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RESEARCH ARTICLE
The Endosymbiotic Bacterium Wolbachia
Selectively Kills Male Hosts by Targeting the
Masculinizing Gene
Takahiro Fukui
1☯
, Munetaka Kawamoto
1☯
, Keisuke Shoji
1☯
, Takashi Kiuchi
1☯
,
Sumio Sugano
2
, Toru Shimada
1
, Yutaka Suzuki
3
, Susumu Katsuma
1
*
1Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, Bunkyo-ku, Tokyo, Japan, 2Department of Medical Genome Sciences, Graduate
School of Frontier Sciences, The University of Tokyo, Minato-ku, Tokyo, Japan, 3Department of
Computational Biology, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba,
Japan
☯These authors contributed equally to this work.
*katsuma@ss.ab.a.u-tokyo.ac.jp
Abstract
Pathogens are known to manipulate the reproduction and development of their hosts for
their own benefit. Wolbachia is an endosymbiotic bacterium that infects a wide range of
insect species. Wolbachia is known as an example of a parasite that manipulates the sex of
its host's progeny. Infection of Ostrinia moths by Wolbachia causes the production of all-
female progeny, however, the mechanism of how Wolbachia accomplishes this male-spe-
cific killing is unknown. Here we show for the first time that Wolbachia targets the host
masculinizing gene of Ostrinia to accomplish male-killing. We found that Wolbachia-infected
O.furnacalis embryos do not express the male-specific splice variant of doublesex, a gene
which acts at the downstream end of the sex differentiation cascade, throughout embryonic
development. Transcriptome analysis revealed that Wolbachia infection markedly reduces
the mRNA level of Masc, a gene that encodes a protein required for both masculinization
and dosage compensation in the silkworm Bombyx mori. Detailed bioinformatic analysis
also elucidated that dosage compensation of Z-linked genes fails in Wolbachia-infected O.
furnacalis embryos, a phenomenon that is extremely similar to that observed in Masc
mRNA-depleted male embryos of B.mori. Finally, injection of in vitro transcribed Masc
cRNA into Wolbachia-infected embryos rescued male progeny. Our results show that Wol-
bachia-induced male-killing is caused by a failure of dosage compensation via repression of
the host masculinizing gene. Our study also shows a novel strategy by which a pathogen
hijacks the host sex determination cascade.
Author Summary
Pathogens are known to manipulate the physiology, behavior, and reproduction of their
hosts for their own benefit. The endosymbiotic bacterium Wolbachia is known to
PLOS Pathogens | DOI:10.1371/journal.ppat.1005048 July 14, 2015 1/14
OPEN ACCESS
Citation: Fukui T, Kawamoto M, Shoji K, Kiuchi T,
Sugano S, Shimada T, et al. (2015) The
Endosymbiotic Bacterium Wolbachia Selectively Kills
Male Hosts by Targeting the Masculinizing Gene.
PLoS Pathog 11(7): e1005048. doi:10.1371/journal.
ppat.1005048
Editor: Elizabeth Ann McGraw, Monash University,
AUSTRALIA
Received: June 12, 2015
Accepted: June 25, 2015
Published: July 14, 2015
Copyright: © 2015 Fukui et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: The authors confirm
that all data underlying the findings are fully available
without restriction. The nucleotide sequence of
Ostrinia Masc has been submitted to the DDBJ/
EMBL/GenBank data bank under the accession
number LC028928. Deep sequencing data obtained
in this study are available under the accession
number DRA003038 (DDBJ).
Funding: This work was supported by grants from
Japan Society for the Promotion of Science grant
number 15K14893, and Science and Technology
Research Promotion Program for Agriculture,
manipulate the sex of its host's progeny. Male-killing is one of the phenotypes that Wolba-
chia induces, but the mechanism of how Wolbachia induces sex-specific death is
unknown. Here we found a marked down-regulation of Masc, a lepidopteran-specific zinc
finger protein gene, in embryos that are produced by Wolbachia-infected Ostrinia moths.
We also observed that dosage compensation fails in Wolbachia-infected Ostrinia embryos.
