The genome sequence of the Figure of Eight, Diloba caeruleocephala (Linnaeus, 1758)

We present a genome assembly from an individual male Diloba caeruleocephala (the Figure of Eight; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 762.5 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.36 kilobases in length.


Background
The Figure of Eight Diloba caeruleocephala (Linnaeus, 1758) is an endangered moth in Britain, having suffered a 96% decrease in abundance between 1970 and 2016 as well as a decline in its distribution (Randle et al., 2019).Within the family Noctuidae, it is the sole British representative of the subfamily Dilobinae, and is a distinctive species named for the white markings on the forewing, the inner of which resembles an '8'.The most likely confusion species in the British Isles are probably the Lasiocampids Tethea ocularis (Linnaeus, 1767), the Figure of Eighty, and Tethea or ([Denis & Schiffermüller], 1775), the Poplar Lutestring, which have similar white markings but otherwise very different forewing patterning and lack serrated antennae in the male.D. caeruleocephala overwinters as an egg, with the larvae hatching in late April.Larvae feed on the leaves of a large variety of trees and shrubs (particularly Prunus spinosa and Crataegus spp.), before pupating on or under the ground in a silken cocoon.The larvae are pale grey with large black pinacula, yellow dorsal and lateral spots and a raised yellow band across the second abdominal segment (Henwood et al., 2020).The life cycle of the species was studied in detail by Bolu & Özgen (2007).
Despite the rapid decline of the species in Britain, in Ireland (where it has a very scattered distribution) it has fared better, seeing a significant increase in records from just five before 1970 to twenty-six between 2000and 2019(Randle et al., 2019)).A synthetic sex-attractant for the species was presented by Subchev et al. (2000).
We present a chromosomally complete genome sequence for Diloba caeruleocephala, based on one male specimen from Wytham Woods, Oxfordshire, as part of the Darwin Tree of Life Project.This project is a collaborative effort to sequence all named eukaryotic species in the Atlantic Archipelago of Britain and Ireland.

Genome sequence report
The genome was sequenced from one male Diloba caeruleocephala (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77,.A total of 30-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 2 missing joins or mis-joins and removed one haplotypic duplication. The final assembly has a total length of 762.5 Mb in 36 sequence scaffolds with a scaffold N50 of 27.9 Mb (Table 1).The snailplot in Figure 2 provides a summary of the assembly statistics, while the distribution of reads 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 31 chromosomal-level scaffolds, representing 30 autosomes and the X 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
A male Diloba caeruleocephala (specimen ID Ox001971, ToLID ilDilCaer1) was collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude -1.34) on 2021-10-10 using a light trap.The specimen was collected and identified by Liam Crowley (University of Oxford) and preserved on dry ice.
The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) includes a sequence of core procedures: sample preparation; sample homogenisation; DNA extraction; HMW DNA fragmentation; and fragmented DNA clean-up.The sample was prepared for DNA  DNA was extracted at the WSI Scientific Operations core using the Qiagen MagAttract HMW DNA kit, according to the manufacturer's instructions.
RNA was extracted from head and thorax tissue of ilDil-Caer1 using the Automated MagMax™ mirVana protocol   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 as described previously (Howe et al., 2021).Manual curation was performed using HiGlass (Kerpedjiev et al., 2018) and Pretext (Harry, 2022).The mitochondrial genome was assembled using MitoHiFi (Uliano-Silva et al., 2023), which runs MitoFinder (Allio et al., 2020) or MITOS (Bernt et al., 2013) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.
A Hi-C map for the final assembly was produced using bwa-mem2 (Vasimuddin et al., 2019) in the Cooler file format (Abdennur & Mirny, 2020).To assess the assembly metrics, the k-mer completeness and QV consensus quality values were calculated in Merqury (Rhie et al., 2020).This work was done using Nextflow (Di Tommaso et al
Table 3 contains a list of relevant software tool versions and sources.

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.
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 In short, this manuscript presents a complete view of the genomic organisation of Diloba caeruleocephala.The rationale and the methodology are very well presented.
However, I found the results part insufficient, I was expecting some stories about the evolutionary pattern of the Diloba caeruleocephala species and the genomic relationship between the other species, as the tree of life tries to reconstruct the evolutionary relationship between species.
I'm also hoping that the authors will explain the figure 5 a bit more in terms of chromosomal rearrangements and genomic structuring.

