Immuno-Enzymatic and Proteomic Approaches for Sexing the African Bonytongue (Heterotis niloticus Cuvier, 1829)
, Joël Henry 2,3, Erwan Corre 4, Julien Pontin 3, Benoît Bernay 3 and Jésus Nunez 5,*
Round 1
Reviewer 1 Report
Very interesting MS. Personally, I think that it should be printed after minor corrections. My comments are included in the text of the work. To see them all, open the file in Acrobat Reader. I believe that the authors should have no problem improving this MS.
Comments for author File: 
Comments.pdf
Author Response
Please see attached pdf file Koua_et_al_rev1_jnr.pdf
Corrections are highlighted in yellow
Author Response File: Author Response.pdf
Reviewer 2 Report
In general, the manuscript is well presented and it is scientifically interesting. I would like to recommend the publication of this study. However, some minor improvements are required in the m+m section, and a high number of formal mistakes should be corrected before considering the publication of this work.
L43, 251, 252, 256, 263, 264 (x3), etc. Species names must be written in italics.
L46 Give a short explanation of which are the anatomical features avoiding the artificial reproduction of this species.
L52 What does it means "open water"?
L71-72 This tissue distribution, it is a natural one?, or is a consequence of a endocrine disruption activity in experiments with zebrafish?
L72-73 Specify which are the 3 species.
L78 Maybe, reference 38 should be added at the end of the previous sentence. Anyway it has no relationship with the hypothesis mentioned in the final sentence (L77-78).
L93 1.5 kg
L109 15 ml
L109-118. Include the composition of the different buffers and solutions.
L110 Give some details regarding the Mini Array membrane (company?, material?, size?).
L120 What does it means "control spot"?
L125 delete first "g"
L126 4 m3
L126 ...with a 20 mM PBS (phosphate buffer saline) solution containing 2 mg/ml of 17B-estradiol and 1% peanut oil. ...??
L129- cumulated dose??...a total of 2 mg of E2 divided in 7 injections... ??
L130 48 h
L130-131 Is there any previous evidence that 48 + 48 h is time enough to induce a significant amount of circulating Vtg in this species?
L134, 158 One hundred µl
L139 Define MW
L145 100 µg
L159 -20 ºC
L165 Delete "by"
L167 Include the volume of each reactive or solution.
L177 25 ± 2 ºC
L183 Revelation/development?
L185 Specify the H2O2 concentration
L185 30 min by
L186 H2SO4
L199 Delete dot
L213 Define ACN
LL213 Confirm nl
L215 Confirm B rebalance
Figures 3 and 5. Use the same order (F,M: F,M) in both graphs.
L302 niloticus
L318 this result/these results?
L361, 363, 379 384, 387 Avoid spaces before or after < or >
L370, 371, + bibliography section. Species names must be written in italics.
L396 100%
L416 mixed / undetermining, not-conclusive?
L518, 521, 524 Toxicology
Author Response
L43, 251, 252, 256, 263, 264 (x3), etc. Species names must be written in italics.
R: All species names are now in italics.
L46 Give a short explanation of which are the anatomical features avoiding the artificial reproduction of this species.
R: A paragraph has been added to give more information on this topic.
L52 What does it means "open water"?
R: the areas were there is no vegetation
L71-72 This tissue distribution, it is a natural one?, or is a consequence of a endocrine disruption activity in experiments with zebrafish?
R: We assume it is "natural" as no chemicals are used in this area. The presence of Vtg in many organs can also be explained by the fact that this molecule is transported in the blood and the blood is present throughout all fish organs.
L72-73 Specify which are the 3 species.
R: the species are listed in the second part of the sentence.
L78 Maybe, reference 38 should be added at the end of the previous sentence. Anyway it has no relationship with the hypothesis mentioned in the final sentence (L77-78).
R: corrected
L93 1.5 kg
R: corrected
L109 15 ml
R: corrected
L109-118. Include the composition of the different buffers and solutions.
R: the buffers are supplied with the commercial kit, and their composition is not specified.
L110 Give some details regarding the Mini Array membrane (company?, material?, size?).
R: info added
L120 What does it means "control spot"?
R: means that if this spot reacts, all reagents of the kit are OK. If this spot is not visible, the reagents are not working properly and test spot result if visible is not valid.
L125 delete first "g"
R: corrected
L126 4 m3
R: corrected
L129- cumulated dose??...a total of 2 mg of E2
R: corrected
L129- divided in 7 injections... ??
R: corrected in the text (285 µl of the emulsified 17ß-estradiol mixture/injection)
L130 48 h
R: corrected
L130-131 Is there any previous evidence that 48 + 48 h is time enough to induce a significant amount of circulating Vtg in this species?
R: no previous data in this species but we have results from previous studies that show a Vtg synthesis in E2 treated fish after 48 h. So we used the same protocol as for other species (Arapaima, Osteoglossum, ...)
L134, 158 One hundred µl
R: corrected
L139 Define MW
R: corrected
L145 100 µg
R: corrected
L159 -20 ºC
R: corrected
L165 Delete "by"
R: corrected
L167 Include the volume of each reactive or solution.
R: corrected
L177 25 ± 2 ºC
R: corrected
L183 Revelation/development?
R: changed to Color development
L185 Specify the H2O2 concentration
R: corrected
L185 30 min by
R: corrected
L186 H2SO4
R: corrected
L199 Delete dot
R: corrected
L213 Define ACN
R: corrected
LL213 Confirm nl
R: Yes, nanoliter
L215 Confirm B rebalance
R: Yes
Figures 3 and 5. Use the same order (F,M: F,M) in both graphs.
R: corrected
L302 niloticus
R: corrected
L318 this result/these results?
R: corrected, ok for "these results"
L361, 363, 379 384, 387 Avoid spaces before or after < or >
R: corrected
L370, 371, + bibliography section. Species names must be written in italics.
R: corrected
L396 100%
L416 mixed / undetermining, not-conclusive?
R: means that the sexing kit for Pirarucu is not effective to sex 100% of Heterotis females. Only 20% were positive with the Pirarucu kit
L518, 521, 524 Toxicology
R: corrected
