Comparative Transcriptome Analysis of Toxic and Non-Toxic Nassarius Communities and Identification of Genes Involved in TTX-Adaptation

Round 1

Reviewer 1 Report

A total of 12 samples of were used for transcriptome sequencing in this study, half out of which were from Nassarius succinctus and half out of which were from N. variciferus. Some specimens of each species are TTX-bearing (toxic) while some are non-toxic. As the result, they report that for both species, the cellular and metabolic process, and binding and catalytic activity accounted for the top classification categories, and the toxic communities generally produced more up-regulated genes than non-toxic communities. In addition, they found a specific mutation in Nav domeinII of N. succinctus, which suggests the TTX-resistant Nav. These data are interesting and worth publishing. However, some points should be revised.

1. In Section 2.4, how was the sequences of domain I and IV? If some sequence data have obtained for them, please add some comments about these regions. I think that the sequences in Figure 10 are for S5-S6 regions of these domains. Such explanation is also needed.

Author Response

Dear Editors and Reviewer-1: Thank you very much for giving us the chance to revise the manuscript for publication. We are very appreciated for you and the reviews. We have modified the manuscript accordingly point by point as below. Comments and Suggestions for Authors: A total of 12 samples of were used for transcriptome sequencing in this study, half out of which were from Nassarius succinctus and half out of which were from N. variciferus. Some specimens of each species are TTX-bearing (toxic) while some are non-toxic. As the result, they report that for both species, the cellular and metabolic process, and binding and catalytic activity accounted for the top classification categories, and the toxic communities generally produced more up-regulated genes than non-toxic communities. In addition, they found a specific mutation in Nav domeinII of N. succinctus, which suggests the TTX-resistant Nav. These data are interesting and worth publishing. However, some points should be revised.  Thank you very much for your good points for our study. 1. In Section 2.4, how was the sequences of domain I and IV? If some sequence data have obtained for them, please add some comments about these regions. I think that the sequences in Figure 10 are for S5-S6 regions of these domains. Such explanation is also needed.  Thanks for the important point. The domain I and IV were not obtained in this study, which have already been mentioned in section 2.4 as “Unfortunately, no perfect matched sodium channels genes were found from unigenes of N. variciferus even though we blasted the clean reads of both species (150bp) to all reference sequences, which meant that the sequences of Domain I and Domain IV could not be obtained in this study (the reason was referred in the discussion)”. The detailed reason for missing the domain I and IV was also referred in the discussion as “Unfortunately, other domains of Nassarius could not be obtained by unigene blasting, reads blasting or RACE-PCR. We infer the reasons may be that the RNA-seq sequences in this study do not completely cover the sodium channel regions of both species due to the complication of sodium channel family. In future studies, we would employ comprehensive samples and deep sequencing to explore the complete sodium channel genes of various toxic and non-toxic communities in more Nassarius species to better understand the adaptive evolution of TTX in Nassarius”. 2. In Figure 1, description “Japan” may be wrong. That area is Taiwan, isn't it? 2. In Figure 1, description “Japan” may be wrong. That area is Taiwan, isn't it?  Again, thanks very much for the important point. Yes, it is absolutely Taiwan, not Japan. Sorry for this sample error. We have revised Figure 1 and changed it with the new one. 3. If TTX concentrations in Nassarius specimens used in this study are quantified, please add such data into the manuscript.  Actually, as revised in the section of “4.1. Sample collection, treatment and toxicity test”: The TTX toxicity of both N. succinctus and N. variciferus was tested in our previous research which included the detailed sample collection and toxicity detection (Zou et al. 2019). Based on the toxicity of all samples of various Nassarius species tested in Zou et al. (2019), we selected samples of N. succinctus and N. variciferus for transcriptional analysis since they included both toxic and non-toxic communities from Dalian and Lianyungang. The samples used in this study were collected together with that in Zou et al. (2019). Since the RNA-seq request high quality of RNA we had to use fresh samples without TTX detection again.” So we just know the TTX toxicity of samples in different communities of Dalian and Lianyungang, but not possible know their detailed TTX concentrations. All the changes in the revised manuscript have been highlighted with red color.

