Abstract
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.
Keywords: Apoda limacodes, the Festoon, genome sequence, chromosomal, Lepidoptera
Species taxonomy
Eukaryota; Metazoa; Ecdysozoa; Arthropoda; Hexapoda; Insecta; Pterygota; Neoptera; Endopterygota; Lepidoptera; Glossata; Ditrysia; Zygaenoidea; Limacodidae; Apoda; Apoda limacodes (Hufnagel, 1766) (NCBI:txid287200)
Background
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.
Genome sequence report
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.
Figure 1. Image of the Apoda limacodes (ilApoLima1) specimen used for genome sequencing.
Figure 2. Genome assembly of Apoda limacodes, ilApoLima1.1: 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 800,350,849 bp assembly. The distribution of sequence lengths is shown in dark grey with the plot radius scaled to the longest sequence present in the assembly (55,212,830 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 sequence lengths (33,043,310 and 23,972,641 bp), respectively. The pale grey spiral shows the cumulative sequence 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/ilApoLima1.1/dataset/CAMJKE01/snail.
Figure 5. Genome assembly of Apoda limacodes, ilApoLima1.1: Hi-C contact map.
Hi-C contact map of the ilApoLima1.1 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=QnKidS0aRnKlj6qLfctIeQ.
Table 1. Genome data for Apoda limacodes, ilApoLima1.1.
| Project accession data | ||
|---|---|---|
| Assembly identifier | ilApoLima1.1 | |
| Species | Apoda limacodes | |
| Specimen | ilApoLima1 | |
| NCBI taxonomy ID | 287200 | |
| BioProject | PRJEB54603 | |
| BioSample ID | SAMEA11025013 | |
| Isolate information | Head/thorax tissue (HI-C and PacBio sequencing) | |
| Assembly metrics * | Benchmark | |
| Consensus quality (QV) | 61.4 | ≥ 50 |
| k-mer completeness | 100% | ≥ 95% |
| BUSCO ** | C:98.4%[S:97.8%,D:0.6%],
F:0.4%,M:1.2%,n:5,286 |
C ≥ 95% |
| Percentage of assembly mapped to chromosomes | 99.98% | ≥ 95% |
| Sex chromosomes | Z chromosome | localised homologous pairs |
| Organelles | Mitochondrial genome assembled | complete single alleles |
| Raw data accessions | ||
| PacificBiosciences SEQUEL II | ERR9959164 | |
| Hi-C Illumina | ERR9930694 | |
| Genome assembly | ||
| Assembly accession | GCA_946406115.1 | |
| Accession of alternate haplotype | GCA_946409105.1 | |
| Span (Mb) | 800.4 | |
| Number of contigs | 90 | |
| Contig N50 length (Mb) | 15.9 | |
| Number of scaffolds | 31 | |
| Scaffold N50 length (Mb) | 33.0 | |
| Longest scaffold (Mb) | 55.2 | |
Assembly metric benchmarks are adapted from column VGP-2020 of “Table 1: Proposed standards and metrics for defining genome assembly quality” from ( Rhie et al., 2021).
BUSCO scores based on the lepidoptera_odb10 BUSCO set using v5.3.2. C = complete [S = single copy, D = duplicated], F = fragmented, M = missing, n = number of orthologues in comparison. A full set of BUSCO scores is available at https://blobtoolkit.genomehubs.org/view/ilApoLima1.1/dataset/CAMJKE01/busco.
Figure 3. Genome assembly of Apoda limacodes, ilApoLima1.1: GC coverage.
BlobToolKit GC-coverage plot. Chromosomes are coloured by phylum. Circles are sized in proportion to chromosome length. Histograms show the distribution of chromosome length sum along each axis. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/ilApoLima1.1/dataset/CAMJKE01/blob.
Figure 4. Genome assembly of Apoda limacodes, ilApoLima1.1: cumulative sequence.
BlobToolKit cumulative sequence plot. The grey line shows cumulative length for all chromosomes. Coloured lines show cumulative lengths of chromosomes assigned to each phylum using the buscogenes taxrule. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/ilApoLima1.1/dataset/CAMJKE01/cumulative.
