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Dear Drs. Foresman and Tartar:
Thanks for revising your manuscript based on the concerns that were raised. I now believe that your manuscript is suitable for publication. Congratulations! I look forward to seeing this work in print, and I anticipate it being an important resource for groups studying entomopathogenic fungi biology and systematics, as well as fungal-derived insecticides for deployment against mosquitoes. Thanks again for choosing PeerJ to publish such important work.
Best,
-joe
[# PeerJ Staff Note - this decision was reviewed and approved by Konstantinos Kormas, a PeerJ Section Editor covering this Section #]
Dear Drs. Foresman and Tartar:
Thanks for revising your manuscript. The reviewers are very satisfied with your revision (as am I). Great! However, there are a few minor edits to make. Please address these ASAP so we may move towards acceptance of your work.
Note that reviewer 2 kindly provided a marked-up version of your manuscript.
Best,
-joe
No comment
No comment
No comment
All of my previous comments have been addressed to my satisfaction
Very good.
-The article is well written and has only minor grammatical errors (please see attached edited tracked changes document for recommended changes.
-Background and literature cited provide sufficient context.
-The hypotheses and questions to investigated are clearly articulated in introduction and addressed in results.
-The methods section has been substantially updated in response to reviewers 1 and 2 comments regarding media used for culturing and methods for RNA extractions, PCR etc. to provide better documentation and sufficient details for replication
-The findings are potentially impactful. While further functional assays are needed, identification of a potential highly virulent toxin that can be used for biological control of mosquitos is an important finding.
-The addition of Fig. 4 and phylogenetic analysis of the actin gene provide some support for grouping Culicinomyces in the Ophiocordycipitaceae and are more convincing than previous manuscript
-Table 1 is simply giving the identity of CAZyme enzymes found in the Culicinomyces transcriptome not really comparing GH families found in Culicinomyces against other entomopathogens. If there are other studies of transcriptomes of entomopathogens that penetrate through the cuticle that these results could be compared with, it would be more meaningful. I didn't find the blast search results against Hirsutella particularly useful. I would recommend revising this table if possible.
-Caveats have been added to the discussion to address the limitations of a transcriptome study and with respect to calling the hirsutellin homolog a ribotoxin prior to functional testing.
The authors have successfully addressed most of the points raised by reviewers with addition of Figure 4 and adding caveats and limitations of the study to the discussion. For minor revisions:
1) Address grammatical errors
2) Revise Table 1 to include GH genes found in one or more other entomopathogens, ideally one known to penetrate through the cuticle (perhaps Pandora formicae?). While mentioned in the text, it might be more impactful to have it in the table.
Dear Drs. Foresman and Tartar:
Thanks for submitting your manuscript to PeerJ. I have now received two independent reviews of your work, and as you will see, the reviewers raised some concerns about the research. Despite this, these reviewers are optimistic about your work and the potential impact it will have on research studying entomopathogenic fungi biology and systematics, as well as fungal-derived insecticides for deployment against mosquitoes. Thus, I encourage you to revise your manuscript, accordingly, taking into account all of the concerns raised by both reviewers.
Per reviewer 1, please provide a rationale for using transcriptomics over comparative genomics to identify toxins. There also seems to be some concern about the approach for generating the transcripts. Please address these concerns. Both reviewers have concerns that some relevant genes may not have been collected by the transcription/growth conditions.
Please provide all of the relevant information in your methods to ensure that your work is repeatable.
Avoid overstating the utility of the ribotoxin. Clearly, there are more experiments that need to be done in order to make the strong claims.
Per reviewer 2, please consider providing a table with comparisons of numbers and types of relevant CAzymes (and/or specific GH families discussed in the text) with other entomopathogenic fungi that are known to infect through the cuticle with secreted enzymes (e.g. Metarhizium and others).