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: Malaria vector bio-ecology and genomic 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.Major concerns: Although this manuscript represents a brief report of the de novo genome assembly, I think that all important information about the generating of the genome assembly has to be provided here.
The missing information in the manuscript text include the following: It would be great if the authors provide description of different steps in the assembly development. 1.
Please, describe how different methods used here were incorporated into developing the genome assembly.What was the purpose of using each method? 2.
The purpose of obtaining RNA-seq data is not explained in the manuscript at all.Please, provide this information.

3.
Please, explain what does it mean the assembly "was not fully phased".Provide, some specific details of "not fully".

4.
It is unclear if cytogenetic evaluation of a chromosome number in this species or related species were conducted previously and does this number matches the number of chromosomal pseudomolecules generated by this project.

5.
Please, also provide information about the companies from which specific reagents and kits were obtained.

6.
I would also suggest to improve the quality of the figures: Figure 1.The purpose of showing microcentrifuge tubes on the figure 1 is unclear.If tubes are shown to provide some scale for the size of the moth specimen, this has to be explained in the figure legend.However, it is better to use a ruler with scale in centimeters here.I would suggest to reformat this figure, provide a scale in centimeters and also remove some empty space around the specimen.Minor concerns.The sex chromosome is indicated as Z chromosome in abstract but as X chromosome in the manuscript text.Please, correct this. 1.
In Figure 5 legend authors wrote "Chromosomes are shown in order of size".It is better to write "in order of decreasing in size".

2.
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? Partly
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Cytogenetics and genomics of mosquitoes.
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.
The manuscript presents a comprehensive genome assembly of an individual male Diloba caeruleocephala, a member of the Noctuidae family.The assembly spans 762.5 megabases and is scaffolded into 31 chromosomal pseudo-molecules, including the Z sex chromosome.Additionally, the mitochondrial genome, spanning 15.36 kilobases, has been assembled.As usual, the manuscript presents a valuable resource in the form of a high-quality genome assembly of Diloba caeruleocephala, shedding light on its chromosomal organization and mitochondrial genome.With clear data visualization and comprehensive assembly statistics, the manuscript is well-positioned to contribute to the field of Lepidoptera genomics.I recommend acceptance of the manuscript as it is.
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: phylogenomics, 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.

Figure 2 .
Figure 2. Genome assembly of Diloba caeruleocephala, ilDilCaer1.1: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 762,531,772 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 (33,052,791 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (27,899,106 and 17,255,644 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/Diloba%20caeruleocephala/dataset/CANHGH01/snail.

(
https://dx.doi.org/10.17504/protocols.io.6qpvr36n3vmk/ v1).The RNA concentration was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit RNA Broad-Range (BR) 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 Tree of Life laboratory are publicly available on protocols.io:https://dx.doi.org/10.17504/protocols.io.8epv5xxy6g1b/v1.SequencingPacific Biosciences HiFi circular consensus and 10X Genomics read cloud 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 remaining head and thorax tissue of ilDilCaer1 using the

Figure 5 .
Figure 5. Genome assembly of Diloba caeruleocephala, ilDilCaer1.1:Hi-C contact map of the ilDilCaer1.1 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=RGHSoMHwRhCT5dxmF8w16w.

Reviewer Report 26
May 2024 https://doi.org/10.21956/wellcomeopenres.22690.r74125© 2024 Sharakhova M. 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.Maria Sharakhova Virginia Polytechnic and State University,, Blacksburg, VA, USA The manuscript of Liam M. Crowley and Finley Hutchinson " The genome of the Figure Eight, Diloba caeruleocephala" represents a brief report of de novo sequenced genome of an endangered in Britain moth species.The genome was generated from a single male using a cutting-edge technology sequencing Pacific Biosciences (HiFi) and Hi-C scaffolding approach.The genome assembly is 762.32 Mb in size and consists of 31 chromosomal pseudomolecules including Z sex chromosome.Mitochondrial genome was also assembled.The genome was sequenced as part of the Darvin Tree of Life project with the purpose of obtaining sequencing data for all named eukaryotic species in Britain and Ireland.

Figure 2 .
The numbers on the scale in the center of the figure (100K, 10.0K) overlap and have to be corrected.In the right top corner, where BUSCO score is shown, it is unclear where different shades of green are shown in the circular diagram.They are simply invisible on the diagram.I would suggest to connect different fractions on the diagram by lines with the squares with different shades of green.Also, the scale which is shown in the left bottom corner of the figure has to be explained in the figure legend.Figure5.I would suggest to add a scale in Mb to indicate the sizes of the chromosomal scaffolds.Please, also indicate which chromosome is considered as Z here.

Table 3 . Software tools: versions and sources. Software tool Version Source was
analysed within the BlobToolKit environment (Challis