Morphometric, Histochemical, and Ultrastructural Analysis of the Reproductive System and Spermatogenic Stages of Male Blue Crab (Callinectessapidus Rathbun, 1896)

Round 1

Reviewer 1 Report

Review for the paper "Morphometric, histochemical, and ultrastructural analysis of the male reproductive system and spermatogenic stages of male blue crab (Callinectes sapidus. Rathbun 1896)" by Awatef Ali, Soheir El Sherif, Jamila Abd-Alla, Sahya Maulu, Ahmed A.Tantawy, Mahmoud F. K. Soliman, Mohammed M. Hassan, Montaser M. Hassan, and Hala Saber Khalil  submitted to "Journal of Marine Science and Engineering".

In this paper, the authors described the morphological features and ultrastructure of the reproductive system, spermatogenesis, and spermatophores structure of male blue crab Callinectes sapidus from the Mediterranean region. They also provided some information about the crabs' size and gonadosomatic index. The authors used an appropriate sample size of crabs and standard methods including electron microscopy. The authors provided excellent photographs and detailed discussions.

General comment.

Figure 2 is inappropriate because the authors presented different parameters (with different units) in one graph. They should provide this information in separated plates or figures.

Figure 7. Consider replacing “Fig.A" and ”Fig.B" with “A" and "B"

Some text revisions are made in the attached PDF.

Comments for author File: Comments.pdf

Author Response

Dear reviewer1,

Thanks a lot for your tremendous efforts and significant comments that enable us to increase the significance of our paper.

We already replied to all comments with "track changes" in the text.

Reviewer 1,

• Figure 2 is inappropriate because the authors presented different parameters (with different units) in one graph. They should provide this information in separated plates or figures.

Reply: We are used to making different measurements with different units, represented by numbers on the x-axis in one graph, and this is accurate because we write each parameter with its standard unit next to it as it appears in (Fig. 2).

• Figure 7. Consider replacing "Fig.A" and" Fig.B" with "A" and "B"

Reply: We consider replacing Fig. A and Fig. B

to A and B.

• All suggestions in PDF:

Replay: We corrected all comments in the pdf file as asked and replaced all word figures in parenthesis (Fig. 7b).

Moreover, species were corrected to italic text. All grammatical issues were corrected as asked. Thus, you can see all corrections in the word file with track changes.

Thank you again for your efforts and great comments, and we are ready for further comments or suggestions.

I am looking forward to hearing from you as soon as possible

Kind regards,

Corresponding author:

Ahmed A. Tantawy

Reviewer 2 Report

Comments about the revised version of the manuscript:

“Morphometric, histochemical, and ultrastructural analysis of the male reproductive system and spermatogenic stages of male blue crab (Callinectes sapidus. Rathbun 1896)”

The manuscript has been seriously improved by the authors. Hanks to the authors for considering my remarks. For me, it is ready to be published after minor corrections:

Page 8, spermatogenesis: put “figure 4” in brackets.

Page 10, 2^nd line: put “figure 5”, “figure 5c”, “figure 5d”, “figure 5e” in brackets.

Page 11, vas deferens: put “figure7A”, “figure 7B” in brackets.

Page 12: put “figure 8”, “figure 8a”, “figure 8c”, “figure 8f” in brackets.

Author Response

Dear reviewer 2,

Thanks a lot for your tremendous efforts and significant comments that enable us to increase the significance of our paper.

We already replied to all comments with "track changes" in the text.

Reviewer 2:

Page 8, spermatogenesis: put "figure 4" in brackets.

We found different corrections in this point between reviewers 1 and 2, so we corrected all figures in parenthesis (Fig. 5d), not brackets [Fig. 5d) to become consistent and performed all corrections in the same way.

Page 10, 2^nd line: put "figure 5", "figure 5c", "figure 5d", "figure 5e" in brackets.

Done

Page 11, vas deferens: put "figure7A", "figure 7B" in brackets.

Done

Page 12: put "figure 8", "figure 8a", "figure 8c", "figure 8f" in brackets.

Done

Thank you again for your efforts and great comments, and we are ready for further comments or suggestions.

I am looking forward to hearing from you as soon as possible.

