The genome sequence of the Four-banded Bee-grabber, Conops quadrifasciatus De Geer, 1776
Steven Falk, Liam M. Crowley, David K. Clements, Joel Gibson, Halide Nihal Açıkgöz, Sam Heraghty
TL;DR
This paper presents the genome sequence of the Four-banded Bee-grabber, a fly species, including its chromosomal structure and gene content.
Contribution
The study provides the first genome assembly for Conops quadrifasciatus, including sex chromosomes and mitochondrial DNA.
Findings
The genome assembly spans 210.4 megabases and is scaffolded into 7 chromosomal pseudomolecules.
The mitochondrial genome is 18.07 kilobases long and has been fully assembled.
Gene annotation identified 23,090 protein-coding genes using Ensembl.
Abstract
We present a genome assembly from an individual male Conops quadrifasciatus (the Four-banded Bee-grabber; Arthropoda; Insecta; Diptera; Conopidae). The genome sequence is 210.4 megabases in span. Most of the assembly is scaffolded into 7 chromosomal pseudomolecules, including the X and Y sex chromosomes. The mitochondrial genome has also been assembled and is 18.07 kilobases in length. Gene annotation of this assembly on Ensembl identified 23,090 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
Figure 2
Figure 3
Figure 4
Figure 5| Project accession data | ||
|---|---|---|
| Assembly identifier | idConQuad1.1 | |
| Species |
| |
| Specimen | idConQuad1 | |
| NCBI taxonomy ID | 2823188 | |
| BioProject | PRJEB59143 | |
| BioSample ID | SAMEA8603151 | |
| Isolate information | idConQuad1, male: thorax (DNA sequencing)
| |
| Assembly metrics
|
| |
| Consensus quality (QV) | 63.6 |
|
|
| 100.0% |
|
| BUSCO
| C:96.5%[S:96.1%,D:0.4%],F:0.7%,M:2.8%,n:3,285 |
|
| Percentage of assembly mapped to chromosomes | 99.96% |
|
| Sex chromosomes | XY |
|
| Organelles | Mitochondrial genome: 18.07 kb |
|
| Raw data accessions | ||
| PacificBiosciences SEQUEL II | ERR10812844 | |
| Hi-C Illumina | ERR10802472 | |
| PolyA RNA-Seq Illumina | ERR11837463 | |
| Genome assembly | ||
| Assembly accession | GCA_949752815.1 | |
|
| GCA_949752755.1 | |
| Span (Mb) | 210.4 | |
| Number of contigs | 48 | |
| Contig N50 length (Mb) | 13.2 | |
| Number of scaffolds | 13 | |
| Scaffold N50 length (Mb) | 39.8 | |
| Longest scaffold (Mb) | 47.95 | |
| Genome annotation | ||
| Number of protein-coding genes | 23,090 | |
| Number of gene transcripts | 23,804 | |
| INSDC accession | Chromosome | Length (Mb) | GC% |
|---|---|---|---|
| 1 | 47.95 | 35.5 | |
| 2 | 41.7 | 36.0 | |
| 3 | 39.76 | 36.0 | |
| 4 | 34.49 | 35.0 | |
| 5 | 33.51 | 36.0 | |
| X | 11.62 | 36.5 | |
| Y | 1.23 | 35.0 | |
| MT | 0.02 | 24.5 |
| Software tool | Version | Source |
|---|---|---|
| BlobToolKit | 4.2.1 |
|
| BUSCO | 5.3.2 |
|
| Hifiasm | 0.16.1-r375 |
|
| HiGlass | 1.11.6 |
|
| Merqury | MerquryFK |
|
| MitoHiFi | 2 |
|
| PretextView | 0.2 |
|
| purge_dups | 1.2.3 |
|
| sanger-tol/genomenote | v1.0 |
|
| sanger-tol/readmapping | 1.1.0 |
|
| YaHS | 1.2a |
|
- —Wellcome Trust
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Taxonomy
TopicsGenomics and Phylogenetic Studies · Genetic diversity and population structure · Insect and Arachnid Ecology and Behavior
Species taxonomy
Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Ecdysozoa; Panarthropoda; Arthropoda; Mandibulata; Pancrustacea; Hexapoda; Insecta; Dicondylia; Pterygota; Neoptera; Endopterygota; Diptera; Brachycera; Muscomorpha; Eremoneura; Cyclorrhapha; Schizophora; Acalyptratae; Conopoidea; Conopidae; Conopinae; Conops; Conops quadrifasciatus De Geer, 1776 (NCBI:txid2823188).
