A chromosome-level genome assembly of Cydia pomonella provides insights into chemical ecology and insecticide resistance

The codling moth Cydia pomonella, a major invasive pest of pome fruit, has spread around the globe in the last half century. We generated a chromosome-level scaffold assembly including the Z chromosome and a portion of the W chromosome. This assembly reveals the duplication of an olfactory receptor gene (OR3), which we demonstrate enhances the ability of C. pomonella to exploit kairomones and pheromones in locating both host plants and mates. Genome-wide association studies contrasting insecticide-resistant and susceptible strains identify hundreds of single nucleotide polymorphisms (SNPs) potentially associated with insecticide resistance, including three SNPs found in the promoter of CYP6B2. RNAi knockdown of CYP6B2 increases C. pomonella sensitivity to two insecticides, deltamethrin and azinphos methyl. The high-quality genome assembly of C. pomonella informs the genetic basis of its invasiveness, suggesting the codling moth has distinctive capabilities and adaptive potential that may explain its worldwide expansion.

In MS# NCOMMS-19-04302-T ("The genetic basis of globalization and destructiveness of codling moth, Cydia pomonella") by Li et al, the authors conduct in-depth sequencing of a globally important pest, the codling moth, Cydia pomonella. The manuscript largely focuses on the sequencing and associated methodologies of this important pest, although the authors leverage functional testing of three identified genes.
Although the methods are robust, the manuscript largely focuses on the genomic sequencing methods to such a degree that the manuscript reads like a methods paper. In parallel, although the authors selectively test some of the identified genes, only three genes are tested and these results are distinct relative to the rest of the manuscript. In other words, the sub-headings are not well-linked, thus making the manuscript read like three or four different studies, rather than a single important manuscript. This significantly lessens my enthusiasm for the manuscript. Beyond this comment, there are a number of major and minor comments that are detailed below.
1. Related to the comments above, detailed methods take up a large part of the "Results" section, such that Lines 132-206 describe little of the results. It's unclear in this section what novel results are presented. For instance, how do the results compare to other lepidopteran species in terms of chromosomal number or sex chromosomes? Although Table 1 shows these results, there is relatively little discussion about these differences. Similarly, 2. Sub-heading connection. The sequencing and olfactory receptor sub-headings could be better linked. The OR section is largely based on previous studies in this domain, and it's unclear what new information the new genomic data provide. What other ORs show strong selection? What others show strong expression levels? What other ORs could candidates for control interventions, and how does the new genomic data provide this insight? These questions are lacking in this section, making it distinct compared to the previous sections.
3. Genetic basis of insecticide resistance. Again, as this section is written, it appears that the authors largely leverage previously published results (51-53) in this section, and find similar results as previous studies(53,54). It's unclear what new functional insights are provided by the genomic studies.
4. There are a number of grammatical and formatting errors in the manuscript. These are detailed below: a. Line 72: Re-write for clarity/grammar. b. Lines 80-84: Re-write for clarity/grammar. c. Line 101: Replace "uncontrolled" for clarity. d. Lines 111-113: Change for grammar / clarity. e. Lines 147-168 should be shortened. f. Sub-heading "Synteny, karyotype and sex chromosomes": This is an interesting section, but given the functional analysis in the other major subsections, it raises the question about the functional aspects or testing of these results. g. Lines 226-229: Place into first subheading. h. Line 297: Change word "exploits" to "detects" or another descriptor. i. Lines 300-303: The previous studies really raises the question what new information this subsection brings. Moreover, it is poorly linked with the previous sections. j. Figure 3: Show data for all ORs, rather than Or3a and b. k. Lines 529-531: Rewrite for grammar and clarity. l. References: Please check references for proper formatting.

Reviewer#1's comments (Richard Newcomb)
Wan et al describe a significant body of work on one of the most significant pests of horticulture worldwide. They have sequenced, assembled and annotated the genome of Cydia pomonella to produce a high quality chromosomal level assembly. They then go onto to describe two pieces of functional genomics research, one on chemical ecology and a second on insecticide resistance. In both cases they use their genome assembly to discover new genes involved in these traits and provide evidence based on RNAi and other functional assays to demonstrate the role of a particular odorant receptor and p450 in chemical ecology and resistance to pesticides, respectively. This work will provide a high quality genome assembly of an important group of moths that includes many pests (Tortricidae) and has significantly advanced the fields of both chemical ecology and insecticide resistance in insects.
