Review History


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Summary

  • The initial submission of this article was received on June 11th, 2020 and was peer-reviewed by 3 reviewers and the Academic Editor.
  • The Academic Editor made their initial decision on July 10th, 2020.
  • The first revision was submitted on November 6th, 2020 and was reviewed by 1 reviewer and the Academic Editor.
  • A further revision was submitted on November 26th, 2020 and was reviewed by 1 reviewer and the Academic Editor.
  • A further revision was submitted on December 16th, 2020 and was reviewed by 1 reviewer and the Academic Editor.
  • A further revision was submitted on December 28th, 2020 and was reviewed by 1 reviewer and the Academic Editor.
  • The article was Accepted by the Academic Editor on December 31st, 2020.

Version 0.5 (accepted)

· Dec 31, 2020 · Academic Editor

Accept

Now that the remaining reviewer recommends its acceptance, I am pleased to inform you that I will recommend its acceptance to the Editor-in-Chief. Congratulations!

Reviewer 1 ·

Basic reporting

non

Experimental design

non

Validity of the findings

non

Additional comments

non

Version 0.4

· Dec 26, 2020 · Academic Editor

Minor Revisions

The remaining referee still points out an issue, as described below. But it might be too late for the reviewer to point out this. Thus, I would like to only suggest to address this problem this time. Thanks for your patience.

Reviewer 1 ·

Basic reporting

non

Experimental design

non

Validity of the findings

The criteria for discriminating genuine and pseudo genes are not consistent. For example, XP_028042624.1 and XP_028042717.1 are respectively only 158 and 125 aa long but counted as genuine gene products. Although the lengths of the neglected genes/pseudogenes are not shown, they are likely to be longer than 200aa as judged from Figure S2 of the first submission. The gene/pseudogene discrimination should be carried out on consistent criteria that are explicitly described.

Version 0.3

· Dec 7, 2020 · Academic Editor

Minor Revisions

Your re-revised manuscript has been reviewed by the same reviewer who reviewed its previous versions. As you see from the comments below, the reviewer only requests one additional minor revision. Since I also believe that this point is important, I would like to ask you for another round of minor revision. Please address the point accordingly in your hopefully final revision.

Reviewer 1 ·

Basic reporting

Line 343:
AKAi -> Akai

Experimental design

no comment

Validity of the findings

Line 22 and others:
Disjointing three artificially fused genes would yield three more predicted genes. Table 1 suggests that at least two of the three affected genes (XP_028036546.1 and XP_028040841.1) appear to respectively correspond to two CYP genes in B. mori. The authors should reexamine all B. mandarina vs. B. mori syntenic regions where presence/absence of CYP genes is discordant.

Version 0.2

· Nov 16, 2020 · Academic Editor

Minor Revisions

Your revised manuscript has been reviewed by one of the three reviewers (the other two declined because they were too busy). Fortunately, the most important reviewer, who recommended its rejection to the original manuscript, agreed. You can read his/her comments below. Based on the reviewer's comments and my own confirmation that you addressed the points raised by the others fairly well, I now recommend its minor revision. Please read the following comments carefully and re-revise your manuscript accordingly.

Reviewer 1 ·

Basic reporting

I appreciate that the authors generally well responded to my and other reviewers’ criticisms and the revised manuscript, table, and figures are considerably improved in various aspects. However, I consider that the following points must be clarified before this manuscript is accepted for publication.

1. Sentences in the following lines should be refined.
Line 227
Line 294
Line 372-374
2. Following references should be modified or completed.
2014. Transdecoder_r20140704
AKAi -> Akai
Harris R. 2007

Experimental design

No comment

Validity of the findings

1. I still doubt the authors’ conclusion that B. mandarina has a significantly smaller CYPome than B. mori. Nearly 5% of BUSCO entries are not found on the B. mandarina assembly, indicating that the number of missing genes is not negligible. As previously pointed out by Reviewer 3, Table S2 and Figure 3 indicate that several chromosomes of B. mandarina are significantly smaller than the counterparts of B. mori. In particular, Chromosome 21 encodes a CYP cluster that is missing in the B. mandarina assembly. The size variation represented in logarithmic scale in Table S2 is somewhat misleading; (wild - domestic) / domestic would be a more natural measure of size variation. Anyway, if these size variations are real, they would be visually detected by cytogenetic analyses. The authors should refer to his point.

2. Table 1 and Figure S5 suggest that XP_028036989.1 (CYP9A20), XP_028036546.1 (CYP367B1), and XP_028040841.1 (CYP4M5) are mis-predicted to generate artificial fusion genes. These mis-predictions should be corrected.

Version 0.1 (original submission)

· Jul 10, 2020 · Academic Editor

Major Revisions

Your manuscript has been reviewed by three experts in the field. As you can see from their comments below, all of them raise rather fundamental criticisms. Particularly, two of them point out the potential unreliability of the data, caused at the time of genome assembly, for example. Although my decision is 'major revision', it would be possible that I would reject its revised version if you cannot persuade the reviewers (and myself) with sufficient evidence next time. Please read their comments below carefully and revise the manuscript accordingly. I hope that you will be able to address all points in the revised version of the manuscript.

