An Optimized Environmental DNA Method to Improve Detectability of the Endangered Sichuan Taimen (Hucho bleekeri)
Round 1
Reviewer 1 Report
Monitoring the occurrence and distribution of rare species using classical collection methods is labor-intensive, inefficient, and could harm individuals of the species of interest. Collection and screening of environmental DNA (eDNA) is often a viable alternative, although it entails development of protocols for collection of water (in this case) and PCR amplification of the DNA of the focal species. Sichuan taimen is an endangered species in China, and Deng et al. have developed protocols for collection of water and specific amplification of its DNA. This is well and good, and worthy of being reported in the literature in a tight, reasonably short publication. The manuscript before me can be shortened by removing unneeded material, its message made more succinct, and its prose polished to a more readable form. Below I elaborate on each of these comments. I also have marked the manuscript to guide revision of the prose.
Title. – Neither the title nor the abstract contains the common name of the species. Mention of the common name would make the report more easily findable by search engines. I suggest that the common name, Sichuan taimen, be included in the title.
Abstract. – eDNA techniques have been reported in the literature for over a decade now, and so the claim that publication of this manuscript will support the field of eDNA analysis is a bit of an oversell. Hence, I suggest deletion of that claim from the abstract at lines 23-24.
Introduction. – At lines 41 and 42, “overfishing” and “overexploitation” are both mentioned as factors affecting taimen habitat. Both can be construed as harvesting too many taimen from the system. I believe that the authors intend “overexploitation” to refer to too much water extraction from the rivers in question, and so “water extraction” might be used there.
At line 79, “PCR amplification system and the amplification process” is a bit redundant, and can be more succinctly started as “PCR protocol”.
Methods. – Addition of a map of the study site, showing its location within China, would add to the effectiveness of the manuscript.
At line 110, the reader would appreciate being told that Brachymystax lenok tsinlingensis is another salmonid, sharp-snouted lenok. Few would know that, and indicating its phylogenetic affinity to the focal species would tell the reader why it was included. At lines 123-124, mention of it as a “proximal” species would be better expressed as it being an “outgroup” species.
At line 136, I don’t know why the authors write that the filter membranes were removed by enzymatic lysis. I think that is in error.
At line 147, in the description of the PCR protocol, the authors fail to mention the Taq polymerase used. This is an important omission. The particular enzyme use has large bearing on the results of the PCR amplification process.
At line 164, what is the 85 C “reading board”? I think we have a bad translation here.
Results. – Figure 2 is not really needed; further, the lanes are not labelled as indicated by the caption.
Figures 3 and 4 are not really needed. The text tells the reader which PCR reaction mixtures and amplification protocols worked best and which reaction volumes worked best.
Similarly, Figure 4 is not really needed. Anyone who has done such work is familiar with the results of a query of GenBank.
In Table 3, all reactions used FAM as the fluorescent marker, and so column 5 tells the reader nothing that cannot be better said in the label for the table or its footnote.
Similarly, all entries in columns 3 and 5 of Table 4 are the same. These columns can be removed and the information conveyed in the label or footnote of the table.
Discussion. – At line 307, the authors should a dd a few words to clarify this key sentence: Wild population distribution surveys are effective for PLANNING CONSERVATION ACTIONS for protecting endangered species. My point is that the surveys themselves do not protect the species; rather the results inform subsequent human actions on behalf of the species.
At line 390, the prose needs sharpening to read IMPROVEMENTS of sequencing technology and REFERENCE DATABASES.
At line 391, the authors should write that they predict that eDNA analysis will provide a more accurate evaluation system – that determination is yet ahead of us, so the qualifying “we predict that” is needed.
Noting that eDNA surveys are now common, the claims made at lines 407-408 are not needed and seem to oversell the value of this particular project.
References. – I’ve marked departures of literature citations from journal stylistics on the manuscript document.
