Combined actions of bacteriophage-encoded genes in Wolbachia-induced male lethality

Hiroshi Arai; Hisashi Anbutsu; Yohei Nishikawa; Masato Kogawa; Kazuo Ishii; Masahito Hosokawa; Shiou-Ruei Lin; Masatoshi Ueda; Madoka Nakai; Yasuhisa Kunimi; Toshiyuki Harumoto; Daisuke Kageyama; Haruko Takeyama; Maki N Inoue

doi:10.1016/j.isci.2023.106842

. 2023 May 10;26(6):106842. doi: 10.1016/j.isci.2023.106842

Combined actions of bacteriophage-encoded genes in Wolbachia-induced male lethality

Hiroshi Arai ^1,^2,^11,^12,^∗, Hisashi Anbutsu ^3,^4,^5,¹¹, Yohei Nishikawa ^3,^5,¹¹, Masato Kogawa ⁵, Kazuo Ishii ¹, Masahito Hosokawa ^3,^5,^6,⁷, Shiou-Ruei Lin ⁸, Masatoshi Ueda ¹, Madoka Nakai ¹, Yasuhisa Kunimi ¹, Toshiyuki Harumoto ^9,¹⁰, Daisuke Kageyama ², Haruko Takeyama ^3,^5,^6,^7,^∗∗, Maki N Inoue ^1,^∗∗∗

PMCID: PMC10209535 PMID: 37250803

Summary

Some Wolbachia endosymbionts induce male killing, whereby male offspring of infected females are killed during development; however, the origin and diversity of the underlying mechanisms remain unclear. In this study, we identified a 76 kbp prophage region specific to male-killing Wolbachia hosted by the moth Homona magnanima. The prophage encoded a homolog of the male-killing gene oscar in Ostrinia moths and the wmk gene that induces various toxicities in Drosophila melanogaster. Upon overexpressing these genes in D. melanogaster, wmk-1 and wmk-3 killed all males and most females, whereas Hm-oscar, wmk-2, and wmk-4 had no impact on insect survival. Strikingly, co-expression of tandemly arrayed wmk-3 and wmk-4 killed 90% of males and restored 70% of females, suggesting their conjugated functions for male-specific lethality. While the male-killing gene in the native host remains unknown, our findings highlight the role of bacteriophages in male-killing evolution and differences in male-killing mechanisms among insects.

Subject areas: Virology, Genetics, Microbiology, Evolutionary biology

Graphical abstract

Highlights

•
The origin and diversity of the male-killing mechanisms of Wolbachia were studied
•
Bacteriophages are involved in evolution and differences in male-killing mechanisms
•
Combinations of wmk genes induce multiple MK mechanisms and lethality variations

Virology; Genetics; Microbiology; Evolutionary biology

Introduction

Wolbachia (Alphaproteobacteria) is a maternally inherited intracellular bacterium that is present in at least 40% of all insect species, making it one of the most widespread endosymbionts.¹^,² A notable feature of Wolbachia is the reproductive manipulation of its host, such as cytoplasmic incompatibility (CI), male killing (MK), parthenogenesis, and feminization, which have contributed to the spread of Wolbachia in nature.¹^,³^,⁴^,⁵^,⁶

The molecular mechanisms underlying Wolbachia-induced phenotypes have attracted extensive attention for decades, and the prophage WO present in the Wolbachia genome is suggested as an important factor for the Wolbachia-induced reproductive manipulations.⁷^,⁸^,⁹^,¹⁰^,¹¹^,¹² For example, WO encodes CI-inducing factors (CifA and CifB), which inhibit normal embryonic development by preventing chromosome segregation after fertilization.¹⁰^,¹¹^,¹² Recently, an MK Wolbachia strain wFur was found to induce MK in the moth Ostrinia furnacalis through an Oscar protein that contains a CifB C-terminus-like domain and ankyrin repeats.¹³ Oscar is thought to achieve MK by disrupting the functions of the protein Masculinizer (Masc), a critical regulator of the sex determination systems of lepidopteran insects. Because Masc is not conserved in insect taxa outside Lepidoptera (e.g., Drosophilidae in Diptera),¹³ whether Oscar is conserved among MK Wolbachia and broadly functions in various insect taxa remains unclear. In addition, the WO-mediated killing (wmk) gene, a putative transcriptional regulator located in the prophage, has been proposed as a candidate MK gene.⁷^,⁸ The transgenically overexpressed wmk gene, derived from the non-MK Wolbachia strain wMel, induces weak male lethality (ca. 30% of males die) in the fruit fly Drosophila melanogaster.⁷ Moreover, overexpressed wmk homologs derived from a non-MK strain, wSuz, and an MK strain, wRec,¹⁴^,¹⁵ killed all males and females of D. melanogaster.⁸ These results show various toxicities of wmk homologs in D. melanogaster; however, whether wmk is involved in MK has not been clarified. Insects have diverse sex determination systems, and microbes have been thought to accomplish MK by targeting any molecular mechanisms involved in sex determination and differentiation.¹⁶^,¹⁷^,¹⁸^,¹⁹ Considering that the expression of Wolbachia-induced MK depends on the host’s genetic background,¹⁴^,²⁰^,²¹^,²² it is possible that Wolbachia has acquired multiple mechanisms (i.e., causative genes of different evolutionary origin) to induce MK in a diverse range of host insects.

Here, we identified an MK-associated prophage WO region encoding an Oscar and two tandemly arrayed wmk homologs by comparing the genomes of the MK and non-MK Wolbachia strains in the tea tortrix moth Homona magnanima (Tortricidae, Lepidoptera).²³^,²⁴ We previously found three phylogenetically distinct non-MK Wolbachia strains (wHm-a, wHm-b, and wHm-c) from the Japanese population of H. magnanima. Strain wHm-a does not induce CI or any other apparent phenotypes, wHm-b induces CI, and wHm-c does not induce CI but has positive effects on hosts (i.e., pupal weight gains).²³ We also found an MK Wolbachia strain (wHm-t) that induces early MK (i.e., embryonic MK) from the Taiwanese population of H. magnanima. Intriguingly, non-MK wHm-c and MK wHm-t are phylogenetically closely related.²⁴ The expression of a single wmk gene in the WO region specific to the wHm-t either caused complete death or had no effect on D. melanogaster; however, co-expression of the tandemly arrayed wmk genes induced strong male lethality and recovered female survival in D. melanogaster. Conversely, the oscar homolog did not induce any lethal effects in Drosophila. Our studies revealed the presence of multiple MK mechanisms and a complex pattern of varying male and female lethality owing to the combination of wmk genes. Based on these results, we propose a hypothesis on the mechanism underlying Wolbachia-induced MK and the evolutionary origin of MK.

