The genome sequence of Zethes insularis Rambur, 1833 (Lepidoptera: Erebidae)

TL;DR

This paper presents the genome sequence of the moth Zethes insularis, including detailed assembly of its chromosomes and mitochondrial DNA.

Contribution

The study provides a high-quality genome assembly for Zethes insularis, including haplotype-specific scaffolding and mitochondrial genome data.

Findings

The genome assembly includes two haplotypes scaffolded into 31 chromosomal pseudomolecules each.

The mitochondrial genome was assembled with a length of 15.77 kilobases.

The work is part of Project Psyche, aiming to generate genomes for European Lepidoptera.

Abstract

We present a genome assembly from a male specimen of
Zethes insularis (Arthropoda; Insecta; Lepidoptera; Erebidae). The assembly contains two haplotypes with total lengths of 862.07 megabases and 869.20 megabases. Most of haplotype 1 (99.0%) is scaffolded into 31 chromosomal pseudomolecules, including the Z sex chromosome. Most of haplotype 2 (95.76%) is scaffolded into 31 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled, with a length of 15.77 kilobases. This work is part of Project Psyche, a collaborative programme generating genomes for European butterflies and moths.

Figures

Voucher photograph of the Zethes insularis (ilZetInsu1) specimen used for genome sequencing.

Frequency distribution of k-mers generated using GenomeScope2.The plot shows observed and modelled k-mer spectra, providing estimates of genome size, heterozygosity, and repeat content based on unassembled sequencing reads.

Hi-C contact map of the Zethes insularis genome assembly.Assembled chromosomes are shown in order of size and labelled along the axes. The plot was generated using PretextSnapshot.

Merian elements painted across chromosomes in the ilZetInsu1.hap1.1 assembly of Zethes insularis.Chromosomes are drawn to scale, with the positions of orthologues shown as coloured bars. Each orthologue is coloured by the Merian element that it belongs to. All orthologues which could be assigned to Merian elements are shown.

Evaluation of k-mer completeness using MerquryFK.This plot illustrates the recovery of k-mers from the original read data in the final assemblies. The horizontal axis represents k-mer multiplicity, and the vertical axis shows the number of k-mers. The black curve represents k-mers that appear in the reads but are not assembled. The green curve (the homozygous peak) corresponds to k-mers shared by both haplotypes and the red and blue curves (the heterozygous peaks) show k-mers found only in one of the haplotypes.

Assembly metrics for ilZetInsu1.hap1.1.The BlobToolKit snail plot provides an overview of assembly metrics and BUSCO gene completeness. The circumference represents the length of the whole genome sequence, and the main plot is divided into 1,000 bins around the circumference. The outermost blue tracks display the distribution of GC, AT, and N percentages across the bins. Scaffolds are arranged clockwise from longest to shortest and are depicted in dark grey. The longest scaffold is indicated by the red arc, and the deeper orange and pale orange arcs represent the N50 and N90 lengths. A light grey spiral at the centre shows the cumulative scaffold count on a logarithmic scale. A summary of complete, fragmented, duplicated, and missing BUSCO genes in the set is presented at the top right. An interactive version of this figure can be accessed on the BlobToolKit viewer.

BlobToolKit GC-coverage plot for ilZetInsu1.hap1.1.Blob plot showing sequence coverage (vertical axis) and GC content (horizontal axis). The circles represent scaffolds, with the size proportional to scaffold length and the colour representing phylum membership. The histograms along the axes display the total length of sequences distributed across different levels of coverage and GC content. An interactive version of this figure is available on the BlobToolKit viewer.

Tables

Table 1.. Specimen and sequencing data for BioProject PRJEB87483.

