The Effect of Glucosinolates on the Growth and Development of Helicoverpa armigera Larvae and the Expression of Midgut Sulfatase Genes
Round 1
Reviewer 1 Report
The emergence of pests adapted to defensive secondary metabolites secreted by various pants seems to be a hot topic in insect biology due to agricultural implications. The more data is available, the better we can understand how to overcome these anti-defensive mechanisms. That is why, over the last five years we see a burst of genomic, proteomic, and physiological studies devoted to the dynamics of these anti-defensive systems in insects in response to plant-derived chemical compounds. In this context, the current study is of interest. Multiple genes encoding sulfatases in Helicoverpa armigera imply differential response in terms of gene expression as well as activity of different isoforms. Thus, the article adds new information to the existing body of evidence pertaining to sulfatases in particular and the insects’ transcriptome in general.
Nonetheless, despite a valid experimental framework, the current article could not be recommended for publications in its current form for it should be refined. My main concern refers to the bioinformatic part of the article. What was the point of it? Prediction of isoelectric points, hydrophobicity of proteins, and so on does not say anything itself but is really useful in the proper context. The same refers to phylogeny. It is not in the context, thus, it does not provide any biologically relevant insights itself. What authors discuss when presenting results actually looks like a tautological expression (“The HaSulf of H. armigera has homology with other insects, but there are some differences between different HaSulfs”, lines 210-211). That is why these results are not included in the discussion. If authors really want to gain insights from bioinformatic analysis, they can, for instance, use molecular docking to predict the patterns of protein binding to the substrate. Apart from it, if authors propose a cooperative expression of different genes (line 310), they can predict transcription factors binding sites in upstream promoter regions. There is not a necessary analysis that should be performed, however, my point was that any analysis should be interconnected, but in the current form, this part looks extraneous and irrelevant.
The second major point refers to the Discussion section. It looks more like a part of the Results section, containing one reference and no comparison with other studies. Of course, the topic is poorly investigated, but a simple quick search in PubMed can provide novel articles devoted to sulfatases activity in other insects, and discussing differences in observed patterns of expression could be of interest. By the way, when discussing these differences, authors can compare sequences of sulfatases and perform phylogeny reconstruction, thus, it will have some reliable context for the analysis. The reviewer is not a specialist in this theme, thus the list of references is surely incomplete, however, these are some articles that could be added to the Discussion section:
Beran F, Sporer T, Paetz C, et al (2018) One Pathway Is Not Enough: The Cabbage Stem Flea Beetle Psylliodes chrysocephala Uses Multiple Strategies to Overcome the Glucosinolate-Myrosinase Defense in Its Host Plants. Front Plant Sci 9:1754. https://doi.org/10.3389/fpls.2018.01754
Chen W, Dong Y, Saqib HSA, et al (2020) Functions of duplicated glucosinolate sulfatases in the development and host adaptation of Plutella xylostella. Insect Biochem Mol Biol 119:103316. https://doi.org/https://doi.org/10.1016/j.ibmb.2020.103316
Ma X-L, He W-Y, Wang P, You M-S (2019) Cell lines from diamondback moth exhibiting differential susceptibility to baculovirus infection and expressing midgut genes. Insect Sci 26:251–262. https://doi.org/10.1111/1744-7917.12533
Manivannan A, Israni B, Luck K, et al (2021) Identification of a Sulfatase that Detoxifies Glucosinolates in the Phloem-Feeding Insect Bemisia tabaci and Prefers Indolic Glucosinolates. Front Plant Sci 12:671286. https://doi.org/10.3389/fpls.2021.671286
Ren J, Peng Z-K, Yang Z-Z, et al (2021) Genome-wide identification and analysis of sulfatase and sulfatase modifying factor genes in Bemisia tabaci (Hemiptera: Aleyrodidae). Insect Sci. https://doi.org/10.1111/1744-7917.12898
Sun R, Jiang X, Reichelt M, et al (2019) Tritrophic metabolism of plant chemical defenses and its effects on herbivore and predator performance. Elife 8:e51029. https://doi.org/10.7554/eLife.51029
There are also some minor points but it would be more convenient to discuss them in detail after the revision. Nevertheless, there are of them:
- Something is wrong with fonts throughout the text, e.g., the font in tables and the Reference list differ from the main text;
- The reference list is poorly formatted. Please italicise al, species names, delete links, provide DOI, and unify the style (e.g., in 33, volume and pages are missed, in 28 the colon is missed, and so on);
- Please check the text for spelling and grammatic errors, as well as improper formatting, such as missed spaces (line 14), missed hyphens (sulfur containing -> sulfur-containing, line 51), and missed articles (same age -> the same age, line 102), misprints (sulftases -> sulfatases, line 107), and so on. These are only certain examples but there is a lot more of these issues;
- What was the point of dividing concentration impact into two figures (7 and 8)? As for me, they describe the same type of data;
- Please do not use the expression “protein expression”, this is an improper jargonistic, for expression refers to genes. You can use the term “protein production” instead;
- The font type in figures is not unified it is especially noticeable in the first figure;
- Authors should explain in the Method section what was the point of the usage of gossypol and methyl jasmonate in comparison with glucozinolates;
- Subsection 3.4 in the Results section contains way toomuch monotonous passages andconpletely tha same sentence structures. Please, consider refining the style in this section to improve readability.
Author Response
Point-by-point response to reviewers’ comments
Reviewer: 1
The emergence of pests adapted to defensive secondary metabolites secreted by various pants seems to be a hot topic in insect biology due to agricultural implications. The more data is available, the better we can understand how to overcome these anti-defensive mechanisms. That is why, over the last five years we see a burst of genomic, proteomic, and physiological studies devoted to the dynamics of these anti-defensive systems in insects in response to plant-derived chemical compounds. In this context, the current study is of interest. Multiple genes encoding sulfatases in Helicoverpa armigera imply differential response in terms of gene expression as well as activity of different isoforms. Thus, the article adds new information to the existing body of evidence pertaining to sulfatases in particular and the insects’ transcriptome in general.
Nonetheless, despite a valid experimental framework, the current article could not be recommended for publications in its current form for it should be refined. My main concern refers to the bioinformatic part of the article. What was the point of it? Prediction of isoelectric points, hydrophobicity of proteins, and so on does not say anything itself but is really useful in the proper context. The same refers to phylogeny. It is not in the context, thus, it does not provide any biologically relevant insights itself. What authors discuss when presenting results actually looks like a tautological expression (“The HaSulf of H. armigera has homology with other insects, but there are some differences between different HaSulfs”, lines 210-211). That is why these results are not included in the discussion. If authors really want to gain insights from bioinformatic analysis, they can, for instance, use molecular docking to predict the patterns of protein binding to the substrate. Apart from it, if authors propose a cooperative expression of different genes (line 310), they can predict transcription factors binding sites in upstream promoter regions. There is not a necessary analysis that should be performed, however, my point was that any analysis should be interconnected, but in the current form, this part looks extraneous and irrelevant.
The second major point refers to the Discussion section. It looks more like a part of the Results section, containing one reference and no comparison with other studies. Of course, the topic is poorly investigated, but a simple quick search in PubMed can provide novel articles devoted to sulfatases activity in other insects, and discussing differences in observed patterns of expression could be of interest. By the way, when discussing these differences, authors can compare sequences of sulfatases and perform phylogeny reconstruction, thus, it will have some reliable context for the analysis. The reviewer is not a specialist in this theme, thus the list of references is surely incomplete, however, these are some articles that could be added to the Discussion section:
Ma X L, He W Y, Wang P, You M-S. Cell lines from diamondback moth exhibiting differential susceptibility to baculovirus infection and expressing midgut genes. Insect Sci .2019,26(2):251–262. https://doi.org/10.1111/1744-7917.12533.