Kind regards,

Corresponding author:

Ahmed A. Tantawy

Author Response File: Author Response.docx

Reviewer 3 Report

v

The manuscript by Ali et al. is interesting and overall well conducted. It deals with the reproductive biology of males of the blue crab (Callinectes sapidus), a commercial value species that it is also an invasive species in some areas. So, a good knowledge about its biology is crucial. My main concern is that the ms. has a lot of typos, errors and misconceptions that detract from its quality and make it unkempt.

Specific comments:

-tittle: delete the dot between ‘sapidus’ and ‘Rathbun’ and add a comma between ‘Rathbun’ and ‘1896’.

- Abstract, line 1, the crab’s authority is without parenthesis

- keywords: most of the presented keywords are also included on the title, please change them by others that are not on the title.

- Introduction: please, have a though about it in order to ensure that the work is placed in as wide a geographical context as possible and that you compare and contrast your findings with elsewhere.

- Introduction, 3^rd paragraph, ?Nobilis pinnotheres… it is Pinnotheres nobilis…

- Introduction, 3rd paragraph, brachyurans without italics

- the specific name of the crab can be abbreviated after its first mention on the text but NOT when it is the first word of a paragraph… Please check it along the whole manuscript (e.g. M&M, page 3)

- Fig 1 has not good quality, the names of the different parts are illegible.

- Results, 3.1, the mean data lack the standard deviation, please add them (surprisingly SD are included in the conclusion section).

- Fig 2, it is ‘autumn’ NOT ‘Autmen’

- 3.2. Spermatophore, the start of this section is weird… please check it

- page 8, are you sure that the wrinkled surface that we can see on the SEM images is not an artifact. Did you see the wrinkles on other spermatophores or only in one? It is an important point to check!

-Figure 8, the image C looks like a hematoxylin-eosin-stained section… are you sure that it was stained with the Mallory trichrome?

- Discussion, 4.1, ‘C. Danai’ is not correct!. Please all scientific along the whole text.

- Discussion 4.3., this section is very repetitive if we compared it with the presented text of the Results. Also, some scientific names are wrong (e.g. ‘C. Danae’)…

-Conclusions: they are not well presented… please have a thought about them. Think about the underlying fundamental processes. Focus on the 'so what' and 'what if' questions, i.e. what are the repercussions of your findings.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The MS by Ali et al. deals about the male reproductive system of Callinectes sapidus collected in Egypt where it is an invasive species.  The natomy of the male reproductive system of this species was previous done by Cronin, L. E. (1947). Anatomy and histology of the male reproductive system of Callinectes sapidus Rathbun. Journal of Morphology, 81(2), 209-239; and the revised by Johnson (1980) and Carvalho-Saucedo et al. (2015). Authors should read this first article.

The MS is a bit confusing and do not remark what are the new contributions for the male reproduction in this species. TEM images regarding spematogenesis are mainly of good quality, although that should increase the micrograph related to spermiogenesis (mid-stage spermatid for instance) and improve the spermatozoa images.  The spermatophore formation is also a new aspect for this species but authors should clarify: 1) where starts the spermatophore formation in testes or it is all done in the vas deferens?; 2) It is difficult to follow how the authors differentiate secretions in the vas deferens from the images they show. Epithelial cells of the vas deferens could be described, that will help also to identify the type of secretion. In the conclusions they report: the Histochemical study was detected 2 testicular secretions and 6 vas deferens secretions. It was not possible for me to reach this conclusion. Summarizing, when and where are the spermatophores constituted? More detail is necessary.

Beside this main concern, another aspect is related to the gonadosomatic index. The gonadosomatic index usually referenced to gonads, not including the vas deferens. The production od spermatozoa is realized in the gonads not in the vas deference and that’s why the gonadosomatic index do not include de vas deferens. If you wanted to check the increase of weight for the formation of spermatophore, then you could have weighted the vas deferens aside from testes. Although I am not sure you will get the information of spermatophore production with the weight of the vas deferens.

Moreover, the MS need to be better organized and English improved, but data, in particular the spermatogenesis is worthy to get published. Some suggestions are included in the MS.

Comments for author File: Comments.pdf

Author Response

Dear reviewer 1, 

I hope this e-mail finds you well.

Firstly, many thanks for your great efforts and your awesome suggestions which make our manuscripts better than the previous version.

Secondly, your great comments were taken considerably, and you can find our reply in the attached file.

Thank you again
We are looking forward to hearing from you as soon as possible, and we are ready for further comments.

Kind regards,
Corresponding author

Secondly, your great comments were taken considerably, and you can find our reply in the attached file.