I have only some minor suggestions and a number of suggested improvements to the wording of the manuscripts. As such I strongly recommend this manuscript for publication in Nature Communications. Response: We greatly appreciate your time and expertise in reviewing our manuscript. We have carefully revised the manuscript according to your constructive comments, which significantly improve our manuscript. Thanks again for your positive enthusiasm for our work.
Minor points: Line 137: I presume the 42 females were all adults? Response: Yes, they were adults. We now indicate this in the revision.
You should use the genera names in full the first time you use them in figure legends in case the figure is used in another publication. Response: corrected.
Alternate title suggestion? I think this better reflects what you have actually achieved. "A chromosome level assembly of the genome of the codling moth (Cydia pomonella) provides insights into its chemical ecology and insecticide resistance mechanisms." Response: It is a great suggestion, and we change the title to "A chromosome-level genome assembly of the codling moth (Cydia pomonella) provides insights into its chemical ecology and insecticide resistance".
When you talk about globalization do you really mean invasiveness? Response: Yes, we mean invasiveness. This has been corrected.
Line 312: Would be good to know the amino acid and nucleotide identity between the OR3 paralogues up front. If the identity is high how did you mitigate algorithms such as bowtie getting the mapping incorrect (check the methods). Might be good to reinforce that the RT-PCR is gene-specific by adding gene-specific before "reverse transcriptase-PCR". In your resequenced moths did you check to make sure the OR3 duplication is fixed and not segregating with some moths having one copy and some two? Response: The OR3a and OR3b share 94% identity in nucleotide and 89% identity in amino acids. Thus, it is feasible to distinguish these two paralogues when using bowtie to map the reads to scaffolds. We revise the methods to reinforce that RT-PCR is gene-specific. We confirmed that in all re-sequenced moths, the OR3 duplication is fixed.
Line 327: I'm not quite sure what you mean by divergent regulatory potentials. Do you simply mean that because they are separate genes they can evolve distinct expression patterns across tissues and development. If so you could simply refer to their potential to evolve new roles (neofunctionalisation) simply by changing expression. Although I note their ligand specificity is not identical so it looks like they are also undergoing neofunctionalisation by changing their ligand specificity presuming due to coding region changes. Response: Changed as suggested.
Line 374: What is the evidence that OR3a and 3b are not expressed in sensilla triciodea as well as sensilla auricillica. Is this a possibility? Or is OR1 the codlemone receptor in sensilla tricoidea? Response: Our FISH experiments showed that OR3a and 3b are expressed in sensilla auricillica. In our experiments, we did not see any labelling in sensilla trichodea, although we cannot completely exclude this. This topic is interesting and deserves further studies, for instance co-labelling of OR1 with sensilla trichodea markers. Nevertheless, we have revised this section to better clarify the current evidence pertaining to sensilla-specific expression and implications thereof.
I also think you can make more of the fact that the detection of mates and sites to oviposit seem to be linked within a single receptor able to detect compounds involved in both in the case of OR3. Certainly, it looks as though pheromone receptors have evolved from receptors involved in detecting plant volatiles. Response: We have updated this section of the manuscript to reflect this suggestion.
When you talk about 5 prime and 3 prime this should be annotated 5' and 3', not 5' and 3'. Response: Corrected, thank you.
Where did the 10-4 significance level come from in the GWAs screen. This seems quite low to me give the limited power you have in this analysis. Response: We did a reference mining for GWAS cutoff and found that the cutoff 10-4 is commonly used. Here is the table for GWAS P-value. So, we keep the cutoff as 10 -4 in this work. There is no validation of the genome assembly expect for comparing of two methods. Is it possible for you to provide any further confidence in the assembly? For example, BAC sequencing or similar?
Response: It is difficult to find BAC sequencing service in China now, so we have used additional data from Nanopore and PacBio sequencing platforms to validate the genome assembly quality (please see responses to Editor comments for further details). We added the following data and analyses: 1) Sequencing the genomic DNA with Oxford Nanopore platform. In total, we obtained ~71 Gb Nanopore long reads with mean length greater than 23 Kb; the longest read is 223 Kb (Table 1). Aligning the reads to the reference genomes showed that 99.96% reads can be mapped to the genome assembly. There are 6,070 reads whose length is larger than or equal to 100 Kb, and these ultra-long reads can be uniquely mapped to the genome scaffolds with high consistency, suggesting the high reliability of the genome assembly.  We greatly appreciate the careful reading and supportive editing of our manuscript that is reflected in this thorough list of corrections.