Chen W, Dong Y, Saqib H S A, et al. Functions of duplicated glucosinolate sulfatases in the development and host adaptation of Plutella xylostella[J/OL]. Insect Biochem Molecul Biol, 2020, 119: 103316. DOI: 10.1016/j.ibmb.2020.103316. Epub 2020 Jan 14.
Beran F, Sporer T, Paetz C, et al. One Pathway Is Not Enough: The Cabbage Stem Flea Beetle Psylliodes chrysocephala Uses Multiple Strategies to Overcome the Glucosinolate-Myrosinase Defense in Its Host Plants. Front Plant Sci.2018,9:1754. https://doi.org/10.3389/fpls.2018.01754.
Ren J, Peng Z-K, Yang Z-Z, et al. Genome-wide identification and analysis of sulfatase and sulfatase modifying factor genes in Bemisia tabaci (Hemiptera: Aleyrodidae). Insect Sci. 2021,https://doi.org/10.1111/1744.
Manivannan A, Israni B, Luck K, et al. Identification of a Sulfatase that Detoxifies Glucosinolates in the Phloem-Feeding Insect Bemisia tabaci and Prefers Indolic Glucosinolates. Front Plant Sci.2021, 12:671286. https://doi.org/10.3389/fpls.2021.671286.
Answer: Yes, we agree and thank the reviewers for their comments and suggestions. We have made corresponding changes in the article and summarized some thoughts here.
（1）Sulfatase is a hydrolase that can decompose sulfate from a variety of substrates, including glycosaminoglycans, sulfolipids, and steroid sulfates [1]. The function of insect sulfatase is related to the gradual adaptation of the host plant's defense system in the co-evolutionary arms race of cruciferous plants. The specialist pest Plutella xylostella can use its own glucosinolate sulfatase (GSSs) to competitively combine with the glucosinolates of the host plant to quickly desulfurize glucosinolates, so that the host plant myrosinase cannot recognize and hydrolyze the substrate glucosinolates, and cannot produce toxic secondary metabolic defense substances [1, 2]. Generalist pests also contain sulfatases. However, the function of sulfatase genes and the molecular mechanism of how to detoxify host plant glucosinolates in generalist pests are still unclear.
（2）In our previous study, we identified eight Hasulf genes and 1 modifier gene HaSumf1. Different HaSulf sequences show substantial similarity at the C/SXPXRXTG site (Figure 5). Each amino acid column has a distinct height, showing variances in the amount of amino acid information and conservation of each HaSulf. The HaSulf has a certain degree of homology with other insect HaSulf enzymes. The HaSUMF1 is similar to the SUMF2 of Tetranychus urticae and Drosophila melanogaster through evolutionary trees (Figure 6). Differences in amino acids and conserved motifs may affect the biological functions of sulfatase and the binding and hydrolytic activity of substrates to a certain extent [2]. From the similarity of the sulfatase genes between H. armigera and other insects, we speculate that H. armigera sulfatase also has the function of detoxifying host plant glucosinolates. But further, the sequence expansion and differences in the members of the H. armigera sulfatase family may be related to their feeding habits. The sulfatase family members have a certain degree of synergy and new functionalization to adapt to the secondary metabolites of different host plants [3].
（3）By predicting the transcription binding site of the promoter region, it is interesting to find that HaSumf1 has high similarity with the transcription factors of HaSulf2, HaSulf4 and HaSulf5a in the sulfatase gene family (Table 4)，and we also found HaSulf2 and HaSulf4 are co-located on the SulfC evolutionary branch [4]. This phenomenon provides an important theoretical basis for subsequent studies on the regulation mechanism of sulfatase genes and their modifier genes.
（5）We have rewritten the discussion section based on the reviewers’ comments and introduced the references mentioned by the reviewers and other useful references to make the discussion clearer.
Other comments:
There are also some minor points but it would be more convenient to discuss them in detail after the revision. Nevertheless, there are of them:
1) Something is wrong with fonts throughout the text, e.g., the font in tables and the Reference list differ from the main text;
Answer: We have made changes in the article.