Platform	PacBio HiFi	Hi-C
ToLID	ilZetInsu1	ilZetInsu1
Specimen ID	LUNDPSY000082	LUNDPSY000082
BioSample (source individual)	SAMEA116129931	SAMEA116129931
BioSample (tissue)	SAMEA116130182	SAMEA116130176
Tissue	thorax	head
Instrument	Revio	Illumina NovaSeq X
Run accessions	ERR14835544	ERR14782893
Read count total	8.13 million	836.79 million
Base count total	81.10 Gb	126.35 Gb

Table 2.. Genome assembly statistics.

Assembly name	ilZetInsu1.hap1.1	ilZetInsu1.hap2.1
Assembly accession	GCA_965239635.1	GCA_965239655.1
Assembly level	chromosome	chromosome
Span (Mb)	862.07	869.20
Number of chromosomes	31	31
Number of contigs	307	296
Contig N50	12.72 Mb	13.86 Mb
Number of scaffolds	214	200
Scaffold N50	29.55 Mb	29.75 Mb
Longest scaffold length (Mb)	40.53	40.41
Sex chromosomes	Z	Z
Organelles	Mitochondrion: 15.77 kb	-

Table 3.. Chromosomal pseudomolecules in the haplotype 1 genome assembly of Zethes insularis ilZetInsu1 (haplotype 2 also at chromosome level).

INSDC accession	Molecule	Length (Mb)	GC%	Assigned Merian elements
OZ250797.1	1	37.50	37	M8
OZ250798.1	2	32.96	37.50	M11
OZ250799.1	3	32.10	37.50	M3
OZ250800.1	4	32.07	37.50	M2
OZ250801.1	5	32.06	37.50	M7
OZ250802.1	6	31.59	37	M9
OZ250803.1	7	31.02	37	M12
OZ250804.1	8	30.95	37.50	M17;M20
OZ250805.1	9	30.79	37.50	M23
OZ250806.1	10	30.48	37.50	M18
OZ250807.1	11	29.83	37.50	M1
OZ250808.1	12	29.66	37.50	M5
OZ250809.1	13	29.55	37.50	M14
OZ250810.1	14	29.11	37	M16
OZ250811.1	15	29.06	37.50	M21
OZ250812.1	16	28.89	37.50	M6
OZ250813.1	17	28.39	37	M13
OZ250814.1	18	28.18	37.50	M15
OZ250815.1	19	28.15	37.50	M10
OZ250816.1	20	26.94	37.50	M22
OZ250817.1	21	26.38	37.50	M4
OZ250818.1	22	25.57	37.50	M19
OZ250819.1	23	24.73	38	M24
OZ250820.1	24	24.39	38.50	M27
OZ250821.1	25	21.06	38	M26
OZ250822.1	26	20.56	39	M29
OZ250823.1	27	20.52	38	M28
OZ250824.1	28	17.29	39.50	M30
OZ250825.1	29	13.50	39	M25
OZ250826.1	30	9.67	39.50	M31
OZ250827.1	Z	40.53	36.50	MZ

Table 4.. Earth Biogenome Project summary metrics for the Zethes insularis assembly.

Measure	Value	Benchmark
EBP summary (haplotype 1)	7.C.Q62	6.C.Q40
Contig N50 length	12.72 Mb	≥ 1 Mb
Scaffold N50 length	29.55 Mb	= chromosome N50
Consensus quality (QV)	Haplotype 1: 62.3; haplotype 2: 62.3; combined: 62.3	≥ 40
k-mer completeness	Haplotype 1: 93.27%; Haplotype 2: 93.33%; combined: 99.79%	≥ 95%
BUSCO	C:98.7% [S:97.8%; D:0.9%]; F:0.4%; M:0.9%; n:5 286	S > 90%; D < 5%
Percentage of assembly assigned to chromosomes	99%	≥ 90%

Full text

Species taxonomy

Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Ecdysozoa; Panarthropoda; Arthropoda; Mandibulata; Pancrustacea; Hexapoda; Insecta; Dicondylia; Pterygota; Neoptera; Endopterygota; Amphiesmenoptera; Lepidoptera; Glossata; Neolepidoptera; Heteroneura; Ditrysia; Obtectomera; Noctuoidea; Erebidae; Erebinae; Zethes; Zethes insularis Rambur, 1833 (NCBI:txid1973593)

Background

The moth Zethes insularis Rambur, 1833 belongs to the family Erebidae, and is found in the Mediterranean region in Europe, with its distribution stretching through the Middle East to the Caucasus mountains ( GBIF Secretariat, 2025). The genus comprises some seven species, but Z. insularis is the only European representative ( Goater et al., 2003).