Thank you again
I am looking forward to hearing from you as soon as possible, and we are ready for further comments.

 

Author Response File: Author Response.docx

Reviewer 2 Report

The blue crab, Callinectes sapidus (Rathbun, 1896) contains more than 20 types of amino acids and it is used as food. This manuscript is about the study of several structural aspects of the male reproductive system of this crab. For this, the authors studied a lot of mature adult males obtained from Abu-Qir bay during one year, from November 2018 to October 2019. They calculated gonadosomatic index of male maturation seasons, they studied the morphology of reproductive system using histology, histochemistry, scanning and transmission electron microscopy.

This study is interesting and useful, and could be published after some improvements.

Lines 39, 42 and other lines: replace “&” with “and”.

Line 48: in the sentence “ellipsoidal in its outline with o terminal stalk”, what is “o”?

Line 66 and other lines among the text: use italics for genera and species: Callinectes sapidus at line 66, Eriphia verrucosa at line 105

Line 113: write in all letters the genus of N. pinnotheres and P. pisum

Line 137: write “crabs” instead “carbs”

Lines 144 to 146: the sentence “In this study, the adult male crabs were used where the adult sexually matured male crab has an external skeleton with a hard 106 carapace shell and five pairs of 146 pereopods” is difficult to understand, and I did not understand what it means. Please, re-write it more simply.

Line 170, Histochemical studies:

“Small fragments taken from isolated parts of testis and vas deferens taken from different stages of maturity were processed for histochemical studies;”: how many crabs were studied? How many fragments were studied from testis? From vas deferens?

Line 174: write “Mallory” with a capital letter.

Line 175: write “Masson`s trichrome” instead “masson`s trichrome stain”

Line 175: Is not “Heidenhain’s iron haematoxylin” instead “Heidenhain iron”?

Line 178: add a full-stop at the end of sentence.

Lines 183 and 184: the tissues were posted "fixed in 1% osmium tetroxide. In the same buffer for 1h at 4ºC": why quotation marks?

Line 187: “the organ was dehydrated in graded ethanol procedures”. This part needs some specifications. How were the tissues dried out? With bypassing critical point? How were they metallized? With gold, gold-palladium, whatever?

Line 193: delete “F1G4-” at the beginning of the line.

Line 195: washed multiple times: how many?

Line 293 what is E.R.? Endoplasmic reticulum? Write all letters.

Lines 359, 340: use italics for “Libinia emarginata” and “Libinia dubia”.

Line 374: “Ulmeritoides uruguayensis” instead “Ulmeritoides Uruguayensis”.

Line 379: “Portunus pelagicus” instead “Portunus-pelagicus”.

Line 388: use italics for “Ovalipes ocellatus”.

Line 440: use italics for “Libinia emarginata”.

Line 543: replace “&” with “and”.

Illustrations

Line 764: the map is not numbered such as a figure and it is not referred in the text.

Lines 770 and 772: figures A and B are not referred in the text.

Lines 774-775: use italics for “Callinectes sapidus”.

Line 778: use “and” instead “&”.

Figure C: this figure is not referred in the text and it needs some explanations. What is a? b? ab?

Page 30: Why figures A, B, C and figures 1 to 26? The figures need to be reorganized with a single numeration with number without forgetting the map. One figures could group figures 1 to 6, another figure could group 7 to 12, and so on…

Line 818: write “PAS” instead “PAS stain” (PAS is not a staining but a reaction).

Line 820: write “Mallory’s triple stain”.

Line 825: write “Masson`s trichrome”

References: check the reference and present them according to the standards of the journal: “In the text, reference numbers should be placed in square brackets [ ], and placed before the punctuation; for example [1], [1–3] or [1,3]. For embedded citations in the text with pagination, use both parentheses and brackets to indicate the reference number and page numbers; for example [5] (p. 10). or [6] (pp. 101–105).“ https://www.mdpi.com/journal/jmse/instructions

Author Response

Dear reviewer 2, 

I hope this e-mail finds you well. Firstly, many thanks for your great efforts and your awesome suggestions which make our manuscripts better than the previous version.

Secondly, your great comments were taken considerably, and you can find our reply in the revised manuscript.

Thank you again.

We are looking forward to hearing from you as soon as possible, and we are ready for further comments.

Kind regards,

corresponding author

Author Response File: Author Response.pdf