The findings of this study and our previous study using a lepidopteran model insect Bom-
byx mori indicate that Wolbachia has evolved to hijack the Masc-dependent, lepidopteran
insect-specific sex determination system by capturing an unknown factor during Wolba-
chia-host coevolution.
Introduction
Wolbachia is a genus in Rickettsiales, a diverse order of intracellular bacteria. Recent meta-
sequencing analysis shows that over 65% of insect species possess Wolbachia [1], indicating
that Wolbachia is the most widespread and common intracellular bacterium that infects
insects. Wolbachia is a well-known example of a parasite that alters host reproduction to facili-
tate its own propagation. Wolbachia-induced phenotypes include parthenogenesis, feminiza-
tion, cytoplasmic incompatibility, and male-killing, each of which is supposed to be adaptive
for Wolbachia by enhancing the production of infected females [2].
Wolbachia-induced male-killing has been reported in three insect orders, Coleoptera [3],
Diptera [4], and Lepidoptera [5], in which male-killing occurs mainly during embryogenesis.
Recent advances in Wolbachia-induced male-killing have been made mainly from studies
using Ostrinia moths [5–7]. Ostrinia species (corn borer) have a WZ/ZZ sex chromosome sys-
tem and all of the progeny of Wolbachia-infected mother moths have a W chromosome, indi-
cating the existence of male-killing [7]. The male-type splice variant of doublesex (dsx), a gene
that acts at the downstream end of the sex differentiation cascade [8–9], is not detected in
Ostrinia late embryos that originate from Wolbachia-infected mothers [7], suggesting that
Wolbachia’s sexual manipulation is presumably established at an early embryonic stage. How-
ever, the molecular mechanism(s) by which Wolbachia manipulates sex and male lethality in
Ostrinia moths has remained elusive.
The sex determination cascade in lepidopteran insects has been studied mainly using silk-
worm Bombyx mori as a model insect [10–11]. In B.mori, females have ZW sex chromosomes
and males have two Z chromosomes. B.mori femaleness is strongly determined by the presence
of the W chromosome irrespective of the Z chromosome number, suggesting that there is a
dominant feminizing gene (Fem) on the W chromosome [12–13]. In 2014, we discovered that
Fem is a precursor of a single W chromosome-derived PIWI-interacting RNA (piRNA) [14].
We also identified the target gene of Fem-derived piRNA (Fem piRNA), which is located on
the Z chromosome. Depletion of this Z-linked gene, Masculinizer (Masc), in male embryos
leads to the production of the female-type splicing of B.mori dsx, indicating that the product of
Masc is a masculinizing factor. These results revealed that sex of B.mori is determined by the
Fem piRNA-Masc cascade [14].
Further experiments showed that silencing of Masc in B.mori embryos results in male-spe-
cific lethality [14]. Deep sequencing (RNA-seq) of Masc mRNA-depleted embryos revealed
that the Masc protein is required for the repression of global transcription from the Z chromo-
some in male embryos, and that a failure of this dosage compensation causes male-specific
embryonic death [14]. Bioinformatic analysis also revealed that most of the Z-linked genes are
dosage compensated by 72 hours post-oviposition (hpo) [15]. These results indicate that
How Does Wolbachia Kill Male Moths Selectively?
PLOS Pathogens | DOI:10.1371/journal.ppat.1005048 July 14, 2015 2/14
Forestry, Fisheries and Food Industry grant number
26034A. The funders had no role in study design,
data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared
that no competing interests exist.
Masc-mediated control of dosage compensation at an early embryonic stage is essential for
male development of B.mori.
In this study, we attempted to explore the mechanism by which Wolbachia accomplishes
sexual manipulation using Wolbachia-infected Ostrinia moths as a model. We previously
showed that artificial depletion of Masc mRNA in B.mori early embryos results in male-spe-
cific embryonic lethality [14]. This phenomenon likely mimics the way that Wolbachia induces
male-killing in Ostrinia [7]. In the light of these facts, we first focused on the expression of
Ostrinia homologue of Masc in Wolbachia-infected embryos. Transcriptome analysis by RNA-
seq revealed that Wolbachia-induced male-killing is established by a failure of dosage compen-
sation through Masc mRNA depletion. Furthermore, injection of Masc cRNA into Wolbachia-
infected Ostrinia embryos prevented males from being killed at embryonic stages. This is the
first study to identify the Wolbachia’s target that is utilized for sexual manipulation.