Not much is known about the biology of Z. insularis. It is found in dry, hot scrub habitats of the Mediterranean region. Its larvae have been recorded feeding on Pistacia terebinthus (Annacardiaceae), a small tree growing in Mediterranean scrub ( Wagner, 2025). The adults come to light and bait traps quite readily and can be quite common where the species is found. The adults fly from March to September in two or three generations ( Goater et al., 2003).

We present a chromosomally complete genome sequence for Zethes insularis, based on a male specimen collected near the town of Novigrad, Zadar County, Croatia ( Figure 1). The genome was sequenced as part of Project Psyche, a collaborative effort to sequence all named species of Lepidoptera found in Europe.

Voucher photograph of the Zethes insularis (ilZetInsu1) specimen used for genome sequencing.

Methods

Sample acquisition

The specimen used for genome sequencing was an adult male Zethes insularis (specimen ID LUNDPSY000082, ToLID ilZetInsu1; Figure 1), collected from Novigrad, Zadar, Croatia (latitude 44.1863, longitude 15.5421) on 2024-07-10. The specimen was collected and identified by Niklas Wahlberg (Lund University).

Nucleic acid extraction

Protocols for high molecular weight (HMW) DNA extraction developed at the Wellcome Sanger Institute (WSI) Tree of Life Core Laboratory are available on protocols.io ( Howard et al., 2025). The ilZetInsu1 sample was weighed and triaged to determine the appropriate extraction protocol. Tissue from the thorax was homogenised by powermashing using a PowerMasher II tissue disruptor.

HMW DNA was extracted in the WSI Scientific Operations core using the Automated MagAttract v2 protocol. DNA was sheared into an average fragment size of 12–20 kb following the Megaruptor®3 for LI PacBio protocol. Sheared DNA was purified by automated SPRI (solid-phase reversible immobilisation). The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit dsDNA High Sensitivity Assay kit. Fragment size distribution was evaluated by running the sample on the FemtoPulse system. For this sample, the final post-shearing DNA had a Qubit concentration of 33.23 ng/μL and a yield of 1 561.81 ng, with a fragment size of 15.6 kb.

PacBio HiFi library preparation and sequencing

Library preparation and sequencing were performed at the WSI Scientific Operations core. Libraries were prepared using the SMRTbell Prep Kit 3.0 (Pacific Biosciences, California, USA), according to the manufacturer’s instructions. The kit includes reagents for end repair/A-tailing, adapter ligation, post-ligation SMRTbell bead clean-up, and nuclease treatment. Size selection and clean-up were performed using diluted AMPure PB beads (Pacific Biosciences). DNA concentration was quantified using a Qubit Fluorometer v4.0 (ThermoFisher Scientific) and the Qubit 1X dsDNA HS assay kit. Final library fragment size was assessed with the Agilent Femto Pulse Automated Pulsed Field CE Instrument (Agilent Technologies) using the gDNA 55 kb BAC analysis kit.

The sample was sequenced on a Revio instrument (Pacific Biosciences). The prepared library was normalised to 2 nM, and 15 μL was used for making complexes. Primers were annealed and polymerases bound to generate circularised complexes, following the manufacturer’s instructions. Complexes were purified using 1.2X SMRTbell beads, then diluted to the Revio loading concentration (200–300 pM) and spiked with a Revio sequencing internal control. The sample was sequenced on a Revio 25M SMRT cell. The SMRT Link software (Pacific Biosciences), a web-based workflow manager, was used to configure and monitor the run and to carry out primary and secondary data analysis.

