The genome sequence of the Four-banded Bee-grabber, Conops quadrifasciatus De Geer, 1776

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.


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 nonessential 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 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,.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%. 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.
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 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.Table 3. Software tools: versions and sources.

Software tool Version
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.
Table 3 contains a list of relevant software tool versions and sources.

Genome annotation
The BRAKER2 pipeline (Brůna et al., 2021)  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

Halide Nihal Açıkgöz
Ankara University,, Ankara, Turkey The manuscript discussed the genome sequence of the Conops quadrifasciatus species in detail.This has provided important information about the biology and genetics of the species.The manuscript makes an original contribution, especially regarding genome sequencing.
Genomic analysis of taxonomically complex groups such as Conops quadrifasciatus can help better understand and classify these groups.The manuscript enables comparison of the genome of this species with other Diptera species, allowing to identify genetic differences within and between species.
The methods used are appropriate and meticulous.
The results are important so that other researchers can reuse and verify them.
The manuscript is generally well written and of scientific value.It should be indexed.

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?
be needed from one of the tissue types?
In the first paragraph of the methods section " 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." The order of the above writing is somewhat confusing.The first sentence could be omitted to make things clearer.
Also in regard to the above section, Hi-C sequencing is still ultimately DNA sequencing.It would be a little clearer to say the male sample was used for Pacbio sequencing to differentiate how male and female DNA sequencing was different.
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: bioinformatics, population 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 2 .
Figure 2. 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.

Figure 3 .
Figure 3. 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.

Figure 4 .
Figure 4. 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.

Figure 5 .
Figure 5. 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.

Darwin Tree of Life Project Sampling Code of Practice', which
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 GovernanceThe 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 '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.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
No competing interests were disclosed.
YesCompeting Interests: https://doi.org/10.21956/wellcomeopenres.23346.r79722©2024 Açıkgöz H.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.