The genome sequence of the Emperor moth, Saturnia pavonia (Linnaeus, 1758)

We present a genome assembly from an individual male Saturnia pavonia (the Emperor moth; Arthropoda; Insecta; Lepidoptera; Saturniidae). The genome sequence is 489.9 megabases in span. Most of the assembly is scaffolded into 30 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.29 kilobases in length. Gene annotation of this assembly on Ensembl identified 11,903 protein coding genes.


Background
The family Saturniidae, named after the ringed planet Saturn, includes over 2000 species of moth worldwide and includes emperor moths and giant silk moths.Two species of Saturniidae have been recorded in Britain: Saturnia pavonia (the Emperor moth or Small Emperor moth, a resident breeding species) and S. pyri (the Giant peacock moth, the few records likely to be escapees from captive breeding).As befits the family and genus name, S. pavonia has large eyespots in the centre of all four wings, each with a black centre surrounded by a buff yellow ring and arcs formed from blue or red scales.The four coloured eyespots give the moth a striking appearance, especially as the moth is one of the largest in Britain or Northern Europe with a wingspan of 60 mm in males or 80 mm in females.Ford (1967) speculated that a hypothesised role of the eye-spots in predator deterrence could be accentuated immediately after eclosion from the pupa, when the wings are still puckered.
The Emperor moth S. pavonia is found widely across Europe, from the north of Finland and Norway to the south of Spain and Italy.The range also extends further east in Eurasia to Russia, Kazakhstan and Mongolia (GBIF Secretariat, 2023).In Britain and Ireland, the moth has been recorded from the south coast of England to the north of Scotland, but is commonest on acid heathlands, moorlands and coastal sand dunes (NBN Atlas Partnership, 2023).
Adult males are day-flying and particularly active on bright warm days in April and May, when they may be seen flying in search of scent trails released by receptive females.Much has been written about the potency of the S. pavonia female sex pheromones and the distances over which males detect females, but many estimates are based conjecture rather than controlled experimentation.After mating, eggs are laid on the foodplant and larvae develop rapidly though summer.On heathlands, the preferred larval foodplant is heather, Calluna vulgaris, but the larvae will also eat leaves of hawthorn, apple, bramble and many other shrubs and trees (South, 1961).Late instar larvae are bright green with black hoops studded with yellow globular outgrowths (scoli); a genetic variant with pink-coloured scoli has also been reported (Laussmann et al., 2012).When physically stimulated, hollow spines on the scoli secrete a blend of proteins and aromatic compounds thought to deter pathogens or predators; benzonitrile is the dominant small molecule but the secreted proteins have not been identified (Deml & Dettner, 1990;Laussmann et al., 2012).Larvae pupate inside a tough silken cocoon attached to stems and twigs of the foodplant near the ground.The species overwinters at the pupal stage, sometimes for two or more winters (Laussmann et al., 2012).
Here we report a complete genome sequence for the Emperor moth Saturnia pavonia determined as part of the Darwin Tree of Life project.The genome sequence of S. pavonia will facilitate research into the biochemical basis of chemical defence and pheromone communication in insects, and contribute to the growing set of resources for studying molecular evolution in the Lepidoptera.

Genome sequence report
The genome was sequenced from one male Saturnia pavonia (Figure 1) reared from a larva collected in Wytham Woods, Oxfordshire, UK (51.77,.A total of 50-fold coverage in Pacific Biosciences single-molecule HiFi long reads was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 6 missing joins or mis-joins and removed 2 haplotypic duplications, reducing the scaffold number by 13.51%. The final assembly has a total length of 489.9 Mb in 31 sequence scaffolds with a scaffold N50 of 17.7 Mb (Table 1).The snailplot in Figure 2 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3.The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla.Most (99.98%) of the assembly sequence was assigned to 30 chromosomal-level scaffolds, representing 29 autosomes and the Z sex chromosome.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 2).While not   fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission.