Specimen details, sequencing platforms, and data yields are summarised in Table 1.

Hi-C

** Sample preparation and crosslinking **

The Hi-C sample was prepared from 20–50 mg of frozen tissue from the head of the ilZetInsu1 sample using the Arima-HiC v2 kit (Arima Genomics). Following the manufacturer’s instructions, tissue was fixed and DNA crosslinked using TC buffer to a final formaldehyde concentration of 2%. The tissue was homogenised using the Diagnocine Power Masher-II. Crosslinked DNA was digested with a restriction enzyme master mix, biotinylated, and ligated. Clean-up was performed with SPRISelect beads before library preparation. DNA concentration was measured with the Qubit Fluorometer (Thermo Fisher Scientific) and Qubit HS Assay Kit. The biotinylation percentage was estimated using the Arima-HiC v2 QC beads.

** Hi-C library preparation and sequencing **

Biotinylated DNA constructs were fragmented using a Covaris E220 sonicator and size selected to 400–600 bp using SPRISelect beads. DNA was enriched with Arima-HiC v2 kit Enrichment beads. End repair, A-tailing, and adapter ligation were carried out with the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs), following a modified protocol where library preparation occurs while DNA remains bound to the Enrichment beads. Library amplification was performed using KAPA HiFi HotStart mix and a custom Unique Dual Index (UDI) barcode set (Integrated DNA Technologies). Depending on sample concentration and biotinylation percentage determined at the crosslinking stage, libraries were amplified with 10–16 PCR cycles. Post-PCR clean-up was performed with SPRISelect beads. Libraries were quantified using the AccuClear Ultra High Sensitivity dsDNA Standards Assay Kit (Biotium) and a FLUOstar Omega plate reader (BMG Labtech).

Prior to sequencing, libraries were normalised to 10 ng/μL. Normalised libraries were quantified again to create equimolar and/or weighted 2.8 nM pools. Pool concentrations were checked using the Agilent 4200 TapeStation (Agilent) with High Sensitivity D500 reagents before sequencing. Sequencing was performed using paired-end 150 bp reads on the Illumina NovaSeq X.

Specimen details, sequencing platforms, and data yields are summarised in Table 1.

Genome assembly

Prior to assembly of the PacBio HiFi reads, a database of k-mer counts ( k = 31) was generated from the filtered reads using FastK. GenomeScope2 ( Ranallo-Benavidez et al., 2020) was used to analyse the k-mer frequency distributions, providing estimates of genome size, heterozygosity, and repeat content.

The HiFi reads were assembled using Hifiasm in Hi-C phasing mode ( Cheng et al., 2021; Cheng et al., 2022), producing two haplotypes. Hi-C reads ( Rao et al., 2014) were mapped to the primary contigs using bwa-mem2 ( Vasimuddin et al., 2019). Contigs were further scaffolded with Hi-C data in YaHS ( Zhou et al., 2023), using the --break option for handling potential misassemblies. The scaffolded assemblies were evaluated using Gfastats ( Formenti et al., 2022), BUSCO ( Manni et al., 2021) and MERQURY.FK ( Rhie et al., 2020).

The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al., 2023), which runs MitoFinder ( Allio et al., 2020) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.

Assembly curation

The assembly was decontaminated using the Assembly Screen for Cobionts and Contaminants ( ASCC) pipeline. TreeVal was used to generate the flat files and maps for use in curation. Manual curation was conducted primarily in PretextView and HiGlass ( Kerpedjiev et al., 2018). Scaffolds were visually inspected and corrected as described by Howe et al. (2021). Manual corrections included 19 breaks and 66 joins. This reduced the scaffold count by 34.2%, increased the scaffold N50 by 2.3%, and reduced the total assembly length by 2.3%. The curation process is described at https://gitlab.com/wtsi-grit/rapid-curation. PretextSnapshot was used to generate a Hi-C contact map of the final assembly.

