Skip to main content
NCBI home page
UKPMC Funders Author Manuscripts logoLink to UKPMC Funders Author Manuscripts
. Author manuscript; available in PMC: 2018 Nov 2.
Published in final edited form as: Nature. 2018 May 2;557(7704):252–255. doi: 10.1038/s41586-018-0086-2

Male-killing toxin in a Drosophila bacterial symbiont

Toshiyuki Harumoto 1,*, Bruno Lemaitre 1,*
PMCID: PMC5969570  EMSID: EMS76821  PMID: 29720654

Abstract

Several lineages of symbiotic bacteria in insects selfishly manipulate host reproduction to spread in a population1, often by distorting host sex ratios. Spiroplasma poulsonii2,3 (hereafter Spiroplasma) is a helical and motile, Gram-positive symbiotic bacterium, that resides in a wide range of Drosophila species4. A striking feature of Spiroplasma is male killing, whereby the sons of infected female hosts are selectively killed during development1,2. Although male killing caused by Spiroplasma has been studied since the 1950s, the underlying mechanism is unknown. Here, we identify a Spiroplasma protein, designated SpAID, whose expression induces male killing. Overexpression of SpAID in D. melanogaster kills males but not females, and induces massive apoptosis and neural defects, recapitulating the pathology observed in Spiroplasma-infected male embryos5–11. Our data suggest that SpAID targets the dosage compensation machinery on the male X chromosome to mediate its effects. SpAID contains ankyrin repeats and a deubiquitinase domain, which are required for its subcellular localization and activity. Moreover, we found a laboratory mutant strain of Spiroplasma with reduced male-killing ability and a large deletion in the SpAID locus. Collectively, our study has uncovered a novel bacterial protein that affects host cellular machinery in a sex-specific way, which is likely to be the long-searched-for factor responsible for Spiroplasma-induced male killing.

Endosymbiotic bacteria have evolved sophisticated strategies to manipulate their hosts to increase their transmission, and sex ratio distorters of arthropods are perhaps the champions of manipulation. These bacteria are transmitted exclusively through female hosts, and as a result, several lineages have evolved the ability to bias infections towards females, either by turning males into females (feminization), causing clonal reproduction (parthenogenesis), or eliminating males (male killing)1. Male killing has independently evolved in at least six bacterial taxa, including Spiroplasma, Wolbachia, and Rickettsia1. The genetic basis of male killing is one of the biggest longstanding mysteries in the field of insect symbiosis. Male killing by Spiroplasma (Fig. 1a) was described as early as the 1950s in Drosophila2. Past studies in Spiroplasma attributed the selective killing of male progeny to an unknown substance called “androcidin”, assumed to be secreted by the bacterium12. The identification of this toxin has been hampered by the lack of practical methods for molecular biology as with the case of other symbiotic bacteria.

Figure 1. Expression of SpAID selectively eliminates male offspring.

Figure 1

Open in a new tab

a, Spiroplasma-induced male killing in Drosophila. Infected females (top) produce only female offspring (bottom). A picture shows a male-killing Spiroplasma of D. melanogaster detected by DNA staining. b, Protein structure of SpAID, which contains ankyrin repeats (ANK, red), the OTU deubiquitinase domain (blue), an N-terminal signal peptide (SP, black), and a C-terminal hydrophobic region (HR, green). SpAID ΔC of the partial male-killing strain encodes a protein with an amino acid substitution (Q787C) and C-terminal truncation. The structures of two deletion constructs of SpAID (ΔANK and ΔOTU) are also indicated. The numbers represent amino acid (aa) residues. c, The number of adult progeny obtained from crosses between the Actin-GAL4 line and four UAS transgenic lines (GFP, SpAID, ΔANK, and ΔOTU; n = 10 independent crosses for each transgene). The UAS-GFP line was used as a negative control. We counted the number of resultant offspring (females, red; males, blue) expressing the transgenes (+, having both Actin-GAL4 and UAS transgenes) and siblings not expressing the transgenes (-, having only UAS transgenes) as internal controls. Different characters indicate statistically significant differences (P < 0.0001, P < 0.05 for ΔANK; N.S., not significant, P > 0.05; Steel-Dwass test; see Supplementary Table 2). Box plots indicate the median (bold line), the 25th and 75th percentiles (box edges), and the range (whiskers). Dot plots show all data points individually. The total numbers of adult counts for each genotype and sex are shown at the bottom.

Spiroplasma symbionts of D. melanogaster (strain MSRO for “melanogaster sex ratio organism”) kill all male progeny (e.g., MSRO-H99; Extended Data Fig. 1a). We unexpectedly identified a Spiroplasma mutant strain that shows reduced male-killing ability (MSRO-SE; the “partial male-killing” strain), where almost half of the male progeny survived (Extended Data Fig. 1a-c). The reduced male killing was not due to host genetic background or low bacterial titre (Extended Data Fig. 1b, d). To identify the genetic basis of reduced male killing, we sequenced the genome of MSRO-SE and compared it with that of MSRO-H99 (Extended Data Fig. 2). We found an intriguing candidate gene that was altered in the partial male-killing strain, encoding a 1,065 amino acid protein with ankyrin repeats and the OTU (ovarian tumour) deubiquitinase domain. We named this Spiroplasma protein SpAID (S. poulsonii androcidin; Fig. 1b) based on our subsequent functional studies. We focused our analysis on this gene, because (i) both ankyrin repeats and the OTU domain are conserved in proteins across eukaryotes and are also present in some bacterial effectors that manipulate host cellular processes13,14 and (ii) SpAID was located on a putative plasmid (Extended Data Fig. 2b) like other bacterial virulence factors15. Further analysis predicted an N-terminal signal peptide for secretion and a C-terminal hydrophobic region (Fig. 1b). The SpAID locus in the partial male-killing strain contained an 828-bp deletion (Extended Data Fig. 3), resulting in a truncated protein lacking the hydrophobic region, as well as a single amino acid substitution (Q787C) (ΔC; Fig. 1b). Of note, this gene was not present in an earlier published version of the Spiroplasma MSRO genome16 (Supplementary Data), and we found no obvious homologous proteins in our BLAST searches.

To test whether SpAID is responsible for male killing, we utilized the GAL4/UAS system to overexpress the gene as a C-terminal GFP fusion protein in D. melanogaster. Strikingly, SpAID expression with the Actin-GAL4 ubiquitous driver eliminated all male offspring, while it had no impact on female emergence (Fig. 1c). Thus SpAID kills flies in a sex-dependent manner.

Spiroplasma-induced male killing in Drosophila is associated with abnormal apoptosis7,9 and neural disorganization5,8,9 during embryogenesis; the mechanism of neural defects is not known, but is suggested to be independent of apoptosis8,9. If SpAID was the bona fide male-killing factor, its expression in embryos should phenocopy the above pathology. We employed the nanos-GAL4 maternal driver to express SpAID during early embryogenesis and monitored apoptosis (Fig. 2; with TUNEL staining) and neural organization (Extended Data Fig. 4; with the neuron-specific marker Elav17). The sex of embryos was determined by antibody staining for the Sex lethal (Sxl), a protein expressed only in females18. We found that SpAID expression induces strong apoptosis and neural disorganization in male but not female embryos (Fig. 2 and Extended Data Fig. 4). The level of apoptosis in SpAID-expressing male embryos increased as their development proceeded (Fig. 2c), similar to Spiroplasma-infected embryos9. Although numerous cells underwent apoptosis in SpAID-expressing male embryos, the neural cells did not appear to suffer ectopic cell death (Extended Data Fig. 4c). Thus, the expression of SpAID is sufficient to induce the two different pathologies caused by male-killing Spiroplasma.

