The genome sequence of an ichneumonid wasp, Gelis areator (Panzer, 1804)
James McCulloch, Liam M. Crowley, Gavin R. Broad, Natalia Araujo, Danilo Trabudo do Amaral, Sarah Inwood, Andrew Legan
TL;DR
This paper provides the genome sequence of the ichneumonid wasp Gelis areator, including a detailed assembly of its chromosomes and mitochondrial DNA.
Contribution
The novel contribution is the first genome assembly for Gelis areator, including chromosomal pseudomolecules and mitochondrial DNA.
Findings
The genome assembly is 228.10 megabases in total length.
98.97% of the assembly is organized into 12 chromosomal pseudomolecules.
The mitochondrial genome is 25.02 kilobases in length.
Abstract
We present a genome assembly from an individual female Gelis areator (ichneumonid wasp; Arthropoda; Insecta; Hymenoptera; Ichneumonidae). The genome sequence has a total length of 228.10 megabases. Most of the assembly (98.97%) is scaffolded into 12 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 25.02 kilobases in length.
Genes, proteins, chemicals, diseases, species, mutations and cell lines named across the full text — each resolved to its canonical identifier and authoritative record.
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Figure 1
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Figure 5| Project information | |||
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| PRJEB70759 | ||
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| SAMEA112232712 | ||
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| 2163653 | ||
| Specimen information | |||
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| iyGelArea1 | SAMEA112233198 | Whole organism |
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| iyGelArea1 | SAMEA112233198 | Whole organism |
| Sequencing information | |||
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| ERR12342503 | 7.22e+08 | 109.06 |
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| ERR12340116 | 1.91e+06 | 19.38 |
| Genome assembly | ||
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| Assembly name | iyGelArea1.hap1.1 | |
| Assembly accession (haplotype 1) | GCA_964059375.1 | |
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| Span (Mb) | 228.10 | |
| Number of contigs | 480 | |
| Number of scaffolds | 139 | |
| Longest scaffold (Mb) | 27.56 | |
| Assembly metrics for GCA_964059375.1
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| Contig N50 length (Mb) | 1.3 |
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| Scaffold N50 length (Mb) | 19.4 |
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| Consensus quality (QV) | 62.1 |
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| 100.0% |
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| BUSCO
| C:95.4%[S:94.9%,D:0.5%],
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| Percentage of assembly
| 98.97% |
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| Sex chromosomes | None |
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| Organelles | Mitochondrial genome:
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| INSDC
| Name | Length
| GC% |
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| 1 | 27.51 | 39.0 | |
| 2 | 24.35 | 39.5 | |
| 3 | 25.98 | 38.5 | |
| 4 | 22.74 | 38.5 | |
| 5 | 19.4 | 39.0 | |
| 6 | 18.36 | 40.0 | |
| 7 | 17.25 | 38.5 | |
| 8 | 15.96 | 39.0 | |
| 9 | 15.44 | 39.5 | |
| 10 | 14.1 | 39.0 | |
| 11 | 10.42 | 39.0 | |
| 12 | 8.55 | 38.5 | |
| MT | 0.03 | 16.0 |
| Software
| Version | Source |
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| BEDTools | 2.30.0 |
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| BLAST | 2.14.0 |
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| BlobToolKit | 4.3.7 |
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| BUSCO | 5.4.3 and 5.5.0 |
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| bwa-mem2 | 2.2.1 |
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| Cooler | 0.8.11 |
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| DIAMOND | 2.1.8 |
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| fasta_windows | 0.2.4 |
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| FastK | 427104ea91c78c3b8b8b49f1a7d6bbeaa869ba1c |
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| Gfastats | 1.3.6 |
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| GoaT CLI | 0.2.5 |
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| Hifiasm | 0.19.8-r603 |
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| HiGlass | 44086069ee7d4d3f6f3f0012569789ec138f42b84a
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| Merqury.FK | d00d98157618f4e8d1a9190026b19b471055b22e |
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| MitoHiFi | 3 |
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| MultiQC | 1.14, 1.17, and 1.18 |
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| NCBI Datasets | 15.12.0 |
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| Nextflow | 23.04.0-5857 |
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| PretextView | 0.2 |
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| samtools | 1.16.1, 1.17, and 1.18 |
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| sanger-tol/ascc | - |
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| sanger-tol/
| 1.1.1 |
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| sanger-tol/
| 1.2.1 |
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| Seqtk | 1.3 |
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| Singularity | 3.9.0 |
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| TreeVal | 1.0.0 |
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| YaHS | 1.2a.2 |
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- —Wellcome Trust
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Taxonomy
TopicsHymenoptera taxonomy and phylogeny · Insect Resistance and Genetics · Insect-Plant Interactions and Control
Species taxonomy
Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Ecdysozoa; Panarthropoda; Arthropoda; Mandibulata; Pancrustacea; Hexapoda; Insecta; Dicondylia; Pterygota; Neoptera; Endopterygota; Hymenoptera; Apocrita; Ichneumonoidea; Ichneumonidae; Phygadeuontinae; Phygadeuontini; Gelina; Gelis; Gelis areator (Panzer, 1804) (NCBI:txid2163653).