Assembly quality assessment

The Merqury.FK tool ( Rhie et al., 2020), run in a Singularity container ( Kurtzer et al., 2017), was used to evaluate k-mer completeness and assembly quality for both haplotypes using the k-mer databases ( k = 31) computed prior to genome assembly. The analysis outputs included assembly QV scores and completeness statistics.

The genome was analysed using the BlobToolKit pipeline, a Nextflow ( Di Tommaso et al., 2017) implementation of the earlier Snakemake version ( Challis et al., 2020). The pipeline aligns PacBio reads using minimap2 ( Li, 2018) and SAMtools ( Danecek et al., 2021) to generate coverage tracks. It runs BUSCO ( Manni et al., 2021) using lineages identified from the NCBI Taxonomy ( Schoch et al., 2020). For the three domain-level lineages, BUSCO genes are aligned to the UniProt Reference Proteomes database ( Bateman et al., 2023) using DIAMOND blastp ( Buchfink et al., 2021). The genome is divided into chunks based on the density of BUSCO genes from the closest taxonomic lineage, and each chunk is aligned to the UniProt Reference Proteomes database with DIAMOND blastx. Sequences without hits are chunked using seqtk and aligned to the NT database with blastn ( Altschul et al., 1990). The BlobToolKit suite consolidates all outputs into a blobdir for visualisation. The BlobToolKit pipeline was developed using nf-core tooling ( Ewels et al., 2020) and MultiQC ( Ewels et al., 2016), with containerisation through Docker ( Merkel, 2014) and Singularity ( Kurtzer et al., 2017).

We used lep_busco_painter to paint Merian elements along chromosomes ( Wright et al., 2024). Merian elements represent the 32 ancestral linkage groups in Lepidoptera. The painting process utilised BUSCO gene locations from the lepidoptera_odb10 set ( Kriventseva et al., 2019) and chromosome lengths from NCBI Datasets. Each complete BUSCO gene (both single-copy and duplicated) was assigned to a Merian element based on a reference database, then coloured and plotted along chromosomes drawn to scale.

Genome sequence report

Sequence data

PacBio sequencing of the Zethes insularis specimen generated 81.10 Gb (gigabases) from 8.13 million reads, which were used to assemble the genome. GenomeScope2.0 analysis estimated the haploid genome size at 867.89 Mb, with a heterozygosity of 0.24% and repeat content of 28.14% ( Figure 2). These estimates guided expectations for the assembly. Based on the estimated genome size, the sequencing data provided approximately 91× coverage. Hi-C sequencing produced 126.35 Gb from 836.79 million reads, which were used to scaffold the assembly. Table 1 summarises the specimen and sequencing details.

Frequency distribution of k-mers generated using GenomeScope2.The plot shows observed and modelled k-mer spectra, providing estimates of genome size, heterozygosity, and repeat content based on unassembled sequencing reads.

Assembly statistics

The genome was assembled into two haplotypes using Hi-C phasing. Haplotype 1 was curated to chromosome level, while haplotype 2 was assembled to scaffold level. The final assembly has a total length of 862.07 Mb in 214 scaffolds, with 93 gaps, and a scaffold N50 of 29.55 Mb ( Table 2).

Most of the assembly sequence (99.0%) was assigned to 31 chromosomal-level scaffolds, representing 30 autosomes and the Z sex chromosome. These chromosome-level scaffolds, confirmed by Hi-C data, are named according to size ( Figure 3; Table 3). Chromosome painting with Merian elements illustrates the distribution of orthologues along chromosomes and highlights patterns of chromosomal evolution relative to Lepidopteran ancestral linkage groups ( Figure 4). This genome has been assembled using PacBio and Hi-C data and phased. The result is two curated haplotypes.Z chromosome identified by Merian painting. A haplotypic inversion was observed in the region on chromosome 1 (34.8-37.4 Mbp), chromosome 6 (22.4–31.5 Mbp), and chromosome 9 (3.5-6.1 Mbp). The exact order and orientation of the contigs on chromosome 24 (12.0–18.3 Mbp) are unknown.