Results and Discussion
Characterization of a male-killing Wolbachia found in Ostrinia moths
We collected over 80 Ostrinia moths in the field and found 3 female moths that were infected
with Wolbachia (Fig 1A). One of these moths produced only female progeny. By sex phero-
mone analysis, this female was found to be O.furnacalis. Subsequently, when the females were
mated with other Wolbachia-free O.furnacalis males only female progeny were produced.
There was no deviation in this pattern of female only progeny through 5 generations (in total
302 females and no males judged by external morphology at the adult stage) (Fig 1B).
We established the quantitative PCR (qPCR)-based molecular sexing method for O.furna-
calis (S1A Fig), and verified that Wolbachia-infected moths were all female (S1B Fig). Using
this method, we found that Wolbachia-infected embryos (just prior to hatching) contained
both female and male individuals (S1C Fig). However, the hatched larvae were all female (S1C
Fig), indicating that this Wolbachia induces male-specific embryonic lethality. In addition,
when Wolbachia was eliminated from infected individuals by tetracycline treatment and Wol-
bachia-eliminated female moths were mated with other Wolbachia-free males, only male prog-
eny were produced (S1D Fig). Taken together with these results, we concluded that this
Wolbachia strain induces male-killing in O.furnacalis, which is similar to the phenotype
observed in Wolbachia-infected O.scapulalis [7].
Sugimoto and Ishikawa [7] reported that the male-type splice variants of Ostrinia dsx [6]is
not expressed in all 5-day-old O.scapulalis embryos (just prior to hatching) that are infected
with a male-killing Wolbachia. In order to determine the precise developmental stage at which
Wolbachia starts sexual manipulation in Ostrinia, we examined the splicing patterns of dsx in
Wolbachia-infected Ostrinia embryos starting immediately after oviposition. Both male- and
female-type variants of dsx were observed in uninfected embryos (Fig 1C). The male-type vari-
ant in uninfected embryos was detected from 12 hpo, indicating that the sex determination sig-
nal is transmitted prior to 12 hpo in Ostrinia. In contrast, the male-type dsx variant was not
detected in Wolbachia-infected embryos throughout embryogenesis (Fig 1C). These results
indicate that Wolbachia manipulates the sex of Ostrinia from the beginning of the sex determi-
nation period.
Marked down-regulation of Masc in Wolbachia-infected Ostrinia
embryos
In order to investigate what occurs during Wolbachia-induced sexual manipulation, we per-
formed RNA-seq experiments using RNAs prepared from Wolbachia-infected and-uninfected
How Does Wolbachia Kill Male Moths Selectively?
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Fig 1. Characterization of a male-killing Wolbachia found in adult Ostrinia.(A) Wolbachia-infected
female moth of Ostrinia. Bar, 5 mm. (B) Brood sex ratios in a Wolbachia-infected matriline through 5
generations. The female:male ratio of each mating is shown. (C) Splicing patterns of Ostrinia dsx during
embryonic development (0–120 hpo) of embryos that were infected with Wolbachia (wol+) or uninfected (wol-
). Total RNA was prepared from Ostrinia embryos (25–50 embryos at each time point) and subjected to
RT-PCR for dsx and actin. The F and M indicate female- and male-type splicing of Ostrinia dsx, respectively.
actin was used as an internal control. Wolbachia infection was verified by PCR using wsp-specific primers.
Note that in other experiments (S1E Fig), the female-type splicing variants were clearly detected when the
same RNA pool prepared from Wolbachia-uninfected embryos at 60 hpo was used, although that was not
observed in this case.
doi:10.1371/journal.ppat.1005048.g001
How Does Wolbachia Kill Male Moths Selectively?
