The genome sequence of an ichneumonid wasp, Amblyjoppa proteus (Christ, 1791)
Liam M. Crowley, Gavin R. Broad, Terrence Sylvester, Shan Gao
TL;DR
This paper provides the genome sequence of the ichneumonid wasp Amblyjoppa proteus, including chromosomal and mitochondrial assemblies.
Contribution
The study presents a high-quality genome assembly and gene annotation for Amblyjoppa proteus, a species in the Hymenoptera order.
Findings
The genome assembly is 290.11 megabases long with 99.64% scaffolded into 9 chromosomal pseudomolecules.
The mitochondrial genome is 30.41 kilobases in length.
Gene annotation identified 12,770 protein-coding genes.
Abstract
We present a genome assembly from a female specimen of Amblyjoppa proteus (ichneumonid wasp; Arthropoda; Insecta; Hymenoptera; Ichneumonidae). The genome sequence has a total length of 290.11 megabases. Most of the assembly (99.64%) is scaffolded into 9 chromosomal pseudomolecules. The mitochondrial genome has also been assembled, with a length of 30.41 kilobases. Gene annotation of this assembly on Ensembl identified 12,770 protein-coding genes.
Genes, proteins, chemicals, diseases, species, mutations and cell lines named across the full text — each resolved to its canonical identifier and authoritative record.
Click any figure to enlarge with its caption.
Figure 1
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Figure 5| Project information | |||
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| Amblyjoppa proteus | ||
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| PRJEB65739 | ||
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| SAMEA10979054 | ||
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| 1905387 | ||
| Specimen information | |||
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| iyAmbProt1 | SAMEA10979446 | thorax |
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| iyAmbProt1 | SAMEA10979444 | head |
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| iyAmbProt1 | SAMEA10979448 | abdomen |
| Sequencing information | |||
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| ERR12035319 | 8.75e+08 | 132.17 |
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| ERR12015778 | 1.68e+06 | 20.84 |
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| ERR12035320 | 7.59e+07 | 11.46 |
| Genome assembly | ||
|---|---|---|
| Assembly name | iyAmbProt1.1 | |
| Assembly accession | GCA_963922035.1 | |
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| Assembly level for primary assembly | chromosome | |
| Span (Mb) | 290.11 | |
| Number of contigs | 155 | |
| Number of scaffolds | 44 | |
| Longest scaffold (Mb) | 49.23 | |
| Assembly metric | Measure |
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| Contig N50 length | 9.75 Mb |
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| Scaffold N50 length | 29.48 Mb |
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| Consensus quality (QV) | Primary: 61.0; alternate: 59.9; combined: 60.3 |
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| Primary: 95.79%; alternate: 68.95%; combined: 99.33% |
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| BUSCO
| C:96.2%[S:96.0%,D:0.2%],
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| Percentage of assembly assigned to chromosomes | 99.64% |
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| Organelles | Mitochondrial genome: 30.41 kb |
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| INSDC accession | Name | Length (Mb) | GC% |
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| 1 | 49.23 | 43 | |
| 2 | 46.54 | 43.5 | |
| 3 | 41.42 | 42.5 | |
| 4 | 29.48 | 43 | |
| 5 | 28.88 | 42 | |
| 6 | 28.49 | 43 | |
| 7 | 26.07 | 43 | |
| 8 | 24.69 | 42 | |
| 9 | 14.28 | 40.5 | |
| MT | 0.03 | 14.5 |
| Software tool | Version | Source |
|---|---|---|
| BLAST | 2.14.0 |
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| BlobToolKit | 4.3.3 |
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| BUSCO | 5.5.0 |
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| bwa-mem2 | 2.2.1 |
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| DIAMOND | 2.1.8 |
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| fasta_windows | 0.2.4 |
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| FastK | 666652151335353eef2fcd58880bcef5bc2928e1 |
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| GenomeScope2.0 | 2.0.1 |
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| Gfastats | 1.3.6 |
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| GoaT CLI | 0.2.5 |
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| Hifiasm | 0.16.1 |
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| HiGlass | 44086069ee7d4d3f6f3f0012569789ec138f42b84aa44357826c0b6753eb28de |
