Identification and Expression Analysis of the FAD Gene Family in Anoplophora glabripennis (Coleoptera: Cerambycidae) Based on Genome-Wide Data

Round 1

Reviewer 1 Report

Xue Song et al have investigated expression of fatty acid desaturase genes in Anoplophora glabripennis that may contribute to pheromone production. The paper is well written and the data will be interesting to those concerned with this invasive forest pest.

I have one question concerning the qPCR results. Line 173 says that 3 biological replicates and 3 technical replicates were done. The error bars shown in Figures 7-9 are in some cases quite small. Did the authors use n=3 in calculating error bars or n=6? Since biological variation is almost always much higher than technical variation, n=3 is appropriate here. If n=6 was used, I suggest the authors redraft the 3 figures with the wider error bars found by using n=3. This may also affect p values found.

The English is generally quite good. I mention the following small change:

Line 17-18 "based on genome-wide level" should be deleted.

Author Response

Dear reviewer

Thank you very much for your comments and professional advice. These opinions help to improve academic rigor of our article. Based on your suggestion and request, we have made corrected modification on the revised manuscript. We hope that our work can be improved again. Furthermore, we would like to show the details as follows:

Comments 1：

I have one question concerning the qPCR results. Line 173 says that 3 biological replicates and 3 technical replicates were done. The error bars shown in Figures 7-9 are in some cases quite small. Did the authors use n=3 in calculating error bars or n=6? Since biological variation is almost always much higher than technical variation, n=3 is appropriate here. If n=6 was used, I suggest the authors redraft the 3 figures with the wider error bars found by using n=3. This may also affect p values found.

Response 1:

Three biological replicates and three technical replicates were performed, but the error bars shown in Figures 7-9 are in some cases quite small. Because we removed the data that was too large and too small as outliers. Based on your comments, we have added the previously deleted data, re-plotted, and you can see that some of the error bars have changed significantly.

Comments 2：

Line 17-18 "based on genome-wide level" should be deleted.

Response 2:

Line 17-18 "based on genome-wide level" has been deleted.

Thank you very much for your attention and time. Look forward to hearing from you.

Yours sincerely,

Xue Song

29 March 2024

Reviewer 2 Report

Review for the manuscript entitled “Identification and expression analysis of the FAD gene family in Anoplophora glabripennis (Coleoptera: Cerambycidae) based on genome-wide data” by Song et al.

The manuscript concentrated on identification of AglaFAD genes based on genome-wide level in Anoplophora glabripennis. Based on the results, it is reported that six genes are defined all of which mapped to the endoplasmic reticulum and shared a highly similar distribution of conserved domains. These results may help in understanding pheromones synthesis and get interest in community working insect biochemistry. However, presentation of the manuscript give the sense that it has been prepared by an “artificial intelligence” as many expressions, especially in Materials and Methods section, are very superficial. There are some other flaws requiring a considerable revision. Thus, the fair decision for the version is major revision.

Title: The title seems to be appropriate.  

Introduction: This section is relatively well prepared regarding the content of the manuscript. The following points need correction. I suggest to give the authors of the taxa mentioned, at least on the first case.

Line 51- “Drosophila” should be italic

Line 61- Give the full name of the genus at first occasion.

Line 74, 79, 86, 89- The reference number should be along the names of the authors, not at the end of the sentence.

Line 102- “A. glabripennis” should be italic.

Material and Methods: This section seems the most carelessly prepared section of the manuscript and requires a considerable revision, at least for clarity.

 Line 115-116-The sentence is not clear! What kind of information was obtained from NCBI?

Line 118-  “… to obtain candidate protein sequences” what kind of sequences? Not clear!

Line 118-119- The sentence is not clear! What is Pfam database? HMM is something to be calculated! When, how and for which dataset this HMM was calculated?

Line 131-132- It is mentioned that they obtained a phylogenetic tree. In fact, it is a dendrogram, not a phylogenetic tree, as NJ dendrograms are based on similarity. Further, what was the dataset subjected to NJ analysis? What was the characteristics of the dataset?

