Gavin R. Broad,,,,,,
The genome sequence of the Festoon, Apoda limacodes (Hufnagel, 1766) [version 1; peer review: 2 approved]
Apoda limacodes,the Festoon,genome sequence,chromosomal,Lepidoptera
We present a genome assembly from an individual male Apoda limacodes (the Festoon; Arthropoda; Insecta; Lepidoptera; Limacodidae). The genome sequence is 800 megabases in span. Most of the assembly is scaffolded into 25 chromosomal pseudomolecules, including the assembled Z sex chromosome. The mitochondrial genome has also been assembled and is 15.4 kilobases in length.
The genome sequence of the Festoon, Apoda limacodes (Hufnagel, 1766) [version 1; peer review: 2 approved]

We present a genome assembly from an individual male Apoda limacodes (the Festoon; Arthropoda; Insecta; Lepidoptera; Limacodidae). The genome sequence is 800 megabases in span. Most of the assembly is scaffolded into 25 chromosomal pseudomolecules, including the assembled Z sex chromosome. The mitochondrial genome has also been assembled and is 15.4 kilobases in length.

Eukaryota; Metazoa; Ecdysozoa; Arthropoda; Hexapoda; Insecta; Pterygota; Neoptera; Endopterygota; Lepidoptera; Glossata; Ditrysia; Zygaenoidea; Limacodidae; Apoda; Apoda limacodes (Hufnagel, 1766) (NCBI:txid287200)

The larva of the Festoon, Apoda limacodes, is an odd caterpillar. It has no visible legs and no obvious body segmentation, hence its Latin name, ‘legless, slug-like’. The Festoon and the Triangle ( Heterogenea asella) are the only British species of Limacodidae, a cosmopolitan family of about 1,700, mainly tropical, described species. They are often called slug moths and many species have spectacular larvae, sometimes with stinging setae. Larvae of the Festoon are harmless, and the adults are relatively sombre in colour, although nothing else in Britain looks like them. Adults have a characteristic posture with the wings arranged in a tent-like fashion and the abdomen pointing upwards. Females lack the darker band across the middle of the wing. Larvae of the Festoon feed mainly on oak and beech; caterpillars can be found most easily at night as they fluoresce under the beam of a UV torch. Adults come to light or can sometimes be seen flying in the canopy. As with other limacodids, instead of abdominal prolegs, there are sucker-like discs which enable the larvae of limacodids to seem to glide along the leaf surface. Adults are on the wing in June and July with larvae feeding from late summer then spending the winter as cocooned prepupae in the leaf litter, pupating the following spring, e.g. Henwood et al., 2020. Often associated with mature woodlands, the Festoon has increased significantly in range in recent years ( Fox et al., 2014), although is still mainly a species of south-east England (NBN Atlas), but this probably follows a period of much reduced abundance. The larvae of A. limacodes support a suite of highly specialised parasitoid wasps (e.g., ( Shaw & Voogd, 2016) and one, Sphinctus serotinus Gravenhorst, was recently rediscovered in England after an apparent absence of over a century ( Thornhill, 2020). This is the first genome for a species of the family Limacodidae, so will be invaluable in comparative genomics across Lepidoptera. As a family which includes several notable agricultural pests ( Cock et al., 1987), a limacodid reference genome should be useful in studying the genetics of related pest species.

The genome was sequenced from a male A. limacodes ( Figure 1) collected from Tonbridge, UK. A total of 23-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 or mis-joins, reducing the scaffold number by 3.13%. The final assembly has a total length of 800.4 Mb in 31 sequence scaffolds with a scaffold N50 of 33.0 Mb ( Table 1). Most (99.98%) of the assembly sequence was assigned to 25 chromosomal-level scaffolds, representing 24 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). The assembly has a BUSCO v5.3.2 ( Manni et al., 2021) completeness of 98.4% (single 97.8%, duplicated 0.6%) using the OrthoDB v10 lepidoptera reference set. While not fully phased, the assembly deposited is of one haplotype. Contigs corresponding to the second haplotype have also been deposited.

A male A. limacodes (ilApoLima1) was collected and identified by Gavin Broad (Natural History Museum). The sample was caught in a garden in Tonbridge, UK (latitude 51.19, longitude 0.29) by using an actinic light and preserved by freezing on dry ice. DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI). The ilApoLima1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing. Head and thorax tissue was disrupted using a Nippi Powermasher fitted with a BioMasher pestle. 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 and Qubit dsDNA High Sensitivity Assay kit. Fragment size distribution was evaluated by running the sample on the FemtoPulse system.

Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers’ instructions. DNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi). Hi-C data were also generated from tissue of ilApoLima1 using the Arima v2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

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., 2022). The assembly was checked for contamination and corrected using the gEVAL system ( Chow et al., 2016) as described previously ( Howe et al., 2021). Manual curation was performed using gEVAL, HiGlass ( Kerpedjiev et al., 2018) and Pretext ( Harry, 2022). The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al., 2021), which performed annotation using MitoFinder ( Allio et al., 2020). The genome was analysed and BUSCO scores generated within the BlobToolKit environment ( Challis et al., 2020). Table 3 contains a list of all software tool versions used, where appropriate.

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. 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. Each transfer of samples is further undertaken according to a Research Collaboration Agreement or Material Transfer Agreement entered into by the Darwin Tree of Life Partner, Genome Research Limited (operating as the Wellcome Sanger Institute), and in some circumstances other Darwin Tree of Life collaborators.