Results

Comparative genomics identified an MK-associated prophage region, WOwHm-t76

Genome assembly and polishing using PacBio RSII and Illumina data generated a 1,542,158 bp complete genome of the MK-inducing wHm-t isolated from an isofemale line W^T12 of H. magnanima (Table S1). Single-cell analysis using an isofemale line of H. magnanima (collected from Yakushima Is, Japan, female ratio = 0.52, n = 1542) harboring non-MK wHm-a, wHm-b, and wHm-c strains generated a complete genome of wHm-c (1,447,668 bp), as well as draft genomes of wHm-a (1,174,342 bp, 20 contigs) and wHm-b (1,302,323 bp, 9 contigs; Table S1). A high level of genome rearrangements was observed among wHm-t, wHm-a, and wHm-b (<50% homologies), whereas phylogenetically related wHm-t and wHm-c showed a high level of syntenic conservation (>92% homology) in most parts of the genomes and were almost perfectly colinear with several inversions (Figure S1). wHm-t and wHm-c had identical genes (84.9%, n = 1210) and orthologs (>90% identity, 94.9%, n = 1353; Table S2). In addition, bacterial density is a crucial factor for the expression of the Wolbachia phenotype. Indeed, Arai et al. (24) identified that wHm-t can induce MK only when its density is sufficiently high in H. magnanima (Figure S2). In this study, wHm-t in the MK matrilines typically showed higher titers than non-MK wHm-c in H. magnanima (p = 0.003 by Steel–Dwass test; Figure S2), but wHm-c did not induce MK even when its density was comparable to or higher than that of wHm-t, suggesting that a high bacterial dose is not sufficient to induce MK in H. magnanima.

Notably, a prophage-associated 76 kb insertion (hereafter referred to as WOwHm-t76) was present in the wHm-t genome but not in the closely related wHm-c (Figures 1A and S1). In addition, the WOwHm-t76 region was absent in wHm-a and wHm-b (Figure S1). The WOwHm-t76 region was annotated to harbor 83 genes (wHmt_12340 to wHmt_13160), which include Wolbachia density/virulence-associated gene homologs such as octomom genes (n = 7, Chrostek et al.²⁵), ankyrin repeat-containing genes (n = 6), and tandemly arrayed wmk homologs (n = 4; Figure 1B). Of the four wmk homologs, wmk-1 (wHmt_12830, 561 bp) and wmk-2 (wHmt_12840, 361 bp) had one helix-turn-helix (HTH) domain. Conversely, wmk-3 (wHmt_12920, 897 bp) and wmk-4 (wHmt_12930, 1006 bp) harbored two HTH domains, as originally found in the wmk (WD0626) of the wMel strain (Figure 1C).⁷ Although the sequence lengths of these genes differ, the four wmk homologs have highly homologous nucleotide and amino acid sequences (Figure S3, Table S3). Notably, the wHm-t specific ankyrin repeat- and papain-like proteinase domain-containing phage gene wHmt_13140 (3558 bp, Figures 1B and 1C) showed high homology to the Wolbachia oscar gene (WP250126898.1, Katsuma et al.¹³) that induces MK in O. furnacalis (Figures 1B–C, Table S3). Further resequencing analysis confirmed that the WOwHm-t76 region was conserved in wHm-t (n = 15; Taiwan) but not in wHm-c (n = 9; Japan; Figure 1D). Other than WOwHm-t76, the deleted or inserted loci in wHm-t genome were IS5 family transposases. To confirm whether WOwHm-t76 exists consistently in wHm-t, the field-collected H. magnanima were subjected to PCR targeting the gene wHmt_13140 (i.e., Hm-oscar) located in the WOwHm-t76 region. The 32 MK matrilines established from a single population of Taiwan were all positive, whereas the 13 non-MK matrilines established from the same population were negative for the Hm-oscar gene. Conversely, all the Japanese H. magnanima derived from 18 populations consistently infected with the non-MK wHm-c were negative (116 females and 129 males) for the Hm-oscar gene (Figure 1E).

Genomic comparisons of *Wolbachia* strains in *Homona magnanima*

(A) An insertion, the WOwHm-t76 region, in the wHm-t genome. The wHmc_11920 of the wHm-c encodes the IS110 family of transposase and has identical sequences of wHmt_12330 of the wHm-t. wHmc_11930 of wHm-c, encoding cytoplasmic incompatibility factor A (CifA), is identical to wHmt_13170 of wHm-t. The area highlighted by (B) and surrounded by a dashed red line indicates the locus encoding pathogenesis-related genes referred to in the following section.

(B) Virulence-associated genes in the WOwHm-t76 region.

(C) The domain structures of *wmk* and *Hm-oscar* genes annotated by Inter-Pro. HTH: helix-turn-helix. ANK: ankyrin repeat. PP: papain-like proteinase domain.

(D) Genomic loci, absent from wHm-c genomes (n = 9) but conserved in wHm-t genomes (n = 15), are highlighted in red on the wHm-t genome. Homologous genes or loci between wHm-c and wHm-t are not highlighted in red. (E) Detection of the WOwHm-t76 region (i.e., *Hm-oscar* gene) from *Wolbachia* infecting Japanese and Taiwanese *H. magnanima*.

Comparison of wmk, oscar, and cif homologs between Wolbachia strains

MK wHm-t as well as three non-MK Wolbachia (wHm-a, wHm-b, and wHm-c) encoded multiple wmk homologs regardless of their phenotype (Figure 2A). Other than the four wmk homologs present in the WOwHm-t76 region (i.e., wmk-1, wmk-2, wmk-3, and wmk-4), closely related wHm-t and wHm-c strains shared six other wmk homologs (100% nucleotide identity for each gene) (Figure 2A). Intriguingly, although absent in the non-MK strains (wHm-a, wHm-b, and wHm-c), the WOwHm-t76-associated wmk homologs were found in the genome of the MK wBol1 strain in Hypolimnas bolina (GenBank accession: ASM33377v1; Duplouy et al.²⁶), with 100% nucleotide identity for each gene (LC701684-LC701687). We confirmed that the wmk genes with identical nucleotide sequences were also conserved in the wBol1 strain in a field-collected adult female of Japanese H. bolina.

Phylogenies and toxicities of *wmk* and *Hm-oscar* genes in the WOwHm-t76 region

(A and B) Phylogenetic trees of *wmk* gene homologs (nucleotide) (A) and Oscar and CifB protein homologs (amino acids) (B). Phylogenetic trees were constructed based on maximum likelihood with bootstrap re-sampling of 1,000 replicates using MEGA7.²⁷ Homologs of *Wolbachia* strains were quoted from the NCBI database, and those of wHm-a, wHm-b, and wHm-t strains were manually annotated by BLAST and HHpred searches. The *wmk* and Oscar homologs present in wHm-t but absent in the wHm-a, wHm-b, and wHm-c are highlighted in red (*wmk-1*, *wmk-2*, *wmk-3*, *wmk-4,* and Hm-Oscar). *wmk-1*, *wmk-2*, *wmk-3*, and *wmk-4* in the wBol1 genome are also highlighted in red.

(C) RT-PCR detection for the *wmk* and Hb-*oscar* genes. 12, 36, 60, 84, and 108 indicate hours post oviposition of *Homona magnanima* egg masses. Ov+: female ovary of *H. magnanima* (W^T12 line); Ov-: non-reverse-transcribed samples (RT minus, control) using RNA extracted from the female ovary of *H. magnanima* (W^T12 line); Hb: female ovary of *Hypolimnas bolina*; P: DNA extracted from an *H. magnanima* W^T12 adult female (positive control); N: negative control (water). M: DNA ladders. 1000 bp ladder for *Hm-oscar,* and 100 bp ladder for other genes.