Figure 2. Expression of SpAID reproduces male-killing phenotypes during embryogenesis.

Figure 2

Open in a new tab

a, b, Representative images ofstages 13-14 female (a, n = 14) and male (b, n = 16) embryos maternally expressing SpAID, stained for apoptosis (TUNEL; green), Sxl (magenta), and DNA (blue). Single-channel images of TUNEL and Sxl are also shown. c, Quantification of TUNEL signals in stages 11-12 and 13-14 embryos (females, red; males, blue). Different characters indicate statistically significant differences (P < 0.0001; Steel-Dwass test; see Supplementary Table 2). Box and dot plots are as in Fig. 1c. Sample sizes (n, number of embryos) are shown at the bottom. Embryos were co-stained for Elav, TUNEL, Sxl, and DNA, and selected channels are shown in a, b and Extended Data Figure 4.

In Drosophila (XX female, XY male), the single male X chromosome is hyper-transcribed by two-fold to equalize gene expression levels between sexes. This dosage compensation system is mediated by a protein-RNA complex called the male-specific lethal (MSL) complex, which is selectively recruited to the male X chromosome19. Prior studies have uncovered a link between the male-killing action of Spiroplasma and the host’s dosage compensation machinery6,10,11. Genetic experiments revealed that Spiroplasma fails to kill males lacking the MSL components, while it can induce death in females ectopically expressing the MSL complex6,10,11. This suggests that Spiroplasma targets either the MSL complex directly or its downstream chromatin modifications (e.g. acetylation of histone H4 on lysine 16)19. Remarkably, we found that the expression of SpAID triggers massive apoptosis in transgenic female embryos engineered to express the MSL complex (Extended Data Fig. 5a-d), indicating that this toxin mediates its effects through the dosage compensation machinery. We previously showed that Spiroplasma infection triggers DNA damage and segregation defects on the male X chromosome, thereby activating male-specific apoptosis11. To test whether SpAID expression can reproduce these phenotypes, we monitored DNA damage on the male X chromosome by antibody staining for the phosphorylated histone H2Av (pH2Av)20 and MSL1, a component of the MSL complex. In GFP-expressing control and SpAID-expressing female embryos, a few H2Av foci were detected, whereas SpAID-expressing male embryos exhibited numerous bright foci (Fig. 3a-c) that frequently overlapped with MSL1 signals (Fig. 3d). We also found that SpAID expression in male embryos causes the formation of chromatin bridges, of which 87.1% contained MSL1 signals (Fig. 3e-g). The MSL1-labelled chromosomes were often fragmented and unevenly distributed after mitosis (Fig. 3f; 47.7% of the MSL1-containing bridges). In addition, MSL1 signals were reduced in SpAID-expressing male embryos, and we observed this phenotype at an even earlier time point (Fig. 3h and Extended Data Fig. 5e, f; stages 8-10) than has been described for Spiroplasma-infected embryos (from stage 13 onward)10, probably because the high expression of SpAID by the GAL4/UAS system results in much more severe effects than Spiroplasma infection. Finally, we examined the subcellular distribution of SpAID in larval salivary glands, whose large cellular sizes facilitate detailed cytological analyses. In both sexes, SpAID-GFP was found at plasma membranes, in the cytoplasm, and throughout nuclei, but was absent from the nucleolus (Fig. 4a, b). Strikingly, SpAID-GFP was enriched on MSL1-labelled chromosomes in male nuclei (Fig. 4b). This result is consistent with its reliance on the dosage compensation machinery, even though equivalent localization analyses in embryos are needed.

Figure 3. SpAID acts through the MSL complex.

Figure 3

Open in a new tab

a, b, Epithelial cells in stage 9 male embryos expressing GFP (a) and SpAID (b), stained for pH2Av (green), MSL1 (magenta), and DNA (blue). c, Quantification of pH2Av foci in embryos expressing GFP and SpAID. d, Percentage of pH2Av foci overlapping with MSL1 signals in male embryos expressing GFP [median (interquartile range): 6.3% (0-19.3%)] and SpAID [52.9% (46.2-64.2%)]. e, f, Dividing cells in stage 9 male embryos expressing GFP (e, proper segregation) and SpAID (f, a broken bridge) stained for DNA (green) and MSL1 (magenta). g, The number of chromatin bridges containing (black; numbers on the left) or not containing (grey; numbers on the right) MSL1 signals in embryos expressing GFP and SpAID. h, The number of MSL1 focal signals in male embryos expressing GFP (black) and SpAID (green). The same datasets of stages 8-10 embryos were analysed in a-h (n = 50 or 48 images per condition). Different characters or asterisks indicate significant differences in c (P < 0.001; Steel-Dwass test), d (P < 0.0001; χ2 test), and h (P < 0.0001; two-tailed Mann-Whitney U test) (see Supplementary Table 2). Box and dot plots in c, d, h are as in Fig. 1c and sample sizes are shown at the bottom in c. All UAS transgenes were expressed maternally.

Figure 4. Subcellular localization of SpAID.

Figure 4

Open in a new tab

a-f, Larval salivary glands expressing SpAID-GFP (a, female, n = 13; b, male, n = 17), ΔANK-GFP (c, female, n = 9; d, male, n = 16), and ΔOTU-GFP (e, female, n = 15; f, male, n = 12) stained for MSL1 (magenta) and DNA (blue). For GFP (green), raw fluorescent signals were detected. Magnified views of nuclei are shown. Dark spots inside nuclei in GFP images represent the nucleolus. Arrowheads indicate GFP signals associated with plasma membranes.

To better characterize the action of SpAID, we made deletion constructs lacking either ankyrin repeats (ΔANK) or the OTU domain (ΔOTU) (Fig. 1b). Expression of ΔANK with Actin-GAL4 did not kill males, pointing to a pivotal role of ankyrin repeats in SpAID activity (Fig. 1c). The expression of ΔOTU with Actin-GAL4 still eliminated adult males, but they were killed later in development (pupal stage) compared with wild-type SpAID (second instar larval stage) (Fig. 1c). Furthermore and in contrast to wild-type form SpAID, weaker expression of ΔOTU with armadillo-GAL4 failed to kill males (Extended Data Fig. 6), indicating that the OTU domain is required for full activity. Interestingly, although its distribution pattern was otherwise indistinguishable from that of SpAID-GFP in salivary gland cells, ΔANK-GFP was not enriched on MSL-labelled chromosomes (Fig. 4c, d). On the other hand, ΔOTU-GFP was only weakly detected within nuclei regardless of sex, and colocalization with MSL was not apparent (Fig. 4e, f). ΔOTU's male killing activity when overexpressed may be due to excess amounts of protein overriding its localization defect. All of these observations can be reconciled in a model of chromosomal targeting of SpAID in which the OTU domain promotes nuclear localization and ankyrin repeats interact with the MSL complex or downstream histone modifications (Extended Data Fig. 7).

In summary, we have discovered a novel Spiroplasma gene that is likely to be responsible for male killing. Remarkably, expression of this single gene was sufficient to recapitulate the phenotypes associated with male killing. Our analysis revealed that SpAID is the sole ankyrin repeat protein in the Spiroplasma genome (Supplementary Data). This contrasts with the case of Wolbachia, whose genomes encode more than 20 ankyrin repeat proteins21,22. Interestingly, the Wolbachia strain wMel has a prophage-associated gene WD0633, that contains ankyrin repeats and the OTU domain23. Nevertheless, its transgenic expression in D. melanogaster produced no obvious phenotypes in a previous report24. Recent studies discovered Wolbachia genes that cause cytoplasmic incompatibility, a reproductive manipulation whereby symbiont-free females are unable to reproduce when mated with infected males25–27. Intriguingly, some of the genes that cause cytoplasmic incompatibility and SpAID contain deubiquitinase domains, raising the possibility that host ubiquitination pathways are a common target in distinct strategies. Future research will focus on the identification of host cellular targets of SpAID, thereby deciphering the principle of its sex-specific activity. A thorough understanding of symbiont-induced reproductive manipulations would not only provide novel insights into fundamental aspects of development, sex determination, and their evolution in insects, but could also provide significant clues to control insect populations.