Sample acquisition and nucleic acid extraction
The Saturnia pavonia specimen used for genome sequencing and Hi-C data (specimen ID Ox002139, ToLID ilSatPavo1) was collected as a larva from Wytham Woods, Oxfordshire   et al., 2023).Tissue from the thorax was homogenised using a PowerMasher II tissue disruptor (Denton et al., 2023a).HMW DNA was extracted in the WSI Scientific Operations core using the Automated MagAttract v2 protocol (Oatley et al., 2023).HMW DNA was sheared into an average fragment size of 12-20 kb in a Megaruptor 3 system with speed setting 31 (Bates et al., 2023).Sheared DNA was purified by solid-phase reversible immobilisation (Strickland et al., 2023): in brief, the method employs a 1.8X ratio of AMPure PB beads to sample to eliminate shorter fragments and concentrate the DNA.The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer and Qubit dsDNA High Sensitivity Assay kit.Fragment size distribution was evaluated by running the sample on the FemtoPulse system.
RNA was extracted from abdomen tissue of ilSatPavo1 in the Tree of Life Laboratory at the WSI using the RNA Extraction: Automated MagMax™ mirVana protocol (do Amaral et al., 2023).The RNA concentration was assessed using a Nanodrop spectrophotometer and a Qubit Fluorometer using the Qubit RNA Broad-Range Assay kit.Analysis of the integrity of the RNA was done using the Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.Further, the Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use.
The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible.The overarching areas of consideration are: • Ethical review of provenance and sourcing of the material The manuscript by Crowley and colleagues reports the genome assembly of the Emperor moth, Saturnia pavonia.This resource was obtained as a part of the Darwin Tree of Life project, through the use of a highly standardized and reproducible pipeline.Therefore, there are no significant methodological concerns, and the quality of the assembled genome appears to be very high.I just have a very few minor comments dealing with cosmetic changes that the authors may consider including in a revised version."(51.77,-1.33)" -> I am not sure this is the standard or most common way of reporting geographical coordinates.Please add "latitude" and "longitude".Genome annotation report: this was obtain though the Ensembl rapid annotation pipeline, which in other phyla (I am not really sure about arthropods) performs rather poorly.Do the BUSCO metrics obtained by analyzing the annotated gene set match those reported in the previous paragraph by analyzing the genome assembly?Data availability: consider adding here the link to the Ensembl rapid annotation report.

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: non-model species genomics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Peter Dearden
University of Otago, Dunedin, Otago, New Zealand This report describes the genome sequencing of a large day-flying moth; the Emperor moth ( Saturnia pavonia).The report describes the key biological details of the moth well, including the relationships between the sampled British populations, and those in the rest of Europe.Photos are provided of the sampled animal from both larval and adult stages.
The genome report indicates that this is a very high-quality, contiguous, uncontaminated genome.the BUSCO scores show the genome is highly complete, and indicates the high quality of the assembly.
The data generated is available, and the tools used to carry out the work, are well described and available to others.I strongly support this report.I would ask that in the section where the authors are describing the correction of scaffolds that they state the numbers of scaffolds affected, rather than just a percentage.

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Insect Genomics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Jaakko Pohjoismäki
University of Eastern Finland, Joensuu, Finland The genome report by Liam M Crowley, Ellen Baker, Peter WH Holland and the DToL consortium presents the reference genome assembly of the Emperor moth.It is a nice addition of an iconic species to the growing number of Lepidopteran genomes from the DToL.