(D) The male ratio of adult progeny obtained from crosses between the *actin–GAL4 Drosophila melanogaster* line and seven *UAS* transgenic fly lines (*wmk*-1, *wmk*-2, *wmk*-3, *wmk*-4, *cifB*-like, *wmk*-1 and *wmk*-2, and *wmk*-3 and *wmk*-4; n = 11–33 independent crosses for each transgene). We counted the number of resulting offspring (females, orange; males, blue) with both *actin–GAL4* and *UAS* (+) and siblings with *UAS* and *Cyo (CyO)* as internal controls (−). The total numbers of adult counts for each genotype and sex are shown at the bottom. Different letters indicate statistically significant differences (Steel–Dwass test, p < 0.05). Dot plots show all data points individually. Exp.: expression; (−): not expressed; (+): expressed. Female: adult female progeny; Male: adult male progeny.

MK wHm-t harbored no CifB homologs except for the Hm-Oscar (Figure 2B, Table S3), although it encoded a CifA homolog (wHmt_13170) in a region adjacent to the Hm-Oscar (Figures 1A and 1B). This CifA homolog (wHmt_13170) was present as an identical orphan CifA (whm-c_11930) in the wHm-c genome (Figures 1A and 1B) that has neither Oscar nor CifB. The cifA and cifB gene pairs are involved in CI induction.¹¹^,¹²^,¹³^,²⁸^,²⁹^,³⁰^,³¹^,³²^,³³ The orphan cifA genes of wHm-c and wHm-t are likely remnants of the common cifA-cifB gene pair that was broken through evolutionary processes. The ancestral wHm-c type Wolbachia with the orphan cifA gene appears to have acquired the WOwHm-t76 region harboring Oscar and wmk through the integration of a certain WO phage, which gave rise to the MK-induced wHm-t. Compared with wHm-t and wHm-c, the CI-inducing wHm-b encoded the cifA-cifB genes (Figure 2B, Table S4). Intriguingly, wHm-a, which does not induce CI in H. magnanima, also had an intact cifA-cifB gene pair. Although its functions remain unclear, the Cifs in wHm-a are insufficient to induce CI in H. magnanima but may have a rescue function for CI induced by other Wolbachia strains (as observed in Bourtzis et al.³⁴ and Merçot and Poinsot³⁵). PCR assays confirmed that the four wHm-t-specific wmk genes as well as the Hm-oscar genes were expressed during embryogenesis and in the ovary of H. magnanima (Figure 2C). In addition, the wmk genes were also expressed in the ovary of H. bolina.

Single-gene expression of certain wmk genes killed both males and females, and dual expression of tandemly arrayed wmk genes resulted in MK in Drosophila

To assess whether the four wmk genes (wmk-1, wmk-2, wmk-3, and wmk-4) and the Hm-oscar gene on the WOwHm-t-76 region affect insect viability, we synthesized these genes with codon optimization and overexpressed them in Wolbachia-free D. melanogaster (Figure S4) using the GAL4/UAS system.⁷^,¹¹^,³⁶ When wmk-1 was overexpressed (12 replicates), no adult males and only a small number of adult females (n = 52 in total, ranging from 0 to 21 individuals per replicate) emerged, which is in sharp contrast with the non-expressing control groups (male ratio: 51.6% ± 5.11; 982 females and 1014 males in total) (p = 0.0007 for sex ratio, Steel–Dwass test, Figure 2D; p = 0.0003 for the number of females and p = 0.0001 for the number of males; Figure S4). Notably, when wmk-3 was overexpressed (11 replicates), almost no adults emerged (no males and females in 10 replicates and only one female in 1 replicate) in contrast with the control group (793 females and 888 males in total). Conversely, sex ratio and viability were not affected by the overexpression of wmk-2 (male ratio: 49.2% ± 4.42, 12 replicates), wmk-4 (male ratio: 50.6% ± 3.38, 12 replicates), and Hm-oscar genes (male ratio: 49.8% ± 5.57, 12 replicates) (sex ratio: p = 0.8932 for wmk-2; p = 0.9999 for wmk-4; p = 0.4618 for Hm-oscar gene; Figure 2D; number of emerged females: p = 0.9206 for wmk-2, p = 0.9971 for wmk-4, and p = 0.5815 for Hm-oscar; number of emerged males: p = 0.9534 for wmk-2, p = 0.9998 for wmk-4, and p = 0.9999 for Hm-oscar) (Figure S4).

To assess whether the dual expression of tandemly arrayed wmk pairs has additive or different effects on host viability similar to the cifA- and cifB-involving CI phenotype of Wolbachia, we simultaneously overexpressed each pair of the wmk genes (i.e., wmk-1 and wmk-2 or wmk-3 and wmk-4) in Drosophila, as described by Beckmann et al.¹¹ In contrast to the single expression of wmk-1 or wmk-2, dual expression of wmk-1 and wmk-2 did not bias the sex ratio (male ratio: 47.5% ± 3.6, 12 replicates, p = 0.1817) and generated numbers of adults (935 females and 845 males in total) comparable to the control groups (902 females and 992 males) (p = 0.9776624 for females; p = 0.5997 for males). Conversely, dual expression of tandemly arrayed wmk-3 and wmk-4 led to strongly female-biased sex ratios (female ratio: 89.1% ± 7.84, 33 replicates) compared to the control groups (50.1% ± 3.00, 33 replicates) (p = 0.0000; Figure 2D) and generated larger numbers of adults (2564 females and 395 males) than the flies singly expressing wmk-3 (p = 0.0004, Figure S4). Dead pupae (n = 2641 in total, 33 replicates) were conspicuously identified in the cohorts including those that should co-express wmk-3 and wmk-4. Among the 94 dead pupae harboring wmk-3, wmk-4, and Gal4, 77 were found to be males through Y chromosome-specific PCR (male ratio: 81.9%) (Figure S4).

Discussion

By comparing the genomes of an MK Wolbachia strain and three non-MK Wolbachia strains derived from H. magnanima, we identified a 76 kb prophage region WOwHm-t76, which was specifically present in the MK Wolbachia. Transgenic overexpression of one of the wmk gene pairs (wmk-1 or wmk-3) encoded by WOwHm-t76 in the non-native host D. melanogaster induced complete male lethality and high female lethality. This is similar to the previous finding that overexpression of each wmk gene from wSuz and wRec killed all D. melanogaster individuals.⁸ Compared with such strong lethality in D. melanogaster, when wmk homologs derived from Drosophila MK Wolbachia (e.g., wBif and wInn) and the butterfly H. bolina (wBol1) were overexpressed, no effect was observed in D. melanogaster.³⁷ The wmk homolog of wBol1 tested in Perlmutter et al.³⁷ (the sequence therein not available) is not likely to be identical in sequence to those tested in our study (wmk-1 to wmk-4), which should otherwise induce strong lethality; indeed, wBol1 harbors many wmk homologs outside the WOwHm-t76 region. We further demonstrated the recovery of flies by the additional expression of wmk-2 or wmk-4 in addition to wmk-1 or wmk-3, which induce strong lethality. Collectively, the virulence of wmk in males and females can be attributed to the difference in wmk sequences. However, recovery of viability by the co-expression of the tandemly arrayed wmk homologs may be explained by the combined action, wherein one wmk is toxic to both sexes and the other pair acts as its suppressor. Many microbes, including Wolbachia, and some animals employ toxin–antitoxin (TA) systems.³⁷^,³⁸^,³⁹^,⁴⁰^,⁴¹^,⁴²^,⁴³^,⁴⁴^,⁴⁵^,⁴⁶^,⁴⁷ By definition, TA modules encode a toxin that can globally downregulate metabolism and an antitoxin that counteracts this action.⁴⁴ Some type II TA modules encode HTH domain-containing proteins that form a stable complex and thus inactivate the toxin.⁴³^,⁴⁵^,⁴⁶^,⁴⁷ Although it remains to be elucidated, we propose that the adjacent wmk pairs form a complex (i.e., a dimer), as reported in the Wolbachia CifA-CifB pair,¹¹^,²⁸^,²⁹^,³⁰^,³¹^,³²^,³³ which can mitigate the virulence of wmk.