Background
Conops quadrifasciatus is a member of the acalyptrate family Conopidae within the Diptera, or ‘true flies’. As far as is known, all members of this family develop as endoparasitoids in other insects, chiefly the adults of aculeate Hymenoptera ( Stuke, 2016).
Conops quadrifasciatus is a widespread and common Palaearctic species which occurs in most countries of Europe as well as in Russia, Ukraine, Türkiye and Iran, extending as far east as Kyrgyzstan and Novosibirsk ( GBIF Secretariat, 2024; iNaturalist, 2024; Stuke, 2016). The adult is one of several rather similar black and yellow species of rather elongated, wasp-like appearance, which probably benefit from mimicry of aposematic Hymenoptera. The nectar-feeding adults are chiefly found in warm, sunlit and flower-rich habitats, often on umbels and other flower-heads amongst swarms of similar-looking insects including various hoverflies, bees and wasps.
Females of C. quadrifasciatus lay their eggs directly into the abdominal cavities of adult bees, primarily members of the bumblebee genus Bombus ( Stuke, 2016). Host bees are usually intercepted in flight, the conopid grabbing the bee with its long legs and wrestling with it while tumbling to the ground. Female conopids are characterised by specialised adaptations of the abdomen which create a pincer-like mechanism formed by the elongated final segment of the abdomen and a corresponding bulge on the underside of the fifth segment called the ‘theca’. This mechanism is probably used to pry apart the abdominal segments of the host to allow an egg to be inserted through the intersegmental membrane ( Clements, 1997). Oviposition usually occurs very rapidly, however, and the mechanics of it are still not well understood.
Within a few days the conopid egg hatches and the larva attaches itself to the host’s tracheal system for oxygenation. The larva initially devours the host’s fat-bodies and other non-essential organs. Usually there is only one conopid larva per host. The depredations of the conopid larva make the host increasingly unable to function normally leading to alterations in its behaviour, for example by focusing only on easily foraged flowers or undertaking shorter foraging journeys ( Schmid-Hempel, 1998; Schmid-Hempel & Schmid-Hempel, 1990). Infested hosts may also remain out of the nest at night, which has been shown to slow down the development of the conopid larva through exposure to lower temperatures. In some cases, this may allow the host to complete most of its natural lifespan before the conopid larva is able to complete its development. In many cases, however, the conopid larva ultimately kills the host ( Clements, 1997; Schmid-Hempel, 1998).
In its final instar the conopid larva develops a long, attenuated anterior section with the mouth at its tip. Having consumed the abdominal contents, the larva probes through the host’s narrow petiole to consume the thoracic organs, causing its death. Prior to host death, the conopid will often invoke fossorial behaviour, causing the host to dig itself into the soil before it dies. The conopid larva then pupates in the buried husk where it is protected from adverse weather and predation by scavengers ( Müller, 1994). After overwintering, the adult conopid hatches and digs its way to the surface.
Whilst adult conopids tend to be found only at relatively low densities, research has shown that larval infestation in host bees can occur at rates of 30–70% ( Schmid-Hempel, 1998). Conopids are therefore considered to represent potentially significant predators of bees and wasps, with some species being significant pests in commercial honey production worldwide ( Tingek et al., 2007).
Conopid species are taxonomically complex with many forms of uncertain taxonomic status, including within the genus Conops. The whole genome sequence present here may assist in resolving these taxonomic uncertainties ( Stuke, 2016).
Genome sequence report
The genome was sequenced from one specimen of Conops quadrifasciatus ( Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77, –1.31). A total of 95-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 37 missing joins or mis-joins and removed 8 haplotypic duplications, reducing the assembly length by 0.12% and the scaffold number by 12.50%, and increasing the scaffold N50 by 15.24%.
Photograph of the Conops quadrifasciatus (idConQuad1) specimen used for genome sequencing.
The final assembly has a total length of 210.4 Mb in 13 sequence scaffolds with a scaffold N50 of 39.8 Mb ( Table 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. Most (99.96%) of the assembly sequence was assigned to 7 chromosomal-level scaffolds, representing 5 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). The sex chromosomes identified using coverage information. Chromosome Y scaffold was determined by mapping Hi-C data from a female sample to the male assembly. 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.
Table 1.: Genome data for Conops quadrifasciatus, idConQuad1.1.
