Review Reports - The Natural Infection of Freshwater Snails with the Avian Air Sac Fluke, <i>Cyclocoelum mutabile</i> (Trematoda: Cyclocoelidae), in Brazil

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

It's a valuable study, but I think it needs a few improvements.

1. Abstract could better reflect the significant results of this study.

I think instead of the uninformative "The obtained molecular sequences revealed a high degree of similarity", the differences between the larval and adult stages of each gene should be described.

2. Morphological characterization and Remarks would be easier to read if the main morphological measurements are presented here, but not only in the tables.

3. In the Table 1 should be measurements of oral suckers.

4. For the clarity, in the Results, the identity of the Nad-1 sequences of the larval stages and their difference from the adult should be noted.

5. In the Discussion, I miss analyzes of cox-1 and Nad-1 differences between larval stage and adult. Could cox-1 and Nad-1 be population genetic markers?

Author Response

RESPONSE LETTER

Reviewer 2

It's a valuable study, but I think it needs a few improvements.

Authors’ response

Thank you very much for the positive feedback.

Abstract could better reflect the significant results of this study.

I think instead of the uninformative "The obtained molecular sequences revealed a high degree of similarity", the differences between the larval and adult stages of each gene should be described.

Authors’ response

The Abstract was changed as requested. The similarities between larval and adult stages were disclosed. Thank you.

Morphological characterization and Remarks would be easier to read if the main morphological measurements are presented here, but not only in the tables.

Authors’ response

We thank this suggestion. However, we opt by maintain the morphometric data only in the Tables.

In the Table 1 should be measurements of oral suckers.

Authors’ response

The anterior organ in the cercariae was not well visible. We include this information in the description.

For the clarity, in the Results, the identity of the Nad-1 sequences of the larval stages and their difference from the adult should be noted.

Authors’ response

We improved this stretch of the molecular results. In fact, the haplotype found in the mollusks is different from that found in G. galeata.

In the Discussion, I miss analyzes of cox-1 and Nad-1 differences between larval stage and adult. Could cox-1 and Nad-1 be population genetic markers?

Authors’ response

Yes, these mitochondrial markers can be used in the future in a genetic population genetic study. Howerver, we consider this discussion is out of the scope of the present MS.

Reviewer 2 Report

Comments and Suggestions for Authors

The article deals with the study of the larval stages of the cosmopolitan trematode Cyclocoelum mutabile ex freshwater gastropods in Belo Horizonte (Brazil). Additionally, the authors consider adult digeneans from the final host (Gallinula galeata) in the same area. The authors' merits include the joint use of traditional morphological and contemporary molecular genetic methods as it should be in serious parasitological studies. Congratulations to the authors. The well-conducted research is of undoubted interest. The article theme is relevant to Diversity.

I have only one important note about this work.

1)There is only one standard flaw in the study. Illustrations of phylogenetic trees show Bayesian support for all clades, including polytomous ones. This does not make sense since Bayesian supports are calculated for dichotomies. Therefore, it is necessary to either remove supports in polytomous clades in the figures, or display hidden (“collapsed”) dichotomous ones where they are not currently visible, using the “contype=allcompat” function when executing the “sumt” command in MrBayes. And accordingly replace the figures.

2)And I would advise changing the morphological descriptions in style somewhat. For example, for adult trematodes (lines 172-184): “Body elongated, lanceolate, with smooth tegument (Fig. 3A). Oral sucker absent. Prepharynx short. Pharynx developed, muscular. Esophagus short. Cecum

smooth, joint near the posterior body forming cyclocoel. Genital pore prepharyngeal (Fig.

3B). Cirrus sac piriform; seminal vesicle present. Cirrus small, located laterally to the pharynx above the cecal bifurcation. Two testes, round to oval, located in the posterior quarter of the body, in intercecal position, lie diagonally to each other, forming a triangle with the ovary. Ovary small, spherical, located on the right side of the body in intertesticular position. Vitellaria follicular, in cecal and extracecal position, extending from anterior second half of body to posterior fourth of body, non-confluent. Vitelline ducts extend from the vitelline in the posterior quarter of the body, uniting into a single canal. Uterus with sinuous curvatures, filling most of the intercecal space, extending from the anterior border of the posterior testis to the anterior body quarter. Egg elliptical to oval-shape, operculated, golden in color, mature, with occulate miracidium presenting a redia inside.” It looks better this way.

What can you say about the Mehlis’ gland? Is there a vitelline reservoir?

Descriptions of the larval stages of the trematode should also be restructured.

