The genome sequence of the Field Cuckoo-bee, Bombus campestris (Panzer, 1801)

We present a genome assembly from an individual male Bombus campestris (the Field Cuckoo-bee; Arthropoda; Insecta; Hymenoptera; Apidae). The genome sequence is 275 megabases in span. Most of the assembly is scaffolded into 25 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 24.7 kilobases in length. Gene annotation of this assembly on Ensembl identified 12,993 protein coding genes.


Background
The Field Cuckoo-bee, Bombus campestris, is one of six 'cuckoo bumblebee' species in the UK.It is a social parasite of Bombus pascuorum, and probably also all four other UK carder bees (B. humilis, B. muscorum, B. ruderarius and B. sylvarum), usurping colonies of these species and using the workers to raise its own offspring.This species does not produce workers.Females search out and enter a host nest, before dominating or killing the host queen.The host workers may kill the cuckoo bee, but if the usurpation is successful, they will rear the B. campestris offspring (Fisher, 1988).Cuckoo bumblebees were formerly placed in their own genus Psithyrus, which has subsequently been sunk to the rank of subgenus (Pedersen, 1996).
It is common and widespread across most of Europe, being found in a wide variety of habitats along with the host species (Pekkarinen & Teräs, 1993), although it has been found to have declined significantly since 1990 (Antonovics & Edwards, 2011).It is a medium sized bumblebee (~18 mm) covered in black hairs with two stripes of yellow hairs on the thorax and often extensive areas of greenish-yellow hairs on the apical half of the abdomen (Edwards & Jenner, 2005).The wings are usually strongly dark-tinged, especially on the female.Males can look similar to B. sylvestris, although the very tip of the abdomen is always black haired (red-haired in B. sylvestris) and with a pair of large hair tufts on sternite six (Falk & Lewington, 2015).
Overwintered females emerge from April, linked to the phenology of the host species.New females and males are produced from July into August.This species does not collect pollen, although females may feed on it to facilitate ovary development.A wide range of flowers are visited for nectar, including thistles and knapweeds.
A complete genome sequence for this species will facilitate studies into the evolution social parasitism and reproductive systems, as well as conservation of pollinator species.

Genome sequence report
The genome was sequenced from one male B. campestris specimen (Figure 1) collected from Wytham Woods, Oxfordshire (latitude 51.76, longitude -1.33).A total of 83-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 128-fold coverage in 10X Genomics read clouds were generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 48 missing joins or mis-joins, increasing the assembly length by 1.65% and the scaffold number by 24.32%, and increasing the scaffold N50 by 77.22%.
The final assembly has a total length of 275.3 Mb in 92 sequence scaffolds with a scaffold N50 of 12.2 Mb (Table 1).Most (98.28%) of the assembly sequence was assigned to 25 chromosomal-level scaffolds.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 2-Figure 5; Table 2).The assembly has a BUSCO v5.3.2 (Manni et al., 2021) completeness of 97.7% (single 97.4%, duplicated 0.3%) using the hymenoptera_odb10 reference set (n = 5,911).

Genome annotation report
Annotation of the B. campestris GCA_905333015.1 assembly was generated using the Ensembl genome annotation pipeline (Table 1; https://rapid.ensembl.org/Bombus_campestris_GCA_905333015.1/).The resulting annotation includes 12,993 protein coding genes with an average length of 13,241.70 and an average coding length of 1,416.02,and 5,328 non-protein coding genes.There is an average of 6.15 exons and 5.15 introns per canonical protein coding transcript, with an average intron length of 1,721.82.A total of 7,584 gene loci have more than one associated transcript.

Sample acquisition and nucleic acid extraction
A single male B. campestris specimen (iyBomCamp1) was collected by netting in Wytham Woods, Oxfordshire (biological vice-county: Berkshire), UK (latitude 51.76, longitude -1.33) on 13 August 2019.The specimen was collected and identified by Liam Crowley (University of Oxford) and then snap-frozen on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute.The iyBomCamp1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C and RNA sequencing.Whole organism tissue was cryogenically disrupted to a fine powder using a Covaris cryoPREP Automated Dry Pulveriser, receiving multiple impacts.High molecular weight  RNA was extracted from tissue of iyBomCamp1 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.et al., 2020).The genome was analysed and BUSCO scores generated within the BlobToolKit environment (Challis et al., 2020).Table 3 contains a list of all software tool versions used, where appropriate.

Genome annotation
The Ensembl gene annotation system (Aken et al., 2016) was used to generate annotation for the B. campestris assembly   GCA_905333015.1.Annotation was created primarily through alignment of transcriptomic data to the genome, with gap filling via protein to-genome alignments of a select set of proteins from UniProt (UniProt Consortium, 2019).

Ethics and compliance issues
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.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

Cheng Sun
College of Life Sciences, Capital Normal University, Beijing, China This study provided a high-quality reference genome for the field cuckoo bumblebee, which will be useful for understanding the evolution of social parasitism and the conservation of this species.
In general this is a well-organized manuscript, and I only have the following suggestions: Double check the number of genes in hymenoptera_odb10 reference set.It should 5991 other than 5911. 1.
It will be better to use a color different from red to show the border of scaffold in Figure 5.For me, it's hard to count how many scaffolds there are.

2.
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: bumblebee genome evolution 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 Bombus campestris, iyBomCamp1.3: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 275,276,780 bp assembly.The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest sequence present in the assembly (17,090,360 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (12,249,729 and 7,492,178 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/iyBomCamp1.2/dataset/CAJOSK02/snail.

Figure 3 .
Figure 3. Genome assembly of Bombus campestris, iyBomCamp1.3:GC coverage.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/iyBomCamp1.2/dataset/CAJOSK02/blob.

Figure 4 .
Figure 4. Genome assembly of Bombus campestris, iyBomCamp1.3:cumulative sequence.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/iyBomCamp1.2/ dataset/CAJOSK02/cumulative.

Figure 5 .
Figure 5. Genome assembly of Bombus campestris, iyBomCamp1.3:Hi-C contact map.Hi-C contact map of the iyBomCamp1.3assembly, 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=UF88I5HARxe-eYswgcm2bQ.

Table 1 . Genome data for Bombus campestris, iyBomCamp1.3. Project accession data
ing.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

INSDC accession Chromosome Size (Mb) GC%
. 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, Genome Research Limited (operating as the Wellcome Sanger Institute), and in some circumstances other Darwin Tree of Life collaborators.The genome sequence is released openly for reuse.The Bombus campestris 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.

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.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
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.