The genome sequence of the Cinnabar Moth, Tyria jacobaeae (Linnaeus, 1758) [version 1; peer review: awaiting peer review]

We present a genome assembly from an individual male Tyria jacobaeae (the Cinnabar Moth


Background
An evolutionary arms race can occur between plants and herbivores. Many plants produce toxic chemicals to deter predators, but some insects have evolved mechanisms to detoxify chemicals or sequester and deploy them for their own use. For example, moths of the subfamily Arctiinae (family Erebidae) can sequester pyrrolizidine from food plants to use as a defence. The chemistry of this phenomenon has been studied in the cinnabar moth Tyria jacobaeae.
Tyria jacobaeae is an easily recognised moth found across Europe and Asia: the conspicuous red and black adults are a familiar sight in well-drained grassland, heathland, woodland rides and occasionally gardens in spring and summer, flying from May to July in southern England (Waring et al., 2017). The adults are primarily active at night, but they are easily disturbed in the daytime and the species is often incorrectly described as a day-flying moth. The yellow and black striped larvae are equally distinctive, feeding openly on ragwort (Senecio jacobaea) and grounsel (S. vulgaris). Writing seventy years ago, Ford said that "country folk" refer to the striped larvae as "wort maggots with football jerseys on" (Ford, 1952).
The food plants produce toxic pyrrolizidine alkaloids as defence chemicals. Analysis has shown that T. jacobaeae larvae take up and store many of these alkaloids, which are then retained in the bodies of the larvae, pupae and adults (Ehmke et al., 1990). This retention requires the larva to express an enzyme (senecionine N-oxygenase) for converting the toxic chemicals into N-oxide forms that are non-toxic to insects; a gene encoding this enzyme has been cloned (Naumann et al., 2002;Sehlmeyer et al., 2010). Pupae and adults of T. jacobaeae (but not larvae) also contain an additional alkaloid, not present in the food plant, given the name 'callimorphine' (Edgar et al., 1980). Around the larval-pupal transition stage, T. jacobaeae synthesise callimorphine from smaller breakdown products of plant-derived alkaloids (Ehmke et al., 1990). The genes encoding the enzymatic pathway have not been characterised. Adult and larval cinnabar moths are avoided or rejected by most vertebrate predators (Aplin et al., 1968); juvenile cuckoos are an exception and feed extensively on cinnabar moth larvae (Mills et al., 2020).
Ragwort has become invasive in several countries causing problems to livestock farming due to its toxic effects. The Cinnabar Moth was introduced as a biological control agent in New Zealand in 1929, on the west coast of the United States in 1959 and in Canada in the 1960s; it is now well established in these countries and has proved successful at ragwort control (Frick & Holloway, 1964;Harris et al., 1975;Harman et al., 1990;Markin & Littlefield, 2008).
The genome sequence of Tyria jacobaeae was determined as part of the Darwin Tree of Life project. The assembled genome sequence will aid development of genetic markers for monitoring population spread and greatly facilitate research into the biochemical pathways underpinning toxin chemistry in insects.

Genome sequence report
The genome was sequenced from one male Tyria jacobaeae ( Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77, -1.33). A total of 31-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 24 missing joins or mis-joins and removed 4 haplotypic duplications, reducing the assembly length by 0.2% and the scaffold number by 11.11%.
The final assembly has a total length of 589.7 Mb in 55 sequence scaffolds with a scaffold N50 of 21.0 Mb (Table 1). Most (99.8%) of the assembly sequence was assigned to 31 chromosomal-level scaffolds, representing 30 autosomes and the Z sex chromosome. Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 2- 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.   A second T. jacobaeae specimen (specimen number NHMUK01411107, individual ilTyrJaco2) was collected in Wigmore Park, Luton (latitude 51.88, longitude -1.37) on 2020-06-23 by netting. The specimen was collected and identified by Olga Sivell (Natural History Museum), and then preserved on dry ice. This specimen was used for Hi-C scaffolding and RNA sequencing.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI). The ilTyrJaco4 sample was weighed and dissected on dry ice. Whole organism tissue was cryogenically disrupted to a fine powder using a Covaris cryoPREP Automated Dry Pulveriser, receiving multiple impacts. High molecular weight (HMW) DNA was extracted using the Qiagen MagAttract HMW DNA extraction kit. HMW DNA was sheared into an average fragment size of 12-20 kb in a Megaruptor 3 system with speed setting 30. Sheared DNA was purified by solid-phase reversible immobilisation using AMPure PB beads with a 1.8X ratio of beads to sample to remove the shorter fragments and concentrate the DNA sample. The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer   Illumina HiSeq 4000 (RNA-Seq) instruments. Hi-C data were also generated from head tissue of ilTyrJaco2 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 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., 2022), 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., 2017) DSL2 pipelines "sanger-tol/readmapping" (Surana et al., 2023a) and "sanger-tol/genomenote" (Surana et al., 2023b). The genome was analysed within the BlobToolKit environment (Challis et al., 2020) and BUSCO scores (Manni et al., 2021;Simão et al., 2015) were calculated. 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 genome sequence is released openly for reuse. The Tyria jacobaeae genome sequencing initiative is part of the Darwin Tree of Life (DToL) project. All raw sequence data and the assembly have been deposited in INSDC databases. The genome will be annotated using available RNA-Seq data and presented through the Ensembl pipeline at the European Bioinformatics Institute. Raw data and assembly accession identifiers are reported in Table 1.