The process of lateral gene transfer is as equally parsimonious as shared ancestry between species with similar toxin profiles. You do not seem to have enough analyses that can argue for or against either phenomenon for the observation of similar genes in two species. Thus, more in silico experiments (comparative genomics and/or phylogeny estimations) would very much help your arguments and make the paper more robust.
There are many comments by both reviewers that ask for more information on specific issues; please address these.
I look forward to seeing your revision, and thanks again for submitting your work to PeerJ.
Good luck with your revision,
-joe
[# PeerJ Staff Note: Please ensure that all review and editorial comments are addressed in a response letter and any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate. #]
• The language used in the manuscript is of appropriate quality and is easy to comprehend.
• The introduction section is appropriately rigorous and gives a good level of background on the field. A minor quibble is that the authors state there is a “limited toolbox of vector management strategies”, which may have been true a decade ago before the development of novel mosquito control techniques which are becoming more widely used.
• The article structure is appropriate for Peer Journal
• The figures are of good quality.
• Raw data were not provided with the manuscript I received but raw sequences are available and accessible through the sequence read archive.
• The study is novel and appropriate for Peer Journal
• The research question is meaningful in that it attempts to identify a novel mosquitocidal active ingredient, that could feasibly be used to generate novel mosquito control strategies. The authors make a clear statement describing how their proposed investigation of Culicinomyces clavisporus transcriptomics will address this knowledge gap in an attempt to identify the active ingredient. However, what they do not address is why they chose the approach they did over other approaches (i.e., transcriptomics over genomics).
• Rigor: I have some concerns about the rigor of the investigation:
o Only one culturing condition has been used to generate the material used for the RNA-Seq assay. For a genomics-based assay this would not be a concern, but with transcriptomics it is possible that some genes were not transcribed under the culturing conditions utilized, and that these could have been toxins involved in mosquitocidal activity. No rationale for the specific culturing conditions (media choice, temperature and light conditions, agitation speed) utilized in the study has been provided in the methods section.
o No information on the quantity of mycelial tissue or age of the culture used to generate the RNA sample was provided.
o There is no information on replication (biological or technological) for the RNA-Seq assay.
o Additional details on the process for annotating gene function are required.
• At the level of detail provided in the methods section, it would not be possible for anyone to replicate the study. Key details are lacking throughout. i.e., which reagents were used to conduct PCR? My suggestion to the authors is to revise and expand this section so that all relevant details are included.
• Identifying a mosquitocidal active ingredient from a fungus with an oral delivery route is something unique and potentially valuable to the mosquito control research field. The discovery could also be leveraged to generate novel mosquito control tools and strategies which could supplement and improve current strategies, subject to future evaluation of the toxin and its effects on mosquitoes, non-target species, and the environment. The authors make note of the former in the text, but they could also remark on the caveat.
• One thing missing from the manuscript is a description of the annotated transcriptome. Ideally this should come in the form of a table describing transcript numbers in different functional classes. It is important for your readers to be able to parse these data for themselves, so that they can see whether there are other candidates for the mosquitocidal factor in addition to the Hirsutellin-like gene. The presence of these data will also help to rationalize the choice of the Hirsutellin-like gene as the subject of additional characterization over other potential candidates.
• The authors have perhaps overinterpreted their data. Yes, the Hirsutellin-like gene has a high degree of homology to other fungal toxins, but that does not definitively imply that it is a mosquitocidal factor, or that it is the only mosquitocidal factor produced by Culicinomyces clavisporus. To do that would require both of the following:
o Produce a Hirsutellin-like gene knock-out Culicinomyces clavisporus and demonstrate that it has no mosquitocidal activity, or perform a comparative assay of closely related fungi with and without that gene to demonstrate that only the fungi with the gene are mosquitocidal
o Synthesize or purchase the gene product and demonstrate that it is a mosquitocidal factor.
While completing those assays is perhaps beyond the scope of this article, it is important for the authors to avoid overinterpretation of their findings and make sure that the text clearly specifies that it is still not clear whether this gene is the cause of mosquitocidal activity.