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| MerquryFK | d00d98157618f4e8d1a9190026b19b471055b22e |
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| Minimap2 | 2.24-r1122 |
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| MitoHiFi | 3.2 |
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| MultiQC | 1.14, 1.17, and 1.18 |
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| Nextflow | 23.04.1 |
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| PretextView | 0.2.5 |
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| purge_dups | 1.2.3 |
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| samtools | 1.18 |
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| sanger-tol/ascc | 0.1.0 |
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| sanger-tol/blobtoolkit | 0.3.0 |
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| Seqtk | 1.3 |
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| Singularity | 3.9.0 |
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| TreeVal | 1.2.0 |
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| YaHS | 1.1a.2 |
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- —Wellcome Trust
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Taxonomy
TopicsHymenoptera taxonomy and phylogeny · Insect-Plant Interactions and Control · Insect symbiosis and bacterial influences
Species taxonomy
Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Ecdysozoa; Panarthropoda; Arthropoda; Mandibulata; Pancrustacea; Hexapoda; Insecta; Dicondylia; Pterygota; Neoptera; Endopterygota; Hymenoptera; Apocrita; Ichneumonoidea; Ichneumonidae; Ichneumoninae; Heresiarchini; Amblyjoppa; Amblyjoppa proteus (Christ, 1791) (NCBI:txid1905387)
Background
Amblyjoppa proteus is a comparatively large and striking ichneumonid wasp, up to 25mm long and basically black, with restricted white markings, including on the inner orbits of the eye and the scutellum. It can be separated from other Ichneumoninae with a similar colour pattern by its large size, dusky edges to the wings, and the shape of the area superomedia of the propodeum, which is small, D-shaped and entirely ‘filled in’. Amblyjoppa can be identified using Perkins (1959), where A. proteus is separated from the only other European species, A. fuscipennis (Wesmael). The two have different colour patterns; A. fuscipennis has a reddish metasoma.
In Europe, Amblyjoppa proteus is a specialised parasitoid of Elephant Hawk-moth ( Deilephila elpenor) (Lepidoptera: Sphingidae) ( Perkins, 1959). Uchida (1930) and Kusigemati (1981) reports two different hosts in Japan, both in the same subfamily (Macroglossinae) as Deilephila, Theretra japonica (Boisduval) and T. oldenlandiae (Fabricius), although it is quite possible that there are two species of Amblyjoppa involved. Oviposition is into the host larva ( Broad et al., 2018) then, as with almost all other Ichneumoninae, emergence is from the host pupa, with the emerging adult A. proteus cutting a neat ‘cap’ from the head end of the host pupa ( Shaw et al., 2015).
As with its host, A. proteus is widespread across Europe and in Britain. Adult females over-winter and are on the wing in late May and June, with the next generation emerging in late summer, with females over-wintering. Deilephila elpenor has usually been univoltine in Britain but a second generation is becoming more regular ( Waring et al., 2017), so it will be interesting to see whether A. proteus takes advantage of this second generation.
Here we present a chromosomally complete genome sequence for Amblyjoppa proteus, based on a specimen from Wytham Woods, Oxfordshire, United Kingdom ( Figure 1). The genome of was sequenced 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.
Photograph of the Amblyjoppa proteus (iyAmbProt1) specimen used for genome sequencing.
Genome sequence report
Sequencing data
The genome of a specimen of Amblyjoppa proteus ( Figure 1) was sequenced using Pacific Biosciences single-molecule HiFi long reads, generating 20.84 Gb from 1.68 million reads, which were used to assemble the genome. GenomeScope analysis estimated the haploid genome size at 277.09 Mb, with a heterozygosity of 0.33% and repeat content of 20.98%. These estimates guided expectations for the assembly. Based on the estimated genome size, the sequencing data provided approximately 71 coverage. Hi-C sequencing produced 132.17 Gb from 875.30 million reads, and was used to scaffold the assembly. RNA sequencing data were also generated and are available in public sequence repositories. Table 1 summarises the specimen and sequencing details.
Table 1.: Specimen and sequencing data for Amblyjoppa proteus.
