The genome sequence of a conopid fly, Thecophora atra (Fabricius, 1775)

We present a genome assembly from an individual male Thecophora atra (a Conopid fly; Arthropoda; Insecta; Diptera; Conopidae). The genome sequence is 354.2 megabases in span. Most of the assembly is scaffolded into 5 chromosomal pseudomolecules, including the X and Y sex chromosomes. The mitochondrial genome has also been assembled and is 17.3 kilobases in length. Gene annotation of this assembly on Ensembl identified 30,620 protein coding genes.


Background
Thecophora atra (Fabricius, 1775) is a medium sized black fly from the family Conopidae, occasionally called thick-headed flies.It is one of three species from genus Thecophora Rondani, 1845 occurring in Britain (Ransom et al., 2015;Whitehead, 2022).It differs from Thecophora fulvipes (Robineau-Desvoidy, 1830) by being slightly smaller in size (T.atra is 4-7 mm, in contrast to 5-9 mm of T. fulvipes) and having scarcer dusting on the abdomen.The legs of T. atra are usually black with yellow "knees" (distal tip of the femora and proximal tip of the tibia) and a yellow basal half of the hind femora, while in T. fulvipes all the femora are entirely yellow or orange-brown (the hind one at least in basal two-thirds).The leg colouration is somewhat variable and some T. atra may have some yellow at the base or apex of front and/or mid femora (Ransom et al., 2015;Smith, 1969).
Thecophora cinerascens (Meigen, 1804) was discovered in the Channel Islands in 2015 and in Wales in 2019, adding another species to the British list.Although similar in size and appearance to T. atra, the females of both species can be reliably separated by the shape of theca (Ransom et al., 2015;Whitehead, 2022).Male T. atra and T. cinerascens are very difficult to separate.The specimen used for sequencing was confirmed as T. atra using DNA barcodes.
Thecophora atra is oviparous and its larvae are internal parasites of halictid bees.Usually, a single egg is laid in flight into a host's abdomen.The white and smooth larva feeds on the insides of the abdomen during which time the host is active.The larva then becomes tapered anteriorly (in the third instar) and feeds on the contents of the thorax through the petiole resulting in the death of the host.Pupation occurs soon after, inside the host's abdomen (Smith, 1966).
The hosts recorded outside Britain include Halictus and Lasioglossum species.In Britain, T. atra has been observed around colonies of Lasioglossum morio and Halictus spp., but parasitism of these species has not been confirmed through rearing (Baldock & Early, 2015;Smith, 1969).
The high-quality genome of a conopid fly Thecophora atra was sequenced based on one male specimen from Hartslock Nature Reserve.It will aid research on the taxonomy, phylogeny and biology of this and related taxa.The genome of T. atra 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.

Genome sequence report
The genome was sequenced from one male Thecophora atra (Figure 1) collected from Hartslock Reserve, Oxfordshire (51.51, -1.11).A total of 44-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 46 missing joins or misjoins and removed two haplotypic duplications, reducing the assembly length by 0.66% and the scaffold number by 52.17%, and increasing the scaffold N50 by 122%.
The final assembly has a total length of 354.2 Mb in 22 sequence scaffolds with a scaffold N50 of 94.1 Mb (Table 1).Most (99.94%) of the assembly sequence was assigned to 5 chromosomal-level scaffolds, representing three autosomes and the X and Y sex chromosomes.The Y chromosome was identified based on read coverage against the Hi-C reads from a different specimen.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 2-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.
Metadata for specimens, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/1219171.

Sample acquisition and nucleic acid extraction
Thecophora atra specimens (idTheAtra2 and idTheAtra3 NHMUK014444821) were collected from Hartslock Nature Reserve, Oxfordshire, UK (latitude 51.51, longitude -1.11) on 2020-08-20 using an aerial net.The specimens were collected and identified by Ryan Mitchell (Natural History Museum) and preserved on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The idTheAtra2 sample was weighed and dissected on dry.Head 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.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    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.
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 whole organism tissue of idTheAtra1 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 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

Genome annotation
The BRAKER2 pipeline (Brůna et al., 2021) was used in the default protein mode to generate annotation for the Thecophora atra assembly (GCA_937620795.1) in Ensembl Rapid Release.