Background
Gelis areator is a rather small (3–4 mm long), strikingly patterned ichneumonid wasp. The wings are black-banded, and the body has a variable amount of red, contrasting with the black ground colour. As with many other species of the subfamily Phygadeuontinae , G. areator is an idiobiont ectoparasitoid of cocooned hosts: the host is permanently paralysed and eaten from the outside by the wasp larva ( Broad et al., 2018). G. areator can often be found on tree trunks and foliage, searching for hosts, and are frequent at light, being at least partly nocturnal. It parasitises a wide range of hosts, primarily small Lepidoptera in cases or cocoons, such as Coleophora species, but also including a range of parasitoids Hymenoptera cocoons and sometimes other insect orders, as summarised by Schwarz and Shaw (1999) Gelis areator is found across the Palaearctic and into the Oriental region in China ( Schwarz, 2009). This species has also been accidentally introduced to other regions (e.g. Mason, 1978); Schwarz and Shaw (1999) recorded G. areator from across Britain and Ireland and from spring through to autumn in several generations.
Most Gelis species have wingless (apterous) females but usually fully winged (macropterous) males. The relatively small number of species with macropterous females can be identified as Gelis by the mandibles, which are strongly swollen proximally, with a vertical groove at the base, and the fore wing pterostigma, which is broadly triangular with a white apex. Many macropterous species are classified in the areator species group, most with red markings and dark patches on the wings. Species can be identified using Schwarz (2009) The extent of red patterning is very variable and identification not always straightforward, but the exact pattern of red, together with the extent of the black wing bands and the shape of the area superomedia on the propodeum, are usually sufficient to identify G. areator.
Host ranges within Gelis, and across the subfamily Phygadeuontinae as a whole, are fascinatingly varied. Here we present a chromosomally complete genome sequence for Gelis areator, based on a specimen from Wytham Woods, Berkshire, United Kingdom. The increasing availability of genomes should allow for better understanding of the drivers behind the massive success of these parasitoid wasps.
Genome sequence report
The genome of an adult specimen of Gelis areator ( Figure 1) was sequenced using Pacific Biosciences single-molecule HiFi long reads, generating a total of 19.38 Gb (gigabases) from 1.91 million reads, providing approximately 86-fold coverage. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data, which produced 109.06 Gb from 722.28 million reads, yielding an approximate coverage of 478-fold. Specimen and sequencing details are provided in Table 1.
Photograph of the Gelis areator (iyGelArea1) specimen used for genome sequencing.
Table 1.: Specimen and sequencing data for Gelis areator.
Assembly errors were corrected by manual curation, including 317 missing joins or mis-joins and 11 haplotypic duplications. This reduced the assembly length by 2.51% and the scaffold number by 49.46%, and increased the scaffold N50 by 22.01%. The final assembly has a total length of 228.10 Mb in 139 sequence scaffolds, with 340 gaps, and a scaffold N50 of 19.4 Mb ( Table 2).
Table 2.: Genome assembly data for Gelis areator, iyGelArea1.hap1.1.
The snail plot 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.
Genome assembly of Gelis areator, iyGelArea1.hap1.1: metrics.The BlobToolKit snail plot 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 228,171,769 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 (27,505,580 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (19,395,723 and 10,418,536 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/GCA_964059375.1/dataset/GCA_964059375.1/snail.
Genome assembly of Gelis areator: Blot plot of base coverage in the raw data against GC proportion for sequences in iyGelArea1.hap1.1.Sequences are coloured by phylum. Circles are sized in proportion to sequence length. Histograms show the distribution of sequence length sum along each axis. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/GCA_964059375.1/dataset/GCA_964059375.1/blob.