2) The reference list is poorly formatted. Please italicise al, species names, delete links, provide DOI, and unify the style (e.g., in 33, volume and pages are missed, in 28 the colon is missed, and so on);
Answer: We have made changes in the article.
3) Please check the text for spelling and grammatic errors, as well as improper formatting, such as missed spaces (line 14), missed hyphens (sulfur containing -> sulfur-containing, line 51), and missed articles (same age -> the same age, line 102), misprints (sulftases -> sulfatases, line 107), and so on. These are only certain examples but there is a lot more of these issues;
Answer: We have made changes in the article.
4) What was the point of dividing concentration impact into two figures (7 and 8)? As for me, they describe the same type of data;
Answer: Yes, it is the same type of data from the perspective of gene expression. Our perspective is that the biological function of H. armigera sulfatase requires HaSumf1 to activate, and HaSumf1 also has different responses to different concentrations of glucosinolates, which may also be one of the reasons for the different expression of sulfatase. The cooperative expression of sulfatase genes seems to be consistent with the results in the evolutionary tree (Figure 4). The sulfatase gene family is mainly composed of four branches, namely SulfA, B, C and D [4]. In the evolutionary tree, HaSulf1, HaSulf2 and HaSulf4 are on the C branch, so here we separate the expression of these four genes from the others.
5) Please do not use the expression “protein expression”, this is an improper jargonistic, for expression refers to genes. You can use the term “protein production” instead;
Answer: We have made changes in the article.
6) The font type in figures is not unified it is especially noticeable in the first figure;
Answer: We have made changes in the article.
7) Authors should explain in the Method section what was the point of the usage of gossypol and methyl jasmonate in comparison with glucozinolates;
Answer: We have made changes in the article.
8) Subsection 3.4 in the Results section contains way toomuch monotonous passages andconpletely tha same sentence structures. Please, consider refining the style in this section to improve readability.
Answer: We have made changes in the article. 4 ppm of gossypol and methyl jasmonate were fed to H. armigera as controls for xenobiotics, and the production of HaSulf protein was measured.
References
[1] Parenti G, Meroni G, Ballabio A. The sulfatase gene family. Curr. Opin. Genet. Devol., 1997, 7(3):386-391.DOI 10.1016/S0959-437X(97)80153-0
[2] Chen W.; Dong Y.; Saqib H.S.A.; et al. Functions of duplicated glucosinolate sulfatases in the development and host adaptation of Plutella xylostella. Insect Biochem. Mol. Biol. 2020, 119:103316. DOI 10.1016/j.ibmb.2020.103316
[3] Ren J.; Peng Z.K, Yang Z.Z.; et al. Genome-wide identification and analysis of sulfatase and sulfatase modifying factor genes in Bemisia tabaci (Hemiptera: Aleyrodidae). Insect Sci. 2021, 28(6):1541-1552. DOI 10.1111/1744-7917.12898
[4] Heidel-Fischer Hanna M, Kirsch Roy, Reichelt Michael, et al. An Insect Counteradaptation against Host Plant Defenses Evolved through Concerted Neofunctionalization. Mol. Biol. Evol. 2019, 36(5): 930-941. DOI 10.1093/molbev/msz019
Author Response File: 
Author Response.docx
Reviewer 2 Report
In this research paper, authors discussed the effect of glucosinolates on the growth and development of Helicoverpa armigera larvae and the expression of midgut sulfatase genes. In this study, authors characterized HaSulfs and HaSumf1 genes. They have checked the mRNA expression profile and protein expression profiles. The have checked the development period of the H. armigera at different instars under different concentrations of glucosinolate. They used precise calculation method to compare the effects. Specific comments as follows
The following change should be made throughout the manuscript
Abstract
HaSulf family genes in the midgut of 5th instar larvae of H. armigera
>
HaSulf family genes in the midgut of fifth instar larvae of H. armigera
Introduction
worldwide generalist pest
>
Worldwide pest
Throughout manuscript:
This reviewer recommended to use below instar writing format
5th instar > fifth instar
Figure
If possible, can you please repeat the SDS PAGE and western blotting experiment and replace the figure 10 and 11. Protein bands are not so clear.