Table 1- The table needs some revisions. Are these FAD proteins belonging to the same gene family including the AglaFADs studied here? If yes why the names used here are not given, at least along the already given names? Why there is no reference for the accession numbers?

Line 139- “..the gff file” what is gff file!

Line 178- What was the parameters used in the statistical analyses or these test were analyses for which kind of data?

Results: In general results of all conducted analyses are presented. However, in parallel with the unclear points in “Material and Methods” section there are some unclear points in the section requiring clarification (some specific points are indicated below).

Line 196 – Phylogenetic tree should be replaced by dendrogram!

Figure 1- The name of the species and proteins, not the accessions numbers, should be given at tip of the branches.

Figures 2, 3, 4, 5 and 6- The legend is not sufficiently explanatory. For example, provide an explanation for these abbreviation along the legend. In figure 6 some letters/words cannot be read due to size.

Discussion: In general, this section is well organised and sufficient. It would be better to modify “Results” like sentences.

Conclusions: This section is totally repetition of some earlier sentences.

References: This section has not been checked.

Author Response

Dear reviewer

Thank you very much for your comments and professional advice. These opinions help to improve academic rigor of our article. Based on your suggestion and request, we have made corrected modification on the revised manuscript. We hope that our work can be improved again. Furthermore, we would like to show the details as follows:

Comments 1：

Line 51- “Drosophila” should be italic

Response 1:

“Drosophila” was changed to “Drosophila”.

Comments 2：

Line 61- Give the full name of the genus at first occasion.

Response 2:

Line 61 “A. glabripennis” was changed to “Anoplophora glabripennis (Motschulsky, 1853)”.

Comments 3：

Line 74, 79, 86, 89- The reference number should be along the names of the authors, not at the end of the sentence.

Response 3:

Line 74, 79, 86, 89- The reference number has been changed along the names of the authors, respectively.

Comments 4：

Line 102- “A. glabripennis” should be italic.

Response 4:

“A. glabripennis” was changed to “A. glabripennis”.

Comments 5：

Line 115-116-The sentence is not clear! What kind of information was obtained from NCBI?

Response 5:

The information is the genome sequence data for A. glabripennis. The modification is as follows:

The genome sequence data for A. glabripennis were obtained from the NCBI database  (https://www.ncbi.nlm.nih.gov/) (accessed on 3 April 2023).

Comments 6：

Line 118-  “… to obtain candidate protein sequences” what kind of sequences? Not clear!

Response 6:

“… to obtain candidate protein sequences” means to collect genome sequences of A. glabripennis that meet the requirements of the calculation. The modification are as follows:

“… to collect all sequences with an E-value below 1e^-5.”

Comments 7：

Line 118-119- The sentence is not clear! What is Pfam database? HMM is something to be calculated! When, how and for which dataset this HMM was calculated?

Response 7:

Pfam database (http://pfam-legacy.xfam.org/) is a large collection of protein families, each represented by multiple sequence alignments and hidden Markov models (HMMs). The HMM profile for FA_desaturase conserved domain (PF00487) was downloaded from the Pfam database and used to search against the protein sequence data for A. glabripennis using HMMER 3.0 to acquire putative A. glabripennis FAD genes with an E-value below 0.05.  

Thank you for pointing this out. We have described it in more detail in the text, adding links and operating times.

Comments 8：

Line 131-132- It is mentioned that they obtained a phylogenetic tree. In fact, it is a dendrogram, not a phylogenetic tree, as NJ dendrograms are based on similarity. Further, what was the dataset subjected to NJ analysis? What was the characteristics of the dataset?

Response 8:

1.Line 131-132- It is mentioned that they obtained a phylogenetic tree. In response to this problem, I consulted other articles and found the same figure, they all write “phylogenetic tree”, so I haven't changed it yet. We will learn more about this in the future.

2.The dataset subjected to NJ analysis are the amino acid sequences of FADs listed in Table 1.

3.All FADs listed in Table 1 belong to the First Desaturases, a subfamily of desaturases.    

