Ingestion of Species-Specific dsRNA Alters Gene Expression and Can Cause Mortality in the Forest Pest, Ips calligraphus
Round 1
Reviewer 1 Report
The manuscript written by Wallace and Rieske identified effective RNAi path-way genes in Ips calligraphus transcriptome, and demonstrated changes in gene expression following oral ingestion of exogenous dsRNAs, which may facilitate integrated control of this pest species in the future. While I think there are several aspects of this manuscript that can be improved. My general concerns and suggestions are listed as follow.
In Materials and Methods, the authors used BLASTx and BLASTp search to obtain target sequences, but the key parameters (e.g. E-value, which is important for getting appropriate result) were not clearly mentioned.
For transcriptomic sequencing, what is the size of your raw data and clean data? Please provide the information in the manuscript.
In Discussion, I found this part is not well-organized and a bit unconcentrated. For instance, in the second paragraph (line 274-288), the authors mentioned the genomes from forest pests are limited, and tried to emphasize the importance of Ips calligraphus transcriptome provided in present study. While, I think this is unnecessary. In addition, except for generating the data to obtain the targeted genes, no other result was yield based on this RNA-seq here.
A similar problem is existed in the fifth (line 315-322) paragraph of Discussion. In this part, I suggest the authors to analyze the results focusing on present study, other than providing general information which was already discussed by previous studies.
Line 257, “in an Ips spp.” --> “in an Ips species”.
Data Availability Statement, the accession numbers are missing.
References, there are several formatting inconsistencies. Please check and revise carefully. For example, the authors provided the literature titles at least in two formats, one has every word starts with a capital letter, and the other one only has the first word with a capital letter.
Author Response
In Materials and Methods, the authors used BLASTx and BLASTp search to obtain target sequences, but the key parameters (e.g. E-value, which is important for getting appropriate result) were not clearly mentioned.
- Done, e-value threshold was added to both the BLASTp search (line 191): “ORFs were queried using a BLASTp search with an e-value threshold of 1e-5…” and the tBLASTx/BLASTx searches (lines 203-206): “…using tBLASTx with the predicted adult I. calligraphus CDS as the subject (e-value<1e-5). Resulting matching sequences were used to perform a BLASTx to the NCBI non-redundant (nr) protein database to corroborate that the RNAi gene query was the best match (e-value<1e-5)…”
For transcriptomic sequencing, what is the size of your raw data and clean data? Please provide the information in the manuscript.
- Number of raw and processed reads were added, from lines 236-238: “NovaSeq6000 sequencing produced 430,208,758 raw reads, and after read correction and removal of low-quality reads, the 420,255,024 remaining high quality trimmed reads were used for the final assembly.”
In Discussion, I found this part is not well-organized and a bit unconcentrated. For instance, in the second paragraph (line 274-288), the authors mentioned the genomes from forest pests are limited, and tried to emphasize the importance of Ips calligraphus transcriptome provided in present study. While, I think this is unnecessary. In addition, except for generating the data to obtain the targeted genes, no other result was yield based on this RNA-seq here. A similar problem is existed in the fifth (line 315-322) paragraph of Discussion. In this part, I suggest the authors to analyze the results focusing on present study, other than providing general information which was already discussed by previous studies.
- The genomic information generated in this work will significantly contribute to our understanding of other scolytines, and potentially open avenues for molecularly based bark beetle management technologies. We made no change - editor?
Line 257, “in an Ips spp.” --> “in an Ips species”.
- Change made, on line 261 “…gene silencing in an Ips species…”
Data Availability Statement, the accession numbers are missing.
- Sequencing data have been submitted to NCBI, Bioproject number added, waiting on accession numbers for the GenBank sequences.
References, there are several formatting inconsistencies. Please check and revise carefully. For example, the authors provided the literature titles at least in two formats, one has every word starts with a capital letter, and the other one only has the first word with a capital letter.
- References have been edited for capitalization and consistency throughout.