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear Authors,

This work is interesting and the results obtained with comparative transcriptome analysis of toxic and non-toxic Nassarius seem quite promising. Nevertheless, some suggestions of change and remarks are presented below.

In general:

Avoid beginning sentences with a numeral, so consider rewording the sentence or spelling out the number (e.g., page 15, line 303, “13 random differentially expressed gene”) and avoid using final full stops in subtitles (e.g., page 2, line 72, “2.1. Quality of RNA-seq data and de novo transcriptome assembly.”).

As you are not referring to Class Gastropoda of Phylum Mollusca, please harmonize terminology and replace “marine gastropoda mollusk” by “marine gastropod mollusks” in Abstract (Page 1, line 19) and in Introduction (Page 2, lines 64 ).

Specific comments:

Abstract

Page 1, line 2 - Please consider replacing “provide us” by “lead to”

Introduction

In order to improve comprehension, please consider referring in the first sentences that Nassarius are marine gastropod mollusks, as stated in abstract (Page 1, line 19).

Page 1, lines 28 & 34 - “nassariids of Nassarius” - Consider rephrasing in order to avoid redundancy.

Page 1, line 37 – consider replacing “ accumulated in Nassarius sp’s body” by “accumulated in the edible tissues of individuals of the genus Nassarius“

Page 1, lines 40-41 - Please verify citation “(Wang et al. 2008)”, as it is lacking in references.

Page 2, lines 44-45 - Replace “(Soong et al. 2006)” by “(Soong and Venkatesh, 2006)”.

Page 2, line 48 - Replace “(Sheumack et. 1984)” by “(Sheumack et al. 1984)” and verify extra space in “(Tarvin et al. 2017)”.

Page 2, line 53 - Replace “analysis to revealed some gene” by “analysis to reveal some genes”.

Page 2, lines 61 & 63- Replace “(Hille et al. 2001)” & ”(Kaneko et al. 10997;” by “(Hille, 2001)” & ”(Kaneko et al. 1997;”.

Results

Page 2, line 82 - According to Table 1 “mean lenght from 430bp to 476bp” should be replaced by “mean lenght from 430bp to 576bp”.

Page 2, line 83 - Replace “153357 nigenes” be  “153357 unigenes”.

Discussion

As the origin of samples seemed to influence some results (such as expressed genes, e.g. page 6, lines 127-129), please elaborate if/how the different origin of toxic and non-toxic specimens (Dalian versus Lianyungang) could influence results.

Page 12, line 191 – “like PSP, TSX”. As this is the first time PSP and TSX abbreviations are used, please also write out the terms (e.g., paralytic shellfish poisoning [PSP]). Please verify if “TSX” should be replaced by “STX”, already mentioned in Page 2, line 43 “as Saxitoxin (STX)”.

Page 13, line 224 – consider replacing “the TTX in Nassarius’s body” by “the TTX present in the tissues of Nassarius”.

Page 13, line 234 – please include a reference for the statement “ should be further verified by electrophysiology” or rephrase.

Materials and Methods

Please detail the description of the sample sites and collection shown in Fig. 1. Also describe the conditions (e.g., time/temperature, preservatives?) used for the preservation of the tested tissues (liver and pancreas) until treatment for RNA extraction and sequencing .

Page 14, line 251 – “since they contained more toxin” please base this statement on references. And what about gonads?

Page 14, Fig. 1 – replace “N. Variciferus “ by “N. variciferus”

Page 15, line 290, 297 & 300 – replace “Li et al., 2011”, “McKenna et al., 2010”, “Thiel et al. 2003” by “Li and Dewey, 2011”, “McKenna, 2010”, “Thiel, 2003”.

Page 15, line 308 – harmonize presentation of values with units and the usage of space between them, e.g. “95ºC” versus “95 ºC”.

