DOI: 10.5061/dryad.j9kd51cjh
title: Data from: Two male-killing Wolbachia from Drosophila birauraia that are closely related but distinct in genome structure

description: Insects harbour diverse maternally inherited bacteria and viruses, some of
 which have evolved to kill the male progeny of their hosts (male killing:
 MK). The fly species Drosophila biauraria carries a maternally transmitted
 MK-inducing partiti-like virus, but it was unknown if it carries other
 MK-inducing endosymbionts. Here, we identified two male-killing Wolbachia
 strains (wBiau1 and wBiau2) from D. biauraria and compared their genomes
 to elucidate their evolutionary processes. The two strains were
 genetically closely related but had exceptionally different genome
 structures with considerable rearrangements compared with combinations of
 other Wolbachia strains. Despite substantial changes in the genome
 structure, the two Wolbachia strains did not experience gene losses that
 would disrupt the male-killing expression or persistence in the host
 population. The two Wolbachia-infected matrilines carried distinct
 mitochondrial haplotypes, suggesting that wBiau1 and wBiau2 have invaded
 D. biauraria independently and undergone considerable genome changes owing
 to unknown selective pressures in evolutionary history. This study
 demonstrated the presence of three male-killers from two distinct origins
 in one fly species and highlighted the diverse and rapid genome evolution
 of MK Wolbachia in the host.
description: <strong>Methods</strong>
 <strong>Collection and rearing of <em>Drosophila
 biauraria</em></strong> <em>D.
 biauralia</em> samples were collected from the Field Science Center
 for Northern Biosphere, Hokkaido University, Tomakomai, Hokkaido, Japan in
 2015 and 2017. Flies were collected by sweeping and banana traps. The
 collected females were individually maintained at 19 °C with the standard
 banana medium [24]. The sex ratios of the lines derived from
 field-collected females were determined at the adult stage. The normal sex
 ratio (NSR) isofemale line SP11-20 [25] was maintained for more than 70
 generations. The all-female matrilines (W1 and W2), each derived from a
 single female, were maintained by crossing with males of the SP11-20 line.
 <em>Wolbachia</em> and DbMKPV1 infections were detected by
 PCR, as described previously [24-25].
 <strong>Tetracycline treatment</strong>
 All-female matrilines (W1 and W2) were reared on
 tetracycline-containing banana medium (0.05% [w/v]) [24] for two
 generations. <strong>Egg hatching
 rates</strong> Egg-hatching rates were estimated
 by counting the number of hatched and unhatched larvae. A total of 50–100
 females of either W1 or NSR (SP11-20) were allowed to oviposit on grape
 juice agar medium for 1 d [25]. The eggs were collected and maintained in
 phosphate-buffered saline with Tween 20 (PBST; 137 mmol/l NaCl, 8.1 mmol/l
 Na<sub>2</sub>HPO<sub>4</sub>, 2.68 mmol/l KCl,
 1.47 mmol/l KH<sub>2</sub>PO<sub>4</sub>, 0.02%
 Tween 20, pH7.4) for 4 d. The number of neonates and remaining embryos
 were counted manually under a microscope. This treatment was repeated at
 least four times. <strong>Sex determination of
 embryos and hatchlings of <em>D.
 biauraria</em></strong> We determined the
 sex of embryos and hatchlings by PCR targeting a male-specific Y
 chromosome marker. Briefly, each embryo and neonate was squashed in 20 μL
 of PrepMan™ Ultra Sample Preparation Reagent (ThermoFisher). Samples were
 then incubated at 100°C for 10 min, vortexed for 15 s, centrifuged at
 20,000 × <em>g</em> for 2 min, and finally subjected to PCR. A
 Y chromosome-linked male-specific marker for <em>D.
