The genome sequence of the Common White Wave, Cabera pusaria (Linnaeus, 1758)

We present a genome assembly from an individual male Cabera pusaria (the Common White Wave; Arthropoda; Insecta; Lepidoptera; Geometridae). The genome sequence is 794.3 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.64 kilobases in length.


Background
Cabera pusaria is a moth in the family Geometridae, the second largest family of macro-moths in the British Isles, and like most of the members of this family has a thin body and triangular wings (Waring & Townsend, 2017).It has white wings with grey cross-lines, and can be distinguished from the similar Common Wave Cabera exanthemata by the straightness of the outer cross-lines towards the leading edge of the wing, compared to the curved lines in C. exanthemata (Skinner, 2009;Waring & Townsend, 2017).
C. pusaria is found throughout the British Isles where it is often disturbed from vegetation during the day and comes to light in small numbers, flying between late-May and early-September in the south where it is double-brooded and from May to July in the north where it is single-brooded.The larvae feed on a range of trees -including Downy Birch Betula pubescens, Silver Birch Betula pendula, Sallow Salix spp., Alder Alnus spp., Oak Quercus spp., and Sweet Chestnut Castanea sativa -from July to September, before pupating to overwinter (Skinner, 2009;Waring & Townsend, 2017).
While trace elements are important in the diet, high doses of the heavy metal manganese are fatal to the larvae and pupae of C. pusaria (Martinek et al., 2020), however, the larvae were the most numerous species on Birch Betula pendula in an area of the Czech Republic polluted by sulphur dioxide where they are considered pests (Kula et al., 2005).
We present a chromosomally complete genome sequence for Cabera pusaria, based on one male specimen collected using a mercury vapour light trap in a rural garden in the hamlet of Bratton, near Minehead, in Somerset, as part of the Darwin Tree of Life Project.This project is a collaborative effort to sequence all named eukaryotic species in the Atlantic Archipelago of Britain and Ireland.

Genome sequence report
The genome was sequenced from one male Cabera pusaria (Figure 1) collected from Bratton, Somerset, UK (51.20,.A total of 27-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 30 missing joins or mis-joins, reducing the assembly length by 0.75% and the scaffold number by 5.08%. The final assembly has a total length of 794.3 Mb in 55 sequence scaffolds with a scaffold N50 of 27.5 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 (99.72%) of the assembly sequence was assigned to 31 chromosomal-level scaffolds, representing 30 autosomes and the Z sex chromosome.Chromosome Z was assigned based on synteny to Petrophora chlorosata (GCA_951640565.1).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.

Sample acquisition and nucleic acid extraction
The specimens used in this study were collected in a light trap in a garden in Bratton, Somerset, UK (latitude 51.20, longitude -3.51) on 2022-06-20.The specimens were collected and identified by Denise Wawman (University of Oxford) and were snap-frozen on dry ice.The specimen with ID Ox002230 (ToLID ilCabPusa1) was used for DNA sequencing, while the specimen with ID Ox002236 (ToLID ilCabPusa2) was used for Hi-C analysis and RNA sequencing.The ilCabPusa1 sample was prepared for DNA extraction at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The specimen 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.DNA was extracted at the WSI Scientific Operations core using the Qiagen MagAttract HMW DNA kit, according to the manufacturer's instructions.
RNA was extracted from head tissue of ilCabPusa2 in the Tree of Life Laboratory at the WSI using TRIzol, according to the manufacturer's instructions.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 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 A Hi-C map for the final assembly was produced using bwa-mem2 (Vasimuddin et al., 2019) in the Cooler file format    Table 3. Software tools: versions and sources.

Software tool Version
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  Once, gene annotations will be available, it will be possible to determine which detoxification pathway are involved in the excretion of manganese.What genome modification have led to the capacities of some C pusaria populations to survive on high concentration of heavy metals?Are these genomic regions already involved in adaptation of lepidopteran to anthropisation of the environment?
I have other questions that could help understand the data presented in this manuscript.Could you explain why some "no hit data" in figure3 have the same GC contents as C pusaria?How could you differentiate these scaffolds from the C pusaria genome scaffold?Where you able to find DNA from bacteria, fungi or viruses that could also give information of this insect physiology?Could you give the main origine of these contaminants.

Suriya Narayanan Murugesan
National University of Singapore,, Singapore, Singapore Denise C. Wawman presents a report on the complete genome assembly of Cabera pusaria.The assembly was generated from a single male individual, with 99.72% of the sequences assigned to 31 chromosomes using Hi-C data.This work is a valuable addition to the growing collection of Lepidoptera genome assemblies and will be very helpful to the community.I have only a few minor suggestions: 1.The author mentioned that annotation will be done using the Ensembl pipeline with the collected RNA-Seq data.It would be appreciated if the author could include the annotation information once it's ready, along with the BUSCO score obtained using protein sequences from the annotation, in the revision.
2. The author mentioned that Chromosome Z was assigned based on synteny to Petrophora chlorosata.It would be helpful to include a line or two on how the synteny analysis was conducted and the level of synteny observed between the two species.
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: Evo-Devo, Genetics 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 Cabera pusaria, ilCabPusa1.1: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 794,294,107 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 (35,978,249 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (27,510,719 and 19,118,028 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/ilCabPusa1.1/dataset/CATOZX01/snail.

Figure 5 .
Figure 5. Genome assembly of Cabera pusaria, ilCabPusa1.1:Hi-C contact map of the ilCabPusa1.1 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=ZJRZI5v_TS6R5ve-N46dqA.
Sophia Agrobiotech, Universite Cote d'Azur, Nice, Provence-Alpes-Côte d'Azur, France The manuscript describes the obtention of the genome of Cabera pusaria at the chromosome level.The genome of C pusaria could be used for the study of lepidopteran adaptation to heavy metal.Genomic comparison of the populations showing different level of tolerance to heavy metal could also be performed.Martinek et al 2020 [Ref-1] described a population of C pusaria able to eliminate excess of the heavy metal manganese.More genomic formation are needed to understand how insect populations adapt to polluted environment (Kozlov,2022) [Ref-2] A complete genome publication should propose a gene annotation that is still missing here.I suggest to update the article with this data.

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? Partly Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests:
No competing interests were disclosed.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
Reviewer Report 02 May 2024 https://doi.org/10.21956/wellcomeopenres.22297.r79544© 2024 Murugesan S. 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.