Methods

Fly stocks and genetics

Laboratory stocks of D. melanogaster were maintained at 25°C with standard cornmeal medium. The fly stocks used in this study were cultured in tetracycline containing medium (0.7-0.8 mg/ml) for one generation to eliminate possible contamination with Wolbachia and Spiroplasma. After treatment, about 10 females and/or males were checked by diagnostic PCR with specific primers for Wolbachia (wsp_81F/691R)28 and Spiroplasma (16SA1F/TKSSsp)29,30 (Supplementary Table 1). Treated flies were maintained in the normal medium for at least two generations before using them in experiments. The following lines were obtained from the Bloomington Drosophila Stock Center at Indiana University (BDSC) and the Department of Drosophila Genomics and Genetic Resources at Kyoto Institute of Technology (DGGR): Actin5C-GAL4 (Actin-GAL4; BDSC #4414), nanos-GAL4::VP16 (nanos-GAL4; BDSC #4937), tubulin-GAL80ts (BDSC #7108), armadillo-GAL4 (BDSC #1560), UASp-EGFP (DGGR #116071), and CyO, ActGFP (the green balancer; DGGR #107783). The nanos-GAL4 flies, found to be Wolbachia/Spiroplasma-free by PCR, were not treated with antibiotics, because they became sick after treatment. Oregon-R (used as a wild-type line) and msl1L60/CyO; H83M2 (ref. 31) flies were generously provided by Takehide Murata (RIKEN) and Mitzi Kuroda (Harvard Medical School), respectively.

To express the SpAID-encoding gene, we employed the GAL4/UAS system32 (see below). For the zygotic expression of SpAID, Actin-GAL4/CyO (Fig. 1c) or armadillo-GAL4 (homozygous; Extended Data Fig. 6) flies were crossed to homozygous UAS transgenic flies. To determine the lethal phase during larval stages, we analysed the number of teeth present in mouth hooks of killed larvae33. For maternal expression34, UAS females were crossed to nanos-GAL4 males, and the resultant female progeny were mated with Oregon-R males (Figs 2, 3, and Extended Data Figs 4 and 5e, f). For the ectopic formation of the MSL complex in females, msl1L60/CyO, ActGFP; H83M2 flies were used instead of Oregon-R. The H83M2 transgene expresses the msl2 coding sequence (lacking Sxl-binding sites and resistant to translational repression) under the control of a heat shock-inducible promoter31, but we used its leaky basal expression. The resultant embryos were distinguished by GFP staining and only GFP-positive embryos (wild-type for msl1) were analysed (Extended Data Fig. 5a-d). For the expression of SpAID in larval salivary glands (Fig. 4), we used a recombined Actin-GAL4, tubulin-GAL80ts/CyO line to avoid male lethality and let them grow until the third instar larval stage. Crosses maintained at 18°C for 7-8 days were shifted to 29°C and kept for 1 day before dissection. Only GFP-positive wandering third instar larvae were dissected.

Construction of transgenic fly lines

For the gene synthesis of the SpAID coding sequence by the GeneArt service (Thermo Fisher Scientific), TGA stop codons, which encode tryptophan in Spiroplasma, were mutated into TGG. The codon usage was optimization for the expression in D. melanogaster. Consequently, GC content was modified from 23.3% to 50.4%. We divided the SpAID coding sequence into two parts and synthesized them separately: one was a 2,367-bp fragment that corresponds to SpAID ΔC and the other was an 838-bp fragment for the remaining 3’ portion of full length SpAID. To obtain the full length SpAID coding sequence (3,195 bp, without a stop codon), two fragments were fused by PCR with a 30-bp overlap and cloned into the pENTR vector by the pENTR/D-TOPO cloning kit (Thermo Fisher Scientific). To generate SpAID deletion constructs, we amplified two PCR fragments (nucleotide positions 1-276 and 787-3,195 for ΔANK; nucleotide positions 1-789 and 1,396-3,195 for ΔOTU) from the synthetic SpAID coding sequence and fused by PCR with a 24-bp overlap and cloned into the pENTR vector. We utilized PrimeSTAR HS DNA Polymerase (Takara Bio) for all PCR reactions. The Gateway cassette containing the SpAID fragments was transferred into the pPWG destination vector (The Drosophila Genomics Resource Center #1078; The Drosophila Gateway Vector Collection by Terence Murphy) by the LR clonase II enzyme mix kit (Thermo Fisher Scientific) to construct pUASp-SpAID-EGFP, pUASp-SpAID.ΔANK-EGFP, and pUASp-SpAID.ΔOTU-EGFP plasmids. Transgenic fly lines were generated by the standard microinjection method for P-element transformation (BestGene).

Identification and characterization of the partial male-killing Spiroplasma strain

Male-killing Spiroplasma can be easily transferred to other fly stocks by hemolymph injection2. Spiroplasma containing hemolymph was collected from a naturally infected D. melanogaster line Ug-SR (ref. 35) provided by John Jaenike (University of Rochester), and transferred to wild-type Oregon-R flies that were used as the source of hemolymph for subsequent artificial infection. We established two artificially infected D. melanogaster stocks, Df(3L)H99 (DGGR #106395) and Sxl-EGFP (BDSC #24105) in 2012 (Extended Data Fig. 1a). These infected stocks had shown perfect male killing (no emergence of adult males) for a period of at least 20 generations after the establishment of stable infection (they were used in ref. 9). Afterwards, however, the infected Sxl-EGFP line started to produce male escapers, while the Df(3L)H99 line showed complete male killing. We collected hemolymph from these fly stocks and injected them into Oregon-R flies to exclude the effect of host genetic background. We confirmed that only the Spiroplasma strain collected from the Sxl-EGFP line showed partial male killing (Extended Data Fig. 1b, c). The male-killing Spiroplasma strain in D. melanogaster is conventionally designated as MSRO (melanogaster sex ratio organism). Therefore, we call these Spiroplasma strains MSRO-H99 (the complete male killer as a control) and MSRO-SE (the partial male killer) after the genotypes of the host fly lines from which they were derived [Df(3L)H99 and Sxl-EGFP, respectively]. We also refer to the original male-killing strain from the Ug-SR line as MSRO-Ug according to the previous study35.

We suspect that the partial male-killing strain might have resulted from genome rearrangements as reported in other Spiroplasma species maintained in the laboratory36. Repeat-rich sequences like viral fragments are often associated with genomic instability of Spiroplasma37, and accordingly, the SpAID gene of the partial male-killing strain seemed to be truncated by rearrangements between genes containing repetitive sequences (e.g. integrase and p58; Extended Data Fig. 3b). To keep the partial male-killing strain, we used aged females (7-11 days) harbouring high bacterial titres to prevent the loss of the symbiont during vertical transmission. Since the titre of the partial male-killing strain appears to be higher than the original strain in some aged females, this may have led the propagation of mutant bacteria and promoted their transmission (Extended Data Fig. 1d).