3)Lines 86,138,139,202 – first mentions of species (parasites, intermediate and final hosts) in the text. According to the rules of International Code of Zoological Nomenclature (ICZN), at the first mention of species or genera, their full Latin name with the author and year of description should be given. This should be done for all species first mentioned in the text.

4) According to recent concepts, trematode Morishitium polonicum is a junior synonym of Morishitium dollfusi.

See Heneberg, P., Sitko, J. Morishitium polonicum (Machalska, 1980) is a junior synonym of Morishitium dollfusi (Timon-David, 1950) (Trematoda: Cyclocoeliidae). Parasitol. Res. 2023. 122. 3159–3168. https://doi.org/10.1007/s00436-023-08006-y

5)Lines 83,84 – correct references Assis et al. [21]. Paraense [22] and Paraense & Pointier [23].

The same should be corrected in the lines 121,122,124,127, etc. Throughout the text. The year of reference should not be included.

6)Line 143 – second mention of the species, therefore – C. mutabile

7)Line 146,203,219 – italics.

8) lines 351,356,515 – Correct name of scientist – Feyzullaev.

The manuscript can be published, but minor corrections are needed.

Comments for author File: Comments.pdf

Author Response

Authors’ response

We thank the Reviewer for this very positive feedback.

I have only one important note about this work.

Authors’ response

We thank the Reviewer for this correction. The Figure was edited and the supports of clades with polytomies removed.

Authors’ response

The description of adult and larval stages were improved. Thank you very much.

Authors’ response

We revised all the text to follow this rule of ICZN

4) According to recent concepts, trematode Morishitium polonicum is a junior synonym of Morishitium dollfusi. See Heneberg, P., Sitko, J. Morishitium polonicum (Machalska, 1980) is a junior synonym of Morishitium dollfusi (Timon-David, 1950) (Trematoda: Cyclocoeliidae). Parasitol. Res. 2023. 122. 3159–3168. https://doi.org/10.1007/s00436-023-08006-y

Authors’ response

We now use the correct name, Morishitium dollfusi. in the trees and text. Thank you for this update.

5)Lines 83,84 – correct references Assis et al. [21]. Paraense [22] and Paraense & Pointier [23].

The same should be corrected in the lines 121,122,124,127, etc. Throughout the text. The year of reference should not be included.

6)Line 143 – second mention of the species, therefore – C. mutabile

7)Line 146,203,219 – italics.

8) lines 351,356,515 – Correct name of scientist – Feyzullaev.

Authors’ response

The correction 5-8 were all accepted.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The MS still needs some fixes.

1. The whole description of the anterior organ should be placed before the description of the ventral sucker.

2. Ln 205 - Sentence about Tables 1 and 2 should be removed. Table numbers should be indicated at the beginning of the morphological characterization of larval and adult stages, respectively.

3. Ln 276 - Should be Table 2.

Again, for me, is not comfortable reading morphological description without measurements in the text. Please add the main measurements in the text of the Morphological characterization and Remarks.

Author Response

The whole description of the anterior organ should be placed before the description of the ventral sucker.

Authors' Response

We carried out this correction. Thank you.

Ln 205 - Sentence about Tables 1 and 2 should be removed. Table numbers should be indicated at the beginning of the morphological characterization of larval and adult stages, respectively.

Authors' Response

We add the call for the Tables in the descriptions.

Ln 276 - Should be Table 2.

Authors' Response

We change Table 3 to Table 2

4. Again, for me, is not comfortable reading morphological description without measurements in the text. Please add the main measurements in the text of the Morphological characterization and Remarks.

Authors' Response

We add a call for the measures in Tables in the description of each developmental stage.

However, as we are not describing a new species, we consider the inclusion of the measures in the text is not mandatory and could be considered as a personal preference. Moreover, for cercariae for instance, data were collected from different mollusk species and fixation techniques. We respectfully disagree with the Reviewer and consider that replaying the measures in the text will add unnecessary complexity and redundancy.

Reviewer 2 Report

Comments and Suggestions for Authors

Congratulations to the authors on their high-quality work. There is only one remark left. Please check the spelling of the Latin names as in the case of Cyclocoelum (lines 48, 73). The manuscript is ready for publication.

Author Response

Congratulations to the authors on their high-quality work.

Authors' Response

We thank the REviewer for this positive feedback.

There is only one remark left. Please check the spelling of the Latin names as in the case of Cyclocoelum (lines 48, 73). The manuscript is ready for publication.

Authors' Response

We changed all Cyclocoelium by Cyclocoelum. Sorry for this mistake.

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

I marked "Accept present form", but I suggest describing the anterior organ in one sentence.