers to move this manuscript closer to publication in Fishes.
The manuscript has been rechecked and the necessary changes have been made in accordance with the reviewer’ suggestions. The responses to all comments have been prepared and attached herewith below.
Thank you for your consideration. I look forward to hearing from you.
Sincerely,
Hiroki Fujita
R1
This paper discussed the life stages of Cymothoa pulchrum and its genetic sequence. The information is needed and valuable for future research.
Some of the images are blurry and should be replaced or deleted.
There is some repetition and unnecessary information in the methods section that needs to be addressed. Some clarity is needed in the descriptions too.
The different life stages are clearly distinguished and drawings are provided of each. It would be helpful to know how many of each life stage were observed.
More small edits are provided in the pdf.
Response: Thank you for your helpful comments and suggestions. We responded to each of the points raised as follows.
Comments in PDF
L34: The
Response: I don't think articles should be added to scientific names.
L76: Under the tongue?
Response: Fish don't have tongue, they just have basihyal. I added an explanation.
L81: A lot of this information is repeated in the discussion. A more condensed version needs to be provided here to avoid repetition. Just numbers of hosts and how long they lived or something similar.
Response: We have removed repetition in the discussion.
L95: This reference is not in the reference list
Response: Agreed and revised as your suggestion.
L104: This seems like results and should be lower down
Response: Agreed and revised as your suggestion.
Figure 3: Some of these images are very blurry. Can you maybe get better images? Otherwise remove this plate as it is not helpful at this point.
Response: A high quality version was inserted.
L211: What about the mouthparts?
Response: The information was omitted to match the information listed in this taxonomy.
L221: What is a pleopod gill?
Response: It was not appropriate and has been removed.
L404: Is a pearl yellow?
Response: We think this is pearl yellow.
L427: I assume this was manca II?
Response: If pre-manca is called manca I, manca is called manca II. We added the explanation in the first mention (L56). The figure of pereopod 7 has also been added.
L429: In what way is it underdeveloped?
Response: We added the explanation in the first mention (L402).
L459: How many observations of each stage were made?
Response: Individuals observed are listed in Material examined.
L500; Are these all accurate identifications? Would you move some of the life stages into different life stages?
Response: I confirmed the life cycle stages described in each paper.
L569: Looking at this paper I see the original name was also Aulopus japonicus and this is most likely a taxonomic misapplied name problem (where the "new" species name was not correct at the time).
Response: Although I was unable to find any taxonomic history that would explain why the two species were confused, I changed the expression to a milder one.
R2
I have reviewed the manuscript titled “Description of life cycle stages of the fish parasite Cymothoa pulchrum (Isopoda: Cymothoidae), with DNA barcode linked to morphological details.” The manuscript is well-written and provides valuable taxonomic information on this parasitic isopod species. The illustrations are of high quality, and the study utilizes two relevant molecular markers for species identification. This work contributes to a better understanding of the biodiversity of isopod parasites in fish. Below, I provide some minor comments for consideration.
Response: Thank you for your helpful comments and suggestions. We responded to each of the points raised as follows.
Introduction
Line 48. Do all males transition to females, or do some remain male throughout their entire lives?
Response: The exact details are not known, so we will avoid mentioning them.
Line 58. The presence of C. pulchrum in Tetraodontiformes is mentioned twice in consecutive lines. Please reword to avoid repetition.
Response: Agreed and revised as your suggestion.
Methods
Lines 146 – 150. In this paragraph, it would be helpful to specify how many other species were included in the phylogenetic trees. You could also mention that the GenBank accession numbers are provided in the respective section below. If deemed useful, the authors may consider providing a table with relevant information on these species, such as their locality, hosts, and references if previously published.
Response: Agreed and revised as your suggestion.
R3
- lines 125-126. The amount of samples: only 3 parasites (each of them is different ) is too small to get DNA information.
Response: These populations are considered to be sufficient for DNA barcoding.
- line 196. If the female was damaged what kind of precise morphological information could be achieved?
Response: As written in the material examibed, that individual was not used for detailed morphological observations.
- lines 514-521. The information in this paragraph is not convincing enough. It consist only of suppositions. I suggest that the authors study it more carefully before including it in the article or exclude it from the article.
Response: The relevant discussion has been deleted.
R4
The manuscript provides a comprehensive morphological description of Cymothoa pulchrum across its life stages, supported by molecular phylogenetic analysis using COI and 16S rRNA sequences. This research contributes significantly to cymothoid taxonomy by addressing the lack of morphological data beyond adult stages. However, substantial revisions are needed to improve data presentation, methodological justification, and phylogenetic interpretation.
Response: Thank you for your helpful comments and suggestions. We responded to each of the points raised as follows.
The genetic division of C. pulchrum into two groups in COI and 16S rRNA trees raises critical taxonomic concerns, yet the manuscript does not assess whether this divergence indicates cryptic speciation. It remains unclear whether genetic distance analyses, such as pairwise K2P distances, were conducted to evaluate intra- and interspecific variation or whether additional nuclear markers (e.g., 18S rRNA, ITS) were used to validate species delimitation.
Response: We added analysis of perawise intra- and inter specific genetic distances.