Genome assembly of Gelis areator iyGelArea1.hap1.1: 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/GCA_964059375.1/dataset/GCA_964059375.1/cumulative.
Most (98.97%) of the assembly sequence was assigned to 12 chromosomal-level scaffolds. Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size ( Figure 5; Table 3). During manual curation, it was observed that the order and orientation of scaffolds are uncertain on Chromosome 2 in the region from ~4.10–5.41Mb.
Genome assembly of Gelis areator iyGelArea1.hap1.1: Hi-C contact map of the iyGelArea1.hap1.1 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=JclKKjSsSiOJzmP9tCGRTg.
Table 3.: Chromosomal pseudomolecules in the genome assembly of Gelis areator, iyGelArea1.
Hi-C data allowed phasing into two haplotypes. Haplotype 1 was curated to chromosome level, while Haplotype 2 was deposited at scaffold level. 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 62.1 with k-mer completeness of 100.0%, and the assembly has a BUSCO v5.4.3 completeness of 95.4% (single = 94.9%, duplicated = 0.5%), using the hymenoptera_odb10 reference set ( n = 5,991).
Metadata for specimens, BOLD barcode results, spectra estimates, sequencing runs, contaminants and pre-curation assembly statistics are given at https://links.tol.sanger.ac.uk/species/2163653.
Methods
Sample acquisition and DNA barcoding
An adult Gelis areator (specimen ID Ox002512, ToLID iyGelArea1) was collected from Wytham Woods, Oxfordshire, United Kingdom (latitude 51.78, longitude –1.33) on 2022-07-20, using a sweep net. The specimen was collected by James McCulloch and Liam Crowley (University of Oxford), identified by James McCulloch and then preserved on dry ice.
The initial identification was verified by an additional DNA barcoding process according to the framework developed by Twyford et al. (2024). A small sample was dissected from the specimens and stored in ethanol, while the remaining parts were shipped on dry ice to the Wellcome Sanger Institute (WSI). The tissue was lysed, the COI marker region was amplified by PCR, and amplicons were sequenced and compared to the BOLD database, confirming the species identification ( Crowley et al., 2023). Following whole genome sequence generation, the relevant DNA barcode region was also used alongside the initial barcoding data for sample tracking at the WSI ( Twyford et al., 2024). The standard operating procedures for Darwin Tree of Life barcoding have been deposited on protocols.io ( Beasley et al., 2023).
Nucleic acid extraction
The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) Tree of Life Core Laboratory includes a sequence of core procedures: sample preparation and homogenisation, DNA extraction, fragmentation and purification. Detailed protocols are available on protocols.io ( Denton et al., 2023b). The iyGelArea1 sample was prepared for DNA extraction by weighing and dissecting it on dry ice ( Jay et al., 2023). Tissue from the whole organism was homogenised using a PowerMasher II tissue disruptor ( Denton et al., 2023a).
HMW DNA was extracted in the WSI Scientific Operations core using the Automated MagAttract v2 protocol ( Oatley et al., 2023). The DNA was sheared into an average fragment size of 12–20 kb in a Megaruptor 3 system ( Bates et al., 2023). Sheared DNA was purified by solid-phase reversible immobilisation, using AMPure PB beads to eliminate shorter fragments and concentrate the DNA ( Strickland et al., 2023). The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit dsDNA High Sensitivity Assay kit. Fragment size distribution was evaluated by running the sample on the FemtoPulse system.
Hi-C preparation
Tissue from the iyGelArea1 sample was processed at the WSI Scientific Operations core, using the Arima-HiC v2 kit. In brief, frozen tissue (stored at –80 °C) was fixed, and the DNA crosslinked using a TC buffer with 22% formaldehyde. After crosslinking, the tissue was homogenised using the Diagnocine Power Masher-II and BioMasher-II tubes and pestles. Following the kit manufacturer's instructions, crosslinked DNA was digested using a restriction enzyme master mix. The 5’-overhangs were then filled in and labelled with biotinylated nucleotides and proximally ligated. An overnight incubation was carried out for enzymes to digest remaining proteins and for crosslinks to reverse. A clean up was performed with SPRIselect beads prior to library preparation.
Library preparation and sequencing
Pacific Biosciences SMRTbell libraries were constructed using the Revio HiFi prep kit, according to the manufacturers’ instructions. DNA sequencing was performed by the Scientific Operations core at the WSI on a Pacific Biosciences Revio instrument.