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embryos (of both sexes) at 0, 12, 24, 36, and 48 hpo. We recently showed that Masc protein
encodes a lepidopteran-specific CCCH-tandem zinc finger protein, which is required for both
masculinization and dosage compensation in B.mori [14]. RNA interference-mediated knock-
down experiments also show that depletion of Masc mRNA in B.mori early embryos results in
male-specific death via a failure of dosage compensation [14]. In order to test whether this
male-specific death in B.mori is related to the Wolbachia-induced male killing in Ostrinia,we
first identified and characterized the Masc homolog of Ostrinia. Using RNA-seq data, we iden-
tified contigs that potentially encode a protein with significant homology to Masc proteins
(Fig 2A and S2 Fig). Ostrinia Masc was composed of 583 amino acid residues and showed
28.1% identity to B.mori Masc (S2 Fig). Phylogenetic analysis based on the amino acid
sequences of zinc finger domains revealed that Ostrinia Masc was closely related to that of
Chilo suppressalis (Fig 2A).
The silkworm ovarian cell line BmN4 expresses the female-type Bmdsx variant only,
whereas transfection of B.mori Masc cDNA results in the production of the male-type splice
variant [14]. Using this system, we examined whether Ostrinia Masc protein also has a mascu-
linizing activity. As shown in Fig 2B, we observed the expression of the male-type variant of
Bmdsx in BmN4 cells when transfected with Ostrinia Masc cDNA as well as B.mori Masc
cDNA. In addition, we examined the expression of B.mori insulin-like growth factor II mRNA-
binding protein (BmIMP), a factor that is involved in the male-specific splicing of Bmdsx [16],
in Ostrinia Masc cDNA-transfected cells. We found that either B.mori or Ostrinia Masc cDNA
markedly enhanced BmIMP expression (Fig 2C). These results strongly suggest that Ostrinia
Masc encodes a masculinizing protein and may be required for masculinization in Ostrinia sex
determination pathway.
We next compared the level of Masc mRNA in Wolbachia-infected and-uninfected embryos
by mapping RNA-seq tags onto the Ostrinia Masc coding sequence. We found a significant
decrease in Masc mRNA in Wolbachia-infected embryos prior to 12 hpo (Fig 3A). To elucidate
this reduction in greater detail, we performed reverse transcription-qPCR (RT-qPCR) using
total RNA isolated from Wolbachia-infected and uninfected embryos at 0, 6, 12, and 18 hpo. In
uninfected embryos, Masc expression peaked at 6 hpo and decreased rapidly. In contrast, Masc
expression in Wolbachia-infected embryos declined by 6 hpo, and remained at a low level com-
pared with that observed in uninfected embryos (Fig 3B). These results clearly showed that
Wolbachia infection markedly reduces Masc mRNA level during embryogenesis of Ostrinia.
Together with transfection results (Fig 2B and 2C) and our previous results showing that
knock down of Masc mRNA results in the production of the female-type dsx in male embryos
of B.mori [14], we conclude that the lack of the male-type dsx in Wolbachia-infected Ostrinia
embryos (Fig 1C) was caused by down-regulation of Masc mRNA (Fig 3A and 3B).
Failure of dosage compensation in Wolbachia-infected Ostrinia embryos
Considering our recent finding that depletion of Masc mRNA in early embryos of B.mori
results in male-specific embryonic lethality due to a failure of dosage compensation [14], we
hypothesized that a decrease in Masc mRNA in Wolbachia-infected Ostrinia embryos also
affects dosage compensation, presumably resulting in a male-specific embryonic death. To test
this hypothesis, we examined dosage compensation effects in Wolbachia-infected Ostrinia
embryos using RNA-seq data. As reported previously [14], Z-linked transcripts are expressed
at higher levels in Masc mRNA depleted B.mori embryos than in control embryos (Fig 4A, left
panel). A similar transcriptional bias in putative Z-linked genes in Wolbachia-infected Ostrinia
embryos at 48 hpo was also found (Fig 4A, right panel). A failure of dosage compensation of Z-
linked genes was detected from 24 hpo and continued to 48 hpo (Fig 4B and S3 Fig). These
How Does Wolbachia Kill Male Moths Selectively?
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How Does Wolbachia Kill Male Moths Selectively?
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results strongly support our hypothesis that Wolbachia infection leads to abnormally enhanced
expression of Z-linked genes in male embryos via Masc mRNA down-regulation, resulting in a
male-killing phenotype.