Assembly statistics
The primary haplotype was assembled, and contigs corresponding to an alternate haplotype were also deposited in INSDC databases. The assembly was improved by manual curation, which corrected 98 misjoins or missing joins and removed one haplotypic duplication. These interventions decreased the scaffold count by 62.5% and increased the scaffold N50 by 2.08%. The final assembly has a total length of 290.11 Mb in 44 scaffolds, with 111 gaps, and a scaffold N50 of 29.48 Mb ( Table 2).
Table 2.: Genome assembly data for Amblyjoppa proteus.
The snail plot in Figure 2 provides a summary of the assembly statistics, indicating the distribution of scaffold lengths and other assembly metrics. Figure 3 shows the distribution of scaffolds by GC proportion and coverage. Figure 4 presents a cumulative assembly plot, with separate curves representing different scaffold subsets assigned to various phyla, illustrating the completeness of the assembly.
Genome assembly of Amblyjoppa proteus, iyAmbProt1.1: metrics.The BlobToolKit snail plot provides an overview of assembly metrics and BUSCO gene completeness. The circumference represents the length of the whole genome sequence, and the main plot is divided into 1,000 bins around the circumference. The outermost blue tracks display the distribution of GC, AT, and N percentages across the bins. Scaffolds are arranged clockwise from longest to shortest and are depicted in dark grey. The longest scaffold is indicated by the red arc, and the deeper orange and pale orange arcs represent the N50 and N90 lengths. A light grey spiral at the centre shows the cumulative scaffold count on a logarithmic scale. A summary of complete, fragmented, duplicated, and missing BUSCO genes in the hymenoptera_odb10 set is presented at the top right. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/GCA_963922035.1/dataset/GCA_963922035.1/snail.
Genome assembly of Amblyjoppa proteus, iyAmbProt1.1: BlobToolKit GC-coverage plot.Blob plot showing sequence coverage (vertical axis) and GC content (horizontal axis). The circles represent scaffolds, with the size proportional to scaffold length and the colour representing phylum membership. The histograms along the axes display the total length of sequences distributed across different levels of coverage and GC content. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/GCA_963922035.1/dataset/GCA_963922035.1/blob.
Genome assembly of Amblyjoppa proteus, iyAmbProt1.1: 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_963922035.1/dataset/GCA_963922035.1/cumulative.
Most of the assembly sequence (99.64%) was assigned to 9 chromosomal-level scaffolds. These chromosome-level scaffolds, confirmed by Hi-C data, are named according to size ( Figure 5; Table 3). During curation, it was noted that the order of the contigs in the region 13.4 Mbp to 29.7 Mbp of chromosome 2 is uncertain.
Hi-C contact map of the iyAmbProt1.1 assembly.The map was generated using PretextSnapshot. Chromosomes are shown in order of size and labelled with chromosome accession numbers (left) and names (top).
Table 3.: Chromosomal pseudomolecules in the genome assembly of Amblyjoppa proteus, iyAmbProt1.
The mitochondrial genome was also assembled. This sequence is included as a contig in the multifasta file of the genome submission and as a standalone record.
Assembly quality metrics
The estimated Quality Value (QV) and k-mer completeness metrics, along with BUSCO completeness scores, were calculated for each haplotype and the combined assembly. The QV reflects the base-level accuracy of the assembly, while k-mer completeness indicates the proportion of expected k-mers identified in the assembly. BUSCO scores provide a measure of completeness based on benchmarking universal single-copy orthologues.
The combined primary and alternate assemblies achieve an estimated QV of 60.3. The k-mer recovery for the primary haplotype is 95.79%, and for the alternate haplotype 68.95%; the combined primary and alternate assemblies have a k-mer recovery of 99.33%. BUSCO v.5.5.0 analysis using the hymenoptera_odb10 reference set ( n = 5,991) identified 96.2% of the expected gene set (single = 96.0%, duplicated = 0.2%).
Table 2 provides assembly metric benchmarks adapted from Rhie et al. (2021) and the Earth BioGenome Project Report on Assembly Standards September 2024. The assembly achieves the EBP reference standard of 6.C.Q61.