We can raise two distinct mechanistic possibilities for the wmk-induced strong male lethality in Drosophila. One possibility is that Wolbachia produces wmk regardless of its host’s sex and the strong male lethality is represented by the different levels of susceptibility to the wmk genes between the sexes. The other possibility is that the wmk gene functions differently depending on the host sex. Moreover, the level of affinity between the wmk pairs may differ depending on the physiological environment, which could account for the differences in virulence between the sexes. These potential mechanisms of wmk-induced male lethality appear to be different from that of Spiroplasma-induced MK in D. melanogaster, where the spaid toxin specifically damages the male X chromosome.¹⁹^,³⁶ Further investigation is needed to determine whether wmk replicates the effects observed with MK Wolbachia in Drosophila (e.g., male-specific cell death and malfunctioning of dosage compensation³⁶^,⁴⁸).

Intriguingly, we found that the overexpression of Hm-oscar did not induce lethality in D. melanogaster. MK has been hypothesized to target molecular mechanisms involved in sex determination and differentiation.¹⁶^,¹⁷^,¹⁸^,¹⁹ Insect sex-determining systems are diverse; for example, Lepidoptera (including H. magnanima) and Diptera (including D. melanogaster) do not share any known sex-determining genes other than the widely conserved doublesex (dsx) gene. The oscar protein encoded by the wFur strain induces embryonic MK in Ostrinia moths by targeting the host factor Masc, an essential protein for male sex determination.¹³^,⁴⁹ Since Masc is conserved in Lepidoptera but not in Diptera,¹³ Hm-Oscar may not be able to induce MK in Drosophila. Similar to the effects of wFur in Ostrinia moths,¹³^,⁴⁹ wHm-t impairs the expression of masc and sex determination cascades in H. magnanima.⁵⁰ This finding suggests that Hm-oscar in the WOwHm-t76 region may be involved in the wHm-t-induced MK in H. magnanima. Considering that oscar homologs have only been identified from the MK Wolbachia in lepidopteran insects (i.e., wFur, wBol, and wHm-t),¹³ the mechanisms (i.e., causative genes) of Wolbachia-induced MK may differ between insect taxa.

We suspect that Wolbachia strains have acquired multiple MK genes through their evolution. Generally, virulent genes often undergo duplications and substitutions through strong selective pressure.⁵¹^,⁵² Regardless of their phenotype, MK Wolbachia strains (e.g., wRec, wBol1, and wHm-t) and non-MK Wolbachia strains (e.g., wMel, wHm-a, wHm-b, and wHm-c) generally possess multiple wmk gene cassettes, some of which showed virulence toward the non-native host D. melanogaster. The wmk homologs encoded by WOwHm-t76 may be able to induce MK in H. magnanima by targeting common machinery present in insects. Actually, wmk genes of wHm-t were expressed throughout embryogenesis, which is when MK occurred in H. magnanima (Figure 2C). We speculate that the wmk gene may have originally caused lethality in arthropods without preference for sex. The duplications and nucleotide substitutions in the wmk genes may have reduced the virulence and allowed the offspring of Wolbachia-infected individuals to survive. As mentioned above, the dual expression of wmk gene pairs resulted in high male lethality. An excessively female-biased sex ratio selects for host suppressors against MK in various insects.¹⁶ If MK was quantitatively suppressed by detoxification of wmk, the production of a higher amount of wmk via the multiplication of wmk gene cassettes would counteract the action of host suppressors. The subsequent development of host suppressors would then lead to an increased number of wmk genes in the Wolbachia genome.¹⁶^,²²^,³⁷^,⁵³ An intense evolutionary arms race between Wolbachia and its host³⁷ may have increased the diversity of MK-associated genes (i.e., wmk gene pairs and oscar) in the Wolbachia genome.

In addition, although the two MK strains wBol1 and wHm-t are phylogenetically distantly related,²³^,²⁴ they harbor four wmk genes in the WO phage region, each of which is identical in nucleotide sequence. As molecular phylogenetic studies have shown previously in WO phages,⁵⁴^,⁵⁵^,⁵⁶^,⁵⁷^,⁵⁸ our data suggest that the WO phage may have moved between wHm-t and wBol1. Prophage regions such as WOwHm-t76, which include multiple virulence genes, may be conserved among MK Wolbachia strains regardless of their phylogenetic relationships. While the origin (source) of WOwHm-t76 remains unclear, further genome analysis of wBol1 as well as other Wolbachia strains will shed light on the evolutionary processes of MK Wolbachia. The diversification of the virulence-associated genes, such as the wmk and oscar genes, as well as rampant horizontal transmission of WO phages between different Wolbachia strains may have contributed to the prominent success of Wolbachia. Our findings offer insights into the molecular mechanisms and evolutionary associations of MK Wolbachia, WO phages, and their host insects.

Limitations of the study

Our study highlights the diversity in MK mechanisms and the bacteriophage as a critical driver of the evolution of MK Wolbachia. A limitation of this study is that we did not elucidate the functions and toxicities of the wmk and oscar homologs in the native host H. magnanima. Although our study could highlight the virulence and conjugated functions of the wmk genes in the non-native host D. melanogaster, the higher expression levels achieved by the Gal4-UAS system do not necessarily recapitulate the natural effects produced by the expression state of Wolbachia in insects. Future genome comparisons and functional validation of wmk and oscar homologs in H. magnanima will contribute to an integrated understanding of the diversity of the Wolbachia-induced MK mechanisms.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Biological samples

Homona magnanima, NSR line, Taiwan	Arai et al.²³	https://doi.org/10.1007/s00248-019-01469-6
H. magnanima, WT12 line, Taiwan	Arai et al.²³	https://doi.org/10.1007/s00248-019-01469-6
H. magnanima, Yakushima line, Japan	This paper	N/A
Drosophila melanogaster, actin5C–GAL4 [actin–GAL4/CyO]	BDSC	Stock number: 4144 (https://flybase.org/reports/FBst0004414.html)
D. melanogaster, UAS-wmk-1	This paper	N/A
D. melanogaster, UAS-wmk-2	This paper	N/A
D. melanogaster, UAS-wmk-3	This paper	N/A
D. melanogaster, UAS-wmk-4	This paper	N/A
D. melanogaster, UAS-wmk-1-t2a-wmk-2	This paper	N/A
D. melanogaster, UAS-wmk-3-t2a-wmk-4	This paper	N/A
D. melanogaster, UAS-Hm-oscar	This paper	N/A