Rescue of male progeny by injection of Masc cRNA into Wolbachia-
infected Ostrinia embryos
To obtain direct evidence that down-regulation of Masc mRNA in Wolbachia-infected Ostrinia
embryos results in the male-killing phenotype, we performed rescue experiments by injecting
in vitro synthesized capped, poly(A)-tailed Masc cRNA into Wolbachia-infected embryos. As
shown in Fig 5, the hatched larvae injected with control (GFP) cRNA were all female, whereas
both male and female larvae were observed when injected with Masc cRNA. This clearly indi-
cates that introduction of Masc cRNA into Wolbachia-infected embryos can rescue male
embryos. Together with the results of transcriptome data, we conclude that a decrease in Masc
mRNA in Wolbachia-infected embryos causes a male-killing phenotype via a failure of dosage
compensation.
Fig 2. Identification and characterization of Ostrinia Masc protein. (A) Phylogenetic analysis of Ostrinia
Masc protein. The neighbor-joining tree was generated using the amino acid sequences corresponding to the
zinc finger domains from 43 proteins showing homology to B.mori Masc protein. The numbers on the internal
branches represent the support value following bootstrap analysis (1,000 replicates). (B) Effect of Ostrinia
Masc cDNA transfection on the Bmdsx splicing in BmN4 cells. The splicing patterns of Bmdsx in BmN4 cells
transfected with Ostrinia or B.mori Masc expression vectors or empty vector were examined by RT–PCR.
The F and M indicate female- and male-type splicing of Bmdsx, respectively. (C) Effect of Ostrinia Masc
cDNA transfection on BmIMP expression. BmIMP expression was examined by RT-qPCR. rp49 was used as
a normalization control. Data shown are means + standard deviation of triplicates.
doi:10.1371/journal.ppat.1005048.g002
Fig 3. A marked decrease in Masc mRNA was observed in Wolbachia-infected embryonic Ostrinia.(A) Number of Ostrinia Masc coding sequence-
derived tags found in RNA-seq library generated from Wolbachia-infected (wol+) and-uninfected (wol-) embryos at 0, 12, 24, 36, and 48 hpo. (B) Expression
profile of Ostrinia Masc mRNA during early embryogenesis (0, 6, 12, and 18 hpo). Total RNA was prepared from embryos (25–50 embryos at each time point)
that were wol+ or wol-. Rps3 was used as a normalization control for RT-qPCR.
doi:10.1371/journal.ppat.1005048.g003
How Does Wolbachia Kill Male Moths Selectively?
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Fig 4. A failure of dosage compensation in Wolbachia-infected embryos. (A) Chromosomal distribution of differentially expressed transcripts in B.mori
and Ostrinia embryos. The B.mori RNA-seq data of Masc RNAi experiments (GFP and Masc RNAi embryos of mixed sex, 72 h post-injection) were used as
a control experiment (left panel). The Ostrinia RNA-seq data (Wolbachia-infected and uninfected embryos of both sexes at 48 hpo were used to draw the
panel on the right. The chromosome number for each Ostrinia transcript-derived contig was assigned using B.mori gene models. The data are shown by
box-and-whisker diagrams. The boxes represent the median and 25–75 percentile ranges of the expression ratios. (B) Time-course and Z chromosome-
biased distribution of differentially expressed transcripts in Wolbachia-infected and uninfected Ostrinia embryos. The data are separately shown in Z
How Does Wolbachia Kill Male Moths Selectively?
PLOS Pathogens | DOI:10.1371/journal.ppat.1005048 July 14, 2015 8/14
Conclusion and perspective
In conclusion, our study answered the question of how Wolbachia manipulates sex ratios in
moths: in Ostrinia,Wolbachia targets Masc, a masculinizing gene that was originally character-
ized in B.mori, to establish male-killing (Fig 6). In Drosophila, the Sex lethal protein functions
as a master switch for sex determination, and also controls dosage compensation by inhibiting
translation of male-specific lethal 2 (msl-2)[17]. Veneti et al. reported that a male-killing Spiro-
plasma targets the dosage compensation complex, including msl-2, to kill male D.melanogaster
[18]. Our current results demonstrate that a similar event occurs in Wolbachia-infected lepi-
dopteran insects; Wolbachia infection leads to male-killing by down-regulating Masc (Fig 3A
and 3B), which is an essential factor controlling both sex determination and dosage compensa-
tion pathways in lepidopteran insects [14]. This analogy comes from the fact that the sex deter-
mination cascade is often tightly associated with the control of dosage compensation in insects.
