The genome sequence of the Green Silver-lines, Pseudoips prasinana (Linnaeus, 1758)

We present a genome assembly from an individual female Pseudoips prasinana (the Green Silver-lines; Arthropoda; Insecta; Lepidoptera; Nolidae). The genome sequence is 1,125.7 megabases in span. Most of the assembly is scaffolded into 33 chromosomal pseudomolecules, including the Z and W sex chromosomes. The mitochondrial genome has also been assembled and is 15.23 kilobases in length. Gene annotation of this assembly on Ensembl identified 20,065 protein coding genes.


Background
The Green Silver-lines Pseudoips prasinana is a nocturnal moth in the family Nolidae, superfamily Noctuioidea, found in woodland habitats across the Palaearctic from Portugal to Japan (GBIF Secretariat, 2023).The adult has orange antennae, pink front legs and bright green forewings crossed by a series of silver diagonal lines.Adults of P. prasinana are attracted to light, and can also be found resting in the foliage of trees or on low growing woodland plants (South, 1961).In Britain, the species is widely distributed across England and Wales, but more local in Scotland and Northern Ireland (NBN Atlas Partnership, 2023).It is locally common in Ireland (MothsIreland, 2024).
In Britain, adults of P. prasinana have been recorded primarily from May to June (NBN Atlas Partnership, 2023), with larvae developing through late summer and early autumn.The larvae feed on foliage of oak Quercus spp.and beech Fagus sylvatica trees, with pupation occurring inside a silken cocoon spun by the larva on the underside of leaves.The pupal stage overwinters.The similarity of the cocoon to those produced by silkmoths has prompted research into biochemistry of P. prasinana cocoon silks.A combination of X-ray analysis, proteomics and larval transcriptomics has revealed that P. prasinana produces some silk proteins with similar core amino acid composition to those of Bombyx mori, plus many additional silk proteins (Rindos et al., 2021).
Male P. prasinana produce ultrasonic and audible clicks from tymbal organs in a cleft on the second abdominal segment; these consist of a disk of flexible cuticle, an air-filled cavity and large fan-shaped muscles (Skals & Surlykke, 1999).The soundproducing organ is only found in males suggesting a role in sexual communication rather than as a bat defence system.
A genome sequence of the Green Silver-lines Pseudoips prasinana was determined as part of the Darwin Tree of Life project.The genome sequence will facilitate research into host plant specialisation and silk biochemistry, and will contribute to the growing set of resources for studying molecular evolution in insects.

Genome sequence report
The genome was sequenced from one female Pseudoips prasinana (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77,.A total of 33-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 41 missing joins or mis-joins and removed 14 haplotypic duplications, reducing the assembly length by 0.73% and the scaffold number by 26.32%, and decreasing the scaffold N50 by 1.03%.
The final assembly has a total length of 1125.7 Mb in 55 sequence scaffolds with a scaffold N50 of 37.0 Mb (Table 1).The snailplot in Figure 2 provides a summary of the assembly statistics, while the distribution of assembly scaffolds 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 (99.88%) of the assembly sequence was assigned to 33 chromosomallevel scaffolds, representing 31 autosomes and the Z and W sex chromosomes.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.

Genome annotation report
The Pseudoips prasinana genome assembly (GCA_951640165.1)was annotated at the European Bioinformatics Institute (EBI) using the Ensembl rapid annotation pipeline (  In sample preparation, the ilPsePras1 sample was weighed and dissected on dry ice (Jay et al., 2023).Tissue from the thorax and abdomen was homogenised using a PowerMasher II tissue disruptor (Denton et al., 2023a).HMW DNA was

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 a Pacific Biosciences SEQUEL II (HiFi) instrument.Hi-C data were also generated from thorax tissue of ilPsePras1 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.
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 Pretex-tView (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.
Table 3 contains a list of relevant software tool versions and sources.

Genome annotation
The BRAKER2 pipeline (Brůna et al., 2021) was used in the default protein mode to generate annotation for the Pseudoips   Douglas Boyes et.al report the sequencing, genome assembly and annotation of Pseudoips prasinana (a moth known the the Green Silver-lines, which reflect its forewing colour pattern).The natural history of this moth is engagingly presented and the genomic resources generated are of excellent quality, employed appropriate and well-documented methods with accessions available as stated in public databases.
I second the suggestion of the previous reviewer to add "moth" to the title.
Additional minor comments relate to templated parts of the report that I have raised previously.Namely, (1) that the geographical co-ordinates should be noted as latitude and longitude in the Genome Sequence Report section; (2) that kmer length should be reported for the MerquryFK evaluations and (3) that the bead:sample ratio used for AMPure cleanup should be 0.6x not 1.8x.

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: Genomics, Evolution, Bioinformatics 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.

Are the datasets clearly presented in a useable and accessible format? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Evolutionary biology of insects, phylogenomics 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 Pseudoips prasinana, ilPsePras1.1: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 1,125,692,588 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(46,196,481 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths(36,985,250 and 26,292,167 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/ilPsePras1_1/dataset/ilPsePras1_1/snail.
A female Pseudoips prasinana (specimen ID Ox000409, ToLID ilPsePras1) was collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude -1.34) on 2020-05-22 using a light trap.The specimen was collected and identified by Douglas Boyes (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, fragmentation, and clean-up.

Figure 3 .
Figure 3. Genome assembly of Pseudoips prasinana, ilPsePras1.1:BlobToolKit GC-coverage plot.Scaffolds are coloured by phylum.Circles are sized in proportion to scaffold length.Histograms show the distribution of scaffold length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/ilPsePras1_1/dataset/ilPsePras1_1/blob.

Figure 4 .
Figure 4. Genome assembly of Pseudoips prasinana, ilPsePras1.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all scaffolds.Coloured lines show cumulative lengths of scaffolds assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/ilPsePras1_1/dataset/ilPsePras1_1/cumulative.

Figure 5 .
Figure 5. Genome assembly of Pseudoips prasinana, ilPsePras1.1:Hi-C contact map of the ilPsePras1.1 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=SgMxdIG5RIWvn6p8zxCpMQ.

Table 2 . Chromosomal pseudomolecules in the genome assembly of Pseudoips prasinana, ilPsePras1.
Wellcome Sanger Institute -Legal and GovernanceThe 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.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.