Chemicals, peptides, and recombinant proteins

SilkMate 2S	Nosan Co., Yokohama, Japan	https://www.nosan.co.jp/english/
H. magnanima sex pheromone	Sumitomo Chemical Co., Ltd., Tokyo, Japan	https://jppaonlinestore.raku-uru.jp/item-detail/972069
SE traps	Sankei Chemical, Kagoshima, Japan	https://jppaonlinestore.raku-uru.jp/item-detail/972059
Universal SYBR Green Master Mix ROX	F. Hoffmann-La Roche, Ltd., Basel, Switzerland	https://www.n-genetics.com/products/1295/1023/13822.pdf
5.0-μm filter	Sartorius AG, Göttingen, Germany	https://www.sartorius.com/en/products-jp/lab-filtration-purification-jp/syringe-filters-jp
1.2-μm filter	Sartorius AG, Göttingen, Germany	https://www.sartorius.com/en/products-jp/lab-filtration-purification-jp/syringe-filters-jp
dsDNA HS quantification kit	Invitrogen, MA, USA	https://www.thermofisher.com/order/catalog/product/jp/ja/Q32851
REPLI-g Mini Kit	Qiagen, Hilden, Germany	https://www.qiagen.com/jp/listpages/ez1-cards/repli-g-mini-kit/
QIAseq FX DNA Library Kit	Qiagen, Hilden, Germany	https://www.qiagen.com/ja-us/products/discovery-and-translational-research/next-generation-sequencing/metagenomics/qiaseq-fx-dna-library-kit
MiSeq Reagent Kit v3	Illumina, CA, USA	https://jp.illumina.com/products/by-type/sequencing-kits/cluster-gen-sequencing-reagents/miseq-reagent-kit-v3.html
Rapid Sequencing Kit (SQK-RAD004)	Oxford Nanopore Technologies, Oxford, UK	https://store.nanoporetech.com/rapid-sequencing-kit.html
NEBNext dsDNA Fragmentase	New England BioLabs, MA, USA	https://international.neb.com/products/m0348-nebnext-dsdna-fragmentase#Protocols,%20Manuals%20&%20Usage
AMPure XP beads	Beckman Coulter, CA, USA	https://www.beckman.jp/reagents/genomic/cleanup-and-size-selection/pcr
GenNext NGS Library Prep Kit	TOYOBO Co Ltd., Osaka, Japan	https://www.toyobo-global.com/seihin/xr/lifescience/support/manual/NLQ-101.pdf
NEBNext Multiplex Oligos for Illumina	New England BioLabs, MA, USA	https://international.neb.com/products/e7335-nebnext-multiplex-oligos-for-illumina-index-primers-set-1#Product%20Information
Emerald Amp Max Master mix	TaKaRa Bio Inc., Shiga, Japan	https://www.takarabio.com/products/pcr/pcr-master-mixes/dye-added/emeraldamp-max-products/emeraldamp-max-premix
ISOGEN II	Nippongene, Tokyo, Japan	https://www.nippongene.com/siyaku/product/extraction/isogen2/isogen2.html
PrimeScript™ II Reverse Transcriptase	TaKaRa Bio Inc., Shiga, Japan	https://catalog.takara-bio.co.jp/product/basic_info.php?unitid=U100007713
pUASz1.1	DGRC, Kyoto, Japan	https://dgrc.bio.indiana.edu//stock/1433
NEBuilder® HiFi DNA Assembly	New England BioLabs, MA, USA	https://international.neb.com/applications/cloning-and-synthetic-biology/dna-assembly-and-cloning/nebuilder-hifi-dna-assembly

Deposited data

Raw sequence reads	This paper	GenBank BioProject: PRJDB13119, SAMD00445397 - SAMD00445400
wHm-a genome data	This paper	GenBank accession number: BQXF01000001 - BQXF01000020
wHm-b genome data	This paper	GenBank accession number: BQXG01000001 - BQXG01000009
wHm-c genome data	This paper	GenBank accession number: AP025639
wHm-t genome data	This paper	GenBank accession number: AP025638
Wolbachia wmk, cif, oscar genes	This paper	GenBank accession number: LC701647 - LC701694
Other datasets (Wolbachia density and fly data)	This paper	https://data.mendeley.com/datasets/jpvk5rjksk/1

Experimental models: Organisms/strains

Homona magnanima, NSR line, Taiwan	Arai et al.²³	https://doi.org/10.1007/s00248-019-01469-6
H. magnanima, WT12 line, Taiwan	Arai et al.²³	https://doi.org/10.1007/s00248-019-01469-6
H. magnanima, Yakushima line, Japan	This paper	N/A
Drosophila melanogaster, actin5C–GAL4 [actin–GAL4/CyO]	BDSC	Stock number: 4144 (https://flybase.org/reports/FBst0004414.html)
D. melanogaster, UAS-wmk-1	This paper	N/A
D. melanogaster, UAS-wmk-2	This paper	N/A
D. melanogaster, UAS-wmk-3	This paper	N/A
D. melanogaster, UAS-wmk-4	This paper	N/A
D. melanogaster, UAS-wmk-1-t2a-wmk-2	This paper	N/A
D. melanogaster, UAS-wmk-3-t2a-wmk-4	This paper	N/A
D. melanogaster, UAS-Hm-oscar	This paper	N/A

Oligonucleotides

PCR primers forwsp	Arai et al.²³	https://doi.org/10.1007/s00248-019-01469-6
PCR primers forHm-oscar	This paper	N/A
PCR primers forwmk-1	This paper	N/A
PCR primers forwmk-2	This paper	N/A
PCR primers forwmk-3	This paper	N/A
PCR primers forwmk-4	This paper	N/A
PCR primers foractin	Arai et al.²³	https://doi.org/10.1007/s00248-019-01469-6
PCR primers for COI	Arai et al.²³	https://doi.org/10.1007/s00248-019-01469-6
PCR primers for D. melanogaster Y chromosome	This study	N/A
PCR primers forD. melanogaster Gal4	This study	N/A
Codon-optimized Hm-oscar	This paper	N/A
Codon-optimized wmk-1	This paper	N/A
Codon-optimized wmk-2	This paper	N/A
Codon-optimized wmk-3	This paper	N/A
Codon-optimized wmk-4	This paper	N/A
Codon-optimized wmk-1-t2a-wmk-2	This paper	N/A
Codon-optimized wmk-3-t2a-wmk-4	This paper	N/A

Software and algorithms

Canu 1.6 and 2.0	Li⁵⁹	https://canu.readthedocs.io/en/latest/
minimap2 v2.17	Walker et al.⁶⁰	https://github.com/lh3/minimap2
Pilon v.1.23	Walker et al.⁶¹	https://github.com/broadinstitute/pilon
DFAST	Arndt et al.⁶²	https://dfast.ddbj.nig.ac.jp/
PHASTER	Li et al.⁶³	https://phaster.ca/
SAMtools v.1.9	Petkau et al.⁶⁴	http://www.htslib.org/
Gview	Sullivan et al.⁶⁵	https://server.gview.ca
Easyfig v2	Darling et al.⁶⁶	https://mjsull.github.io/Easyfig/
Mauve 2.4	Ihaka and Gentleman⁶⁷	https://darlinglab.org/mauve/download.html
R software v4.0	Paysan-Lafosse et al.⁶⁸	https://www.r-project.org
InterPro	Gabler et al.⁶⁹	https://www.ebi.ac.uk/interpro
HHpred	Kumar et al.²⁷	https://toolkit.tuebingen.mpg.de/tools/hhpred
MEGA7	Kumar et al.²⁷	https://www.megasoftware.net/

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Resource availability

Lead contact

Further information and requests for resources and reagents are accessible from the lead contact, Hiroshi Arai (dazai39papilio@gmail.com).

