The genome sequence of the Brown-spot Pinion, Agrochola litura (Linnaeus, 1761)

We present a genome assembly from an individual female Agrochola litura (the Brown-spot Pinion; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 772.2 megabases in span. Most of the assembly is scaffolded into 32 chromosomal pseudomolecules, including the W and Z sex chromosomes. The mitochondrial genome has also been assembled and is 15.55 kilobases in length. Gene annotation of this assembly on Ensembl identified 19,500 protein coding genes.

With a wingspan of 32-39 mm (forewing length 14-17 mm) (Bretherton et al., 1983;Waring et al., 2017), adults usually have brownish chestnut-coloured forewings, greyer towards the base, with five black marks or dashes evenly spaced along the costa, and a brown stain between the stigmata and the costa.
Agrochola litura occurs throughout Britain and the Scilly and Channel Islands but not Ireland (apart from two records in County Down) (Waring et al., 2017), and in Europe from Southern Scandinavia southwest to the Pyrenees and the northern edge of the Mediterranean, in Turkey, and in Western Asia as far east as the Caspian Sea (GBIF Secretariat, 2023).
In Britain the Brown-spot Pinion is found in a wide range of habitats including broad-leaved woodland and scrubland, hedgerows, parks, grasslands, heaths, fenland, and gardens (Waring et al., 2017).
The species is monovoltine, the adult flying in the Autumn (late August to October), relatively later in the South of Britain (Randle et al., 2019).It can be found feeding on overripe blackberries and ivy blossum.The species overwinters as an egg.In a study of noctuid moth fecundity in S. Bohemia (Spitzer et al., 1984), a female had a relatively low number of eggs (201), as well as a relatively low potential population growth rate compared to some migrant species.The larvae hatch in the Spring and are polyphagous on various herbaceous plants and grasses, later sometimes climbing onto trees such as Salix, Crataegus and Quercus to complete their growth (Bretherton et al., 1983).The greenish to pinkish brown larva (Henwood et al., 2020) makes a subterranean cocoon, usually in early June, and pupates within six weeks (Waring et al., 2017).
The species has been classed as vulnerable, with an annual decrease of 3.9%, a dramatic 82% decline in abundance overall, over 35 years in a study of Rothamsted trap catches (Conrad et al., 2006) The nearest species on BOLD appears to be Agrochola luteogrisea (Warren, 1911) (BIN, BOLD:AFE5283) which is around 4.76% divergent and which it closely resembles.This genome could help clarify phylogenetic relationships in this group of moths, and several other genomes of Agrochola (s.l.) are available (e.g. A. circellaris, A. lota, A. macilenta (Boyes et al., 2023a, Boyes et al., 2023b;Lees et al., 2023)).

Genome sequence report
The genome was sequenced from one female Agrochola litura (Figure 1) collected from Restharrow Dunes National Nature Reserve, England.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 36 missing joins or mis-joins and removed 6 haplotypic duplications, reducing the scaffold number by 13.24%.
The final assembly has a total length of 772.2 Mb in 58 sequence scaffolds with a scaffold N50 of 25.3 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 (99.86%) of the assembly sequence was assigned to 32 chromosomal-level scaffolds, representing 30 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 Agrochola litura genome assembly (GCA_949152395.1)was annotated using the Ensembl rapid annotation pipeline (Table 1; https://rapid.ensembl.org/Agrochola_litura_GCA_949152395.1/Info/Index).The resulting annotation includes 19,682 transcribed mRNAs from $PCG protein-coding and $NCG non-coding genes.purified following either the Manual solid-phase reversible immobilisation (SPRI) protocol (https://dx.doi.org/10.17504/protocols.io.kxygx3y1dg8j/v1), or the Automated SPRI protocol for (https://dx.doi.org/10.17504/protocols.io.q26g7p1wkgwz/v1) for higher throughput.In brief, the method employs a 1.8X ratio of AMPure PB beads to sample to eliminate shorter fragments and concentrate the DNA.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.

Sample acquisition and nucleic acid extraction
RNA was also extracted from abdomen tissue of ilAgrLitu1 in the Tree of Life Laboratory at the WSI using TRIzol, as per the protocol (https://dx.doi.org/10.17504/protocols.io.yxmvm334nl3p/v1).RNA was then eluted in 50 μl RNAse-free water and its concentration assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit RNA Broad-Range (BR) Assay kit.Analysis of the integrity of the RNA was done using the Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions.Poly(A) RNA-Seq libraries were constructed using the NEB Ultra II RNA Library Prep kit.DNA and RNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi) and Illumina NovaSeq 6000 (RNA-Seq) instruments.Hi-C data were also generated from remaining head and thorax tissue of ilAgrLitu1 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., 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 Agrochola litura assembly (GCA_949152395.1) in Ensembl Rapid Release.

Wellcome Sanger Institute -Legal and Governance
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', 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.The overarching areas of consideration are: • Ethical review of provenance and sourcing of the material

Muzafar Riyaz
Xavier Research Foundation, St Xavier's College, Palayamkottai, Tamil Nadu, India The paper provides valuable insights into the genomic characterization of A. litura, which could contribute to further research on phylogenetic relationships and conservation efforts within this moth group.Yes, the paper presents a clear rationale for creating the dataset by highlighting the importance of sequencing the genome of Agrochola litura for understanding phylogenetic relationships and aiding conservation efforts.The protocols employed for sample acquisition, nucleic acid extraction, sequencing, genome assembly, and annotation are described meticulously, indicating technically sound work.Furthermore, the paper provides comprehensive details of methods and materials, facilitating replication by others.The datasets, including genome assembly metrics, annotation results, and accession numbers, are presented clearly within the paper and deposited in public databases, ensuring usability and accessibility for other researchers.Therefore, the study appears to be methodologically robust, with transparent documentation of procedures and accessible datasets, enhancing its reliability and potential for scientific advancement.
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.

Simon H Martin
Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, UK This report describes a chromosome-scale genome assembly for the moth Agrochola litura.The approaches used are state-of-the-art and the assembly produced is of high quality.The assembly was produced from a single individual Agrochola litura using pacBio HiFi sequencing, which is excellent for insect genome assembly.Hi-C chromatin conformation sequencing was also used for scaffolding.The resulting assembly is therefore of excellent contiguity and sequence accuracy.I have no major concerns about the essmebly or article, but one minor concern about methodology reporting.
Minor points 1.The report states that RNA-seq data was generated, but it does not mention in the Genome Annotation section whether the RNA-seq data was used to support annotation.This should be clarified in the text.

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: Evolutionary genomics . Randle et al. (2019) characterised the Great Britain abundance decline between 1970 and 2016 of 73% as substantial, and distribution range has also declined in this period.DNA barcode records on BOLD (as of 19/10/2023) comprise a single Barcode Index Number or BIN (BOLD: AAC8167).

Figure 2 .
Figure 2. Genome assembly of Agrochola litura, ilAgrLitu1.1: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 772,191,765 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 (35,726,591 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (25,335,912 and 17,777,047 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/Agrochola%20litura/dataset/CASCJX01/snail.
Figure 3. Genome assembly of Agrochola litura, ilAgrLitu1.1: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/Agrochola%20litura/dataset/CASCJX01/blob.

Figure 5 .
Figure 5. Genome assembly of Agrochola litura, ilAgrLitu1.1:Hi-C contact map of the ilAgrLitu1.1 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=awQT5bdURuSuMjttcRBwEw.

Table 3 . Software tools: versions and sources. Software tool Version Source Open Peer Review Current Peer Review Status: Version 1
This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.