Table 2. Chromosomal pseudomolecules in the genome assembly of Apoda limacodes, ilApoLima1.
| INSDC accession | Chromosome | Size (Mb) | GC% |
|---|---|---|---|
| OX291541.1 | 1 | 54.85 | 35.4 |
| OX291542.1 | 2 | 38.48 | 35.6 |
| OX291543.1 | 3 | 36.79 | 35.7 |
| OX291544.1 | 4 | 35.62 | 35.4 |
| OX291545.1 | 5 | 34.64 | 35.6 |
| OX291546.1 | 6 | 34.32 | 35.7 |
| OX291547.1 | 7 | 34.27 | 35.5 |
| OX291548.1 | 8 | 33.04 | 35.4 |
| OX291549.1 | 9 | 33.18 | 35.7 |
| OX291550.1 | 10 | 33.12 | 35.8 |
| OX291551.1 | 11 | 32.08 | 35.5 |
| OX291552.1 | 12 | 31.42 | 36 |
| OX291553.1 | 13 | 31.11 | 35.4 |
| OX291554.1 | 14 | 30.1 | 35.4 |
| OX291555.1 | 15 | 30.04 | 35.6 |
| OX291556.1 | 16 | 29.02 | 35.3 |
| OX291557.1 | 17 | 28.78 | 35.6 |
| OX291558.1 | 18 | 27.89 | 35.4 |
| OX291559.1 | 19 | 24.43 | 36 |
| OX291560.1 | 20 | 24.33 | 35.6 |
| OX291561.1 | 21 | 23.97 | 35.4 |
| OX291562.1 | 22 | 21.25 | 36.8 |
| OX291563.1 | 23 | 21.13 | 35.5 |
| OX291564.1 | 24 | 20.88 | 35.9 |
| OX291540.1 | Z | 55.21 | 34.9 |
| OX291565.1 | MT | 0.02 | 18.7 |
| - | - | 0.37 | 40.4 |
Methods
Sample acquisition and nucleic acid extraction
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.
Sequencing
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.
Genome assembly
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.
Table 3. Software tools and versions used.
| Software tool | Version | Source |
|---|---|---|
| BlobToolKit | 3.4.0 | Challis et al., 2020 |
| gEVAL | N/A | Chow et al., 2016 |
| Hifiasm | 0.16.1-r375 | Cheng et al., 2021 |
| HiGlass | 1.11.6 | Kerpedjiev et al., 2018 |
| MitoHiFi | 2 | Uliano-Silva et al., 2021 |
| PretextView | 0.2 | Harry, 2022 |
| purge_dups | 1.2.3 | Guan et al., 2020 |
| YaHS | yahs-1.1.91eebc2 | Zhou et al., 2022 |
Ethics/compliance issues
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.
Funding Statement
This work was supported by Wellcome through core funding to the Wellcome Sanger Institute (206194, <a href=https://doi.org/10.35802/206194>https://doi.org/10.35802/206194</a>) and the Darwin Tree of Life Discretionary Award (218328, <a href=https://doi.org/10.35802/218328>https://doi.org/10.35802/218328</a>).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
[version 1; peer review: 2 approved]
Data availability
European Nucleotide Archive: Apoda limacodes. Accession number PRJEB54603; https://identifiers.org/ena.embl/PRJEB54603 ( Wellcome Sanger Institute, 2022).
The genome sequence is released openly for reuse. The Apoda limacodes 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.
Author information
Members of the Natural History Museum Genome Acquisition Lab are listed here: https://doi.org/10.5281/zenodo.4790042.
Members of the Darwin Tree of Life Barcoding collective are listed here: https://doi.org/10.5281/zenodo.4893703.
Members of the Wellcome Sanger Institute Tree of Life programme are listed here: https://doi.org/10.5281/zenodo.4783585.
Members of Wellcome Sanger Institute Scientific Operations: DNA Pipelines collective are listed here: https://doi.org/10.5281/zenodo.4790455.
Members of the Tree of Life Core Informatics collective are listed here: https://doi.org/10.5281/zenodo.5013541.
Members of the Darwin Tree of Life Consortium are listed here: https://doi.org/10.5281/zenodo.4783558.