Comments for author File: 
Comments.pdf
Author Response
Reviewer: 2
In this research paper, authors discussed the effect of glucosinolates on the growth and development of Helicoverpa armigera larvae and the expression of midgut sulfatase genes. In this study, authors characterized HaSulfs and HaSumf1 genes. They have checked the mRNA expression profile and protein expression profiles. The have checked the development period of the H. armigera at different instars under different concentrations of glucosinolate. They used precise calculation method to compare the effects. Therefore, this reviewer would like to suggest that the manuscript is acceptable for publication in the journal after minor revisions. Specific comments as follows.
The following change should be made throughout the manuscript
Abstract
HaSulf family genes in the midgut of 5th instar larvae of H. armigera
HaSulf family genes in the midgut of fifth instar larvae of H. armigera
Answer: We have made changes in the article.
Introduction
worldwide generalist pest
Worldwide pest
Answer: We have made changes in the article.
Throughout manuscript:
This reviewer recommended to use below instar writing format
5th instar > fifth instar
Answer: We have made changes in the article.
Figure
If possible, can you please repeat the SDS PAGE and western blotting experiment and replace the figure 10 and 11. Protein bands are not so clear.
Answer: As the reviewer saw, before glucosinolates were fed to Helicoverpa armigera, our western blot experiment could not detect the band of interest. Then we did a lot of experiments to try to find the relationship between protein yield and glucosinolate concentration, but we found that protein bands do not become obvious with the increase of glucosinolate concentration. The bands in Figure 10 and 11 in the article, is the clearest band we have obtained so far. In the later research, if we can understand the regulatory mechanism between the expression of sulfatase and the host plant glucosinolate, I think we should be able to get a clearer band.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
The authors have sufficiently improved the presentation of their data in accordance with all my major comments. Therefore, the manuscript now could be recommended for publication after some minor refinements.
There are some minor points to address before accepting:
1) please paraphrase "protein expression" in line 314 as done earlier throughout the text;
2) The JASPAR 2022 was used for TF binding sites prediction but the corresponding paper was not cited, please add the citation;
3) Please either italicize gene names in Table 2 or replace the column name to 'Protein name';
4) Please add the version of genome assembly of Helicoverpa armigera that was used to get the location coordinates in Table 2;
5) Please ether remove hyperlinks from Table 2 or add them to the first two genes;
6) Please change the font of the parentheses in the Discussion (lines 289, 294, etc.) to make it uniform with the text below (as in lines 271-273 and others);
7) Parentheses in Figure 1 look too thick, please change the font;
8) Please add letters to Figures 7-11 as in Figure 3;
Author Response
The authors have sufficiently improved the presentation of their data in accordance with all my major comments. Therefore, the manuscript now could be recommended for publication after some minor refinements.
Answer: Thanks again for the valuable comments from the reviewers, we have made corrections one by one in the article. Languages are also corrected by native speakers.
There are some minor points to address before accepting:
1) please paraphrase "protein expression" in line 314 as done earlier throughout the text;
Answer: We have made changes in the article.
2) The JASPAR 2022 was used for TF binding sites prediction but the corresponding paper was not cited, please add the citation;
Answer: We have made changes in the article.
3) Please either italicize gene names in Table 2 or replace the column name to 'Protein name';
Answer: We have made changes in the article.
4) Please add the version of genome assembly of Helicoverpa armigera that was used to get the location coordinates in Table 2;
Answer: We have made changes in the article.
5) Please ether remove hyperlinks from Table 2 or add them to the first two genes;
Answer: We have made changes in the article.
6) Please change the font of the parentheses in the Discussion (lines 289, 294, etc.) to make it uniform with the text below (as in lines 271-273 and others);
Answer: We have made changes in the article.
7) Parentheses in Figure 1 look too thick, please change the font;
Answer: We have made changes in the article.
8) Please add letters to Figures 7-11 as in Figure 3;
Answer: We have made changes in the article.