Analysis of Spiroplasma titres by quantitative PCR

Oregon-R virgin females infected with MSRO-Ug, MSRO-H99, and MSRO-SE were collected and aged for 0 (virgin), 7, and 14 days and individually transferred to a 1.5 ml tube and stored at -80°C. Then, they were homogenized with plastic pestles by hand, and genomic DNA was purified by the DNeasy Blood & Tissue Kit (Qiagen). A portion of the Spiroplasma dnaA gene and the D. melanogaster RpS17 gene were amplified with specific primer sets [dnaA_109F/246R (ref. 38) and rps17_615F/695R (ref. 39), respectively] (Supplementary Table 1) under the following PCR conditions: 95°C for 5 min followed by 45 cycles of 95°C for 10 sec, 55°C for 20 sec and 72°C for 20 sec. The 10-µl PCR mixture contained 2 µl of genomic DNA, 1x LightCycler 480 SYBR Green I master mix (Roche), 0.5 µM primers. Quantitative PCR was performed by LightCycler 480 II (Roche). Cp values were obtained by the second derivative maximum method and the values of technical duplicates were averaged. We excluded samples if their duplicates had a difference in Cp values > 0.5 cycles or if their Cp values were more than 30 cycles (2 out of 63 samples in total). To calculate the relative copy number of Spiroplasma dnaA gene against the host RpS17 gene, we followed the Pfaffl method40. To estimate the PCR efficiency of each primer set, we performed quantitative PCR by using six 10-fold serial dilutions of genomic DNA purified from 10 female adult flies infected with MSRO-Ug, aged for 4-6 days after eclosion. PCR efficiency values were 82% (dnaA_109F/246R) and 98% (rps17_615F/695R), respectively.

Whole-genome sequencing of male-killing Spiroplasma

To collect a sufficient amount of DNA for whole-genome sequencing of Spiroplasma strains MSRO-H99 and MSRO-SE, we recovered fly hemolymph by the centrifugal separation method developed by Laura Musselman (Washington University School of Medicine)41 with minor modification. We first prepared a 0.5 ml polypropylene tube (Sarstedt, 72.704) whose bottom has a slit made by a sharp blade (hereafter called “cartridge”). Under CO2 anesthesia, we pricked the preepisternal area of the thorax of infected female adults and put them into the cartridge. The cartridge containing 30-40 flies was inserted into a 1.5 ml tube and centrifuged at 5,000 rpm (2,300 rcf) for 5 min at 4°C. We usually made 3-10 cartridges at one time and they were kept on ice until centrifugation. After removing cartridges, we checked precipitates that contain host hemolymph and Spiroplasma under a stereomicroscope, and confirmed that there were no large debris (embryos and carcasses etc.). The tubes were washed with 400 µl PBS to resuspend and merge all precipitates. The merged suspension was filtered with a 0.65-µm pore size membrane filter (Ultrafree-MC; Merck, UFC30DV00) by centrifugation at 12,000 rcf for 3 min at 4°C to eliminate large microorganisms including yeasts. The flow-through was centrifuged at the maximum speed for another 10 min and the supernatant was removed. Remaining bacterial pellets were stored at -80°C. After suspending the bacterial pellet in 180 µl buffer ATL (Qiagen), 20 µl proteinase K solution (Qiagen) was added and pulse-vortexed for 10 sec, followed by 1 hr incubation at 56°C. Then, 4 µl RNase A (100 mg/ml; Qiagen) was added and treated for 5 min at room temperature. Genomic DNA was purified by the conventional phenol–chloroform extraction method (see PacBio Shared Protocol online). During the extraction, samples were mixed by pulse-vortexing within 20 sec to prevent shearing of high molecular weight DNA. We collected hemolymph from 1,843 (MSRO-H99) and 1,378 (MSRO-SE) adult females to recover 18.9 µg and 7.56 µg genomic DNA, respectively (quantified by NanoDrop 1000; Thermo Fisher Scientific).

The genomic DNA was purified with Agencourt AMPure XP beads (Beckman Coulter) and was sheared in a Covaris g-TUBE (Covaris) to obtain 20-kb fragments. After shearing, the size distribution of DNA was checked by the Fragment Analyzer (Advanced Analytical Technologies). 4 µg (MSRO-H99) or 5 µg (MSRO-SE) of the sheared DNA was used to prepare a SMRTbell library with the PacBio SMRTbell Template Prep Kit 1.0 (Pacific Biosciences) according to the manufacturer's recommendations. The resulting library was size selected on the BluePippin system (Sage Science) for molecules larger than 16 kb (MSRO-H99) or 18 kb (MSRO-SE). The recovered library bound to MagBeads was sequenced on a single SMRT Cell with P6/C4 chemistry per genome by the PacBio RS II system (Pacific Biosciences) at 240 min movie length. Genome assembly was performed with the HGAP (Hierarchical Genome Assembly Process) software (Pacific Biosciences) version 3 and version 2 for the MSRO-H99 and MSRO-SE genomes, respectively. Library preparation, whole-genome sequencing and genome assembly were performed in the Lausanne Genomic Technologies Facility (GTF) in the University of Lausanne (UNIL).

Genomic data analyses and protein domain searches

Genomic data analysis was performed on the Bio-Linux 8 platform (NERC Environmental Omics)42. The genome sequences of MSRO-H99 and MSRO-SE assembled into five and two contigs, respectively (Extended Data Fig. 2a), and were aligned with the previously published MSRO genome16 and ordered by Mauve v2.4.0 (refs 43,44) (Extended Data Fig. 2b). For the MSRO-H99 genome, three major contigs (#1-3) were assigned to the main chromosome, while the remaining two minor contigs (#4 and #5) were assigned to extra chromosomes. Two contigs of the MSRO-SE genome were assigned to the main chromosome (contig #1; circularized) and the extra chromosome (contig #2), respectively. These extra chromosomes could be plasmids because they contain several proteins, like Soj (the chromosome partitioning protein) and ARPs (adhesion-related proteins; P58, P12, P54, P123 and P18), which are located on plasmids in other Spiroplasma species45.

Whole-genome annotation was conducted by using Prokka v1.11 (ref. 46). We created a custom annotation database by combining published genomic sequences of several Spiroplasma species [S. citri CII3-3X (AM285301)47, S. melliferum KC3 (AGBZ02000000)48, S. melliferum IPMB4A (AMGI01000000)49, S. chrysopicola DF-1 (CP005077)37, S. poulsonii MSRO (NZ_JTLV00000000)16, S. kunkelii CR2-3x (CP010899)50; the numbers in parentheses represent GenBank accession numbers].

To identify conserved protein domains including ankyrin repeats, annotated protein sequences were analysed by the NCBI Conserved Domain Database (NCBI CDD)51,52 with default parameters (Supplementary Data). Besides SpAID (SMH99_26490; 3,198 bp), other genes containing the OTU domain (SMH99_25890; 597 bp) and ankyrin repeats (SMH99_25900; 204 bp) were predicted on an identical contig (#4; 87,892 bp) in MSRO-H99. It is likely that they are derived from the miss or partial assembly of SpAID-encoding reads, because they are located close to the end of the contig (from 4,133 to 5,048 bp) with much lower coverage (below 10x) compared to the SpAID locus (from 51,206 to 54,403 bp) with high coverage (over 330x). These genes were not found in MSRO-SE. Further domains were predicted by the InterPro protein sequence analysis and classification database53. The protein domain structure of SpAID was drawn by the Illustrator for Biological Sequences (IBS) software v 1.0.2 (ref. 54) and modified manually.

Homology searches

For homology searches, we utilized the protein BLAST (blastp) program on the NCBI BLAST website with a non-redundant protein sequence database (nr). After searching the entire database without specifying the organism, we also searched bacteria, viruses, and Drosophila databases separately; however, we found no protein sequences that aligned to the entire SpAID protein sequence. The top hit having the highest score was a hypothetical protein of Spiroplasma kunkelii (WP_053391598), which covers the portion of the N- and C-terminal sequences of SpAID [amino acid positions 1-49 (score: 45.4, E-value: 0.71, identities: 48.98%, positives: 69.39%, gaps: 14.29%) and 704-1,065 (score: 385, E-value: 2.63x10-118, identities: 58.03%, positives: 69.63%, gaps: 14.07%), respectively].