Reviewer #2's comments (Remarks to the Author):
In MS# NCOMMS-19-04302-T ("The genetic basis of globalization and destructiveness of codling moth, Cydia pomonella") by Li et al, the authors conduct in-depth sequencing of a globally important pest, the codling moth, Cydia pomonella. The manuscript largely focuses on the sequencing and associated methodologies of this important pest, although the authors leverage functional testing of three identified genes.
Although the methods are robust, the manuscript largely focuses on the genomic sequencing methods to such a degree that the manuscript reads like a methods paper. Response: We greatly appreciate your time and expertise in reviewing our manuscript. Thanks for your keen eyes and constructive criticism. We concur that the original version of our manuscript contained an excess of methodological detail, and we have worked to substantially streamline this aspect of the manuscript, moving many such details to the methods or supplemental sections, and retaining only the most significant methodological details.
In parallel, although the authors selectively test some of the identified genes, only three genes are tested, and these results are distinct relative to the rest of the manuscript. In other words, the sub-headings are not well-linked, thus making the manuscript read like three or four different studies, rather than a single important manuscript. This significantly lessens my enthusiasm for the manuscript. Beyond this comment, there are a number of major and minor comments that are detailed below. Response: Thank you for pointing out that we had not fully succeeded in generating a manuscript that sufficiently integrates the various research efforts presented here into a coherent research narrative. This sentiment resonates with the editor's suggestion to elaborate on the links between the genomic and functional analysis. With the aim to improve this aspect of the manuscript, we have reworked the text to better integrate the different parts of this manuscript to make a more coherent and consistent final product. We decided that subheading titles we used were too broad and thus changed the sub-heading titles in revision. We have chosen to focus on three genes of particular interest to highlight significant characteristics of the codling moth evolutionary ecology.
In this work, we present a chromosome-level genome assembly for the codling moth, which served as an essential foundation for discovering novel genetic aspects of noteworthy eco-physiological features of codling moth. As we detail in our responses to the Editor's comments (see above), the gene duplication of OR3, which contributes to enhanced abilities for locating food and mates, could not be confidently identified without a high quality assembly. Additionally, through the GWAS analysis, we identified three SNPs in the promoter of CYP6B2 and further confirmed their role in conferring insecticide resistance. Our perspective is that the genome assembly was an essential component of these functional investigations concerning the OR3 duplication and insecticide resistance, and we hope our revisions to the manuscript better capture this integrative nature of our research efforts.
1. Related to the comments above, detailed methods take up a large part of the "Results" section, such that Lines 132-206 describe little of the results. It's unclear in this section what novel results are presented. For instance, how do the results compare to other lepidopteran species in terms of chromosomal number or sex chromosomes? Although Table 1 shows these results, there is relatively little discussion about these differences. Response: As mentioned above, we removed large parts of methods information in results.
We also now discuss chromosome number difference and sex chromosomes between lepidopteran insects. We developed the discussion about the comparison of Cydia pomonella genome with those of other Lepidoptera species (Line 157-161, Line 500-504). other Lepidoptera as well as between chromosomes in the genome. We see this as an important (and interesting) contribution towards fully contextualizing the data provided in the genome assembly. As such, we view these results as an end unto themselves, and not as a means towards subsequent functional analysis. Accordingly, we do not clearly comprehend the nature of "the question" concerning function or testing to which you allude, but do not explicitly articulate. We apologize we cannot better intuit what you are asking for here, and we hope that, in light of clarifying our motivation, this element of the manuscript can satisfactorily stand on its own. g. Lines 226-229: Place into first subheading. Response: Corrected h. Line 297: Change word "exploits" to "detects" or another descriptor. Response: Re-wrote these sentences. i. Lines 300-303: The previous studies really raises the question what new information this subsection brings. Moreover, it is poorly linked with the previous sections. Response: Only one copy of OR3 has been previously found in an antennae transcriptome. The new information this subsection brings is: with the assistance of a high-quality genome assembly, we found two copies of this gene. The duplication of this gene forms a tandem repeat on chromosome 17. We further confirmed that this duplication significantly improves the ability of codling moth to detect pear ester. We rewrite this section to clarify this. j. Figure 3: Show data for all ORs, rather than Or3a and b. Response: Provided and updated k. Lines 529-531: Rewrite for grammar and clarity. Response: Changed l. References: Please check references for proper formatting. Response: Corrected