Materials availability

The Drosophila lines and vectors used in this study are accessible upon request. H. magnanima lines are not authorized to share under legislation by the Ministry of Agriculture, Forestry and Fisheries (permission No. 27 - Yokohama Shokubou 891 and No. 297 - Yokohama Shokubou 1326).

Experimental model and subject details

Homona magnanima

To construct a genome of wHm-t strain, we used the laboratory-maintained all female H. magnanima line, W^T12, which was initially collected in Taiwan (The Tea Research Extension Station, Taoyuan city) in 2015.²³ The W^T12 line was maintained over 50 generations by crossing the males of the normal sex ratio line (NSR), which is negative for Wolbachia.²³ To construct genomes of wHm-a, wHm-b, and wHm-c strain, we used a 1:1 sex ratio line (referred to as a Yakushima line) triply infected with the Wolbachia strains that was newly established from a female collected from Yakushima Island, Kagoshima, Japan in 2018. The Yakushima line was maintained by inbreeding (i.e., sib mating) across more than 20 generations. Larvae were reared using artificial diet SilkMate 2S (Nosan Co., Yokohama, Japan) at 25°C under a long (16L:8D) photoperiodic condition. For the resequencing of Wolbachia, we also used H. magnanima samples collected from fields using sex pheromone (Sumitomo Chemical Co., Ltd., Tokyo, Japan) with SE traps (Sankei Chemical, Kagoshima, Japan) in Japan (Table S5) and the Taiwanese individuals previously obtained by Arai et al.²³

Drosophila melanogaster

D. melanogaster line actin5C–GAL4 (actin–GAL4/CyO; BDSC 4414) was obtained from Bloomington Drosophila Stock Center at Indiana University (BDSC). Those D. melanogaster lines were maintained at 25°C with the standard banana medium. We also maintained transgenic fly lines harboring the codon-optimized wmk-1, wmk-2, wmk-3, wmk-4, and Hm-oscar genes. One of the two tandemly arrayed wmk pairs, either (i) wmk-1 and wmk-2 or (ii) wmk-3 and wmk-4, was conjugated using T2A peptide as described by Beckmann et al.¹¹ Transgenic fly lines were established by the standard microinjection method for phiC31 integrase transformation into the third chromosome at BestGene Inc. (Chino Hills, CA, UAS) using the pUASz 1.1 vector containing the synthesized genes. The newly established transgenic flies shared the genetic background of the fly strain with stock number 24749 (genotype: y1 M{vas-int.Dm}ZH-2A w∗; M{3xP3-RFP.attP}ZH-86Fb), and each wmk and Hm-oscar gene was inserted into the locus (estimated cytological site #86F8) on the right hand side of the third chromosome. Prior to zygotically overexpressing the synthetic gene with a codon optimized for D. melanogaster, fly stocks were reared in a tetracycline-containing medium (0.05% [w/v]) for at least one generation and in a standard banana medium for two generations. We confirmed that all the fly lines were negative for Wolbachia infection by PCR targeting Wolbachia wsp gene (Figure S4).

Method details

Wolbachia quantification

Wolbachia density was quantified as described in Arai et al.²³ Briefly, total DNA was extracted from individual H. magnanima abdomen using cell lysis solution (10 mM Tris-HCl, 100 mM EDTA, and 1% SDS, pH 8.0). DNA concentrations were quantified using Nano Photometer NP 80 (Implen, München, Germany) and were adjusted to a concentration of 10 ng/μL with MilliQ water, mixed with Wolbachia wsp gene primer sets (Table S6) and Universal SYBR Green Master Mix ROX (F. Hoffmann-La Roche, Ltd., Basel, Switzerland), and subjected to real-time PCR assays using Step One Plus Real-Time PCR System (Life Technologies Japan, Tokyo, Japan) as reported by Arai et al.²³ The cycle conditions were as follows: 10 min at 95°C; followed by 40 cycles of 15 s at 95°C, and 1 min at 60°C.

Genome construction of Wolbachia

Wolbachia was purified based on Duplouy et al.²⁶ and Iturbe-Ormaetxe et al.⁷⁰ with several modifications. To pellet wHm-t cells, 20 abdomens of W^T12 females were individually placed in a plastic tube and homogenized in 1,300 μL of phosphate-buffered saline (PBS; 137 mmol/l NaCl, 8.1 mmol/l Na₂HPO₄, 2.68 mmol/l KCl, 1.47 mmol/l KH₂PO₄, pH 7.4). After centrifugation at 4°C, 3,000 g for 5 min, 1,000 μL of the supernatant were filtered using 5.0-μm and 1.2-μm filters (Sartorius AG, Göttingen, Germany). The filtrates were centrifuged at 4°C, 18,000 g, for 20 min. Pellets were dissolved in 50 μL of PBS and subjected to DNA extractions using the cell lysis solution. DNA was quantified with NP80 (Implen) and Qubit v4 and dsDNA HS quantification kit (Invitrogen, MA, USA). Extracted DNA was whole-genome-amplified (WGA) with the REPLI-g Mini Kit (Qiagen, Hilden, Germany) for 16 h at 30°C according to the manufacturer’s protocol. The amplicon was sequenced using the PacBio RSII and Illumina (150 bp paired-end) platforms. PacBio RSII data were assembled using Canu 1.6,⁷¹ and subsequent polishing using Illumina data with minimap2 v2.17⁵⁹ and Pilon v.1.23⁶⁰ generated the complete circular genome of wHm-t.