Sanger sequencing of the SpAID locus

10 adult Oregon-R females uninfected (as a negative control) or infected with MSRO-Ug, MSRO-H99, and MSRO-SE were collected and genomic DNA was purified (see quantitative PCR methods). The 3’ portion of SpAID gene was amplified with forward and reverse primer sets (spaid_1568F with spaid.L_+136R for MSRO-Ug/MSRO-H99 and spaid.S_+362R for MSRO-SE, respectively) (Supplementary Table 1) under the following PCR conditions: 95°C for 2 min followed by 30 cycles of 95°C for 30 sec, 50°C for 30 sec and 60°C for 3 min. Due to the low GC content of the SpAID gene (23.3%), extension at 60°C instead of 72°C was essential for the successful PCR amplification55. The 50-µl PCR mixture contained 2 µl of genomic DNA, 1.25 U of GoTaq G2 DNA polymerase (Promega), 1x green reaction buffer (Promega), 0.5 µM primers, and 0.2 mM dNTP mixture. The PCR fragment was purified from agarose gel by the Wizard SV Gel and PCR Clean-Up System (Promega) and read by direct sequencing. To design sequencing primes, we referred to the genome sequences assembled from the PacBio data. No distinct PCR amplification was detected in uninfected Oregon-R female flies, confirming that the SpAID gene is not encoded by the D. melanogaster genome but by the Spiroplasma genome.

Staining and imaging

To collect embryos, we put female and male flies in culture vials and waited for two days. These adults were transferred to egg collection cages with grape juice agar plates and fed with yeast paste. Developmental staging of embryos was according to refs 56,57. Embryos were collected from grape juice agar plates and dechorionated in 2.8% sodium hypochlorite solution, subsequently fixed in 1:1 mixture of heptane and 4% paraformaldehyde (EM Grade; Electron Microscopy Sciences, 15710) diluted in PBS for 20 min at room temperature, and devitellinized by vigorously shaking in heptane/MeOH. The embryos were washed in MeOH three times and rehydrated through an EtOH series (95%, 70%, 50%, and 35%), and then washed in PBT (PBS containing 0.1% Triton X-100). After treated with a blocking buffer [PBT containing 2% bovine serum albumin (BSA, Fraction V; MP Biomedicals, 02160069)] for 60 min, the embryos were incubated with primary antibodies at 4ºC overnight, washed three times in PBT and incubated with secondary antibodies at room temperature for 90 min. Antibodies were diluted in the blocking buffer. Anti-Sex lethal and anti-MSL1 antibodies were utilized for sexing embryos (see below).

The following primary antibodies were used: mouse anti-Sex lethal [1:20; Developmental Studies Hybridoma Bank (DSHB), M18]58, mouse and rabbit anti-MSL1 [1:200 and 1:500; kindly provided by John Lucchesi (Emory University)], rabbit anti-Histone H2AvD pS137 (pH2Av; 1:1,000; Rockland Immunochemicals, 600-401-914), chicken anti-GFP (1:500; Aves Labs, GFP-1020), rat anti-Elav (1:20; DSHB, 7E8A10)59. Secondary antibodies (1:1,000; Alexa Fluor 488/555/647 conjugate) were purchased from Molecular Probes (Thermo Fisher Scientific). DNA staining was carried out with DAPI (1 µg/ml; Sigma, D9542) for 10 min at room temperature after secondary antibody staining. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling) staining was performed by using the In Situ Cell Death Detection Kit, TMR red (Roche), and the embryos stained with primary antibodies were incubated in 50 µl TUNEL reaction mixture with secondary antibodies at 4ºC overnight. Stained embryos were washed three times in PBT, mounted in FluorSave Reagent (Calbiochem) and observed under a confocal microscope (Zeiss LSM 700).

To detect Spiroplasma in host hemolymph (Fig. 1a), the abdomen of an adult female was dissected in 5 µl PBS with tweezers on a microscope slide. 1 µl SYTO 9 nucleic acid stain solution (0.02 mM; Thermo Fisher Scientific) was added and observed under a widefield microscope equipped with a CCD camera (Zeiss Axio Imager Z1/AxioCam MRm).

To monitor the subcellular distribution of the GFP fusion proteins of SpAID, we dissected out salivary glands from wandering third instar larvae and fixed them in 4% paraformaldehyde diluted in PBS for 15 min at room temperature. Blocking and staining were performed as described above. For GFP, we detected raw fluorescent signals without antibody staining.

Image analysis and processing

Confocal z-sections were max projected by the Fiji software (Fiji Is Just ImageJ)60 with a custom macro. Image analysis was performed by custom R scripts with the EBImage package61. For the quantification of TUNEL signals of whole embryos (Figs 2c and Extended Data Fig. 5d) (acquired by a 20x/0.8 objective with 0.6x zoom scan; frame size: 512 x 512; 1.5 µm, two times optimal intervals), maximum projection images of DAPI and TUNEL staining were binarized and the former was utilized to make an embryonic mask image. TUNEL signals inside a corresponding mask image were measured by image integration. This value was divided by mask image area to normalize the embryonic size.

For the quantification of focal pH2Av signals (Fig. 3c), stages 8-10 embryos were triply stained (pH2Av, MSL1, and DAPI) and two images were acquired per embryo (by a 63x/1.4 oil immersion objective; frame size: 1,024 x 1,024; 0.3 µm optimal intervals). We compiled 20 serial z-sections from the top (among 25 sections in total) to make projected images of pH2Av and MSL1 signals. These images were smoothed by Gaussian filter and binarized by moving average method, respectively. To identify focal pH2Av signals, image objects were extracted from the stacked images by segmentation and labelling, where objects not greater than 20 pixels were eliminated to remove noise and ambiguous signals. To calculate the enrichment of focal pH2Av signals on the MSL1-labelled chromosome (Fig. 3d), overlaps between focal pH2Av signals and MSL1 signals were obtained by image integration. To quantify the number of MSL1 signals with discrete focal shapes (Fig. 3h), objects over 30 pixels were extracted and counted. Above datasets were also utilized to count the number of chromatin bridges (Fig. 3g). The 25 serial z-slice images were visually inspected to detect chromatin bridges in the Fiji software.

The brightness and contrast of the presented images in the manuscript were adjusted by the level tool in the GIMP 2.8 software (the GNU General Public License). The adjustment was performed uniformly on the entire images and only black/white input levels were modified.

Statistics and reproducibility

No statistical methods were applied to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. We used the R software v3.4.1 (the R Foundation) for all statistical analyses. Multiple comparisons in Figs 1c, 2c, 3c and Extended Data Figs 1d, 5d were performed using the Steel-Dwass test by the pSDCFlig function in the NSM3 R package62. We performed the χ2 test in Fig. 3d and the Mann-Whitney U test (two-tailed) in Fig. 3h and Extended Data Fig. 6. P-values less than 0.05 were considered as significant. Exact P-values are listed in Supplementary Table 2. For the zygotic expression of SpAID and its deletion constructs (Fig. 1c and Extended Data Fig. 6), two independently established UAS transgenic lines were used to verify the reproducibility of the results. Other experiments were independently repeated at least two times, except for the qPCR analysis in Extended Data Fig. 1d (one experiment with 6-7 biological replicates).

Data availability

Whole genomic sequence data have been deposited at GenBank under the BioProject number PRJNA416288. Sequence data for synthetic gene fragments have been deposited at GenBank under the accession numbers MG837001 and MG837002. Source data for Figs 1, 2, 3 and Extended Data Figs 1, 5, 6 are available in the online version of the paper. All relevant data supporting the findings of this study are included within the article and its supplementary information files or available upon reasonable request.

