The genome sequence of a woodlouse fly, Phyto melanocephala (Meigen, 1824) [version 1; peer review: awaiting peer review]

We present a genome assembly from an individual female Phyto melanocephala (woodlouse fly; Arthropoda; Insecta; Diptera; Rhinophoridae). The genome sequence is 739.7 megabases in span. Most of the assembly is scaffolded into 6 chromosomal pseudomolecules, including the X sex chromosome. The mitochondrial genome has also been assembled and is 16.3 kilobases in length. Gene annotation of this assembly on Ensembl identified 29,294 protein coding genes.


Background
Phyto melanocephala (Diptera, Calliophoridae) is a small to medium, grey and black fly from the subfamily Rhinophorinae, commonly referred to as woodlouse flies. The status of the group has been recently reviewed by Yan et al. (2021). The family Rhinophoridae has been reclassified as a subfamily and placed within Calliphoridae (Yan et al., 2021). This classification has been accepted in Britain (Chandler, 2023).
In Britain there are eight species of Rhinophorinae currently recorded, including two species in the genus Phyto Robineau-Desvoidy, 1830 (Chandler, 2023). Phyto melanocephala can be distinguished from the other British woodlouse flies by the following characters: the wings are almost clear, the stalk at the end of cell R₄₊₅ is very short; the third and fourth tergites have upright discal setae, vein A₁+CuA₂ is short, ending far from the wing margin, and there are three black stripes on the thorax in front of the suture (Falk, 2016;Sivell, 2020;Van Emden, 1954). The body length has been reported to be 4.5-9.0 mm (Van Emden, 1954) or 3.5-7.5 mm (Falk, 2016).
Adult Rhinophorinae feed on sugars from flowering plants, and their larvae are internal parasites of woodlice (Isopoda, Oniscidea). Phyto melanocephala are ready to copulate soon after eclosion (hours for females, a day for males). The female mates with a single male, while males readily mate with multiple females. Oviposition is stimulated by viscous secretions of woodlice in the substrate. The female lays elongate, spindle-shaped eggs in cracks and crevices, in small batches of 2 to 10 eggs. In laboratory conditions it was observed that 200 to 500 eggs (depending on size of the fly) are laid within 1 to 2 days, after which the female dies (Bedding, 1965).
The first instar larvae of Phyto remain in the moist substrate (they move away from dry sites) awaiting the arrival of a host. The larva moves by somersaulting and attaches itself to the legs, epimerites, uropods, antennae or pleopods of the host. Subsequently it moves to the base of the penis (in males) or between the anterior pleopods (in females), burrows into the woodlouse and begins feeding. The first, second and early third instars feed on the haemolymph (blood), while late third instar larvae also consume the gonads and fat of the host. The woodlouse ultimately dies and its insides are liquified and ingested about a day before the parasite pupates. Pupation occurs inside the host's cuticle and adults emerge after 11 to 12 days (at 25°C) (Bedding, 1965 (Bedding, 1965;Herting, 1961;Pape & Andersen, 1995;Séguy, 1941;Thompson, 1934).
Descriptions of P. melanocephala larvae and pupa are given by Thompson (1934) and the egg and first instar larva by Bedding (1973). Bedding (1973) also provides identification keys for all instars of British Rhinophorinae larvae.
Phyto melanocephala is common and widely distributed in southern Britain, reaching northwards to Yorkshire, but is absent from Scotland. It is on the wing from late April to October (Falk, 2016;Rhinophoridae Recording Scheme, 2023;Van Emden, 1954). Phyto melanocephala prefers open, warm sites and wasteground, particularly coastal sites; this fly has been collected from brownfield land, coastal shingle, chalk downland, coastal dunes, saltmarsh edge, sea walls, gardens and woodland clearings (Falk, 2016;Van Emden, 1954).
Here we present a chromosomally complete genome sequence for Phyto melanocephala, based on one female specimen from Hartslock Nature Reserve, UK.

Genome sequence report
The genome was sequenced from one female Phyto melanocephala ( Figure 1) collected from Hartslock Nature Reserve, UK. A total of 24-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 50-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 76 missing joins or mis-joins and removed five haplotypic duplications, reducing the scaffold number by 87.14%, and increasing the scaffold N50 by 0.5%.
The final assembly has a total length of 739.7 Mb in 9 sequence scaffolds with a scaffold N50 of 136.8 Mb (Table 1). Most (99.99%) of the assembly sequence was assigned to 6 chromosomal-level scaffolds, representing 5 autosomes and the X sex chromosome. Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 2- Figure 5; Table 2). The order and orientation of the regions from 0-1.29 Mb and 2.78-4.50 Mb on the X chromosome are uncertain. 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/1262321.

Sample acquisition and nucleic acid extraction
A female Phyto melanocephala specimen (specimen ID NHMUK014444516, idPhyMeln1) was collected from Hartslock Nature Reserve, UK (latitude 51.51, longitude -1.11) on 20 August 2020, using an aerial net. The specimen was collected and identified by Ryan Mitchell (Natural History Museum). The specimen was preserved in liquid nitrogen.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI). The idPhyMeln1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing. Thorax 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. Low molecular weight DNA was removed from a 20 ng aliquot of extracted DNA using the 0.8X AMpure XP purification kit prior to 10X Chromium sequencing; a minimum of 50 ng DNA was submitted for 10X sequencing. 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 by solidphase 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 abdomen tissue of idPhyMeln1 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 and 10X Genomics read cloud 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 were performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi) and Illumina NovaSeq 6000 (RNA-Seq and 10X) instruments. Hi-C data were also generated from head tissue of idPhyMeln1 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). One round of polishing was performed by aligning 10X Genomics read data to the assembly with Long Ranger ALIGN, calling variants with FreeBayes (Garrison & Marth, 2012). 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 as described previously (Howe et al., 2021). Manual curation was performed using HiGlass (Kerpedjiev et al., 2018) and Pretext (Harry, 2022). The mitochondrial genome was assembled using MitoHiFi (Uliano-Silva et al., 2022), 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 "sangertol/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 Phyto melanocephala assembly (GCA_941918925.1) 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  The genome sequence is released openly for reuse. The Phyto melanocephala genome sequencing initiative is part of the Darwin Tree of Life (DToL) project. All raw sequence data and the assembly have been deposited in INSDC databases. Raw data and assembly accession identifiers are reported in Table 1.