Genome annotation report
The Amblyjoppa proteus genome assembly (GCA_963922035.1) was annotated externally by Ensembl at the European Bioinformatics Institute (EBI). This annotation includes 20,634 transcribed mRNAs from 12,770 protein-coding and 1,077 non-coding genes. The average transcript length is 10,489.70 bp. There are 1.49 coding transcripts per gene and 6.19 exons per transcript. For further information about the annotation, please refer to https://beta.ensembl.org/species/982e1535-98da-4bab-8358-38cdd8aa5db9.
Methods
Sample acquisition and DNA barcoding
The specimen used for genome sequencing was an adult female Amblyjoppa proteus (specimen ID Ox001795, ToLID iyAmbProt1), collected from Wytham Woods, Oxfordshire, United Kingdom (latitude 51.767, longitude –1.311) on 2021-08-09 by netting. The specimen was collected and identified by Liam Crowley (University of Oxford) and 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 specimen and stored in ethanol, while the remaining parts were shipped on dry ice to the Wellcome Sanger Institute (WSI) ( Pereira et al., 2022). 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).
Metadata collection for samples adhered to the Darwin Tree of Life project standards described by Lawniczak et al. (2022).
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 procedures: sample preparation and homogenisation, DNA extraction, fragmentation and purification. Detailed protocols are available on protocols.io ( Denton et al., 2023). The iyAmbProt1 sample was prepared for DNA extraction by weighing and dissecting it on dry ice ( Jay et al., 2023). Tissue from the thorax was cryogenically disrupted using the Covaris cryoPREP ^®^ Automated Dry Pulverizer ( Narváez-Gómez et al., 2023). 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.
RNA was extracted from abdomen tissue of iyAmbProt1 in the Tree of Life Laboratory at the WSI using the RNA Extraction: Automated MagMax™ mirVana protocol ( do Amaral et al., 2023). The RNA concentration was assessed using a Nanodrop spectrophotometer and a Qubit Fluorometer using the Qubit RNA Broad-Range Assay kit. Analysis of the integrity of the RNA was done using the Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.
Hi-C sample preparation and crosslinking
Hi-C data were generated from the head of the iyAmbProt1 sample using the Arima-HiC v2 kit (Arima Genomics) with 20–50 mg of frozen tissue (stored at –80 °C). As per manufacturer’s instructions, tissue was fixed, and the DNA crosslinked using a TC buffer with 22% formaldehyde concentration, and a final formaldehyde concentration of 2%. The tissue was then homogenised using the Diagnocine Power Masher-II. The crosslinked DNA was digested using a restriction enzyme master mix, then biotinylated and ligated. A clean up was performed with SPRIselect beads prior to library preparation. DNA concentration was quantified using the Qubit Fluorometer v4.0 (Thermo Fisher Scientific) and Qubit HS Assay Kit, and sample biotinylation percentage was estimated using the Arima-HiC v2 QC beads.
Library preparation and sequencing
Library preparation and sequencing were performed at the WSI Scientific Operations core.
** PacBio HiFi **
At a minimum, samples were required to have an average fragment size exceeding 8 kb and a total mass over 400 ng to proceed to the low-input SMRTbell Prep Kit 3.0 protocol (Pacific Biosciences), depending on genome size and sequencing depth required. Libraries were prepared using the SMRTbell Prep Kit 3.0 as per the manufacturer's instructions. The kit includes the reagents required for end repair/A-tailing, adapter ligation, post-ligation SMRTbell bead cleanup, and nuclease treatment. Size-selection and clean-up were carried out using diluted AMPure PB beads (Pacific Biosciences). DNA concentration was quantified using the Qubit Fluorometer v4.0 (ThermoFisher Scientific) with Qubit 1X dsDNA HS assay kit and the final library fragment size analysis was carried out using the Agilent Femto Pulse Automated Pulsed Field CE Instrument (Agilent Technologies) and the gDNA 55kb BAC analysis kit.
