The genome sequence of the Elephant Hawk-moth, Deilephila elpenor (Linnaeus, 1758)

We present a genome assembly from an individual female Deilephila elpenor (the Elephant Hawk-moth; Arthropoda; Insecta; Lepidoptera; Sphingidae). The genome sequence is 414.1 megabases in span. Most of the assembly is scaffolded into 30 chromosomal pseudomolecules, including the Z and W sex chromosomes. The mitochondrial genome has also been assembled and is 15.37 kilobases in length. Gene annotation of this assembly on Ensembl identified 11,748 protein coding genes.


Background
The Elephant Hawk-moth Deilephila elpenor gains its common name from the unusual appearance of its larva.The easily recognised larva grows to around 7 cm in length and is either dark brown or bright green.The first few segments behind the head are narrow and can be extended forward; with imagination, this can make the larva resemble an elephant's trunk as it sways side-to-side.Also noticeable are four large eye-spots: two on each side of the larval body.When touched, the larva can enlarge and swell the body region where the eye-spots lie, possibly to deter potential predators (Easterbrook, 1985).A study using artificial caterpillars made of pastry showed that eyespots and expanded anterior segments can reduce bird attack (Hossie & Sherratt, 2013).The larva of D. elpenor was described and illustrated over three centuries ago by Albin (1720) who referred to it simply as 'the elephant'.His suggestion that the larva can swim in water by turning upside down and performing contortions of its body has no modern support.D. elpenor is widely distributed across the Palaearctic, from Portugal and Ireland in the west to China, Korea, Japan and the far east of Russia (GBIF Secretariat, 2023).There are sporadic occurrences outside this range, including several clusters of records from western Canada and the northwest of the United States (GBIF Secretariat, 2023); these are consistent with local establishment following accidental introduction.In Britain, the species is common along river valleys in southern England, including along the River Thames, Kennet and Lea valleys (Allan, 1947), and in the last 40 years its range has expanded greatly across northern England and Scotland (Randle et al., 2019).
Larvae of D. elpenor feed at night on bedstraws Galium sp. and willowherbs including rosebay willowherb Chamaenerion angustifolium and hoary willowherb Epilobium parviflorum (Allan, 1947).Larval development is rapid through July and August in Northern Europe, with the pupal stage overwintering.The pink and olive-green adult is frequently encountered in June and July, when it may be seen on warm nights hovering to take nectar from flowers such as honeysuckle; it is also attracted to light.Experimental studies have shown that adult D. elpenor can discriminate colours even in light conditions comparable to dim starlight: this research was one of the first demonstrations of nocturnal colour vision in animals (Kelber et al., 2002).Electrophysiology of motionsensitive neurons also revealed remarkable visual sensitivity to low velocity motion, necessary for feeding by hovering at night (Theobald et al., 2010).
A genome sequence of Deilephila elpenor was determined as part of the Darwin Tree of Life project.The genome sequence will facilitate research into insect vision, neurobiology and host plant choice, and contribute to the growing set of resources for studying molecular evolution in the Lepidoptera.

Genome sequence report
The genome was sequenced from one female Deilephila elpenor (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77, -1.34).A total of 54-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 10 missing joins or mis-joins and removed one haplotypic duplication, reducing the scaffold number by 15.79%, and increasing the scaffold N50 by 1.00%.
The final assembly has a total length of 414.1 Mb in 31 sequence scaffolds with a scaffold N50 of 14.6 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 (99.99%) of the assembly sequence was assigned to 30 chromosomal-level scaffolds, representing 28 autosomes and the Z and W sex chromosomes.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 2).The Z chromosome identified by coverage and alignment to Deilephila porcellus (GCA_905220455.2) (Boyes et al., 2022).The W chromosome was identified by coverage.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
A female Deilephila elpenor (specimen ID Ox000661, ToLID ilDeiElpe2) was collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude -1.34) on 2020-07-20 using a light trap.The specimen was collected and identified by Douglas Boyes (University of Oxford) and preserved on dry ice.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.
Protocols developed by the WSI Tree of Life laboratory are publicly available on protocols.io(Denton et al., 2023b).

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions.Poly(A) RNA-Seq libraries were constructed using the NEB Ultra II RNA Library Prep kit.DNA and RNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi) and Illumina NovaSeq 6000 (RNA-Seq) instruments.Hi-C data were also generated from head tissue of ilDeiElpe2 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

Genome assembly, curation and evaluation
Assembly was carried out with Hifiasm (Cheng et al., 2021) and haplotypic duplication was identified and removed with purge_dups (Guan et al., 2020).The assembly was then scaffolded with Hi-C data (Rao et al., 2014) using YaHS (Zhou et al., 2023).The assembly was checked for contamination and corrected using the TreeVal pipeline (Pointon et al., 2023).
Table 3 contains a list of relevant software tool versions and sources.

Genome annotation
Ensembl Genebuild (Aken et al., 2016) at the EBI was used to generate annotation for the Deilephila elpenor assembly (GCA_949752805.1).Annotation was created primarily through alignment of transcriptomic data to the genome, with gap filling via protein-to-genome alignments of a select set of proteins from UniProt (UniProt Consortium, 2019).

Wellcome Sanger Institute -Legal and Governance
The materials that have contributed to this genome note have been supplied by a Darwin Tree of Life Partner.The submission of materials by a Darwin Tree of Life Partner is subject to 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.
Table 3. Software tools: versions and sources.

Software tool Version
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

Axel Künstner
University of Lübeck, Lübeck, Germany This report presents the female genome assembly of the Elephant Hawk-moth (Deilephila elpenor), which spans 414.1 megabases of sequence data assembled into 30 chromosomal pseudomolecules, including the Z and W sex chromosomes.Additionally, the mitochondrial genome has also been assembled.
The methods used are sound, software resources used are well described, and the data is available via the European Nucleotide Archive.

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: Cancer genomics, Microbiome, Bioinformatics, Computational Biology 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.Comments: The reported assembly is nearly complete and appears to be the first genome assembly reported for Deilephila elpenor.A thorough functional annotation could improve its quality further as a reference genome.Additional information on genome architecture, like repeats and duplicated regions, could also provide more insights into the reported genome.
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: Bioinformatics, Genomics, Transcriptomics, Metagenomics 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.

Figure 2 .
Figure 2. Genome assembly of Deilephila elpenor, ilDeiElpe2.1: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 414,066,290 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 (22,915,455 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (14,585,127 and 10,162,166 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/ilDeiElpe2_1/dataset/ilDeiElpe2_1/snail.

Figure 3 .
Figure 3. Genome assembly of Deilephila elpenor, ilDeiElpe2.1: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/ilDeiElpe2_1/dataset/ilDeiElpe2_1/blob.

Figure 4 .
Figure 4. Genome assembly of Deilephila elpenor, ilDeiElpe2.1: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/ilDeiElpe2_1/dataset/ilDeiElpe2_1/cumulative.

Figure 5 .
Figure 5. Genome assembly of Deilephila elpenor, ilDeiElpe2.1:Hi-C contact map of the ilDeiElpe2.1 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=f1cVHAvTSO62HOfQfV56FA.

Reviewer Report 16
August 2024 https://doi.org/10.21956/wellcomeopenres.23247.r90853© 2024 Thimmappa B. 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.Bhagya Thimmappa ORISE Postdoctoral Fellow at USDA-ARS, Chatsworth, New Jersey, USA In the work "The genome sequence of the Elephant Hawk-moth, Deilephila elpenor (Linnaeus, 1758)," the authors report a nuclear genome assembly using PacBio sequencing, structural annotation, and mitochondrial genome assembly.