Background
The background is well written and concise.One could add that pavonia is replaced by pavoniella in the east and that the two species have been separated only recently.This close relatedness might then deserve a comment regarding the utility of the genome assembly?Also, the pavonia is fairly early spring species, so I would imagine that May is too late for the flight time in the UK?As of note, there are many old studies on the pheromone attracting marked males, but these will require a bit of digging as they are difficult to find.Main point is that not all estimates are based on "conjecture".The larvae are also sexually dimorphic, but this is maybe an unnecessary detail.
The genome assembly, annotation and completeness looks very good, representing the DToL quality standards.The methods are adequately described and following the DToL genome production pipeline without any issues.
My main complaint is that the genome assembly is from a male and thus lacks the W sex chromosome.I think this is a missed opportunity to collect valuable genome data on the sex chromosome evolution among insects.For Saturnia pavonia missing this opportunity is especially unnecessary, as the females are easily available from light traps, collecting as larvae or bought from a (British) web shop (https://www.wwb.co.uk/emperor-moth-pavonia-cocoons).
However, this detail does not otherwise affect the quality of the work.So far the use of males for the genome assemblies has been a recurrent issue with the Lepidopteran genomes.I hope this will be avoided in the future and females will be used for the coming species.Background.There is little true motivation for this study in the introduction.Nothing wrong is said, but the reader has little idea why the work was done.It's perfectly fine that the work was done in the context of a large survey, but tell the reader that.Almost all the details of its life history are irrelevant to the reader.The paper is not about its ecology or morphology, but about its genetics.That is not mentioned once.
The end paragraph mentions it is an existing or potential model for chemical defenses and pheromone communication but not direct justification or citation of why this species is particularly useful for these is given.All parameter setting if they were different from default of each piece of bioinformatic software needs to be specified.Just put a sentence to begin that everything was default unless specified otherwise.What assessment was done to ensure that default was adequate?
A reasonable amount of genes is predicted.However, the reader has very little understanding of how these were evaluated beyond their initial generation.What QC beyond an automated annotation was done?One annotation run with default parameters should come with a strong disclaimer that any further work on specific gene families requires QC on the part of the user because the current annotation is a guide only.
What is the BUSCO percentage of the predicted gene set?That is an absolute minimum for a reader to be able to assess the minimum quality of the annotation.
Table 3 is of little to no value and contains much duplicated information.Just add the version numbers of software used inline at the appropriate places in the methods section.
Is the Ethics statement needed?It just says the provider of the sample should meet some agreed upon standard, but does not actually say that they did in this case.Either drop the statement or actually say the standard was met for this sample.This is not a paper about the standards of collection.
Add something to the report that contextualize the research for the reader.Is this species the first of a taxon to be assembled, is it an outgroup to some established model species, is it now possible to investigate some interesting aspect of the species biology, etc?This does not to be extensive or highly directed, but there is currently no justification of this work outside of the Data Availability Statement.Even Data Note should contain rationale for the work.

Is the rationale for creating the dataset(s) clearly described? Partly
Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Partly
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Genomics, genetics, epigenetics, behavior, reproduction I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Figure 2 .
Figure 2. Genome assembly of Saturnia pavonia, ilSatPavo1.1:metrics.The BlobToolKit Snailplot shows N50 metrics and BUSCO gene completeness.The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 489,898,868 bp assembly.The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest scaffold present in the assembly (22,760,314 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (17,680,408 and 11,900,431 bp), respectively.The pale grey spiral shows the cumulative scaffold count on a log scale with white scale lines showing successive orders of magnitude.The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot.A summary of complete, fragmented, duplicated and missing BUSCO genes in the lepidoptera_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CANNWM01/dataset/CANNWM01/snail.

Figure 3 .
Figure 3. Genome assembly of Saturnia pavonia, ilSatPavo1.1:BlobToolKit GC-coverage plot.Scaffolds are coloured by phylum.Circles are sized in proportion to scaffold length.Histograms show the distribution of scaffold length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CANNWM01/dataset/CANNWM01/blob.

Figure 4 .
Figure 4. Genome assembly of Saturnia pavonia, ilSatPavo1.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all scaffolds.Coloured lines show cumulative lengths of scaffolds assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CANNWM01/dataset/CANNWM01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Saturnia pavonia, ilSatPavo1.1:Hi-C contact map of the ilSatPavo1.1 assembly, visualised using HiGlass.Chromosomes are shown in order of size from left to right and top to bottom.An interactive version of this figure may be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=SljOrrzkSG67snu-sBNiBg.

Figure 2
Figure 2/3/4/5 titles.These should be informative and tell the reader what you would like them to understand about the data; e.g., Fig 3. Little contamination was found in the final genome assembly.

Figure 4 .
Figure 4. has very little information and can easily be summarized as a sentence in the results.The

Darwin Tree of Life Project Sampling Code of Practice', which
can be found in full on the Darwin Tree of Life website here.By agreeing with and signing up to the Sampling Code of Practice, the Darwin Tree of Life Partner agrees they will meet the legal and ethical requirements and standards set out within this document in respect of all samples acquired for, and supplied to, the Darwin Tree of Life Project.
This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Is the rationale for creating the dataset(s) clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests:
No competing interests were disclosed.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
Reviewer Report 10 May 2024 https://doi.org/10.21956/wellcomeopenres.22857.r80533