The genome sequence of a darkling beetle, Lagria hirta (Linnaeus, 1758)

We present a genome assembly from an individual male Lagria hirta (darkling beetle; Arthropoda; Insecta; Coleoptera; Tenebrionidae). The genome sequence is 336.8 megabases in span. Most of the assembly is scaffolded into 12 chromosomal pseudomolecules, including the X and Y sex chromosomes. The mitochondrial genome has also been assembled and is 16.12 kilobases in length. Gene annotation of this assembly on Ensembl identified 12,850protein coding genes.


Background
Lagria hirta (Linnaeus, 1758) is widely distributed in the western Palaearctic (across the whole of Europe, Algeria and Morocco) and in Asia (Russia: Western and Eastern Siberia, Israel, Cyprus, Turkey, Iran, Iraq, Kazakhstan, Turkmenistan, Uzbekistan and Tajikistan) (Merkl, 2006).
Lagria hirta is the only common species of the subfamily Lagriinae (Coleoptera, Tenebrionidae) in Central Europe (Zhou, 2001), and one of only two species occurring in Britain.They are both elongate beetles, with a black head, pronotum and legs and pubescent elytra that are yellow to dark brown.They are sexually dimorphic, with the males more elongate and parallel sided, the females with the abdomen and elytra broader and more drop shaped.With a body length of 7-10 mm, L. hirta is slightly smaller than the very similar but much rarer Lagria atripes (10-12 mm) and also has proportionately smaller eyes.L. hirta has a strongly punctured pronotum (smoother and more glabrous in L. atripes) and the pubescence of the elytra is simple (arranged in a 'herring bone pattern' in L. atripes) (Buck, 1954).L. atripes is restricted to a few good quality deciduous forests in southern England.
Lagria hirta is crepuscular and nocturnal.The beetles can be encountered on flowers and shrubs.They are polyphagous and feed on young leaves, nectar and pollen (Hůrka, 2005;Zhou, 2001).They were also reported to cause damage to spruce by feeding on young shoots, but are not of economic importance unlike some closely related species from Africa, Australia and Asia (Postner, 1961).The brownish larvae are saprophagous (Zhou, 2001), free living, can be swept from vegetation at night, and have a distinctive appearance with tufts of erect setae on the sides of the abdominal segments, and two small triangular urogomphi at the apex of the abdomen.This species has an annual life cycle, with adults active from late May or June, peaking in July, and with females sometimes surviving well into September.The eggs are laid in batches of approximately 80 at a time into soil or leaf litter.Unmated females can lay a batch of eggs, but these are not fertile and will not hatch (Stammer, 1929;Zhou, 2001).In laboratory conditions egg hatching occurred in temperatures of 10-25 °C, while at 5 °C and 30 °C all eggs failed (Zhou & Topp, 2003).Typically, there are 8-10 larval instars (Zhou, 1996;Zhou & Topp, 2003).The larva requires a period of cooler temperatures to progress to pupation.The experiments conducted in laboratory conditions showed that larvae reared at constant temperatures and without a chilling period were unable to pupate successfully.Some reached the 9-10 th instar, hardly any pupated and all subsequently died (Zhou & Topp, 2003).In natural conditions L. hirta overwinters as a larva and pupation occurs in early May (Postner, 1961).
Throughout their life cycle Lagria beetles live in symbiosis with antibiotic producing bacteria dominated by the genus Burkholderia.They are transferred from female accessory glands to an egg during oviposition and protect it against pathogenic fungi.Subsequently these bacteria colonize specialized dorsal cuticular structures of the mesothorax, metathorax, and the first abdominal segment in both sexes of larvae.They are absent in adult males (Flórez, 2016;Flórez & Kaltenpoth, 2017;Flórez et al., 2017;Janke et al., 2022;Stammer, 1929).The study conducted on closely related L. villosa showed the beetles can also acquire bacteria from dry leaf litter and in turn bacteria can be transferred from a beetle host to the soil, leaf litter or a plant (Wierz et al., 2021).
Lagria hirta is widely distributed in Britain, common in Wales and England north to South Yorkshire, with scattered records up to north-eastern Scotland (UK Beetles, no date).It can be found in woodland, at the edges of broadleaved and coniferous forests, coastal scrub and sand dunes, urban parks and gardens, meadows, clearings, near old hedges, on grasses, flowers and shrubs, and can be attracted to light (Hůrka, 2005;James, 2018).It is the most common and widespread member of the family Tenebrionidae in the United Kingdom.
The high-quality genome sequence described here is the first one reported for Lagria hirta.It was sequenced from one male specimen from Penhale Dunes, England.It will aid research on the biology, phylogeny and ecology of this species.The genome has been generated as part of the Darwin Tree of Life Project, 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 Lagria hirta (Figure 1) collected from Penhale Dunes, England, UK (50.37, -5.14).A total of 72-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 70 missing joins or misjoins and removed 3 haplotypic duplications, reducing the assembly length by 0.25% and the scaffold number by 67.5%, and increasing the scaffold N50 by 4.31%.
The final assembly has a total length of 336.8 Mb in 25 sequence scaffolds with a scaffold N50 of 31.9Mb (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 (99.73%) of the assembly sequence was assigned to 12 chromosomal-level scaffolds, representing 10 autosomes and the X and Y 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.
The estimated Quality Value (QV) of the final assembly is 67 with k-mer completeness of 100%, and the assembly has a BUSCO v5.3.2 completeness of 98.0% (single = 97.1%,Metadata for specimens, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/296003.