Comments 9：

Table 1- The table needs some revisions. Are these FAD proteins belonging to the same gene family including the AglaFADs studied here? If yes why the names used here are not given, at least along the already given names? Why there is no reference for the accession numbers?

Response 9:

These FAD proteins belong to the same gene family, including the AglaFADs. 6 AglaFADs have been added to Table 1.

Comments 10：

Line 139- “..the gff file” what is gff file!

Response 10:

GFF3 (general feature format) is the most commonly used file format for gene structure annotation.It is used for annotating reads that have been mapped to a reference genome in high-throughput sequencing. These files number each chromosome of each species and number each of its base sites, and then people record the known element intervals with the start and end sites.

Comments 11：

Line 178- What was the parameters used in the statistical analyses or these test were analyses for which kind of data?

Response 11:

We analyzed the expression levels of 6 FADs in 6 tissues (head, antennae, thorax, wing, leg, and gonad) of males and females, and tand the changes of their expression levels in 3 developmental stages (primary eclosion, sexual maturity, after mating) of females. This was mentioned in 2.2.1. So I've made a simple addition in line 178.

Comments 12：

Line 196 – Phylogenetic tree should be replaced by dendrogram!

Response 12:

This question is the same as question 8, do I need to change “Phylogenetic tree” to “dendrogram”, if so, look forward to your reply, thank you very much.

Comments 13：

Figure 1- The name of the species and proteins, not the accessions numbers, should be given at tip of the branches.

Response 13:

In Figure 1, accessions numbers have been removed.

Comments 14：

Figures 2, 3, 4, 5 and 6- The legend is not sufficiently explanatory. For example, provide an explanation for these abbreviation along the legend. In figure 6 some letters/words cannot be read due to size.

Response 14:

The legends of Figures 2, 3, 4, 5, and 6 are fully explained, and Figure 6 has been adjusted so that the letters in the figure are more clearly visible.

Comments 15：

Conclusions: This section is totally repetition of some earlier sentences.

Response 15:

Since the content of the conclusion has already been covered in the discussion, we cut it out to make the article look more concise.

Thank you very much for your attention and time. Look forward to hearing from you.

Yours sincerely,

Xue Song

29 March 2024

Reviewer 3 Report

Several revisions need to be done prior acceptance.

Line 51 Drosophila italic

Line 61 and 66 A. In full name of genus.

Line 74 aand 85 Cerambycidae not italic

Line 102 species name italic

Statement for teh objective not clear

The intriduction section too long. Please shortern it.

Line 238 Please avoid We- write in passive form

Fig. 7 No x axis label

Fig.8 No axis label

Fi.g. 9 No axis label

Line 326 Please avoid We-

Line 457 Species name italic

Line 464 Ostrinia italic

Author Response

Dear reviewer

Thank you very much for your comments and professional advice. These opinions help to improve academic rigor of our article. Based on your suggestion and request, we have made corrected modification on the revised manuscript. We hope that our work can be improved again. Furthermore, we would like to show the details as follows:

Comments 1：

Line 51 Drosophila italic.

Response 1:

“Drosophila” was changed to “Drosophila”.

Comments 2：

Line 61 and 66 A. In full name of genus.

Response 2:

Line 61 “A. glabripennis” was changed to “Anoplophora glabripennis (Motschulsky, 1853)”.

Excuse me, Line 66 “A. glabripennis” is the second time it appears, does it also need to be changed to the full name?

Comments 3：

Line 74 and 85 Cerambycidae not italic.

Response 3:

“Cerambycidae” was changed to “Cerambycidae”.

Comments 4：

Line 102 species name italic.

Response 4:

“A. glabripennis” was changed to “A. glabripennis”.

Comments 5：

Statement for teh objective not clear.

Response 5:

Statement was changed to make it clear. As follows:

Data Availability Statement: The data are not publicly available due to the usage for ongoing project.

Comments 6：

The intriduction section too long. Please shortern it.

Response 6:

Some redundant sentences in the introduction have been removed.