For genome sequencing of wHm-a, wHm-b, and wHm-c, Wolbachia cells were purified from the 20 male pupae of Yakushima line as mentioned above. Wolbachia cells in 30 μm diameter droplets (0.3 cell/droplet) were lysed and amplified with REPLI-g Mini Kit (Qiagen) as described by Chijiiwa et al.⁶¹ and Nishikawa et al.⁷² The WGA amplicons were subjected to library preparation using QIAseq FX DNA Library Kit (Qiagen) and sequenced at MiSeq (75 bp paired-end) with MiSeq Reagent Kit v3 (Illumina, CA, USA). The amplicons were further sequenced at GridION using Rapid Sequencing Kit (SQK-RAD004, Oxford Nanopore Technologies, Oxford, UK). The sequence data from GridION were assembled by Canu 2.0⁷¹ and polished via Pilon v.1.23⁶⁰ using MiSeq data. All the Wolbachia genomes were annotated via DFAST.⁷³ Prophage regions were annotated with PHASTER.⁶²

Resequencing analysis and detection of the WOwHm-t76 region

A 1 μg sample of DNA extracted from field-collected H. magnanima (Table S5) harboring either wHm-t (n = 15, Taiwan) or wHm-c (n = 9, Japan) was incubated with NEBNext dsDNA Fragmentase (New England BioLabs, MA, USA) at 37°C for 24 min. After 5 μL of 0.5M EDTA was mixed and incubated at 65°C for 10 min, fragmented DNA was purified with AMPure XP beads (Beckman Coulter, CA, USA) following the manufacturer’s protocol. The purified DNA samples were subjected to library preparation using GenNext NGS Library Prep Kit (TOYOBO Co Ltd., Osaka, Japan) with NEBNext Multiplex Oligos for Illumina (New England BioLabs) for multiplex sequencing. Size selection optimized to 300 bp insert was carried out using AMPure XP beads (Beckman Coulter) following the manufacturer’s protocol. Size selected libraries were amplified using 2x Library Amplification Master Mix (TOYOBO) and NEBNext Universal PCR Primers (New England BioLabs) at 94°C for 2 min, 6 cycles at 98°C for 10 sec, 60°C for 10 sec, and 68°C for 15 sec. Libraries, purified with AMPure XP beads (Beckman Coulter) and eluted in 10 mM Tris-HCl (pH 8.0–8.5), were sequenced at Novogene using Illumina platform (150 bp paired end). Resequenced data, mapped to the wHm-t genome (W^T12) using minimap2 v2.17,⁵⁹ were converted to the consensus genomes using SAMtools v.1.9,⁶³ followed by synteny analysis using GView.⁶⁴ Homologies of the WOwHm-t76 region and other WO phages were visualized using Easyfig v2⁶⁵ and Mauve 2.4.⁶⁶ The presence or absence of the WOwHm-t76 region in field-collected H. magnanima was further assessed using PCR by amplifying a Hm-oscar gene (Table S6) in the WOwHm-t76 region with the Emerald Amp Max Master mix (TaKaRa) at 94°C for 3 min, 35 cycles of 94°C for 30 s, 62°C for 30 s, 72°C for 3 min, and a final extension at 72°C for 7 min.

Gene expression of wmk and oscar homologs of wHm-t

RNA was extracted from the W^T12 egg masses of 12, 36, 60, and 84 h post oviposition (hpo) using ISOGEN II (Nippongene, Tokyo, Japan) following the manufacturer’s protocol. For RT-PCR, the RNA (500–1000 ng/reaction) was reverse-transcribed using PrimeScript™ II Reverse Transcriptase (TaKaRa Bio Inc.) at 30°C for 10 min, 45°C for 60 min, and 70°C for 15 min. The cDNA was then amplified using EmeraldAmp MAX PCR Master Mix with specific primers for wmk (wmk-1, wmk-2, wmk-3, and wmk-4) and Hm-oscar genes (Table S6) at 94°C for 3 min, 35 cycles of 94°C for 30 s, 62°C for 30 s, 72°C for 3 min, and a final extension at 72°C for 7 min. The wmk gene expression levels were also verified using RNA extracted from an adult female ovary of Hypolimnas bolina (collected from Ishigaki Is., Okinawa) harboring wBol1.

Somatic overexpression analysis using D. melanogaster

The wmk and Hm-oscar genes in the WOwHm-t76 region were synthesized with codon optimization for D. melanogaster at Integrated DNA Technologies IDT (Coralville, IA, USA). We used IDT Codon Optimization Tool (https://sg.idtdna.com/pages/tools/codon-optimization-tool) to construct genes optimized for expression in D. melanogaster. To express two tandemly arrayed wmk pairs, either (i) wmk-1 and wmk-2 or (ii) wmk-3 and wmk-4 were conjugated using a T2A peptide as described by Beckmann et al.¹¹ Those synthetic genes were ligated into the plasmid pUASz1.1 (DGRC Stock 1433; https://dgrc.bio.indiana.edu//stock/1433; RRID:DGRC 1433) using NEBuilder® HiFi DNA Assembly (New England Biolabs) following the manufacturer’s protocol. Transgenic fly lines were established using the standard microinjection method for phiC31 integrase transformation into the third chromosome at BestGene Inc. (Chino Hills, CA, UAS) using the synthetic constructs.

To transgenically overexpress the wmk and cifB-like genes, 10 females of D. melanogaster line actin5C–GAL4 (actin–GAL4/CyO; BDSC 4414) were crossed with 5 males of homozygous UAS transgenic flies harboring wmk or Hm-oscar genes. These crossing experiments were replicated at least 11 times (numbers shown in Figure 2D). D. melanogaster lines were maintained at 25°C with standard banana medium and were randomly used for the crossings. Prior to zygotically overexpressing the synthetic gene with a codon optimized for D. melanogaster, fly stocks were reared in a tetracycline-containing medium (0.05% [w/v]) for at least one generation and in a standard banana medium for two generations. We confirmed that all the fly lines were negative for Wolbachia infection via PCR targeting the Wolbachia wsp gene (Figure S3). The numbers of offspring expressing transgenic (actin-GAL4/UAS) and non-expressing genes (CyO/UAS) in each replicate were all counted at the adult stage. The numbers of dead pupae in the lines harboring both wmk-3 and wmk-4 genes were counted and subjected to the detections of wmk-3, wmk-4, Gal4, and Y chromosome genes using specific primer sets (Table S6) at 94°C for 3 min, 35 cycles of 94°C for 30 s, 60°C for 30 s, 72°C for 30 min, and a final extension at 72°C for 7 min.

Quantification and statistical analysis

Wolbachia density

We used the average cycle threshold value (Ct) of two technical replicates for each sample and estimated Wolbachia density in H. magnanima individuals as described by Arai et al.²³ The number of Wolbachia wsp copies in 10 ng of sample DNA (i.e., Wolbachia density) was calculated based on a standard curve generated using 10⁻⁴–10 ng of plasmid DNA harboring a cloned partial wsp sequence used in Arai et al.²³ Wolbachia densities in the field-collected samples (Table S5) were analyzed using Steel–Dwass test [http://aoki2.si.gunma-u.ac.jp/R/Steel-Dwass.html; source("http://aoki2.si.gunma-u.ac.jp/R/src/Steel-Dwass.R", encoding="euc-jp")] in R software v4.0.⁶⁷

Annotations, homology surveys, and phylogenetical analysis of Wolbachia genes

Wolbachia wmk, cifA, cifB and oscar genes (accession numbers are shown in Figures 2A and 2B) were used as queries to identify the homologs from wHm-a, wHm-b, wHm-c, and wHm-t genomes using local BLASTn and BLASTp searches (default parameters). Motifs in the wmk, cifA, cifB, and oscar gene homologs were surveyed using InterPro⁶⁸ (https://www.ebi.ac.uk/interpro/) and HHpred⁶⁹ (https://toolkit.tuebingen.mpg.de/tools/hhpred). Phylogenetic trees of wmk, cifB, and Hm-oscar genes were constructed based on maximum likelihood with bootstrap re-sampling of 1,000 replicates using MEGA7.²⁷