[# PeerJ Staff Note: Please ensure that all review comments are addressed in a rebuttal letter and any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.  It is a common mistake to address reviewer questions in the rebuttal letter but not in the revised manuscript. If a reviewer raised a question then your readers will probably have the same question so you should ensure that the manuscript can stand alone without the rebuttal letter.  Directions on how to prepare a rebuttal letter can be found at: https://peerj.com/benefits/academic-rebuttal-letters/ #]

Reviewer 1 ·

Basic reporting

no comment

Experimental design

no comment

Validity of the findings

This manuscript describes identification of cytochrome P450 genes from draft genomic sequence of wild silkworm (Bombyx mandarina). The authors compare these gene models with those of the well-studied domestic strain Bombyx mori.

1. One of the major claims of this manuscript is that the CYPome of B. mandarina is considerably smaller than that of B. mori (Line 24 and others). However, this claim cannot be accepted immediately. First, the presumable evolutionary time of 5000 years after branching is too short to cause such a large change in gene battery of a wild species. Second, the quality of the primary data, i.e. the assembly of B. mandarina genome, is not high enough to draw any conclusions about the proteome that encodes. Although the authors improved the initial de novo assembly by a template-guided method using B. mori genome as the reference, this attempt cannot recover missing sequences unrepresented in the initial contigs. Third, as detailed in the following item, the authors’ gene models are likely to contain an appreciable number of fusions and fissions of true genes. This increases inaccuracy in the estimated number of P450 genes.
2. The authors use the gene models of B. mori downloaded from Silkbase as the reference. However, more than a half (44/82) of these models appear to be incorrect as Kawamoto et al (2019) reported manually modified versions of them (Table S5), including a total of nine gene models correcting for erroneous gene fusions or fissions. The reliability of the gene models of B. mandarina derived from the Refseq annotation is likely to be even worse than the Silkbase versions, considering the low quality of the genome assembly. Thus, only a very small fraction of 70-80 potential orthologous pairs between the two strains has been legitimately compared based on correct gene models of both strains. The “highly variable intron-exon structures among these genes” (Line 263) most likely reflect the inaccurate gene structure prediction of either or both CYPomes.
3. Another major message of this manuscript is that CYP306A is a candidate gene responsible for the stronger insecticide resistance of the wild strain compared with the domestic one (Line 30 and others). However, I cannot follow the logic described in the paragraph Lines 340-374. Even though CYP306A is differentially expressed between substructures of silk gland, how that phenomenon is relevant to insecticide resistance? Obviously, silk gland is not the major organ involved in insecticide metabolism.
4. In Table 1, the columns of Family and Subfamily are dispensable, as the gene name contains that information. Instead, this table should contain other important information, such as CDS/aa lengths, exon numbers, sequence identities between orthologous B. mandarina and Bombyx mori proteins, etc.
5. Figure 3, Table S6, and S7 (Go and pathway analyses) are not much informative, as they represent the features common among all insects (or even arthropods).
6. The gene structures shown in Fig. S2 are hard to interpret. Exon-intron organization of genes would be better demonstrated in a similar manor as that shown in Fig 1 of Ai et al (2011).

Additional comments

Bioinformatics approaches are valid only when the primary data are of sufficiently high quality. The present stage of the available experimental data does not reach such a standard. Better assembly of the genomic sequence of B. mandarina and more comprehensible expression analyses for various organs, developmental stages, and environmental conditions would be necessary to find any biologically significant differences between wild-type and domestic strains.

Reviewer 2 ·

Basic reporting

In this manuscript, the authors provided P450 monooxygenase genes in wild silkworm, Bombyx mandarina using new reference-guided genome data and RNAseq data from silk grands. They identified 68 P450 genes and determined location of each P450 gene in chromosome. Through KEGG enrichment and transcriptome analysis, they found candidate P450 genes involved in ecdysteroidogenesis and insecticide metabolism.
As the author described, P450 monooxygenase genes have multiple and important roles in biological process such as juvenile hormone biosynthesis, ecdysteroidogenesis and insecticide metabolism. The findings and topics of the manuscript are important for comparative biology and genome biology. So I think the manuscript worth to publishing for “Peer J”. However, there are many points which should be revised before the publishing.
All figure captions is too short to understand the figures. Describe more detail.
Fig. S1 is “Phylogenetic tree of cytochrome P450 genes in the wild and domestic silkworms.”. Although it is important figure, the figure is not clear. I cannot recognize ID of each gene in the figure file. The figure should be replaced to the clear one.

Experimental design

The authors performed several data analysis. The authors utilized many tools of data analysis in this study. However, each tool is not referred property. Tools used in the study should be referred with version, paper, URL of the tools and parameter when you run the tools. So Lasts (line 97), Hisat2 (line 118), Stringtile (line 120), Transdedecoder(line 123), KEGG (line 130), KoBAS (line 131), InterProscan (line 132), MCScanX (line 139), Circos (line 141),GeneWise (line 159) and iTOL (line 165).