 biauraria</em> [25] was amplified using a pair of primers,
 DbY_c52202_F2 (5′-ACCGAGCGCGAAATCATAAAACCAGCATC-3′) and DbY_c52202_R2
 (5′-CTCATATCACTTCATGTATCCCACACTTTTAACAG-3′). Db-actin5C-68-F
 (5′-GGCCATCCAGGCCGTGCTCTC-3′) and Db-actin5C-68-R
 (5′-GCGCTCGGCAGTGGTGGTGAAG-3′) were used to amplify
 <em>actin-5C</em> to confirm proper <em>D.
 biauraria</em> genomic DNA extraction. These markers were amplified
 using the Emerald Amp Max Master mix (TaKaRa) at 94°C for 3 min; the
 cycling conditions were as follows: 35 cycles of denaturation at 94°C for
 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 sec,
 followed by a final extension at 72°C for 7 min. Of the actin-positive
 samples, those that were positive for Y-markers were classified as male.
 Those that were negative for Y-markers were classified as
 female. <strong>Genome sequencing of flies and
 constructions of <em>Wolbachia</em>
 genomes</strong> For genome sequencing of fly
 lines W1 and W2, high molecular weight DNA was extracted from 0.1 g adult
 females (approximately 100–200 individuals) by using Nanobind Tissue Big
 DNA Kit (Circulomics Inc., Baltimore, MD, USA) and was used for library
 construction using Ligation Sequencing Kit v14 (Oxford Nanopore
 Technologies, Oxford, UK) following the manufacturer’s protocol. The
 constructed libraries were sequenced using the ONT MinION flow cell (R
 10.4) (Oxford Nanopore Technologies). The extracted DNA was also subjected
 to Illumina paired-end 150 bp sequencing (PE-150) at the Bioengineering
 Lab. Co., Ltd. (Japan). The obtained nanopore reads were assembled using
 Flye 2.3 [26] in Galaxy Europe (https://usegalaxy.eu/). Homologies between the assembled contigs of W1 and W2 and all <em>Wolbachia</em> genomes available in the NCBI database were assessed using BLASTn searches. Contigs showing homology to known <em>Wolbachia</em> genomes were designated as candidate contigs of <em>Wolbachia</em> strains in <em>D. biauraria</em>. The raw data of W1 and W2 were mapped to <em>Wolbachia</em>-like contigs using minimap2 v2.17-r941 [27], and the mapped reads were extracted using SAMtools v.1.9 [28] and assembled using Flye 2.3 [26]. The circularity of the <em>Wolbachia</em> wBiau1 and wBiau2 genomes was confirmed using Bandage v0.8.1 [29]. Circular <em>Wolbachia</em> genomes were polished against Illumina data using minimap2 [20] and Pilon v. 1.23 [30]. The polished closed genomes of the wBiau1 and wBiau2 strains were annotated via the DFAST web server [31]. Prophage regions were annotated using the PHASTER web server [32]. <em>Wolbachia</em> genes <em>wmk </em>[33], <em>cifs (cifA and cifB) </em>[34-35], and <em>oscar</em> [21, 36] were used to identify homologues in the <em>w</em>Biau1 and <em>w</em>Biau2 genomes using local BLASTn and BLASTp searches (default parameters). Motifs in the <em>wmk</em>, <em>cifA,</em> <em>cifB,</em> and <em>oscar</em> gene homologues were surveyed using InterPro (https://www.ebi.ac.uk/interpro/) and HHpred (https://toolkit.tuebingen.mpg.de/tools/hhpred). Phylogenetic trees of <em>Wolbachia wsp </em>and <em>MLST </em>genes were constructed based on maximum likelihood with bootstrap re-sampling of 1,000 replicates using MEGA7 [37]. <strong>Phylogenetic analysis of mitochondrial CO1</strong> The mitochondrial CO1 of <em>D. biauraria</em> lines was amplified using HCO and LCO primer sets targeting the CO1 gene [38]. Amplicons were purified with Wizard<sup>®</sup> SV Gel and PCR Clean-Up System (Promega), which were subjected to sequencing using BigDye terminator v3.1 (Applied Biosystems) with the following conditions: 96°C for 1 min, followed by 25 cycles of 96°C for 10 sec, 50°C for 5 sec, and 60°C for 4 min. A phylogenetic tree of <em>CO1 </em>was constructed based on maximum likelihood with bootstrap re-sampling of 1,000 replicates using MEGA7 [37].
description: # Massive genomic rearrangement between two closely related male-killing
 Wolbachia in Drosophila biauraria
 We present genome data of
 two male-killing Wolbachia strains and their effects on host survival.