Comments 7：

Line 238 Please avoid We- write in passive form

Response 7:

We- write in passive form was avoid. The sentence was modified to “RT-qPCR was used to detect the expression of......”

Comments 8：

Fig.7, Fig.8, and Fig.9 No x axis label

Response 8:

Fig.7, Fig.8, and Fig.9 x axis labels have been added.

Comments 9：

Line 326 Please avoid We-

Response 9:

We- was avoid. The sentence was modified to “It is speculated that......”

Comments 10：

Line 457 Species name italic

Response 10:

“Saccharomyces cerevisiae” was changed to “Saccharomyces cerevisiae”.

Comments 11：

Line 464 Ostrinia italic

Response 11:

“Ostrinia” was changed to “Ostrinia”.

Thank you very much for your attention and time. Look forward to hearing from you.

Yours sincerely,

Xue Song

29 March 2024

Round 2

Reviewer 2 Report

In the revised version some of my suggestion were considered and manuscript revised accordingly while some others not. Explanations are provided for some, also not for some others! Some points still need revisions are below.

During first review round, it was suggested to give the authors of the taxa mentioned throughout text, on the first case. It has been done for A. glabripennis, but not for many others!

The citation for accession numbers given in Table 1 should be provided, at least in a supplementary file!

The data set used in phylogenetic analysis needs to be defined in the beginning of the respective section of Result. How many protein sequences are used, what was the length and length variation, how many position were constant, how many variable, so on! All need to be define to allow reader to know details. At least to ensure readers! Unless these have been done phylogeny will be unreliable!

In Table 3, it is mentioned that all genes detected are localized in Endoplasmic reticulum! It is not necessary to repeat, instead mention only along the legend!

FAD is italic in some occasions, but not in some others!

Author Response

Dear reviewer

Thanks very much for taking your time to review this manuscript. I really appreciate all your comments and suggestions! Please find my itemized responses in below and my revisions in the re-submitted files.

Comments 1：

During first review round, it was suggested to give the authors of the taxa mentioned throughout text, on the first case. It has been done for A. glabripennis, but not for many others!

Response 1:

We were really sorry for our careless mistakes. Thank you for your reminder. The authors of the taxa were added to the other first-time species in the paper. 

Comments 2：

The citation for accession numbers given in Table 1 should be provided, at least in a supplementary file!

Response 2:

Thank you for your advice. Most of the sequences in Table 1 were cited from reference [33], which has been added to the corresponding section of the article. However, some of the sequences may not have been published in papers, but were directly found in the genome of the species using NCBI, and these sequences have been identified. (https://doi.org/10.3390/f15010169) This paper is also the same, and the sequences used are screened from the NCBI. I have listed all the sequences in Table 1 and uploaded them as supplementary sequences.

Comments 3：

The data set used in phylogenetic analysis needs to be defined in the beginning of the respective section of Result. How many protein sequences are used, what was the length and length variation, how many position were constant, how many variable, so on! All need to be define to allow reader to know details. At least to ensure readers! Unless these have been done phylogeny will be unreliable!

Response 3:

Based on your suggestion, We have defined it in the results section of the phylogenetic analysis, focusing on the branch where AglaFAD is located. Because, we note that the other similar papers have analyzed the genetic relationship with neighboring species by describing the distribution of the gene of interest on the branch, and do not describe the all sequence.

Such as:

https://doi.org/10.3390/f14030540; https://doi.org/10.3390/f14030510; 

https://doi.org/10.3390/f14020207;  https://doi.org/10.3390/f14050961.

Comments 4：

In Table 3, it is mentioned that all genes detected are localized in Endoplasmic reticulum! It is not necessary to repeat, instead mention only along the legend!

Response 4:

Thanks for your suggestion, We've removed the repetitive part of the article.

Comments 5：

FAD is italic in some occasions, but not in some others!

Response 5:

All FADs in the text have been changed to italics.

Thank you very much for your attention and time. Look forward to hearing from you.

Yours sincerely,

Xue Song

6 April 2024