Comments for author File: 
Comments.pdf
The prose must be improved. I have marked the manuscript to guide that process.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 2 Report
Dear Editors,
Dear Authors,
The manuscript entitled: “An optimized environmental DNA method to improve detectability of an endangered salmon species (Hucho bleekeri)” represents valuable study that potentially improves the eDNA analysis approach of rare and endangered aquatic organisms. In the reviewed study, the Authors conducted preliminary eDNA detection of H. bleekeri using water samples collected from breeding farms and form the field. The study aimed to establish a fully operational and reliable eDNA analysis program specifically tailored for stream water samples by optimizing and standardizing various aspects including water sample collection method, sample volume, eDNA extraction method, PCR amplification system, and amplification process. However, the overall quality of information presentation is very low, especially description of Material and Methods as well as Results. Language presentation is also very low. The manuscript bears numerous flaws and major improvements are required. All remarks about this have been placed in the attached file.
In conclusion, I do not recommend the manuscript for the publication in the present form and major revision should be made by authors. After that the manuscript should be evaluated again. All remarks, questions and fixes were placed in the attached pdf file (yellow highlights contain fixes and sentence suggestions, while red highlights contain comments and questions).
Thank you for another interesting manuscript that I could review!
Comments for author File: Comments.pdf
Dear Editors,
Dear Authors,
The manuscript entitled: “An optimized environmental DNA method to improve detectability of an endangered salmon species (Hucho bleekeri)” represents valuable study that potentially improves the eDNA analysis approach of rare and endangered aquatic organisms. In the reviewed study, the Authors conducted preliminary eDNA detection of H. bleekeri using water samples collected from breeding farms and form the field. The study aimed to establish a fully operational and reliable eDNA analysis program specifically tailored for stream water samples by optimizing and standardizing various aspects including water sample collection method, sample volume, eDNA extraction method, PCR amplification system, and amplification process. However, the overall quality of information presentation is very low, especially description of Material and Methods as well as Results. Language presentation is also very low. The manuscript bears numerous flaws and major improvements are required. All remarks about this have been placed in the attached file.
In conclusion, I do not recommend the manuscript for the publication in the present form and major revision should be made by authors. After that the manuscript should be evaluated again. All remarks, questions and fixes were placed in the attached pdf file (yellow highlights contain fixes and sentence suggestions, while red highlights contain comments and questions).
Thank you for another interesting manuscript that I could review!
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Monitoring the occurrence and distribution of rare species using classical collection methods is labor-intensive, inefficient, and could harm individuals of the species of interest. Collection and screening of environmental DNA (eDNA) is often a viable alternative, although it entails development of protocols for collection of water and PCR amplification of the DNA of the focal species. Sichuan taimen is an endangered species in China, and Deng et al. have developed protocols for collection of water and specific amplification of its DNA. This is worthy of being reported in the literature in a tight, reasonably short publication. The manuscript before me is a revised one, and is improved relative to the original version. There are some minor issues yet to be addressed and the prose, while improved, must be further polished. I have marked the manuscript to guide revision of the prose.
Abstract. – eDNA has been screened for about 15 years now, and this manuscript constitutes an incremental contribution to the literature, not a landmark paper. Hence, extravagant claims about this paper providing the basis for (all) work that follows must be carefully qualified. Hence, I have marked sentences at lines 25 and 33 to make only the claim that the tools reported here will affect management of this species.
Methods. – I have marked a sentence at line 92 so much that I’d do well to type it out here: …and one site where Sichuan taimen does not occur but B. lenok tsilingenesis (another salmonid, sharp-snouted lenok, whose distribution partially overlaps with Sichauan taimen) does occur, was used as a negative control.
The addition of the map is nice. Could we make the figure larger and thereby more readable?
In the paragraph starting at line 134, both Taq and Pfu are abbreviations for species names (e.g., Thermophilius aquaticus), and so those three-letter abbreviations should be italicized.
Discussion. – At line 260, this eDNA assay is not useful for monitoring species “diversity”, but rather for monitoring species occurrence.
At line 289, it is not “databases” that are absent, but rather DNA sequences for species of interest.
At line 345, you are not “mitigating” contamination (that is, lessening its impact after it has occurred) but rather preventing or eliminating it.
At line 350, it is important to note that it is AQUATIC animals that are at issue.