-The article structure and writing are clear and concise.
-The literature references are relevant and adequately cover questions being asked in the manuscript and the introduction was well written and did a good job of framing the key questions about this fungus (1) whether oral ingestion is a mechanism of infection, 2) what may contribute to the high and rapid virulence observed against mosquitos
-There are some minor grammatical errors that should be corrected, but overall English language is satisfactory
-Figues are easy to interpret
line 111 - "grounded" --> ground
line 291 - "established" --> establish
1. One main concern about the study was that as I understand, they only sequenced transcriptome from a standard culture media (Yeast Extract Glucose Media). The transcriptome is a valuable contribution, but one should be careful about interpreting the presence of genes such as chitinases or proteases being absent, when conditions required for eliciting them (such as growth on insect cuticle) were not assayed. Some sort of caveat should be added to the discussion to address this.
Other methods seem robust.
-It was nice to see PacBio SMRT sequencing as it appears they recovered mostly full-length reads
-The RACE characterization of the ribotoxin and analysis of its key motifs is solid and a strength of the paper
1. As mentioned in general comments above, it would have been nice to see a table with comparisons of numbers and types of relevant CAzymes (and/or specific GH familites discussed in the text) with other entomopathogenic fungi that are known to infect through the cuticle with secreted enzymes (e.g. Metarhizium and others). I also think you may need to be careful or add a caveat that these genes could have been present in the genome, but were not expressed under the particular conditions you assayed.
2. Lines 286 -290 and 297-299 "the "Culiconimyces/Hirsutella clade"
Phylogenetic placement of Culicinomyces remain uncertain (incertae sedis), so I think it is a little of a stretch to claim that it is closely related to Hirsutella without showing data to support that claim and no discussion of which taxa were included in this preliminary phylogenetic tree. It will also likely require deeper taxon sampling to determine its actual placement. The similarity of the toxins from Hirsutella and Culicinomyces alone is not evidence of a close phylogenetic relationship as horizontal transfer could be at play. The paper could stand alone as characterization of the ribotoxin without making claims/speculations about phylogenetic placement. I think either provide data/evidence in the manuscript to support this phylogenetic claim or just leave it out. I don't think it is necessary to cliam and one could compare the mode of infection and the phylogenetic relationship between the toxins themselves without making phylogenetic claims about the fungi producing these toxins.
Summary or general comments:
This research sequenced the transcriptome of the entomopathogenic Culicinomyces clavisporus, a pathogen of mosquitos that may attack its host via oral ingestion rather than the typical route of entomopathogenic fungi of penetrating the insect cuticle. The researchers found that Culcinomyces lacked the larger repetroire of proteases and carbohydrate active enzymes (especially chitinases) typical of other entomopathogenic fungi and uncovered a homolouge of the ribotoxin. They confirmed the full-length transcript of this ribotoxin using RACE and found that it was most closely related to the Hirsutellin toxin from Hirsutellin rhossilensis. Overall, the manuscript uncovered an interesting toxin that could have a role in the highly virulenct phenotype of this isolate against mostquitos, shows that these toxins more widespread among entomopathogenic fungi than previously realized, and may offer new approaces for biological control. I would have like to have seen a little more robust and quantitative comparison of enzyme families involved in breaking down insect cutitle between this fungus and other sequenced entomopathogens (a table or supplemental table at least) and the authors should be careful to infer that the fungus doesn't have these genes based on transcriptome from a single culture media that may not replicate conditions of infection of an insect. They should also perhaps be careful about making strong conclusions about the evolutionary relationships between the two fungi Culicinomyces and Hirsutella. While the toxins who the closest evolutionary relationship, processes such as horizontal gene transfer, not evolutionary closeness between the fungi could contribute to that and they don't present data/evidence to support phylogenetic relationships between the fungi. I think the phylogenetic claims about the fungi could be removed from the paper without sacrificing its impact.
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