Genome assembly of Conops quadrifasciatus, idConQuad1.1: 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 210,459,157 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 (47,946,236 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (39,758,196 and 33,513,057 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 diptera_odb10 set is shown in the top right. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/idConQuad1_1/dataset/idConQuad1_1/snail.
Genome assembly of Conops quadrifasciatus, idConQuad1.1: BlobToolKit GC-coverage plot.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/idConQuad1_1/dataset/idConQuad1_1/blob.
Genome assembly of Conops quadrifasciatus, idConQuad1.1: BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all sequences. Coloured lines show cumulative lengths of sequences assigned to each phylum using the buscogenes taxrule. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/idConQuad1_1/dataset/idConQuad1_1/cumulative.
Genome assembly of Conops quadrifasciatus, idConQuad1.1: Hi-C contact map of the idConQuad1.1 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=IgeuY3TgSre9iwe0t-9iUg.
Table 2.: Chromosomal pseudomolecules in the genome assembly of Conops quadrifasciatus, idConQuad1.
The estimated Quality Value (QV) of the final assembly is 63.6 with k-mer completeness of 100.0%, and the assembly has a BUSCO v5.3.2 completeness of 96.5% (single = 96.1%, duplicated = 0.4%), using the diptera_odb10 reference set ( n = 3,285).
Metadata for specimens, barcode results, spectra estimates, sequencing runs, contaminants and pre-curation assembly statistics are given at https://links.tol.sanger.ac.uk/species/2823188.
Genome annotation report
The Conops quadrifasciatus genome assembly (GCA_949752815.1) was annotated using the Ensembl rapid annotation pipeline at the European Bioinformatics Institute (EBI). The resulting annotation includes 23,804 transcribed mRNAs from 23,090 protein-coding genes. ( Table 1; https://rapid.ensembl.org/Conops_quadrifasciatus_GCA_949752815.1/Info/Index).
Methods
Sample acquisition and nucleic acid extraction
A male Conops quadrifasciatus (specimen ID Ox000913, ToLID idConQuad1) was netted at Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude –1.31) on 2020-08-03. The specimen was collected and identified by Steven Falk (independent researcher). A male specimen was used for DNA sequencing, and a female specimen was used for Hi-C sequencing. A female specimen, used for Hi-C and RNA sequencing (specimen ID Ox001841, ToLID idConQuad2), was netted at the same location on 2021-08-21. This specimen was collected and identified by Liam Crowley (University of Oxford). Both specimens were snap-frozen on dry ice.
The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) includes a sequence of core procedures: sample preparation; sample homogenisation, DNA extraction, fragmentation, and clean-up. In sample preparation, the idConQuad1 sample was weighed and dissected on dry ice ( Jay et al., 2023), and tissue from the thorax was homogenised using a PowerMasher II tissue disruptor ( Denton et al., 2023a). HMW DNA was extracted using the Automated MagAttract v1 protocol ( Sheerin et al., 2023). DNA was sheared into an average fragment size of 12–20 kb in a Megaruptor 3 system with speed setting 30 ( Todorovic et al., 2023). Sheared DNA was purified by solid-phase reversible immobilisation ( Strickland et al., 2023): in brief, the method employs a 1.8X ratio of AMPure PB beads to sample to eliminate shorter fragments and concentrate the DNA. 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 idConQuad1 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.
Protocols developed by the WSI Tree of Life laboratory are publicly available on protocols.io ( Denton et al., 2023b).
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 head and thorax tissue of idConQuad2 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 as described previously ( Howe et al., 2021). Manual curation was performed using HiGlass ( Kerpedjiev et al., 2018) and PretextView ( 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.
A Hi-C map for the final assembly was produced using bwa-mem2 ( Vasimuddin et al., 2019) in the Cooler file format ( Abdennur & Mirny, 2020). To assess the assembly metrics, the k-mer completeness and QV consensus quality values were calculated in Merqury ( Rhie et al., 2020). This work was done using Nextflow ( Di Tommaso et al., 2017) DSL2 pipelines “sanger-tol/readmapping” ( Surana et al., 2023a) and “sanger-tol/genomenote” ( Surana et al., 2023b). The genome was analysed within the BlobToolKit environment ( Challis et al., 2020) and BUSCO scores ( Manni et al., 2021; Simão et al., 2015) were calculated.
Table 3 contains a list of relevant software tool versions and sources.
Genome annotation
The BRAKER2 pipeline ( Brůna et al., 2021) was used in the default protein mode to generate annotation for the Conops quadrifasciatus assembly (GCA_949752815.1) in Ensembl Rapid Release at the EBI.
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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