Validity of the findings

Authors found candidate P450 genes involved in ecdysteroidogenesis and insecticide metabolism. There are two point which should be addressed. One is to find P450 genes involved in JH synthesis. The authors described “They fulfill many important tasks, from the synthesis and degradation of ecdysteroids and juvenile hormones to insecticide metabolism (Feyereisen 1999).“ (line 316-317). However, they found candidate gene of only ecdysteroidogenesis. Authors must add candidate P450 gene of JH synthesis as they found candidate P450 gene of ecdysteroidogenesis.
Another one point is that the authors concluded that they found candidate P450 gene involved in insecticide metabolism. I think this conclusion did not appropriate. In general, insecticide metabolism could be occurred in other tissues (e.g. fat body). If the authors want to show candidate P450 gene involved in insecticide metabolism, RNAseq data of fat body or other tissues playing insecticide metabolism must be added.

Additional comments

Minor comment
1. Correct “juvenile hormones (JHs)” (line 317)
2. Why “CYP306A1” in bold? (line 332)
3. In figure 4, why one P450 show “CYP306A1”. It makes confused and should be correct to gene ID.

·

Basic reporting

The authors describe the difference in the numbers of P450 genes between the domesticated silkworm (Bombyx mori) and the ancestor wild silkworm (B. mandarina) in the manuscript. It is very interesting that the numbers in B. mandarina is quite different from B. mori, if it is correct, because P450 genes have highly diversified functions and are biologically very important. However, I think that the data are unclear and the discussions are insufficient. The manuscript needs major revision for publishing in this journal. The data, figures and tables should be reconsidered.

Experimental design

The degree of the improvement of the quality of the genome assembly in B. mandarina is essential in this paper. But it is difficult to understand how much the quality was improved. It would be better to summarize the differences between the new B. mandarina genome and the old one in another table (lines 191-207). Chromosomes 4, 6 and 21 on B. mandarina are half in size in comparison with B. mori, as shown in Table S2. Is it correct?

In Materials & Methods, Lastz, HiSAT2, StringTie, TransgDecorder, KEGG, KOBAS, Circos, GeneWise, HTSeq and so on need citations.

In my understanding, insecticides are metabolized mainly in midgut and fat body. RNA-seq data of midgut in B. mandarina seems available in the SilkBase. Checking the P450 genes expression in the midgut should give an important information to this paper (lines 312-).

Validity of the findings

The numbers of 68 P450 genes in B. mandarina is critical, but I am afraid that there is a possibility of missing some P450 genes, because the authors used the fragmental assembly of B. mandarina genome, few RNA-seq data, and B. mori genome as the reference. The authors should describe the manuscript with considering the possibilities of missing P450 genes. In conclusion at line 353, “B. mandarina may have lost many cytochrome P450 genes” sounds overstatement.

The authors should focus on the difference of P450 genes between B. mori and B. mandarina. P450 genes only found in B. mori are listed in Table 1 (CYP365A1, CYP6AB8, 2 CYP6AE5, CYP6AW1, CYP340A1, CYP340A5P, CYP340A6, CYP340E1, CYP341A6, CYP367A1, CYP4AX1, CYP4AX2, CYP4S6, CYP4G23, CYP4M9, CYP3un1, and CYP49A2). CYP6AE7 and CYP6AE3P are duplicated (?) in B. mandarina. However, not all of them are well described. The authors should discuss the possible function of the species-specific P450 genes. Which genes are possible “certain key genes” that are critical for insecticide resistance in B. mandarina (lines 24-25 and 247-250)?

My understanding is that molting and juvenile hormones are synthesized in prothoracic gland or corpus allatum. P450s produced in the silk gland do affect the growth and differentiation of the silk glands (lines 319-347)? CYP306A1 is Phantom, which is an important gene involved in insect hormone biosynthesis as shown in Fig. 3. Does CYP306A1 have some function in the silk gland? Can CYP306A detoxify Phoxim (at lines 340-347)? If so, it is very interesting.

Additional comments

Fig. S1 looks important. I think that this figure can be shown in the text, instead of Fig. 4. When summed up, there seem to be 85 B. mori genes in the figure. Is “KWMTBOMO11023-11024” is in Mito. Clan? or CYP3 Clan?

Figure 4 is the expression analysis of “different tissues in the silk glands”, but it is difficult to understand and not so meaningful. I recommend to improve the figure or I think the figure can be supplemental.

In Table 1, “ ‘*’ and ‘$’ have two counterparts in B. mandarina ” needs more explanation. Are the genes duplicated in B. mandarina?

In Table S1,
- NCBI RefSeq Accession: GCF_003987935.1 is of B. mandarina, not B. mori.
- http://silkbase.ab.a.u-tokyo.ac.jp/cgi-bin/download.cgi is of B. mori, not B. mandarina.

The title would be “Computational functional analysis of --” in line 291.

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