Captions of Figures/Tables

For better understanding, add the meaning of (D) non-toxic and (L) toxic, whenever applicable, and maintain the meaning of (H) refering to N. succinctus and (Z) to N. variciferus.

References

Please remove references which are not mentioned in the text (e.g. references numbers 3, 4, 21, 27, 36, 38, 42).

Comments for author File: Comments.doc

Author Response

Dear Editors and Reviewer-2:

• Thank you very much for giving us the chance to revise the manuscript for publication. We are very appreciated for you and the reviews. We have modified the manuscript accordingly point by point as below.

Comments to the Author

This work is interesting and the results obtained with comparative transcriptome analysis of toxic and non-toxic Nassarius seem quite promising. Nevertheless, some suggestions of change and remarks are presented below.

• Thank you very much for your good points for our study.

In general:

Avoid beginning sentences with a numeral, so consider rewording the sentence or spelling out the number (e.g., page 15, line 303, “13 random differentially expressed gene”) and avoid using final full stops in subtitles (e.g., page 2, line 72, “2.1. Quality of RNA-seq data and de novo transcriptome assembly.”).

As you are not referring to Class Gastropoda of Phylum Mollusca, please harmonize terminology and replace “marine gastropoda mollusk” by “marine gastropod mollusks” in Abstract (Page 1, line 19) and in Introduction (Page 2, lines 64 ).

• Thanks very much for this point. Yes, it is better to avoid using numeral as beginning and using final full stops in subtitiles. We have revised these mistakes throughout the manuscript.

Specific comments:

Abstract

Page 1, line 2 - Please consider replacing “provide us” by “lead to”

• Thanks for this suggestion. We have revised it.

1. Introduction

In order to improve comprehension, please consider referring in the first sentences that Nassarius are marine gastropod mollusks, as stated in abstract (Page 1, line 19).

• We have changed the first sentences as “Nassarius, a species-rich genus of Nassariinae belong to marine gastropod mollusks, is distributed in oceans globally, inhabiting inter- to subtidal shallow marine environments as facultative scavengers”.

Page 1, lines 28 & 34 - “nassariids of Nassarius” - Consider rephrasing in order to avoid redundancy.

• Nassariids of Nassarius means samples of Nassarius. But yes, it is better to avoid redundancy by just using Nassarius as revised.

Page 1, line 37 – consider replacing “ accumulated in Nassarius sp’s body” by “accumulated in the edible tissues of individuals of the genus Nassarius“

• Thanks for the more accurate expression. We have revised it accordingly.

Page 1, lines 40-41 - Please verify citation “(Wang et al. 2008)”, as it is lacking in references.

• Thanks for pointing out this error. We have added another recent reference.

Page 2, lines 44-45 - Replace “(Soong et al. 2006)” by “(Soong and Venkatesh, 2006)”.

• Thanks for this detailed point. The “Soong et al. 2006” has been changed by “Soong and Venkatesh, 2006”.

Page 2, line 48 - Replace “(Sheumack et. 1984)” by “(Sheumack et al. 1984)” and verify extra space in “(Tarvin et al. 2017)”.

• Thanks for this detailed point. This should be chaned accordingly. But based on the standard creteria for the list of reference, the references in the manuscript has been changed as [number order].

Page 2, line 53 - Replace “analysis to revealed some gene” by “analysis to reveal some genes”.

• Thanks for this detailed point. This has also been chaned accordingly.

Page 2, lines 61 & 63- Replace “(Hille et al. 2001)” & ”(Kaneko et al. 10997;” by “(Hille, 2001)” & ”(Kaneko et al. 1997;”.

• Thanks for this detailed point. This should be chaned accordingly. But based on the standard creteria for the list of reference, the references in the manuscript has been changed as [number order].

2. Results

Page 2, line 82 - According to Table 1 “mean lenght from 430bp to 476bp” should be replaced by “mean lenght from 430bp to 576bp”.

• Again, thanks for this detailed point. The 476 has been changed to 576bp.

