The genome sequence of the Swift Louse Fly Crataerina pallida (Latreille, 1812)

We present a genome assembly from an individual female Crataerina pallida (the Swift Louse Fly; Arthropoda; Insecta; Diptera; Hippoboscidae). The genome sequence is 177.0 megabases in span. Most of the assembly is scaffolded into 6 chromosomal pseudomolecules, including the X chromosome. The mitochondrial genome has also been assembled and is 21.57 kilobases in length.


Background
The Swift Louse Fly Crataerina pallida is a haematophagous ectoparasite of birds.It is usually found on the swift Apus apus, Alpine swift A. melba, or pallid swift A. pallidus, and occasionally on Hirundines or Passerines (Hutson, 1981;Hutson, 1984;Oldroyd, 1966).Its range is restricted to Europe with a few records from South Africa (GBIF Secretariat, 2022) presumably from overwintering Swifts.The first record of the species in Britain or Ireland is from Selborne, Hampshire in 1774 (White, 1789).
Swift Louse Flies are highly adapted to their lifestyle: specially adapted feet enable them to cling to their host (Petersen et al., 2018) and flattened bodies and reduced functionless wings help them hide amongst feathers (Hutson, 1984;Oldroyd, 1966).Whereas most flies lay eggs, C. pallida is larviparous.A single larva is fed on a secretion from a gland in the female's uterus and is only deposited into the hosts' nest when it is ready to pupate (Hutson, 1981;Oldroyd, 1966).Despite their large size compared to their hosts, parasite aggregation, and infestation rates of up to 70% of adult swifts and over 90% of nests, there is no evidence that they harm their hosts or influence reproductive success (Hutson, 1981;Lack & Lack, 2018;Lee & Clayton, 1995;Walker & Rotherham, 2010).This is the first complete genome of a member of the family Hippoboscidae to be published, although complete mitochondrial genomes are available for some species (Li et al., 2022;Liu et al., 2017;Wang et al., 2021).
We present a chromosomally complete genome sequence for Crataerina pallida, based on one specimen hatched from a puparium collected from a swift nest in the tower of Oxford University Museum of Natural History, where swifts have been monitored since 1947 (Lack & Lack, 2018;Lack, 1951).

Genome sequence report
The genome was sequenced from one Crataerina pallida (Figure 1) collected from the Oxford University Museum of Natural History tower, Oxfordshire, UK (51.76,.A total of 144-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 51 missing joins or misjoins and removed 8 haplotypic duplications, reducing the assembly length by 0.6% and the scaffold number by 83.72%, and increasing the scaffold N50 by 69.36%. The final assembly has a total length of 177.0 Mb in 6 sequence scaffolds with a scaffold N50 of 31.6 Mb (Table 1).The snailplot 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 (99.98%) 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 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/452744.

Sample acquisition and nucleic acid extraction
Crataerina pallida specimens were collected from the Oxford University Museum of Natural History tower, Oxfordshire, UK (latitude 51.76, longitude -1.25) on 2021-04-17 and 2021-06-17.The specimens were taken as puparia from swift nests by George Candelin (independent researcher) by potting.The specimens were hatched the following spring and identified by Denise Wawman (University of Oxford).They were then preserved on dry ice.The specimen used for DNA  sequencing was idCraPall2 (specimen ID Ox001397), while idCraPall1 (specimen ID Ox001202) was used for Hi-C data.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The idCraPall2 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Head and 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.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 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.

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions.DNA sequencing was performed by the Scientific Operations core at the WSI on the Pacific Biosciences SEQUEL IIe (HiFi) instrument.Hi-C data were also generated from head and thorax tissue of idCraPall1 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 using the gEVAL system (Chow et al., 2016) as described previously (Howe et al., 2021).Manual curation 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 Table 3 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

Software tool
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, 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 Genome Research Limited (operating as the Wellcome Sanger Institute), and in some circumstances other Darwin Tree of Life collaborators.
While the metrics presented here show a nearly complete genome assembly, to which I do not have much to comment, the interest one could have in this work is certainly broader than the sole generation of new sequence data.As the authors emphasize, no genome was available for the family Hippoboscidae until now and it could thus enlighten us on its evolutionary history through the phylogenetic position of Crataerina pallida among the Calyptratae.
Moreover, some of the assembly statistics, like total length, could have been further investigated as compared to genome size of the most closely related available species.Is 177.0 Mb typical of the genome size of Calyptratae species?Crataerina pallida being indeed an haematophagous ectoparasite of birds, it would also be interesting to know how its genome size compares to other sequenced parasitic species that generally show a reduced genome size.On another hand, though I am aware that the authors plan to annotate this genome in another study, this would have had all its place in this paper to check if this ectoparasitic lifestyle has evolved through a reduction of the number of genes as well as a change in the amount and nature of repetitive sequences (transposons family for example).

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.
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.

Jaakko L.O. Pohjoismäki
Department of Environmental and Biological Sciences, University of Eastern Finland, Joensuu, Finland The report by Denise Wawman and coworkers presents the genome of Crataerina pallida, based on a female fly.The species, although abundant in swift colonies, is not trivial to obtain and a nice addition to the growing number of reference genomes from the DToL.As pointed out in the introduction, it is also the first complete genome of a louse fly.It is unfortunate that the specimen was a female, as males represent the heterogametic sex in Diptera.Although the sexual dimorphism is not clear in this species, males could have been possible to differentiate from the females with relative ease, based on the terminal segments of the abdomen, especially when reared material was available.
Of the European species of louse flies, Crataerina pallida could be mistaken with C. hirundinis.
Although the photo of the specimen is not too detailed, it shows enough to confirm the species identity, which is further backed by the host information.I did, however, a pairwise BLAST of the mitochondrial genome against the species Cox1 barcode sequence, which differed by one nucleotide.It would be very informative, if such check was performed always by the genome team.
The genome metrics present the typical high quality of the DToL pipeline, to which I have not much to comment.I do not see the gene annotations in the genome view, not even for the mitochondrial genome, but I assume these will become available (just noted at the end that data availability states that it will be annotated using RNA-seq).I understand the rush to publish, but these would be really interesting to see when browsing through the available genome.
Overall, the published genome will be a nice resource for anybody interested in taxonomy and evolution of Diptera.The published sequences can also contain the genomes of potential pathogens that the louse fly had carried, providing interesting snapshot of this type of dark diversity.

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: Molecular biology, genetics, taxonomy 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 Crataerina pallida, idCraPall2.1: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 177,070,084 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 (37,350,342 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (31,608,065 and 30,943,858 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/idCraPall2.1/ dataset/idCraPall2_1/snail.

Figure 3 .
Figure 3. Genome assembly of Crataerina pallida, idCraPall2.1: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/idCraPall2.1/dataset/idCraPall2_1/blob.

Figure 4 .
Figure 4. Genome assembly of Crataerina pallida, idCraPall2.1: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/idCraPall2.1/dataset/idCraPall2_1/ cumulative.

Figure 5 .
Figure 5. Genome assembly of Crataerina pallida, idCraPall2.1:Hi-C contact map of the idCraPall2.1 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=SK3mzTOGRDukmxsOjOrbIg.