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.

Methods

Collection and rearing of Drosophila biauraria

D. biauralia 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. Wolbachia and DbMKPV1 infections were detected by PCR, as described previously [24-25].

Tetracycline treatment

All-female matrilines (W1 and W2) were reared on tetracycline-containing banana medium (0.05% [w/v]) [24] for two generations.

Egg hatching rates

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 Na₂HPO₄, 2.68 mmol/l KCl, 1.47 mmol/l KH₂PO₄, 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.

Sex determination of embryos and hatchlings of D. biauraria

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 × g for 2 min, and finally subjected to PCR. A Y chromosome-linked male-specific marker for D. biauraria [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 actin-5C to confirm proper D. biauraria 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.

Genome sequencing of flies and constructions of Wolbachia genomes

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 Wolbachia genomes available in the NCBI database were assessed using BLASTn searches. Contigs showing homology to known Wolbachia genomes were designated as candidate contigs of Wolbachia strains in D. biauraria. The raw data of W1 and W2 were mapped to Wolbachia-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 Wolbachia wBiau1 and wBiau2 genomes was confirmed using Bandage v0.8.1 [29]. Circular Wolbachia 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].

Wolbachia genes wmk [33], cifs (cifA and cifB) [34-35], and oscar [21, 36] were used to identify homologues in the wBiau1 and wBiau2 genomes using local BLASTn and BLASTp searches (default parameters). Motifs in the wmk, cifA, cifB, and oscar gene homologues were surveyed using InterPro (https://www.ebi.ac.uk/interpro/) and HHpred (https://toolkit.tuebingen.mpg.de/tools/hhpred). Phylogenetic trees of Wolbachia wsp and MLST genes were constructed based on maximum likelihood with bootstrap re-sampling of 1,000 replicates using MEGA7 [37].

Phylogenetic analysis of mitochondrial CO1

The mitochondrial CO1 of D. biauraria lines was amplified using HCO and LCO primer sets targeting the CO1 gene [38]. Amplicons were purified with Wizard^® 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 CO1 was constructed based on maximum likelihood with bootstrap re-sampling of 1,000 replicates using MEGA7 [37].