Comments for author File: Comments.zip
Extensive editing of English language required
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 2 Report
Dear Editors,
Dear Authors,
The manuscript entitled: “An optimized environmental DNA method to improve detectability of an endangered salmon species (Hucho bleekeri)” has been significantly improve. However, the paper still requires significant revisions. The manuscript still bears a lot of unclear terms that must be clarified. After the reading M&M and Results chapters I am not sure how the detection of the species is carried out. You designed few pairs of primers and one probe based on the information from NCBI. For validation and optimization of PCR conditions you used genetic material extracted from the tissues of taimen and lenok? There is missing information about their specificity to both species. No data about that - why did not you present it? What is the main technique for species detection: (1) amplification of the D-loop mitochondrial fragment, sequencing and detecting species by e.g., NCBI BLAST analysis? or (2) the detection of species-specific target DNA by qPCR from the sample based on the designed primers that are specific for the taimen? I mean species-specific DNA detection and eDNA metabarcoding? Moreover, there is lack of information about the application of tissue DNA. For which amplifications did you use it?
For table 2 and figure 3, I am not sure if Ct good criterium for analysis. Ct value directly depends on the template concentration (starting number of targets). Did you standardize the template DNA amount used for qPCR? How the template DNA concentration differences between species originated, taking into account that DNA extraction was carried out with the same tissues and methods? Moreover, I do not know how differences in Ct can be used for species detection. It could work if you design the primers that are only specific for one species. Then using qPCR you can detect its presence (Ct <35) or absence (Ct>35). But how can it be done when used primers are specific to DNA template from both species? In such situation the results are strongly biased by template concentration used for amplification. Maybe Authors developed such method in fact but the expression of information is misleading. This should be improved.
All remarks about this have been placed in the attached file (yellow highlights contain fixes and sentence suggestions, while red highlights contain comments and questions).
Best regards
Comments for author File: Comments.pdf
Dear Editors,
Dear Authors,
The manuscript entitled: “An optimized environmental DNA method to improve detectability of an endangered salmon species (Hucho bleekeri)” has been significantly improve. However, the paper still requires significant revisions. The manuscript still bears a lot of unclear terms that must be clarified. After the reading M&M and Results chapters I am not sure how the detection of the species is carried out. You designed few pairs of primers and one probe based on the information from NCBI. For validation and optimization of PCR conditions you used genetic material extracted from the tissues of taimen and lenok? There is missing information about their specificity to both species. No data about that - why did not you present it? What is the main technique for species detection: (1) amplification of the D-loop mitochondrial fragment, sequencing and detecting species by e.g., NCBI BLAST analysis? or (2) the detection of species-specific target DNA by qPCR from the sample based on the designed primers that are specific for the taimen? I mean species-specific DNA detection and eDNA metabarcoding? Moreover, there is lack of information about the application of tissue DNA. For which amplifications did you use it?
For table 2 and figure 3, I am not sure if Ct good criterium for analysis. Ct value directly depends on the template concentration (starting number of targets). Did you standardize the template DNA amount used for qPCR? How the template DNA concentration differences between species originated, taking into account that DNA extraction was carried out with the same tissues and methods? Moreover, I do not know how differences in Ct can be used for species detection. It could work if you design the primers that are only specific for one species. Then using qPCR you can detect its presence (Ct <35) or absence (Ct>35). But how can it be done when used primers are specific to DNA template from both species? In such situation the results are strongly biased by template concentration used for amplification. Maybe Authors developed such method in fact but the expression of information is misleading. This should be improved.
All remarks about this have been placed in the attached file (yellow highlights contain fixes and sentence suggestions, while red highlights contain comments and questions).
Best regards
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 3
Reviewer 2 Report
Dear Editors,
Dear Authors,
The manuscript entitled: “An optimised environmental DNA method to improve detectability of the endangered Sichuan Taimen (Hucho bleekeri)” has been significantly improved. All my remarks and questions were addressed. Despite language presentation has been improved minor corrections are still required.
Some few minor fixes were placed in the attached pdf file.
Thank you!
Comments for author File: Comments.pdf
Dear Editors,
Dear Authors,
The manuscript entitled: “An optimised environmental DNA method to improve detectability of the endangered Sichuan Taimen (Hucho bleekeri)” has been significantly improved. All my remarks and questions were addressed. Despite language presentation has been improved minor corrections are still required.
Some few minor fixes were placed in the attached pdf file.
Thank you!
Author Response
Thank you very much for your professional instructions and valuable comments, I have revised them in the original manuscript.
Best regards,