Page 2, line 83 - Replace “153357 nigenes” be  “153357 unigenes”.

• Thanks for this. “153357 nigenes” has been changed to “ 153357 unigenes”.

3. Discussion

As the origin of samples seemed to influence some results (such as expressed genes, e.g. page 6, lines 127-129), please elaborate if/how the different origin of toxic and non-toxic specimens (Dalian versus Lianyungang) could influence results.

• Thanks for this point. Yes, it is better to elaborate the case more clearly. We have added more detailed explanation in the related section of the discussion section (second paragraph).

Page 12, line 191 – “like PSP, TSX”. As this is the first time PSP and TSX abbreviations are used, please also write out the terms (e.g., paralytic shellfish poisoning [PSP]). Please verify if “TSX” should be replaced by “STX”, already mentioned in Page 2, line 43 “as Saxitoxin (STX)”.

• Thanks for this remind. We have changed the first time PSP and STX to their full names. And, yes, the Saxitoxin (STX) is right.

Page 13, line 224 – consider replacing “the TTX in Nassarius’s body” by “the TTX present in the tissues of Nassarius”.

• Thanks for this point. “the TTX in Nassarius’s body” has been changed to “the TTX present in the tissues of Nassarius”.

Page 13, line 234 – please include a reference for the statement “ should be further verified by electrophysiology” or rephrase.

• The reference which is closely related with this sentence is added.

4. Materials and Methods

Please detail the description of the sample sites and collection shown in Fig. 1. Also describe the conditions (e.g., time/temperature, preservatives?) used for the preservation of the tested tissues (liver and pancreas) until treatment for RNA extraction and sequencing .

• Thanks for this point. Actually, as revised in the section of “4.1. Sample collection, treatment and toxicity test”: The TTX toxicity of both N. succinctus and N. variciferus was tested in our previous research which included the detailed sample collection and toxicity detection (Zou et al. 2019). Based on the toxicity of all samples of various Nassarius species tested in Zou et al. (2019), we selected samples of N. succinctus and N. variciferus for transcriptional analysis since they included both toxic and non-toxic communities from Dalian and Lianyungang. The samples used in this study were collected together with that in Zou et al. (2019). Since the RNA-seq request high quality of RNA we had to use fresh samples without TTX detection again. All the samples in this study were stored by liquid nitrogen for next RNA extraction.” We also add the information in the caption of Fig. 10.

Page 14, line 251 – “since they contained more toxin” please base this statement on references. And what about gonads?

• Yes, this point should be referenced. We have added three references and revised this sentence accordingly.

Page 14, Fig. 1 – replace “N. Variciferus “ by “N. variciferus”

• Thanks for this error finding. This has been revised accordingly.

Page 15, line 290, 297 & 300 – replace “Li et al., 2011”, “McKenna et al., 2010”, “Thiel et al. 2003” by “Li and Dewey, 2011” “McKenna, 2010”,. “Thiel, 2003”.

• Thanks for this detailed point. This should be chaned accordingly. But based on the standard creteria for the list of reference, the references in the manuscript has been changed as [number order].

Page 15, line 308 – harmonize presentation of values with units and the usage of space between them, e.g. “95ºC” versus “95 ºC”.

• Thanks for this point. The harmonize presentation of values with units has been changed as universe.

Captions of Figures/Tables

For better understanding, add the meaning of (D) non-toxic and (L) toxic, whenever applicable, and maintain the meaning of (H) refering to N. succinctus and (Z) to N. variciferus.

• Yes, these should be clarified more clearly with species names. We have changed accordingly.

References

Please remove references which are not mentioned in the text (e.g. references numbers 3, 4, 21, 27, 36, 38, 42).

• Sorry for these mistakes. We have deleted them accordingly.

All the changes in the revised manuscript have been highlighted with red color.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors has well answered to the comments and revised the manuscript.

However, Figure 1-5 are not clearly shown. Please replace them with the clear figures to prepare the final version.