The genome sequence of an ichneumon wasp, Clistopyga incitator (Fabricius, 1793)

We present a genome assembly from an individual female Clistopyga incitator (an ichneumon wasp; Arthropoda; Insecta; Hymenoptera; Ichneumonidae). The genome sequence is 260.7 megabases in span. Most of the assembly is scaffolded into 21 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 25.0 kilobases in length.


Background
While most ichneumonids, or Darwin wasps, are parasitoids of immature stages of insects, a few different groups have evolved to take advantage of spiders and their egg sacs.The most species-rich radiation of spider parasitoids and predators within Ichneumonidae is within the subfamily Pimplinae (Gauld et al., 2002;Gauld & Dubois, 2006;Matsumoto, 2016).Clistopyga is a Pimpline genus found across much of the world with several species known to be predators within spider egg sacs (Nielsen, 1929) and some presumed or confirmed to be parasitoids of adult spiders (Fritzén & Sääksjärvi, 2016) in their silk nests.
Clistopyga females are readily recognised by their upcurved ovipositors.They are often attractively patterned, with C. incitator usually being recognisable on colour pattern, with several creamy white marks on the head and thorax and a red and black mesosoma (thorax plus the propodeum).The upcurved ovipositor is used to penetrate dense silken egg sacs of spiders to lay eggs.Clistopyga incitator has been reared from the egg sac of Segestria senoculata (Linnaeus), a spider which spins dense silken tubes, often in stone walls.The sequenced specimen of C. incitator was found walking on a stone wall.The ovipositor of C. incitator might have another function too.In a closely related (unidentified) species, tiny 'teeth' on the lower ovipositor valves pull silk threads when the ovipositor is being withdrawn from the silk nest.Repeated movements act to close up the hole made by the ovipositing female, with the ovipositor acting in a manner analogous to a felting needle (Fritzén & Sääksjärvi, 2016).
Clistopyga incitator is a reasonably common, widespread species, found across Europe and temperate Asia, for example, Kasparyan DR, 2007;Yu, van Achterberg and Horstmann, 2016.At least in southern Britain, there seem to be two broods per year (Fitton et al., 1988).
The subfamily Pimplinae has been the focus of several studies aiming to understand how an amazing variety of life history strategies has evolved in one lineage, for example Wahl and Gauld, 1998;Gauld, Wahl and Broad, 2002;Klopfstein et al., 2019.However, the Pimplinae which belongs to an assemblage (the pimpliformes) which seems to have initially rapidly diversified in the Jurassic, might not be monophyletic (Spasojevic et al., 2021).As the first complete assembly for a species of the subfamily Pimplinae, the genome of Clistopyga incitator should pave the way for new approaches to investigating the evolution of different parasitoid life histories in relatively closely related taxa.

Genome sequence report
The genome was sequenced from one female Clistopyga incitator (Figure 1) collected from Bowcombe Creek, UK (latitude 50.28, longitude -3.76).A total of 68-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 61 missing joins or mis-joins and removed 10 haplotypic duplications, reducing the assembly length by 2.32% and the scaffold number by 50%, and increasing the scaffold N50 by 12.45%.
The final assembly has a total length of 260.7 Mb in 26 sequence scaffolds with a scaffold N50 of 13.7 Mb (Table 1).Most (99.7%) of the assembly sequence was assigned to 21 chromosomal-level scaffolds.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/494138

Sample acquisition and nucleic acid extraction
A female Clistopyga incitator specimen (iyCliInci1) was collected from Bowcombe Creek, UK (latitude 50.28, longitude -3.76) on 20 August 2021.The specimen was collected by hand by Lucy Broad (private address), and then identified by Gavin Broad (Natural History Museum) and frozen at -80°C.
The sample was prepared at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The iyCliInci1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Head and thorax tissue of iyCliInci1 was disrupted using a Nippi Powermasher fitted with a BioMasher pestle.DNA was extracted at the WSI Scientific Operations core using the Qiagen MagAttract HMW DNA kit, according to the manufacturer's instructions.

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 II (HiFi) instrument.Hi-C data were also generated from tissue of iyCliInci1 using the Arima v2 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 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.To evaluate the assembly, MerquryFK was used to estimate consensus quality (QV) scores and k-mer completeness (Rhie et al., 2020).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 software tool versions and sources.

Ethics and compliance issues
The materials that have contributed to this genome   Broad and Broad propose the genome sequence of a Darwin wasp, Clistopyga incitator (Fabricius, 1793) (Pimplinae).This sequence, which will be in a free access database, will be used for future studies using molecular tools.
I consider that the MS can be indexed with minor corrections and I present my observations and suggestions below: Title and abstract: I suggest replacing ichneumon wasp with Darwin wasp.
Backgrounf: "Clistopyga females are readily recognised by their upcurved ovipositors."Except females of C. henryi species-group and C. isayae species-group.Reviewer Expertise: Bioinformatics 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 Clistopyga incitator, iyCliInci1.1: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 260,700,937 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 (19,385,043 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (13,682,479 and 9,078,478 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 hymenoptera_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/iyCliInci1.1/dataset/CANNPW01/snail.
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.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.All efforts are undertaken to minimise the suffering of animals used for sequencing.Each transfer of samples is further undertaken

Figure 5 .
Figure 5. Genome assembly of Clistopyga incitator, iyCliInci1.1:Hi-C contact map of the iyCliInci1.1 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=Kh-LVKesSN6ytn8mcd0U6Q.

Figure 1 :
Figure 1: The specimen is upside down.