Furthermore, the relationship between C. pulchrum and C. eremita is ambiguous, with overlapping COI clades suggesting potential historical misidentifications in GenBank, necessitating further morphological verification of voucher specimens.
Response: MK430019 - MK430026 are sequences obtained at the same time as morphological descriptions by taxonomists, so the risk of misidentification has probably been eliminated. Since there are sequences of eremita that have been reliably identified as species, it is possible to discuss and debate them (L565-587).
The manuscript also inconsistently states that C. pulchrum is specific to Tetraodontiformes while acknowledging its presence in Carangiformes and Acanthuriformes, raising questions about the criteria for determining host specificity.
Response: The claim that it is specific to Tetraodontiformes is that of Martin et al., and we are citing it. The wording has been changed (L570).
Additionally, the proposed life cycle hypothesis lacks direct experimental evidence; stable isotope or gut content analyses would strengthen claims that mancae and juveniles obtain nutrients from their hosts while attached externally. If these concerns cannot be fully addressed, they should be acknowledged as study limitations.
Response: The purpose of this is to present it as a future research topic, so I revised the text.
The writing also lacks clarity and conciseness, with redundant morphological descriptions in the Results and Discussion sections that hinder readability. Summarizing these details in a comparative table would improve organization and enhance academic rigor.
Response: This method of description is a common taxonomic method and should not be changed. If a comparison is needed in the future, the delta file provided as S1 can be used.
Reviewer 4 Report
Comments and Suggestions for AuthorsThe manuscript provides a comprehensive morphological description of Cymothoa pulchrum across its life stages, supported by molecular phylogenetic analysis using COI and 16S rRNA sequences. This research contributes significantly to cymothoid taxonomy by addressing the lack of morphological data beyond adult stages. However, substantial revisions are needed to improve data presentation, methodological justification, and phylogenetic interpretation. The genetic division of C. pulchrum into two groups in COI and 16S rRNA trees raises critical taxonomic concerns, yet the manuscript does not assess whether this divergence indicates cryptic speciation. It remains unclear whether genetic distance analyses, such as pairwise K2P distances, were conducted to evaluate intra- and interspecific variation or whether additional nuclear markers (e.g., 18S rRNA, ITS) were used to validate species delimitation. Furthermore, the relationship between C. pulchrum and C. eremita is ambiguous, with overlapping COI clades suggesting potential historical misidentifications in GenBank, necessitating further morphological verification of voucher specimens. The manuscript also inconsistently states that C. pulchrum is specific to Tetraodontiformes while acknowledging its presence in Carangiformes and Acanthuriformes, raising questions about the criteria for determining host specificity. Additionally, the proposed life cycle hypothesis lacks direct experimental evidence; stable isotope or gut content analyses would strengthen claims that mancae and juveniles obtain nutrients from their hosts while attached externally. If these concerns cannot be fully addressed, they should be acknowledged as study limitations. The writing also lacks clarity and conciseness, with redundant morphological descriptions in the Results and Discussion sections that hinder readability. Summarizing these details in a comparative table would improve organization and enhance academic rigor.
Author Response
Prof. Maria Angeles Esteban
Editor-in-Chief
Fishes
Dear Editor:
We wish to re-submit the manuscript titled “Description of life cycle stages of fish parasite Cymothoa pulchrum (Isopoda: Cymothoidae), with DNA barcode linked to morphological details” The manuscript No. is 3530247.
We thank you and the reviewer for your thoughtful suggestions and insights. The manuscript has benefited from these insightful suggestions. We look forward to working with you and the reviewers to move this manuscript closer to publication in Fishes.
The manuscript has been rechecked and the necessary changes have been made in accordance with the reviewer’ suggestions. The responses to all comments have been prepared and attached herewith below.
Thank you for your consideration. I look forward to hearing from you.
Sincerely,
Hiroki Fujita
R1
This paper discussed the life stages of Cymothoa pulchrum and its genetic sequence. The information is needed and valuable for future research.
Some of the images are blurry and should be replaced or deleted.
There is some repetition and unnecessary information in the methods section that needs to be addressed. Some clarity is needed in the descriptions too.
The different life stages are clearly distinguished and drawings are provided of each. It would be helpful to know how many of each life stage were observed.
More small edits are provided in the pdf.
Response: Thank you for your helpful comments and suggestions. We responded to each of the points raised as follows.
Comments in PDF
L34: The
Response: I don't think articles should be added to scientific names.
L76: Under the tongue?
Response: Fish don't have tongue, they just have basihyal. I added an explanation.
L81: A lot of this information is repeated in the discussion. A more condensed version needs to be provided here to avoid repetition. Just numbers of hosts and how long they lived or something similar.
Response: We have removed repetition in the discussion.
L95: This reference is not in the reference list
Response: Agreed and revised as your suggestion.
L104: This seems like results and should be lower down
Response: Agreed and revised as your suggestion.
Figure 3: Some of these images are very blurry. Can you maybe get better images? Otherwise remove this plate as it is not helpful at this point.
Response: A high quality version was inserted.
L211: What about the mouthparts?
Response: The information was omitted to match the information listed in this taxonomy.