Extended Data

Extended Data Figure 1. Identification and characterization of the partial male-killing Spiroplasma strain.

Extended Data Figure 1

Open in a new tab

a, An illustration showing the origin of the Spiroplasma strains analysed in this study. Pictures show male-killing (MK) Spiroplasma of D. melanogaster. MSRO-Ug is the original male-killing strain maintained in the Oregon-R wild-type fly. Fly stocks [Df(3L)H99 and Sxl-EGFP] artificially infected with this original strain showed complete male killing (100% MK) for the first 20 generations. Afterwards, one strain (MSRO-SE) started to show the partial male-killing phenotype, while the other (MSRO-H99) kept the ability to induce complete male killing. See Methods for more detail. b, c, Sex ratio analysis of the adult progeny obtained from Oregon-R flies infected with MSRO-Ug, MSRO-H99, and MSRO-SE. We repeated experiments for three times at fourth- to sixth-generations (G4-6) after the establishment of infection. In c, the relative number of male offspring (% of females) obtained from Oregon-R female flies infected with MSRO-SE were plotted. The data point was excluded if the total count of flies was below 10. d, Relative titre of Spiroplasma within individual female fly. Adult females infected with three MSRO strains were aged for 0, 7, 14 days after eclosion and analysed by qPCR. Data were normalized with respect to 0 day females infected with MSRO-Ug. Different characters indicate statistically significant differences (P < 0.01; N.S., not significant, P > 0.05; Steel-Dwass test; see Supplementary Table 2). Please note that the titres of three strains are comparable and even the higher titre in old females (see 14 days in d) fails to induce complete male killing in MSRO-SE (c). Box and dot plots are as in Fig. 1c and sample sizes (n, number of adult flies) are shown at the bottom.

Extended Data Figure 2. Whole-genome sequencing studies of the male-killing Spiroplasma variants.

Extended Data Figure 2

Open in a new tab

a, The comparison of genomic features of three Spiroplasma strains. MSRO-H99 and MSRO-SE are newly obtained variants isolated in this study (Extended Data Fig. 1a; see also Methods). As a control, the data of the previously reported male-killing Spiroplasma genome16 is also indicated (MSRO, 2015). b, Whole-genome alignment of the three Spiroplasma strains. To start the alignment from the dnaA gene, the contig #1 of MSRO-H99 was split into two fragments (contig #1.1 and #1.2). The locations of the contigs corresponding to extra chromosomes (putative plasmids; see Methods) are shown as “extra”. SpAID (gene ID: SMH99_26490) and SpAID ΔC (gene ID: SMSE_25110) are located on these extra chromosomes in MSRO-H99 (contig #4) and MSRO-SE (contig #2), respectively.

Extended Data Figure 3. Genetic alterations of the SpAID locus in the partial male-killing Spiroplasma strain.

Extended Data Figure 3

Open in a new tab

The genome structures around the SpAID loci in the male-killing (a, MSRO-Ug and MSRO-H99) and the partial male-killing (b, MSRO-SE) Spiroplasma strains. Genes encoded on opposite strands are shown in different colours (red and blue, respectively). An 828-bp deletion and nucleotide substitutions (coloured in red; corresponding amino acid sequences are presented in one-letter code) in the 3’ region of the SpAID gene are indicated. These sequence alterations were confirmed by the Sanger method (see Methods).

Extended Data Figure 4. Neural defects of SpAID-expressing embryos.

Extended Data Figure 4

Open in a new tab

Representative images of stage 13-14 female (a, n = 14) and male (b, n = 16) embryos maternally expressing SpAID, stained for TUNEL (green) and neural cells (Elav, magenta). Single-channel images of Elav are also shown. The boxed region in b is magnified in c with single-channel images of Elav and TUNEL. Embryos were co-stained for Elav, TUNEL, Sxl, and DNA, and selected channels are shown in a-c and Fig. 2a, b, respectively.

Extended Data Figure 5. SpAID acts through the dosage compensation machinery.

Extended Data Figure 5

Open in a new tab

a-c, Representative images of stages 13-14 embryos ectopically expressing the MSL complex by the H83M2 transgene, stained for TUNEL (green) and Sxl (magenta). GFP-expressing control female (a, n = 15), SpAID-expressing female (b, n = 20), and male (c, n = 19) embryos are shown. d, Quantification of TUNEL signals in H83M2 embryos at stages 13-14. Different characters indicate significant differences (P < 0.001; Steel-Dwass test; see Supplementary Table 2). The box and dot plot (females, red; males, blue) is as in Fig. 1c and sample sizes (n, number of embryos) are shown at the bottom. e, f, Representative images of epithelial cells of stages 8-10 male embryos expressing GFP (e, n = 25) and SpAID (f, n = 25), stained for DNA (green) and MSL1 (magenta) from the datasets analysed in Fig. 3. All UAS transgenes were expressed maternally.

Extended Data Figure 6. Expression of SpAID by using a weak GAL4 driver.

Extended Data Figure 6

Open in a new tab

The number of adult progeny (females, red; males, blue) obtained from crosses between the armadillo-GAL4 driver line (weak and ubiquitous expression) and four UAS transgenic lines (GFP, SpAID, ΔANK, and ΔOTU; n = 6 independent crosses for GFP and SpAID, n = 8 independent crosses for ΔANK and ΔOTU). The UAS-GFP line was used as a negative control. With this weak GAL4 driver, SpAID still eliminated all male progeny, while both ΔANK and ΔOTU had no impact on male viability. An asterisk indicates the statistically significant difference (P < 0.01; N.S., not significant, P > 0.05; two-tailed Mann-Whitney U test; see Supplementary Table 2). Box and dot plots are as in Fig. 1c. The total numbers of adult counts for each genotype and sex are shown at the bottom.

Extended Data Figure 7. A proposed model for SpAID-induced male-killing phenotypes.

Extended Data Figure 7

Open in a new tab

SpAID utilizes the OTU domain and ankyrin repeats (ANK) to target the host nucleus and the male X chromosome. “MSL” and “Ac” indicate the dosage compensation complex and resultant histone acetylation, respectively. See text for other explanations.

Supplementary Material

Supplementary Information is available in the online version of the paper.

SI Guide
Supplementary data
Supplementary tables

Acknowledgements

We thank the Bloomington Stock Center in USA and the Department of Drosophila Genomics and Genetic Resources at Kyoto Institute of Technology in Japan (DGGR) for fly stocks. We also thank the Developmental Studies Hybridoma Bank at the University of Iowa in USA for monoclonal antibodies, John Jaenike, Takehide Murata, and Mitzi Kuroda for providing fly strains, John Lucchesi for providing antibodies, the BioImaging & Optics Platform (BIOP) in EPFL for confocal microscopy, Keith Harshman and the Lausanne Genomic Technologies Facility (GTF) in the University of Lausanne (UNIL) for whole-genome sequencing and draft genome assemblies, Christoph Vorburger and Steve Perlman for comments and suggestions on the manuscript, and the members of the laboratory for discussion and support. We also thank Takema Fukatsu for the Spiroplasma-infected fly stocks that were originally established in his laboratory by T.H. This work was supported by the European Research Council (ERC) Advanced Grant 339970 and the Swiss National Science Foundation (SNSF) Sinergia Grant CRSII3_154396. The purchase of the PacBio RS II system in UNIL was supported in part by the Loterie Romande through the Fondation pour la Recherche en Médecine Génétique.

Footnotes

Toshiyuki Harumoto: ORCID 0000-0003-4394-5883

Bruno Lemaitre: ORCID 0000-0001-7970-1667

Author Contributions

T.H. conceived of and designed the study, performed the experiments and analysed the data, wrote and edited the manuscript. B.L. conceived of and administered the study, wrote and edited the manuscript.