Samples were sequenced using the Sequel IIe system (Pacific Biosciences, California, USA). The concentration of the library loaded onto the Sequel IIe was in the range 40–135 pM. The SMRT link software, a PacBio web-based end-to-end workflow manager, was used to set-up and monitor the run, as well as perform primary and secondary analysis of the data upon completion.
** Hi-C **
For Hi-C library preparation, the biotinylated DNA constructs were fragmented using a Covaris E220 sonicator and size-selected to 400–600 bp using SPRISelect beads. DNA was then enriched using Arima-HiC v2 Enrichment beads. The NEBNext Ultra II DNA Library Prep Kit (New England Biolabs) was used for end repair, A-tailing, and adapter ligation, following a modified protocol in which library preparation is carried out while the DNA remains bound to the enrichment beads. PCR amplification was performed using KAPA HiFi HotStart mix and custom dual-indexed adapters (Integrated DNA Technologies) in a 96-well plate format. Depending on sample concentration and biotinylation percentage determined at the crosslinking stage, samples were amplified for 10–16 PCR cycles. Post-PCR clean-up was carried out using SPRISelect beads. The libraries were quantified using the Accuclear Ultra High Sensitivity dsDNA Standards Assay kit (Biotium) and normalised to 10 ng/μL before sequencing. Hi-C sequencing was performed on the Illumina NovaSeq 6000 instrument using 150 bp paired-end reads.
** RNA **
Libraries were prepared using the NEBNext ^®^ Ultra™ II Directional RNA Library Prep Kit for Illumina (New England Biolabs), following the manufacturer’s instructions. Poly(A) mRNA in the total RNA solution was isolated using oligo(dT) beads, converted to cDNA, and uniquely indexed; 14 PCR cycles were performed. Libraries were size-selected to produce fragments between 100–300 bp. Libraries were quantified, normalised, pooled to a final concentration of 2.8 nM, and diluted to 150 pM for loading. Sequencing was carried out on the Illumina NovaSeq 6000, generating 150-bp paired-end reads.
Genome assembly, curation and evaluation
** Assembly **
Prior to assembly of the PacBio HiFi reads, a database of k-mer counts ( k = 31) was generated from the filtered reads using FastK. GenomeScope2 ( Ranallo-Benavidez et al., 2020) was used to analyse the k-mer frequency distributions, providing estimates of genome size, heterozygosity, and repeat content.
The HiFi reads were first assembled using Hifiasm ( Cheng et al., 2021) with the --primary option. Haplotypic duplications were identified and removed using purge_dups ( Guan et al., 2020). The Hi-C reads ( Rao et al., 2014) were mapped to the primary contigs using bwa-mem2 ( Vasimuddin et al., 2019), and the contigs were scaffolded using YaHS ( Zhou et al., 2023) using the --break option for handling potential misassemblies. 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. Flat files and maps used in curation were generated via the TreeVal pipeline ( Pointon et al., 2023). Manual curation was conducted primarily in PretextView ( Harry, 2022) and HiGlass ( Kerpedjiev et al., 2018), with additional insights provided by JBrowse2 ( Diesh et al., 2023). Scaffolds were visually inspected and corrected as described by Howe et al. (2021). Any identified contamination, missed joins, and mis-joins were amended, and duplicate sequences were tagged and removed. The curation process is documented at https://gitlab.com/wtsi-grit/rapid-curation. PretextSnapshot was used to generate a Hi-C contact map of the final assembly.
** Assembly quality assessment **
The Merqury.FK tool ( Rhie et al., 2020), run in a Singularity container ( Kurtzer et al., 2017), was used to evaluate k-mer completeness and assembly quality for the primary and alternate haplotypes using the k-mer databases ( k = 31) computed prior to genome assembly. The analysis outputs included assembly QV scores and completeness statistics.
The genome was analysed in the blobtoolkit pipeline, a Nextflow ( Di Tommaso et al., 2017) port of the previous Snakemake Blobtoolkit pipeline ( Challis et al., 2020). It aligns the PacBio reads in SAMtools ( Danecek et al., 2021) 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 blastp ( Buchfink et al., 2021). The genome is also divided 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 using DIAMOND blastx. Genome sequences without a hit are chunked using 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 blobtoolkit pipeline was 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.
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