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

Jerome H L Hui
The Chinese University of Hong Kong, Hong Kong, Hong Kong In this data note, Mitchell and colleagues obtained the genomic resource of conopid fly, Thecophora atra (Fabricius, 1775).According to the NatureSpot and NBN Atlas, this species is widespread in Britain.There are only limited molecular data of this species prior to this report.Therefore, this new genome resource will be useful for further studies, ranging from species identification, revealing their population structure and biogeography, unravelling the effect of climate change on them, as well as understanding their evolution with other insects.
This genome resource is excellent from the summary statistics, with high BUSCO scores, high sequence continuity (scaffold N50), and majority of sequences contained on the 3 pseudochromosomes (plus 2 sex chromosomes and mitochondrion).All in all, this is a valuable contribution.

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: I have published with Peter Holland more than three years ago, and confirm that this potential conflict of interest did not affect my ability to write an objective and unbiased review of the article.

Ravikumar Dodiya
Sardarkrushinagar Dantiwada Agricultural University, Sardarkrushinagar, Dantiwada, India Thecophora atra (Fabricius, 1775) is a black fly from the family Conopidae, known as thick-headed flies.It is distinguished from T. fulvipes by its smaller size and unique leg colorations.The species is parasitic, laying eggs in halictid bees, with larvae feeding internally.T. atra inhabits chalky, calcareous grasslands and is distributed widely across Britain and Ireland, especially in southern regions.
A high-quality genome of T. atra was sequenced using one male specimen from Hartslock Nature Reserve.The final assembly spans 354.2 Mb, organized into 22 scaffolds, with 99.94% of sequences mapped to chromosomes.The genome was annotated, identifying 30,620 proteincoding genes.This work, part of the Darwin Tree of Life Project, aids research on taxonomy, phylogeny, and biology of T. atra and related taxa.

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: Biological control 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.
The approach is state-of-the-art, the raw data appear to be of a suitably high quality, and the assembly methods are appropriate.The public availability of raw data and genome assembly are appropriate.The resulting genome is likely to be of very high quality, and I have no doubt that it will be of great value to any researchers working on this fascinating group of flies, or on the comparative or evolutionary genomics of insects more generally.
The introduction is well written, and rich in interesting a relevant biological detail.I particularly like the information on identification and habitat, although I feel it would be additionally valuable to have more information on the geographic distribution and phenology elsewhere in the species range.Is it the same, or different to the British isles?I think there should also be a statement on the conservation status (if any) of the species.As always, I also think it would be nice to have a couple of good photos of male and female in life.
Having read the first reviewer's comments, I echo their call for greater clarity on the collections, sexes, and identification.I am also surprised by the very large number of genes (this seems unprecedented for a dipteran?), and would like to see some comment on what proportion of these are likely to be (e.g.) TEs?.Finally, it seems strange that the associated Spiroplasma endosymbiont assembly (idTheAtra2.Spiroplasma_sp_1) is not mentioned.

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? Partly
Are the datasets clearly presented in a useable and accessible format?Yes

Figure 1 .
Figure 1.Photographs of the Thecophora atra (specimen ID NHMUK014444618, ToLID idTheAtra2) specimen used for genome sequencing.A. The specimen in lateral view.B. The specimen in dorsal view.Photographs by Olga Sivell.

Figure 2 .
Figure 2. Genome assembly of Thecophora atra, idTheAtra2.1: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 354,246,487 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 (98,076,447 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (94,083,503 and 8,213,565 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/Thecophora%20atra/dataset/CALMKQ01/snail.

Figure 3 .
Figure 3. Genome assembly of Thecophora atra, idTheAtra2.1:BlobToolKit GC-coverage plot.Scaffolds are coloured by phylum.Circles are sized in proportion to scaffold length.Histograms show the distribution of scaffold length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Thecophora%20atra/dataset/CALMKQ01/blob.

Figure 4 .
Figure 4. Genome assembly of Thecophora atra, idTheAtra2.1: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/Thecophora%20atra/dataset/CALMKQ01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Thecophora atra, idTheAtra2.1:Hi-C contact map of the idTheAtra2.1 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=LDkQ8LYLT9KdKtYbH37mvw.

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