 This includes genome, sex ratio, and hatchability data. ## Description of
 the data and file structure * Genome data used for the project are offered
 as fasta files. Annotation data for wBiau1 and wBiau2 strains are offered
 as Zip files (annotation genbank file, annotation gff file, coding
 sequences [CDS], protein sequences, RNA sequences, genomic features, and
 statistics data). * Hatch rate data, Y chromosome detection data (i.e.,
 male/female ratio in hatched/unhatched individuals), sex ratio data, data
 for tetracycline treatments, and blast analyses data were presented in an
 excel file 'data_set.v2.xlsx'. The details of each sheet of the
 Excel file are as follows "Fig1 data Hatch rate" sheet
 represents the hatch rate data of D. biauraria. "Fig1 data Y
 chromosome detect" sheet represents the PCR detection data of the Y
 chromosome of D. biauraria. NSR: normal sex ratio line; W1: a male-killing
 host line containing wBiau1; W2: a male-killing host line containing
 wBiau2. "Fig1 dataTetracycline treatment" sheet represents the
 tetracycline treatment data in D. biauraria harbouring Wolbachia. Tet:
 tetracycline treated, Fem: female; Mal: male. "Table S1 bacteria
 classificatio" sheet represents the data of the taxonomy
 classification based on Kraken. taxonomy_lvl: taxonomy level. "Table
 S2 data wBiau2 vs wBiau1" sheet represents the homology data between
 wBiau2 and wBiau1 (wBiau2 as query). "Table S2 data wBiau1 vs
 wBiau2" sheet represents the homology data between wBiau1 and wBiau2
 (wBiau1 as query). "Table S3 IS data" sheet shows the numbers
 and frequencies of the IS genes in the Wolbachia genomes. N: number of
 genes. "Table S4 data" sheet represents the BlastP data between
 proteins encoded by wBiau strains and the phenotype-associated Wolbachia
 proteins. "Table S5 data" sheet represents the homology data of
 wmk and Cif proteins between wBiau1 and wBiau2 strains. * Alignment data
 of MLST genes of Wolbachia is shown as a fasta
 file"Wolbachia_phylogeny.fas" * Alignment data of COI gene of
 Drosophila is shown as a fasta file"CO1.for.phylogeny.fa" *
 Genomic data used for comparative analyses were presented as
 "wXxx(strain name).genome.fasta". Details of Wolbachia strains
 are as follows: wBiau1: Wolbachia endosymbiont in Drosophila biauraria
 wBiau2: Wolbachia endosymbiont in Drosophila biauraria wFur: Wolbachia
 endosymbiont in Ostrinia furnacalis wNo: Wolbachia endosymbiont in
 Drosophila simulans wAu: Wolbachia endosymbiont in Drosophila simulans
 wRi: Wolbachia endosymbiont in Drosophila simulans wHmt: Wolbachia
 endosymbiont in Homona magnanima wPip: Wolbachia endosymbiont in Culex
 pipiens wMel: Wolbachia endosymbiont in Drosophila melanogaster ##
 Sharing/Access information Links to other publicly accessible locations of
 the data: * The sequence read data were deposited in the DDBJ under the
 accession numbers PRJDB16258 (BioProject), SAMD00634859–SAMD00634860
 (BioSample), and DRA016759 (DRA). *Wolbachia* genomes are available in the
 DDBJ database under the accession numbers *w*Biau1 (AP028655) and *w*Biau2
 (AP028656).