References
- Allio R, Schomaker-Bastos A, Romiguier J, et al. : MitoFinder: Efficient automated large-scale extraction of mitogenomic data in target enrichment phylogenomics. Mol Ecol Resour. 2020;20(4):892–905. 10.1111/1755-0998.13160 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Challis R, Richards E, Rajan J, et al. : BlobToolKit - interactive quality assessment of genome assemblies. G3 (Bethesda). 2020;10(4):1361–1374. 10.1534/g3.119.400908 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cheng H, Concepcion GT, Feng X, et al. : Haplotype-resolved de novo assembly using phased assembly graphs with hifiasm. Nat Methods. 2021;18(2):170–175. 10.1038/s41592-020-01056-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chow W, Brugger K, Caccamo M, et al. : gEVAL - a web-based browser for evaluating genome assemblies. Bioinformatics. 2016;32(16):2508–2510. 10.1093/bioinformatics/btw159 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cock MJW, Godfray HCJ, Holloway JD: Slug and Nettle Caterpillars. The biology, taxonomy and control of the Limacodidae of economic importance on palms in South-east Asia. Wallingford: C.A.B. International.1987. Reference Source [Google Scholar]
- Fox R, Oliver TH, Harrower C, et al. : Long-term changes to the frequency of occurrence of British moths are consistent with opposing and synergistic effects of climate and land-use changes. J Appl Ecol. 2014;51(4):949–957. 10.1111/1365-2664.12256 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Guan D, McCarthy SA, Wood J, et al. : Identifying and removing haplotypic duplication in primary genome assemblies. Bioinformatics. 2020;36(9):2896–2898. 10.1093/bioinformatics/btaa025 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Harry E: PretextView (Paired REad TEXTure Viewer): A desktop application for viewing pretext contact maps.2022; (Accessed: 19 October 2022). Reference Source [Google Scholar]
- Henwood B, Sterling P, Lewington R: Field Guide to the Caterpillars of Great Britain and Ireland. London: Bloomsbury.2020. Reference Source [Google Scholar]
- Howe K, Chow W, Collins J, et al. : Significantly improving the quality of genome assemblies through curation. GigaScience. Oxford University Press,2021;10(1):giaa153. 10.1093/gigascience/giaa153 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kerpedjiev P, Abdennur N, Lekschas F, et al. : HiGlass: web-based visual exploration and analysis of genome interaction maps. Genome Biol. 2018;19(1):125. 10.1186/s13059-018-1486-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Manni M, Berkeley MR, Seppey M, et al. : BUSCO Update: Novel and Streamlined Workflows along with Broader and Deeper Phylogenetic Coverage for Scoring of Eukaryotic, Prokaryotic, and Viral Genomes. Mol Biol Evol. 2021;38(10):4647–4654. 10.1093/molbev/msab199 [DOI] [PMC free article] [PubMed] [Google Scholar]
- NBN Atlas: Apoda limacodes (Hufnagel, 1766) . (Accessed: 8 December 2022). (no date). Reference Source [Google Scholar]
- Rao SS, Huntley MH, Durand NC, et al. : A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell. 2014;159(7):1665–1680. 10.1016/j.cell.2014.11.021 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rhie A, McCarthy SA, Fedrigo O, et al. : Towards complete and error-free genome assemblies of all vertebrate species. Nature. 2021;592(7856):737–746. 10.1038/s41586-021-03451-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shaw M, Voogd J: Illustrated notes on the biology of Sphinctus serotinus Gravenhorst (Hymenoptera, Tryphoninae, Sphinctini). J Hymenopt Res. 2016;49:81–93. 10.3897/JHR.49.7705 [DOI] [Google Scholar]
- Thornhill A: Sphinctus serotinus at Rampart's Field. White Admiral. 2020;104:3. [Google Scholar]
- Uliano-Silva M, et al. : MitoHiFi.2021; (Accessed: 19 October 2022). Reference Source [Google Scholar]
- Wellcome Sanger Institute: The genome sequence of the Festoon, Apoda limacodes (Hufnagel, 1766), European Nucleotide Archive [dataset].2022; accession number PRJEB54603.
- Zhou C, McCarthy SA, Durbin R: YaHS: yet another Hi-C scaffolding tool. bioRxiv. 2022. 10.1101/2022.06.09.495093 [DOI] [PMC free article] [PubMed] [Google Scholar]