Competing financial or non-financial interests

The authors declare no competing financial or non-financial interests.

References

  • 1.Hurst GDD, Frost CL. Reproductive parasitism: maternally inherited symbionts in a biparental world. Cold Spring Harb Perspect Biol. 2015;7:a017699. doi: 10.1101/cshperspect.a017699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Williamson DL, Poulson DF. Sex Ratio Organisms (Spiroplasmas) of Drosophila. In: Whitcomb RF, Tully JG, editors. The Mycoplasmas, Volume III: Plant and Insect Mycoplasmas. Ch. 6. Academic Press; 1979. pp. 175–208. [Google Scholar]
  • 3.Williamson DL, et al. Spiroplasma poulsoniisp. nov., a new species associated with male-lethality in Drosophila willistoni, a neotropical species of fruit fly. Int J Syst Bacteriol. 1999;49:611–618. doi: 10.1099/00207713-49-2-611. [DOI] [PubMed] [Google Scholar]
  • 4.Haselkorn TS. The Spiroplasma heritable bacterial endosymbiont of Drosophila. Fly (Austin) 2010;4:80–87. doi: 10.4161/fly.4.1.10883. [DOI] [PubMed] [Google Scholar]
  • 5.Tsuchiyama-Omura S, Sakaguchi B, Koga K, Poulson DF. Morphological features of embryogenesis in Drosophila melanogaster infected with a male-killing Spiroplasma. Zoolog Sci. 1988;5:375–383. [Google Scholar]
  • 6.Veneti Z, Bentley JK, Koana T, Braig HR, Hurst GDD. A functional dosage compensation complex required for male killing in Drosophila. Science. 2005;307:1461–1463. doi: 10.1126/science.1107182. [DOI] [PubMed] [Google Scholar]
  • 7.Bentley JK, Veneti Z, Heraty J, Hurst GDD. The pathology of embryo death caused by the male-killing Spiroplasma bacterium in Drosophila nebulosa. BMC Biol. 2007;5:9. doi: 10.1186/1741-7007-5-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Martin J, Chong T, Ferree PM. Male killing Spiroplasma preferentially disrupts neural development in the Drosophila melanogaster embryo. PLoS ONE. 2013;8:e79368. doi: 10.1371/journal.pone.0079368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Harumoto T, Anbutsu H, Fukatsu T. Male-killing Spiroplasma induces sex-specific cell death via host apoptotic pathway. PLoS Pathog. 2014;10:e1003956. doi: 10.1371/journal.ppat.1003956. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Cheng B, Kuppanda N, Aldrich JC, Akbari OS, Ferree PM. Male-killing Spiroplasma alters behavior of the dosage compensation complex during Drosophila melanogaster embryogenesis. Curr Biol. 2016;26:1339–1345. doi: 10.1016/j.cub.2016.03.050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Harumoto T, Anbutsu H, Lemaitre B, Fukatsu T. Male-killing symbiont damages host’s dosage-compensated sex chromosome to induce embryonic apoptosis. Nat Commun. 2016;7:12781. doi: 10.1038/ncomms12781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Oishi K. Spirochaete-mediated abnormal sex-ratio (SR) condition in Drosophila: a second virus associated with spirochaetes and its use in the study of the SR condition. Genet Res. 1971;18:45–56. doi: 10.1017/s0016672300012404. [DOI] [PubMed] [Google Scholar]
  • 13.Makarova KS, Aravind L, Koonin EV. A novel superfamily of predicted cysteine proteases from eukaryotes, viruses and Chlamydia pneumoniae. Trends Biochem Sci. 2000;25:50–52. doi: 10.1016/s0968-0004(99)01530-3. [DOI] [PubMed] [Google Scholar]
  • 14.Al-Khodor S, Price CT, Kalia A, Abu Kwaik Y. Functional diversity of ankyrin repeats in microbial proteins. Trends Microbiol. 2010;18:132–139. doi: 10.1016/j.tim.2009.11.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Dobrindt U, Hochhut B, Hentschel U, Hacker J. Genomic islands in pathogenic and environmental microorganisms. Nat Rev Microbiol. 2004;2:414–424. doi: 10.1038/nrmicro884. [DOI] [PubMed] [Google Scholar]
  • 16.Paredes JC, et al. Genome sequence of the Drosophila melanogaster male-killing Spiroplasma strain MSRO endosymbiont. MBio. 2015;6:e02437–14. doi: 10.1128/mBio.02437-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Campos AR, Rosen DR, Robinow SN, White K. Molecular analysis of the locus elav in Drosophila melanogaster: a gene whose embryonic expression is neural specific. EMBO J. 1987;6:425–431. doi: 10.1002/j.1460-2075.1987.tb04772.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Salz HK, Erickson JW. Sex determination in Drosophila: the view from the top. Fly (Austin) 2010;4:60–70. doi: 10.4161/fly.4.1.11277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Lucchesi JC, Kuroda MI. Dosage compensation in Drosophila. Cold Spring Harb Perspect Biol. 2015;7:a019398. doi: 10.1101/cshperspect.a019398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Madigan JP, Chotkowski HL, Glaser RL. DNA double-strand break-induced phosphorylation of Drosophila histone variant H2Av helps prevent radiation-induced apoptosis. Nucleic Acids Res. 2002;30:3698–3705. doi: 10.1093/nar/gkf496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Siozios S, et al. The diversity and evolution of Wolbachia ankyrin repeat domain genes. PLoS ONE. 2013;8:e55390. doi: 10.1371/journal.pone.0055390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Jernigan KK, Bordenstein SR. Ankyrin domains across the Tree of Life. PeerJ. 2014;2:e264. doi: 10.7717/peerj.264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Wu M, et al. Phylogenomics of the reproductive parasite Wolbachia pipientis wMel: a streamlined genome overrun by mobile genetic elements. PLoS Biol. 2004;2:E69. doi: 10.1371/journal.pbio.0020069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Yamada R, Iturbe-Ormaetxe I, Brownlie JC, O’Neill SL. Functional test of the influence of Wolbachia genes on cytoplasmic incompatibility expression in Drosophila melanogaster. Insect Mol Biol. 2011;20:75–85. doi: 10.1111/j.1365-2583.2010.01042.x. [DOI] [PubMed] [Google Scholar]
  • 25.Beckmann JF, Fallon AM. Detection of the Wolbachia protein WPIP0282 in mosquito spermathecae: implications for cytoplasmic incompatibility. Insect Biochem Mol Biol. 2013;43:867–878. doi: 10.1016/j.ibmb.2013.07.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Beckmann JF, Ronau JA, Hochstrasser M. A Wolbachia deubiquitylating enzyme induces cytoplasmic incompatibility. Nat Microbiol. 2017;2:17007. doi: 10.1038/nmicrobiol.2017.7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.LePage DP, et al. Prophage WO genes recapitulate and enhance Wolbachia-induced cytoplasmic incompatibility. Nature. 2017;543:243–247. doi: 10.1038/nature21391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Zhou W, Rousset F, O’Neil S. Phylogeny and PCR-based classification of Wolbachia strains using wsp gene sequences. Proc Biol Sci. 1998;265:509–515. doi: 10.1098/rspb.1998.0324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Fukatsu T, Nikoh N. Two intracellular symbiotic bacteria from the mulberry psyllid Anomoneura mori (Insecta, Homoptera) Appl Environ Microbiol. 1998;64:3599–3606. doi: 10.1128/aem.64.10.3599-3606.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Fukatsu T, Nikoh N. Endosymbiotic microbiota of the bamboo pseudococcid Antonina crawii (Insecta, Homoptera) Appl Environ Microbiol. 2000;66:643–650. doi: 10.1128/aem.66.2.643-650.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Kelley RL, et al. Expression of Msl-2 causes assembly of dosage compensation regulators on the X chromosomes and female lethality in Drosophila. Cell. 1995;81:867–877. doi: 10.1016/0092-8674(95)90007-1. [DOI] [PubMed] [Google Scholar]