L221: What is a pleopod gill?
Response: It was not appropriate and has been removed.
L404: Is a pearl yellow?
Response: We think this is pearl yellow.
L427: I assume this was manca II?
Response: If pre-manca is called manca I, manca is called manca II. We added the explanation in the first mention (L56). The figure of pereopod 7 has also been added.
L429: In what way is it underdeveloped?
Response: We added the explanation in the first mention (L402).
L459: How many observations of each stage were made?
Response: Individuals observed are listed in Material examined.
L500; Are these all accurate identifications? Would you move some of the life stages into different life stages?
Response: I confirmed the life cycle stages described in each paper.
L569: Looking at this paper I see the original name was also Aulopus japonicus and this is most likely a taxonomic misapplied name problem (where the "new" species name was not correct at the time).
Response: Although I was unable to find any taxonomic history that would explain why the two species were confused, I changed the expression to a milder one.
R2
I have reviewed the manuscript titled “Description of life cycle stages of the fish parasite Cymothoa pulchrum (Isopoda: Cymothoidae), with DNA barcode linked to morphological details.” The manuscript is well-written and provides valuable taxonomic information on this parasitic isopod species. The illustrations are of high quality, and the study utilizes two relevant molecular markers for species identification. This work contributes to a better understanding of the biodiversity of isopod parasites in fish. Below, I provide some minor comments for consideration.
Response: Thank you for your helpful comments and suggestions. We responded to each of the points raised as follows.
Introduction
Line 48. Do all males transition to females, or do some remain male throughout their entire lives?
Response: The exact details are not known, so we will avoid mentioning them.
Line 58. The presence of C. pulchrum in Tetraodontiformes is mentioned twice in consecutive lines. Please reword to avoid repetition.
Response: Agreed and revised as your suggestion.
Methods
Lines 146 – 150. In this paragraph, it would be helpful to specify how many other species were included in the phylogenetic trees. You could also mention that the GenBank accession numbers are provided in the respective section below. If deemed useful, the authors may consider providing a table with relevant information on these species, such as their locality, hosts, and references if previously published.
Response: Agreed and revised as your suggestion.
R3
- lines 125-126. The amount of samples: only 3 parasites (each of them is different ) is too small to get DNA information.
Response: These populations are considered to be sufficient for DNA barcoding.
- line 196. If the female was damaged what kind of precise morphological information could be achieved?
Response: As written in the material examibed, that individual was not used for detailed morphological observations.
- lines 514-521. The information in this paragraph is not convincing enough. It consist only of suppositions. I suggest that the authors study it more carefully before including it in the article or exclude it from the article.
Response: The relevant discussion has been deleted.
R4
The manuscript provides a comprehensive morphological description of Cymothoa pulchrum across its life stages, supported by molecular phylogenetic analysis using COI and 16S rRNA sequences. This research contributes significantly to cymothoid taxonomy by addressing the lack of morphological data beyond adult stages. However, substantial revisions are needed to improve data presentation, methodological justification, and phylogenetic interpretation.
Response: Thank you for your helpful comments and suggestions. We responded to each of the points raised as follows.
The genetic division of C. pulchrum into two groups in COI and 16S rRNA trees raises critical taxonomic concerns, yet the manuscript does not assess whether this divergence indicates cryptic speciation. It remains unclear whether genetic distance analyses, such as pairwise K2P distances, were conducted to evaluate intra- and interspecific variation or whether additional nuclear markers (e.g., 18S rRNA, ITS) were used to validate species delimitation.
Response: We added analysis of perawise intra- and inter specific genetic distances.
Furthermore, the relationship between C. pulchrum and C. eremita is ambiguous, with overlapping COI clades suggesting potential historical misidentifications in GenBank, necessitating further morphological verification of voucher specimens.
Response: MK430019 - MK430026 are sequences obtained at the same time as morphological descriptions by taxonomists, so the risk of misidentification has probably been eliminated. Since there are sequences of eremita that have been reliably identified as species, it is possible to discuss and debate them (L565-587).
The manuscript also inconsistently states that C. pulchrum is specific to Tetraodontiformes while acknowledging its presence in Carangiformes and Acanthuriformes, raising questions about the criteria for determining host specificity.
Response: The claim that it is specific to Tetraodontiformes is that of Martin et al., and we are citing it. The wording has been changed (L570).
Additionally, the proposed life cycle hypothesis lacks direct experimental evidence; stable isotope or gut content analyses would strengthen claims that mancae and juveniles obtain nutrients from their hosts while attached externally. If these concerns cannot be fully addressed, they should be acknowledged as study limitations.
Response: The purpose of this is to present it as a future research topic, so I revised the text.
The writing also lacks clarity and conciseness, with redundant morphological descriptions in the Results and Discussion sections that hinder readability. Summarizing these details in a comparative table would improve organization and enhance academic rigor.
Response: This method of description is a common taxonomic method and should not be changed. If a comparison is needed in the future, the delta file provided as S1 can be used.
Round 2
Reviewer 4 Report
Comments and Suggestions for AuthorsThanks the author for responding to my previous comments. All the previous concerns have been clarified and well addressed