Sample acquisition and nucleic acid extraction
Lagria hirta specimens collected from Penhale Dunes, Cornwall, England, UK (latitude 50.37, longitude -5.14) on 2021-06-30 were used in this study.The specimen used for DNA sequencing was a male (specimen ID NHMUK014537045, ToLID icLagHirt1), while the specimen used for Hi-C was specimen ID NHMUK014537046 (ToLID icLagHirt2).The specimens were collected by Olga Sivell (Natural History Museum) using an aerial net.The specimens were identified by Brian Levey (National Museums Galleries of Wales) and dry frozen at -80°C.
The female specimen used for RNA sequencing (specimen ID NHMUK014439765, ToLID icLagHirt3), was collected from Fulham Green, London (latitude 51.47, longitude -0.18) on 021-08-04.This specimen was collected and identified by Maxwell Barclay (Natural History Museum) and then dry frozen at -80°C.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The icLagHirt1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Tissue from the whole organism 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 solidphase 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.
RNA was extracted from head and thorax tissue of icLagHirt3 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 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 whole organism tissue of icLagHirt2 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 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., 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 Ensembl gene annotation system (Aken et al., 2016) was used to generate annotation for the Lagria hirta assembly (GCA_947359425.1).Annotation was created primarily through alignment of transcriptomic data to the genome, with gap filling via protein-to-genome alignments of a select set of proteins from UniProt (UniProt Consortium, 2019).

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

Manee M Manee
King Abdulaziz City for Science and Technology, Riyadh, Saudi Arabia The manuscript titled "The Genome Sequence of a Darkling Beetle, Lagria hirta," presents a detailed account of the genomic analysis of Lagria hirta, including the assembly and annotation of its genome.Here are specific areas for improvement: -Enhancing the background with a detailed phylogenetic positioning of Lagria hirta within the Tenebrionidae family, alongside a comprehensive comparative genomic analysis with its close relatives and other family members, could significantly enrich the context.This approach would illuminate evolutionary adaptations, gene family dynamics, and unique genomic features, offering a deeper understanding of Lagria hirta's significance and its comparative evolutionary biology within the beetle order.
-To enhance methodological clarity, detail the bioinformatics workflows, specify parameter settings for genome assembly and annotation, and justify the selections of tools.Include criteria for software choices, details of custom scripts, and their impact on result accuracy and reproducibility.

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?Partly

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

Reinhard Predel
University of Cologne, Cologne, Germany The genome sequence of Lagria hirta is provided, the assembly is of very good quality.It is the first genome of a species from the subfamily Lagriinae, transcriptomes of that species have been previously published.The background provided is restricted to some easily accessible information on morphology and natural history.I miss some more detailed information about the phylogenetic position of the Lagriinae within the hyperdiverse Tenebrionidae.The first beetle whose genome was sequenced, Tribolium castaneum, also belongs to Tenebrionidae, and the genome of this species is now one of the most intensively studied in insects.I think this could be important background information, the report would also benefit from some comparative information, e.g. about genome size of Tribolium compared to Lagria, etc.

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.

Yi-Ming Weng
Department of Entomology, University of Wisconsin Madison, Madison, WI, USA The genome assembly of the darkling beetle by Sivell et al., is reviewed.The report contains sufficient details in describing the general biology, morphology, and genomic feature of this species.The assembly is very complete and well represented and the gene annotation process also includes the transcriptomic information, demonstrating it's high data quality.This will be a good resource for many future studies.

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: Entomology, population genetics, evolutionary genomics 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 1 .
Figure 1.Photograph of the Lagria hirta (Linnaeus, 1758) specimen NHMUK014537045 taken during sample preservation and processing.a) A habitus of the specimen in dorsal view.b) The specimen in ventral view.

Figure 2 .
Figure 2. Genome assembly of Lagria hirta, icLagHirt1.1: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 336,783,501 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 (57,187,386 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (31,908,519 and 21,721,301 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 endopterygota_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Lagria%20hirta/dataset/CANAHW01/snail.

Figure 3 .
Figure 3. Genome assembly of Lagria hirta, icLagHirt1.1: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/Lagria%20hirta/dataset/CANAHW01/blob.

Figure 4 .
Figure 4. Genome assembly of Lagria hirta, icLagHirt1.1: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/Lagria%20hirta/dataset/CANAHW01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Lagria hirta, icLagHirt1.1:Hi-C contact map of the icLagHirt1.1 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=WQn2sjdzQb6h70_XxZBwKQ.
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.Reviewer Report 20 November 2023 https://doi.org/10.21956/wellcomeopenres.22404.r69679© 2023 Weng Y.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.

Table 3 . Software tools: versions and sources. Software tool Version 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.