Fly experiments

The numbers of offspring expressing transgenic (actin-GAL4/UAS) and non-expressing genes (CyO/UAS) in each replicate were all counted at the adult stage. The number of male and female flies expressing or non-expressing each gene (i.e., wmk-1, wmk-2, wmk-3, wmk-4, wmk-1 and wmk-2, wmk-3 and wmk-4, and Hm-oscar) and the male ratio (number of adult males/numbers of all adults) of all the conditions were compared using the Steel–Dwass test in R software v4.0.⁶⁶

Acknowledgments

We thank Greg Hurst (Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool, UK) and Katsuhiko Ito (Tokyo University of Agriculture and Technology, Tokyo, Japan) for their kind advice. We thank Takuhiko Yokoyama (https://www.takuhikoy.com; NARO) for his kind assistance in preparing a graphical abstract of this study. We also thank Hachiman-jyu Tea Factory (Yakushima, Kagoshima) for allowing the sampling of H. magnanima with the help of Hiroki Ishii and Mayu Nishino.

We acknowledge support from the Japan Society for the Promotion of Science (JSPS) Research Fellowships for Young Scientists [Grant Number 19J13123 and 21J00895 to H. Arai], JSPS Grant-in-Aid for Scientific Research [Grant Number 22K14902 to H. Arai], Hakubi Project of Kyoto University (to T. Harumoto), JST ERATO [Grant Number JPMJER1902 to T. Harumoto], Nagase Science Technology Foundation (to T. Harumoto), MEXT KAKENHI Grant-in-Aid for Scientific Research(S) [Grant Number 17H06158 to H. Takeyama], and JSPS Fund for the Promotion of Joint International Research (Fostering Joint International Research (B)) [Grant Number 21KK0105 to H. Arai and MN. Inoue].

H. magnanima was collected from Tea Research and Extension Station (Taoyuan City, Taiwan), and imported with permission from the Ministry of Agriculture, Forestry and Fisheries (No. 27 - Yokohama Shokubou 891 and No. 297 - Yokohama Shokubou 1326). All collaborators are presented as co-authors, and the results have been shared with the provider communities. Moreover, our group is committed to international scientific partnerships as well as institutional capacity building.

Author contributions

H. A. conducted field surveys, genome sequencing, and data analysis; designed fly experiments; wrote the original manuscript; and revised the manuscript. H. A. coordinated the single-cell analysis and presented the entire discussion. Y. N. conducted the single-cell genome sequencing. M. K. conducted the single-cell genome construction. K. I. helped with the genome construction. M. H. supported the entire single-cell analysis. S-R. L. organized the collection of insects in Taiwan and contributed to the discussion. M. U. collected Japanese H. magnanima. M. N. provided assistance during all experiments. Y. K. sampled insects and contributed to the entire discussion. T. H. designed and supported the fly experiments. D. K. supported the fly experiments and revised the manuscript. Lastly, H. A., H. T, and M.N. I. took responsibility for the decision to submit the manuscript for publication and managed the experiments and discussion.

Declaration of interests

The authors declare no competing interests.

Inclusion and diversity

We support inclusive, diverse, and equitable conduct of research.

Published: May 10, 2023

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2023.106842.

Contributor Information

Hiroshi Arai, Email: dazai39papilio@gmail.com.

Haruko Takeyama, Email: haruko-takeyama@waseda.jp.

Maki N. Inoue, Email: makimaki@cc.tuat.ac.jp.

Supplemental information

Document S1. Figures S1–S4 and Tables S1–S6

mmc1.pdf^{(819.5KB, pdf)}

Data and code availability

•
The sequence read data are publicly available in the DDBJ under accession numbers PRJDB13119 (BioProject) and SAMD00445397 to SAMD00445400 (BioSample). Wolbachia genomes (contigs) are available in the DDBJ database under the following accession numbers: wHm-a (BQXF01000001 - BQXF01000020), wHm-b (BQXG01000001 - BQXG01000009), wHm-c (AP025639), and wHm-t (AP025638). Homologs of wmk, cif, Hm-oscar, and Octomom genes and synthetic construct of wmk and Hm-oscar genes are publicly available in the DDBJ under accession numbers LC701647 to LC701694. Other data are publicly available from the Mendeley data (https://data.mendeley.com/datasets/jpvk5rjksk/1).
•
This paper does not report original code.
•
Any additional information required to reanalyze the data reported in this paper is accessible from the lead contact upon request.

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Associated Data

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

Supplementary Materials

Document S1. Figures S1–S4 and Tables S1–S6

mmc1.pdf^{(819.5KB, pdf)}

Data Availability Statement

•
The sequence read data are publicly available in the DDBJ under accession numbers PRJDB13119 (BioProject) and SAMD00445397 to SAMD00445400 (BioSample). Wolbachia genomes (contigs) are available in the DDBJ database under the following accession numbers: wHm-a (BQXF01000001 - BQXF01000020), wHm-b (BQXG01000001 - BQXG01000009), wHm-c (AP025639), and wHm-t (AP025638). Homologs of wmk, cif, Hm-oscar, and Octomom genes and synthetic construct of wmk and Hm-oscar genes are publicly available in the DDBJ under accession numbers LC701647 to LC701694. Other data are publicly available from the Mendeley data (https://data.mendeley.com/datasets/jpvk5rjksk/1).
•
This paper does not report original code.
•
Any additional information required to reanalyze the data reported in this paper is accessible from the lead contact upon request.

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PERMALINK

Combined actions of bacteriophage-encoded genes in Wolbachia-induced male lethality

Hiroshi Arai

Hisashi Anbutsu

Yohei Nishikawa

Masato Kogawa

Kazuo Ishii

Masahito Hosokawa

Shiou-Ruei Lin

Masatoshi Ueda

Madoka Nakai

Yasuhisa Kunimi

Toshiyuki Harumoto

Daisuke Kageyama

Haruko Takeyama

Maki N Inoue

Summary

Graphical abstract

Highlights

Introduction

Results

Comparative genomics identified an MK-associated prophage region, WOwHm-t76

Figure 1.

Comparison of wmk, oscar, and cif homologs between Wolbachia strains

Figure 2.

Single-gene expression of certain wmk genes killed both males and females, and dual expression of tandemly arrayed wmk genes resulted in MK in Drosophila

Discussion

Limitations of the study

STAR★Methods

Key resources table

Resource availability

Lead contact

Materials availability

Experimental model and subject details

Homona magnanima

Drosophila melanogaster

Method details

Wolbachia quantification

Genome construction of Wolbachia

Resequencing analysis and detection of the WOwHm-t76 region

Gene expression of wmk and oscar homologs of wHm-t

Somatic overexpression analysis using D. melanogaster

Quantification and statistical analysis

Wolbachia density

Annotations, homology surveys, and phylogenetical analysis of Wolbachia genes

Fly experiments

Acknowledgments

Author contributions

Declaration of interests

Inclusion and diversity

Footnotes

Contributor Information

Supplemental information

Data and code availability

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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