Reviewer 2 Report
The manuscript by Wallace and Rieske reports on the efficacy of orally-delivered dsRNA in adults of Ips typographus. Three genes were targeted: IAP, shibire and HSP and the outcome of the treatments on gene expression of the targets and adult mortality were measured. mRNA levels were affected differently depending on the target, compared to controls: 1) no change (HSP), 2) decreased levels (IAP) or 3) increased levels (shibire). In line with its lack of effect on HSP mRNA levels, dsHSP dsRNA did not affect survival compared to controls. However, both dsIAP and dsShi dsRNAs trigger lower temporal survival compared to controls. In addition to these findings, a reference transcriptome for I. typographus has been generated.
From my perspective, even though significant, the effects of dsRNA for IAP and SHI on survival are rather muted. I'm wondering if the authors have considered testing the feeding of heat-killed E. coli containing the dsRNAs. For reasons that are unclear this has been done by the same lab for the emerald ash borer but not in this species, why? It could very well be that dsRNA needs to be shielded from the midgut environment (e.g. with a dead bacterial shell) before penetrating midgut cells (or other target tissues).
Beyond that single question, I find the manuscript an acceptable contribution. Minor detail: in figure 1C, the star indication significance should be above the "dsSHI" bar, not the "dsGFP".
Author Response
From my perspective, even though significant, the effects of dsRNA for IAP and SHI on survival are rather muted. I'm wondering if the authors have considered testing the feeding of heat-killed E. coli containing the dsRNAs. For reasons that are unclear this has been done by the same lab for the emerald ash borer but not in this species, why? It could very well be that dsRNA needs to be shielded from the midgut environment (e.g. with a dead bacterial shell) before penetrating midgut cells (or other target tissues).
- The authors agree that including a means for protecting the dsRNA, such as the use of microbes, could enhance RNAi efficiency and potentially increase mortality. We appreciate this suggestion and are taking the use of microbial delivery as a potential avenue of investigation into account for our ongoing work in this system.
Beyond that single question, I find the manuscript an acceptable contribution. Minor detail: in figure 1C, the star indication significance should be above the "dsSHI" bar, not the "dsGFP".
- The star has been moved, and an updated pdf of Figure 1 has been added to the zipped folder of figures.
Reviewer 3 Report
The article properly addresses the issue of new non-invasive methods of protection against forest pests based on molecular tools, including RNA interference. All over the world, many species of forest trees, especially conifers, suffer from bark beetle outbreaks, intensified by climatic disturbances (too long-lasting dry and wet seasons). The most economically valuable forests, very often cultivated as monocultures, are probably even more susceptible to many environmental factors, such as changing climatic conditions, pathogens, or insect infestations.
The presented research is part of the current trend in forest protection. In general, the introduction clearly states the problem, the material and methods, as well as the results are sufficiently described, and the discussion is fully justified in comparison with other research achievements. All discrepancies in the results were commented on, and a possible explanation or need for future research was given. The conclusions are correct, all references are complete and adequate. The authors have extensive experience with the presented methodology and have published another work with a similar approach tested on Dendroctonus frontalis-induced mortality in pine (Scientific Rep. 2019; 9: 5640), but this analysis is the first performed on Ips transcriptome in North America. The results of these analyses may be very useful in new protection strategies against all other bark beetles that destroy coniferous species.
I have only one comment given below:
Line 107 – instead of “Double-stranded green fluorescent protein” it is better to say “Double-stranded RNA coding for green fluorescent protein” because the protein cannot be “double-stranded”, or it needs an explanation
Author Response
Line 107 – instead of “Double-stranded green fluorescent protein” it is better to say “Double-stranded RNA coding for green fluorescent protein” because the protein cannot be “double-stranded”, or it needs an explanation
- Wording has been updated as suggested for clarity. Line 107 now reads “Double-stranded RNA coding for green fluorescent protein (dsGFP) was selected as a negative control…”
