The genome sequence of a digger wasp, Ectemnius lituratus (Panzer,1805)

We present a genome assembly from an individual female Ectemnius lituratus (a digger wasp; Arthropoda; Insecta; Hymenoptera; Crabronidae). The genome sequence is 235.1 megabases in span. Most of the assembly is scaffolded into 13 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 29.67 kilobases in length. Gene annotation of this assembly on Ensembl identified 9,724 protein coding genes.


Background
Ectemnius lituratus is a small to medium sized digger wasp in the family Crabronidae.It is widespread across much of northern Europe, and in the UK it is common across southern England.It is black with yellow markings on the scapes, pronotum, scutellum, tibiae, tarsi and tergites.The clypeus is covered with shining silver hairs, the mesonotum is longitudinally striate, and there is a poorly-developed tooth midway along the inner margin of the mandibles.Unusually for aculeates, the male Ectemnius do not have an additional antennal segment, with both sexes possessing 12 segments.Male E. lituratus are one of two British species with simple untoothed antennae.
E. lituratus occurs in a range of habitats, especially woodlands and forested areas.It is univoltine, with a later flight period than other Ectemnius species, from June to September.Females hunt medium-sized Diptera, typically calypterates including Anthomyiidae and Cordyluridae (Lomholdt, 1975).Nests are constructed in cavities in dead wood, including dead trees, stumps and fence posts.Adults are strongly associated with the flowers of umbellifers, especially wild parsnip, cow-parsley, hogweed, wild angelica and greater burnet-saxifrage, on which they can be abundant (Falk, 1998).
The complete genome sequence for this species will facilitate studies into the evolution of hunting strategies, reproductive systems and Hymenopteran taxonomy.

Genome sequence report
The genome was sequenced from one female Ectemnius lituratus (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77,.A total of 110-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 144-fold coverage in 10X Genomics read clouds were generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 196 missing joins or mis-joins and removed one haplotypic duplication, reducing the scaffold number by 44.17%, and increasing the scaffold N50 by 143.70%. The final assembly has a total length of 235.1 Mb in 115 sequence scaffolds with a scaffold N50 of 16.9 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 (93.16%) of the assembly sequence was assigned to 13 chromosomal-level scaffolds.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 specimen used for genome sequencing and Hi-C data was a female Ectemnius lituratus (specimen ID Ox000161, ToLID iyEctLitu1), which was netted in Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude -1.34) on 2019-08-07.The specimen used for RNA sequencing, also a female (specimen ID Ox000178, ToLID iyEctLitu2) was collected in the same location on 2019-08-13.The specimens were collected and identified by Liam Crowley (University of Oxford) and preserved on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The iyEctLitu1 sample 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.High molecular weight (HMW) DNA was extracted using the Qiagen MagAttract HMW DNA extraction kit.Low molecular weight DNA was removed from a 20 ng aliquot of extracted DNA using the 0.8X AMpure XP purification kit prior to 10X Chromium sequencing; a minimum of 50 ng DNA was submitted for 10X sequencing.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 solid-phase 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 iyEctLitu2 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 and 10X Genomics read cloud 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), Illumina HiSeq 4000 (RNA-Seq) and HiSeq X Ten (10X) instruments.Hi-C data were also generated from abdomen tissue of iyEctLitu1 using the Arima2 kit and sequenced on the HiSeq X Ten 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).One round of polishing was performed by aligning 10X Genomics read data to the assembly with Long Ranger ALIGN, calling variants with FreeBayes   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 Ectemnius lituratus assembly (GCA_910593735.2).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

Natalia de Souza Araujo
Universite Libre de Bruxelles, Brussels, Brussels, Belgium The authors report the sequencing and assembly of the mitochondrial and nuclear genomes of the digger wasp Ectemnius lituratus.The nuclear genome assembly span is ~235Mb, most of which occurs within 13 chromosomal scaffolds and contains 9,724 annotated protein code genes and 1,423 non-coding genes.The mitochondrial genome reported has a size of ~29kb.
The authors give a brief introduction of the species biology, describe the sampling site, and have deposited the data in public sequencing repositories.The extraction and assembly methods are well established within the Darwin Tree of Life pipeline.In conclusion, this is a straightforward assembly report.

Some minor suggestions are:
In the Methods, Genome assembly, curation and evaluation section, the authors say "The mitochondrial genome was assembled using MitoHiFi…, which runs MitoFinder or MITOS…".Please specify which one was used for the annotation in this specific assembly.

1.
The mitochondrial genome annotation is not described in the report, and it is not clear if it contained or not all the expected genes not if the A+T region was recovered.

2.
Figure 5, it would be nice to have the scaffolds delimitation in the plot, it is not clear what in the map indicates the chromosomes delimitations.

3.
Is the rationale for creating the dataset(s) clearly described?Yes The data note is well written and to the point.My only suggestion is as follows: What taxonomic resource was used as the authority to confirm species identity?Report either taxonomic key or original species description.

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.

Figure 2 .
Figure 2. Genome assembly of Ectemnius lituratus, iyEctLitu1.2: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 235,170,256 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 (25,570,032 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (16,908,772 and 8,661,520 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 hymenoptera_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Ectemnius%20lituratus/dataset/CAJVAQ02.1/snail.

Figure 5 .
Figure 5. Genome assembly of Ectemnius lituratus, iyEctLitu1.2:Hi-C contact map of the iyEctLitu1.2assembly, 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=Ilu5QGtMSKO2rcgt0HMfYg.
Are the protocols appropriate and is the work technically sound?YesAre sufficient details of methods and materials provided to allow replication by others?YesAre the datasets clearly presented in a useable and accessible format?YesCompeting Interests: No competing interests were disclosed.Reviewer Expertise: Insect genomics and evolution 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.Reviewer Report 27 March 2024 https://doi.org/10.21956/wellcomeopenres.22520.r77330© 2024 Uhuad Koch J.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.Jonathan Berenguer Uhuad Koch USDA-ARS Pollinating Insect Research Unit, Maryland, USA The authors present a high quality genome assembly of the digger wasp, Ectemnius lituratus (Panzer, 1805).The genome assembly was achieved with HiFi, 10x, and HiC sequencing.Annotation of the genome was achieved through sequencing RNA for annotation with Ensembl pipeline.Then genome assembly is 235.1 Mb and scaffolded into 13 chromosomal pseudo molecules.

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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.