  • 32.Brand AH, Perrimon N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 1993;118:401–415. doi: 10.1242/dev.118.2.401. [DOI] [PubMed] [Google Scholar]
  • 33.Ashburner M, Golic KG, Hawley RS. Drosophila: A Laboratory Handbook. 2nd Edition. Cold Spring Harbor Laboratory Press; 2005. [Google Scholar]
  • 34.Rørth P. Gal4 in the Drosophila female germline. Mech Dev. 1998;78:113–118. doi: 10.1016/s0925-4773(98)00157-9. [DOI] [PubMed] [Google Scholar]
  • 35.Pool JE, Wong A, Aquadro CF. Finding of male-killing Spiroplasma infecting Drosophila melanogaster in Africa implies transatlantic migration of this endosymbiont. Heredity (Edinb) 2006;97:27–32. doi: 10.1038/sj.hdy.6800830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Ye F, Melcher U, Rascoe JE, Fletcher J. Extensive chromosome aberrations in Spiroplasma citri Strain BR3. Biochem Genet. 1996;34:269–286. doi: 10.1007/BF02399947. [DOI] [PubMed] [Google Scholar]
  • 37.Ku C, Lo W-S, Chen L-L, Kuo C-H. Complete genomes of two dipteran-associated spiroplasmas provided insights into the origin, dynamics, and impacts of viral invasion in Spiroplasma. Genome Biol Evol. 2013;5:1151–1164. doi: 10.1093/gbe/evt084. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Anbutsu H, Fukatsu T. Population dynamics of male-killing and non-male-killing spiroplasmas in Drosophila melanogaster. Appl Environ Microbiol. 2003;69:1428–1434. doi: 10.1128/AEM.69.3.1428-1434.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Osborne SE, Leong YS, O’Neill SL, Johnson KN. Variation in antiviral protection mediated by different Wolbachia strains in Drosophila simulans. PLoS Pathog. 2009;5:e1000656. doi: 10.1371/journal.ppat.1000656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001;29:e45. doi: 10.1093/nar/29.9.e45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Musselman LP. Drosophila hemolymph collection procedure. YouTube. 2013 Available at: https://www.youtube.com/watch?v=im78OIBKlPA.
  • 42.Field D, et al. Open software for biologists: from famine to feast. Nat Biotechnol. 2006;24:801–803. doi: 10.1038/nbt0706-801. [DOI] [PubMed] [Google Scholar]
  • 43.Darling ACE, Mau B, Blattner FR, Perna NT. Mauve: multiple alignment of conserved genomic sequence with rearrangements. Genome Res. 2004;14:1394–1403. doi: 10.1101/gr.2289704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Darling AE, Mau B, Perna NT. progressiveMauve: multiple genome alignment with gene gain, loss and rearrangement. PLoS ONE. 2010;5:e11147. doi: 10.1371/journal.pone.0011147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Saillard C, et al. The abundant extrachromosomal DNA content of the Spiroplasma citri GII3-3X genome. BMC Genomics. 2008;9:195. doi: 10.1186/1471-2164-9-195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30:2068–2069. doi: 10.1093/bioinformatics/btu153. [DOI] [PubMed] [Google Scholar]
  • 47.Carle P, et al. Partial chromosome sequence of Spiroplasma citri reveals extensive viral invasion and important gene decay. Appl Environ Microbiol. 2010;76:3420–3426. doi: 10.1128/AEM.02954-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Alexeev D, et al. Application of Spiroplasma melliferum proteogenomic profiling for the discovery of virulence factors and pathogenicity mechanisms in host-associated spiroplasmas. J Proteome Res. 2012;11:224–236. doi: 10.1021/pr2008626. [DOI] [PubMed] [Google Scholar]
  • 49.Lo W-S, Chen L-L, Chung W-C, Gasparich GE, Kuo C-H. Comparative genome analysis of Spiroplasma melliferum IPMB4A, a honeybee-associated bacterium. BMC Genomics. 2013;14:22. doi: 10.1186/1471-2164-14-22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Davis RE, et al. Complete genome sequence of Spiroplasma kunkelii strain CR2-3x, causal agent of corn stunt disease in Zea mays L. Genome Announc. 2015;3 doi: 10.1128/genomeA.01216-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Marchler-Bauer A, et al. CDD: a Conserved Domain Database for the functional annotation of proteins. Nucleic Acids Res. 2011;39:D225–229. doi: 10.1093/nar/gkq1189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Marchler-Bauer A, et al. CDD: NCBI’s conserved domain database. Nucleic Acids Res. 2015;43:D222–226. doi: 10.1093/nar/gku1221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Finn RD, et al. InterPro in 2017-beyond protein family and domain annotations. Nucleic Acids Res. 2017;45:D190–D199. doi: 10.1093/nar/gkw1107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Liu W, et al. IBS: an illustrator for the presentation and visualization of biological sequences. Bioinformatics. 2015;31:3359–3361. doi: 10.1093/bioinformatics/btv362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Su XZ, Wu Y, Sifri CD, Wellems TE. Reduced extension temperatures required for PCR amplification of extremely A+T-rich DNA. Nucleic Acids Res. 1996;24:1574–1575. doi: 10.1093/nar/24.8.1574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Hartenstein V. Atlas of Drosophila Development. Cold Spring Harbor Laboratory Press; 1993. [Google Scholar]
  • 57.Campos-Ortega JA, Hartenstein V. The Embryonic Development of Drosophila melanogaster. Springer-Verlag; 1997. [Google Scholar]
  • 58.Bopp D, Bell LR, Cline TW, Schedl P. Developmental distribution of female-specific Sex-lethal proteins in Drosophila melanogaster. Genes Dev. 1991;5:403–415. doi: 10.1101/gad.5.3.403. [DOI] [PubMed] [Google Scholar]
  • 59.O’Neill EM, Rebay I, Tjian R, Rubin GM. The activities of two Ets-related transcription factors required for Drosophila eye development are modulated by the Ras/MAPK pathway. Cell. 1994;78:137–147. doi: 10.1016/0092-8674(94)90580-0. [DOI] [PubMed] [Google Scholar]
  • 60.Schindelin J, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9:676–682. doi: 10.1038/nmeth.2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 61.Pau G, Fuchs F, Sklyar O, Boutros M, Huber W. EBImage—an R package for image processing with applications to cellular phenotypes. Bioinformatics. 2010;26:979–981. doi: 10.1093/bioinformatics/btq046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62.Hollander MA, Wolfe D, Chicken E. Nonparametric Statistical Methods. 3rd Edition. John Wiley & Sons, Inc; 2013. [DOI] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

SI Guide
NIHMS76821-supplement-SI_Guide.doc (81KB, doc)
Supplementary data
NIHMS76821-supplement-Supplementary_data.xlsx (550.8KB, xlsx)
Supplementary tables
NIHMS76821-supplement-Supplementary_tables.pdf (154.7KB, pdf)

Data Availability Statement

Whole genomic sequence data have been deposited at GenBank under the BioProject number PRJNA416288. Sequence data for synthetic gene fragments have been deposited at GenBank under the accession numbers MG837001 and MG837002. Source data for Figs 1, 2, 3 and Extended Data Figs 1, 5, 6 are available in the online version of the paper. All relevant data supporting the findings of this study are included within the article and its supplementary information files or available upon reasonable request.

RESOURCES