Microscopic Anatomy of the Lining of Hemal Spaces in the Penaeid Shrimp, Sicyonia ingentis

Round 1

Reviewer 1 Report

The paper entitled: "Microscopic anatomy of the lining of hemal spaces…" seems very interesting. Unfortunately my expertise on the crustacean anatomy，and my ability to evaluate this study is very restricted. However at least two suggestions I can give. First: fig. 2. A, in the picture there is an m (muscle layer?), but the explanation in the legend is lacking.

Second, fig. 4 D, in the legend, row 262, …“ovoid pore”, really it seems too large compared to the pores of fig. 3D and 4 F. Please control and eventually explain the so great difference not justified by different enlargement.

Author Response

Responses to Reviewer #1

Thanks for reviewing this paper. We realize it is not a "typical" study on crustacean anatomy but feel the subject has merit for future work. 

• Thanks for catching that we did not mention the muscle layer in the figure legend to 2A. We added a line indicating that the “m” refers to a muscle layer.
• The pores seen in the outer covering of the midgut trunk are significantly larger than we observed in other tissues and we added this comment to the text, original line 229

Reviewer 2 Report

This is an interesting and well written article presenting new data on the lining of hemal spaces in decapod crustaceans. An extensive list of references is included. Just some questions and suggestions for improvement:

Abstract.

Lines 18-20. The abstract ends with a sentence stating the purpose of the study. This statement seems more appropriate for the beginning of the abstract, and the abstract could end with a general conclusion.

Introduction.

Page 2, lines 47-49. The authors mention 3 types of acellular materials, including connective tissue sheaths. But connective tissue sheaths contain cells in addition to the extracellular matrix, therefore are not completely acellular.

Line 58. Please correct the spelling "lamina reticularis" (not reticularicaris)

Line 64. Depending on the vessels: underlying connective or muscular tissues.

Line 67. The paragraph starts mentioning the second type, the first type is the basement membrane?

Line 68. "Shrimp, lobsters and crab" include a very large variety of species, perhaps it would be better to indicate which species were studied because they were not many.

Line 75. This acellular matrix is the connective tissue matrix (?), and not the entire connective tissue sheath (as we currently read in the text) that include cells (as mentioned in line 78).

Materials and Methods.

Page 2, line 91. Can the authors include the SD of the shrimp weight (14.5 g ± ?).

Page 3, lines 98 and 104. Instead of tissues would be better to say "tissues and organs" (stomach, digestive gland, gonads, etc. are organs not just a tissue). Line 106. For the same reason, "Samples" instead of "Tissues" would be better.

Line 112. Please indicate the buffer pH.

Line 113. These are semithin sections, not thick.

Lines 118-122. Measurements. Please give some more information about the measurement procedures in LM and TEM (for example: one or more sections from each block, how many photos were used, how many measurements were made for each structure, etc.)

Results.

In the M&M section the authors say that measurements reported in the text are presented as mean ± SD. But in the results section measurements are presents as a range ± SD. This is unusual and the authors must clarify what these number represent (maximum and minimum?), and indicate the mean.

Line 138. “… stains with eosin and PAS ….” It would be interesting to include a color figure illustrating these results.

Figure 2. Fig2A could be in color, and the small white asterisks and the letter “m” are not mentioned in the legend. 

SEM photos are very good, but TEM imagens are not so clear and some details mentioned in the text are difficult to see in these TEM imagens. Is it possible to improve? Some TEM imagens have very dark corners and the black letters are difficult to see on a dark background. In figure 4 individual photos are separated by black lines, these should be replaced by white lines.

Lines 183-184. Reference to fig. 3 should not be repeated at the beginning and end of the sentence, just in the end is better.

Line 218. A pore is not visible or indicated in figure 4B.

Lines 233-235. It is stated that sheath covering the foregut is similar to the sheath over the gonad, but there are differences regarding the pores.

Line 241 x line 267 – fibroblast or fibroblast-like? It should be consistent along the text.

Discussion.

Line 275. Can it be said that the interna in the dorsal adnominal artery is elastic? Only the presence of fibrilin-like material was shown and its elasticity should be limited and not demonstrated here.

Line 282. Lobster, please indicate the species, many different species can be called lobsters.

Line 296. The reference [40] should be placed immediately after the citation, not at the end of the sentence.

Line 307-309. The sentence alluding to vertebrate kidney charges in the BM is suddenly introduced. A smoother more clear connection should be made to help the readers understands the reasoning.

Line 310. Organs and tissues, not just organs. The abdominal muscle is not and organ.

Institutional Statement (line 355). This is the text with MDPI instruction, not the author’s statement.

Author Response

Thank you for you very helpful suggestions; we have taken almost all of them and feel they improve the paper. Your work is greatly appreciated.

As suggested, we moved the final sentence of the Abstract to the first sentence in the Abstract and added a general suggestion at the end for more research of this type in the future. 

line 47; We agree that the sheaths do include some widely dispersed cells and so are not technically "acellular". To avoid becoming too wordy, we added "primarily" acellular.

line 58: done, reticularis

line 64: added muscular tissue as well

line 47: we added text that the BMs are the first type of acellular material. 

line 68: we agree that few species have been studied but prefer to keep the text as originally written and leave it to readers to look up the species examined in the references cited. Homarus americanus was added later in the text.

line 75: We think we make it clear that there are indeed a few cells in the sheath and adding CT sheath vs CT matrix only further complicates the story at this stage of its development. 

-line 91 SD added

• added tissues and organs to the two sites and replaced tissues with samples in the last suggestion.
• old line 112: pH 4.5
• changed thick to semi-thin
• line 118; expanded explanation of measurements
• for measurements in the text we added a mean with the SD to follow a range
• we chose not to include a color pic as the color is faint and we doubt would copy well. 
• fig 2A we added the explanation of the * and a M for muscle layer to the legend. 
• Currently locked out of Oxy Bio dept so I cannot get back on TEM. I did change the borders on this plate from black to white. Not sure how to submit the new plate but will figure out a way to do so. 
• Line 183: done
• Line 218: changed text to indicate pores were rare and not shown in figure 4B.
• Line 233: we are not clear on the concern here and think the description is appropriate. 
• Line 241: changed fibroblast to fibroblast-like cell throughout the text.
• Line 275: deleted elastic
• Line 282: Homarus americanus added
• Line 296: reference citation moved.
• Line 307: a short transition phrase was added to hopefully smooth out the introduction of charges on the kidney BM.
• Line 310: added tissues
• Line 355: seems NA is the proper response...or advise.