Hi-C contact map of the Zethes insularis genome assembly.Assembled chromosomes are shown in order of size and labelled along the axes. The plot was generated using PretextSnapshot.

Table 3.: Chromosomal pseudomolecules in the haplotype 1 genome assembly of Zethes insularis ilZetInsu1 (haplotype 2 also at chromosome level).

Merian elements painted across chromosomes in the ilZetInsu1.hap1.1 assembly of Zethes insularis.Chromosomes are drawn to scale, with the positions of orthologues shown as coloured bars. Each orthologue is coloured by the Merian element that it belongs to. All orthologues which could be assigned to Merian elements are shown.

The mitochondrial genome was also assembled (length 15.77 kb, OZ250828.1). This sequence is included as a contig in the multifasta file of the genome submission and as a standalone record.

Assembly quality metrics

For haplotype 1, the estimated QV is 62.3, and for haplotype 2, 62.3. When the two haplotypes are combined, the assembly achieves an estimated QV of 62.3. The k-mer completeness is 93.27% for haplotype 1, 93.33% for haplotype 2, and 99.79% for the combined haplotypes ( Figure 5). BUSCO analysis using the lepidoptera_odb10 reference set ( n = 5 286) identified 98.7% of the expected gene set (single = 97.8%, duplicated = 0.9%) in haplotype 1. For haplotype 2, BUSCO analysis identified 98.7% of the expected gene set (single = 98.0%, duplicated = 0.8%).

Evaluation of k-mer completeness using MerquryFK.This plot illustrates the recovery of k-mers from the original read data in the final assemblies. The horizontal axis represents k-mer multiplicity, and the vertical axis shows the number of k-mers. The black curve represents k-mers that appear in the reads but are not assembled. The green curve (the homozygous peak) corresponds to k-mers shared by both haplotypes and the red and blue curves (the heterozygous peaks) show k-mers found only in one of the haplotypes.

The snail plot in Figure 6 summarises the scaffold length distribution and other assembly statistics for haplotype 1. The blob plot in Figure 7 shows the distribution of scaffolds by GC proportion and coverage for haplotype 1.

Assembly metrics for ilZetInsu1.hap1.1.The BlobToolKit snail plot provides an overview of assembly metrics and BUSCO gene completeness. The circumference represents the length of the whole genome sequence, and the main plot is divided into 1,000 bins around the circumference. The outermost blue tracks display the distribution of GC, AT, and N percentages across the bins. Scaffolds are arranged clockwise from longest to shortest and are depicted in dark grey. The longest scaffold is indicated by the red arc, and the deeper orange and pale orange arcs represent the N50 and N90 lengths. A light grey spiral at the centre shows the cumulative scaffold count on a logarithmic scale. A summary of complete, fragmented, duplicated, and missing BUSCO genes in the set is presented at the top right. An interactive version of this figure can be accessed on the BlobToolKit viewer.

BlobToolKit GC-coverage plot for ilZetInsu1.hap1.1.Blob plot showing sequence coverage (vertical axis) and GC content (horizontal axis). The circles represent scaffolds, with the size proportional to scaffold length and the colour representing phylum membership. The histograms along the axes display the total length of sequences distributed across different levels of coverage and GC content. An interactive version of this figure is available on the BlobToolKit viewer.

Table 4 lists the assembly metric benchmarks adapted from Rhie et al. (2021) the Earth BioGenome Project Report on Assembly Standards September 2024. The EBP metric, calculated for the haplotype 1, is 7.C.Q62, meeting the recommended reference standard.

Table 4.: Earth Biogenome Project summary metrics for the Zethes insularis assembly.

Wellcome Sanger Institute – Legal and Governance

The materials that have contributed to this genome note have been supplied by a Tree of Life collaborator. 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 materialLegality of collection, transfer and use (national and international).

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