For Hi-C library preparation, DNA was fragmented to a size of 400 to 600 bp using a Covaris E220 sonicator. The DNA was then enriched, barcoded, and amplified using the NEBNext Ultra II DNA Library Prep Kit following manufacturers’ instructions. The Hi-C sequencing was performed using paired-end sequencing with a read length of 150 bp on an Illumina NovaSeq 6000 instrument.
Genome assembly, curation and evaluation
** Assembly **
The HiFi reads were first assembled using Hifiasm ( Cheng et al., 2021), using the Hi-C phasing mode. The Hi-C reads were mapped to the primary contigs using bwa-mem2 ( Vasimuddin et al., 2019). The contigs were further scaffolded using the provided Hi-C data ( Rao et al., 2014) in YaHS ( Zhou et al., 2023) using the --break option. The scaffolded assemblies were evaluated using Gfastats ( Formenti et al., 2022), BUSCO ( Manni et al., 2021) and MERQURY.FK ( Rhie et al., 2020).
The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al., 2023), which runs MitoFinder ( Allio et al., 2020) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.
** Assembly curation **
The assembly was decontaminated using the Assembly Screen for Cobionts and Contaminants (ASCC) pipeline (article in preparation). Both haplotypes were combined for curation, and one haplotype was curated to chromosome level while the other haplotype was submitted as a scaffold-level assembly. Flat files and maps used in curation were generated in TreeVal ( Pointon et al., 2023). Manual curation was primarily conducted using PretextView ( Harry, 2022), with additional insights provided by JBrowse2 ( Diesh et al., 2023) and HiGlass ( Kerpedjiev et al., 2018). Scaffolds were visually inspected and corrected as described by Howe et al. (2021). Any identified contamination, missed joins, and mis-joins were corrected, and duplicate sequences were tagged and removed. The curation process is documented at https://gitlab.com/wtsi-grit/rapid-curation (article in preparation).
** Evaluation of the final assembly **
The final assembly was post-processed and evaluated using the three Nextflow ( Di Tommaso et al., 2017) DSL2 pipelines: sanger-tol/readmapping ( Surana et al., 2023a), sanger-tol/genomenote ( Surana et al., 2023b), and sanger-tol/blobtoolkit ( Muffato et al., 2024). The readmapping pipeline aligns the Hi-C reads using bwa-mem2 ( Vasimuddin et al., 2019) and combines the alignment files with SAMtools ( Danecek et al., 2021). The genomenote pipeline converts the Hi-C alignments into a contact map using BEDTools ( Quinlan & Hall, 2010) and the Cooler tool suite ( Abdennur & Mirny, 2020). The contact map is visualised in HiGlass ( Kerpedjiev et al., 2018). This pipeline also generates assembly statistics using the NCBI datasets report ( Sayers et al., 2024), computes k-mer completeness and QV consensus quality values with FastK and MERQURY.FK, and runs BUSCO ( Manni et al., 2021) to assess completeness.
The blobtoolkit pipeline is a Nextflow port of the previous Snakemake Blobtoolkit pipeline ( Challis et al., 2020). It aligns the PacBio reads in SAMtools and minimap2 ( Li, 2018) and generates coverage tracks for regions of fixed size. In parallel, it queries the GoaT database ( Challis et al., 2023) to identify all matching BUSCO lineages to run BUSCO ( Manni et al., 2021). For the three domain-level BUSCO lineages, the pipeline aligns the BUSCO genes to the UniProt Reference Proteomes database ( Bateman et al., 2023) with DIAMOND ( Buchfink et al., 2021) blastp. The genome is also split into chunks according to the density of the BUSCO genes from the closest taxonomic lineage, and each chunk is aligned to the UniProt Reference Proteomes database with DIAMOND blastx. Genome sequences without a hit are chunked with seqtk and aligned to the NT database with blastn ( Altschul et al., 1990). The blobtools suite combines all these outputs into a blobdir for visualisation.
The genome assembly and evaluation pipelines were developed using nf-core tooling ( Ewels et al., 2020) and MultiQC ( Ewels et al., 2016), relying on the Conda package manager, the Bioconda initiative ( Grüning et al., 2018), the Biocontainers infrastructure ( da Veiga Leprevost et al., 2017), as well as the Docker ( Merkel, 2014) and Singularity ( Kurtzer et al., 2017) containerisation solutions.
Table 4 contains a list of relevant software tool versions and sources.
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
• Legality of collection, transfer and use (national and international)
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.
The reference list from the paper itself. Each links out to its DOI / PubMed record.
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