In B.mori, femaleness is determined by Fem piRNA-mediated, highly tuned post-transcrip-
tional regulation of Masc mRNA [14,19]. Our findings suggest that Wolbachia has captured
an unknown factor through evolution and succeeded in mimicking this sex determination sys-
tem to execute the male-specific death. Our future goal is to identify a Wolbachia factor that
decreases Masc mRNA post-transcriptionally or that directly inhibits Masc transcription
(Fig 6).
Materials and Methods
Insects
Moths were collected at Matsudo (35.8° N, 139.9° E) and Nishi-Tokyo (35.4° N, 139.3° E),
Japan, in early summer, 2014. GC-MS analysis of the pheromone gland extracts of the moths
used in this study showed the presence of (E)-12- and (Z)-12-tetradecenyl acetates (E12-14:
OAc and Z12-14:OAc). The relative abundance of the two components was 1:1.6 (E12-14:OAc
and Z12-14:OAc) in Wolbachia-infected and 1:2.7 in Wolbachia-uninfected moths, indicating
chromosome (blue)- and autosome (red)-linked genes. The data are shown by box-and-whisker diagrams. The boxes represent the median and 25–75
percentile ranges of the expression ratios.
doi:10.1371/journal.ppat.1005048.g004
Fig 5. Injection of Masc cRNA rescued the male-specific death of Wolbachia-infected embryonic
Ostrinia.Capped, poly(A)-tailed Masc cRNA was synthesized and injected into Wolbachia-infected embryos
immediately after oviposition. The hatched larvae were collected and molecularly sexed. The number
indicates the sample size of each group.
doi:10.1371/journal.ppat.1005048.g005
How Does Wolbachia Kill Male Moths Selectively?
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that they were O.furnacalis (Lepidoptera: Crambidae). Wolbachia-infected strain was main-
tained by crossing with Wolbachia-free O.furnacalis male moths. Ostrinia larvae were reared
on an artificial diet (Insecta LF, Nosan Corp.) at 23°C under a photoperiod of 16 L and 8 D.
Tetracycline treatment was performed as described previously [7].
Molecular sexing
Molecular sexing of Ostrinia moths and embryos was performed by qPCR using two Z-linked
genes triose phosphate isomerase (Tpi) and kettin as described by Kern et al.[20]. The autoso-
mal gene EF-1αwas used for normalization. Primers used for qPCR are listed below:
Tpi_F: 5'-ACGGAGGATCGGTTACTGGAGC-3'
Tpi_R: 5'-CGATGTCAACGAACTCTGGCTTGA-3'
kettin_F: 5'-AGGACTCTGGACGCATGGCT-3'
kettin_R: 5'-TGCAAGGCTATCAACAGGGCA-3'
EF1a_F: 5'-TTGCCACACAGCCCACATTG-3'
EF1a_R: 5'-TTGACAATGGCGGCATCACC-3'
RNA-seq analysis
Total RNA and genomic DNA were prepared simultaneously from Ostrinia embryos (25–50
embryos at each time point) using TRIzol reagent (Invitrogen) according to the manufacturer’s
protocol. Libraries for RNA-seq were generated from 0, 12, 24, 36, 48 hpo embryos using the
TruSeq RNA Sample Preparation kit (Illumina). The cDNAs were analyzed using the Illumina
HiSeq 2500 platform with 100-bp paired-end reads according to the manufacturer's protocol
[21]. De novo assembly of RNA-seq data from 10 data sets was performed as described
Fig 6. A proposed model for Wolbachia-induced male-killing in Ostrinia.In uninfected Ostrinia (left panel), Masc protein is expressed and utilized for
dosage compensation and masculinization in male development. By contrast, in Wolbachia-infected Ostrinia (right panel), Wolbachia reduces Masc mRNA
level in early embryos in an unknown manner. Down-regulation of Masc mRNA inhibits a masculinizing pathway, resulting in loss of the male-type variants of
dsx. Simultaneously, dosage compensation fails to establish and male-specific embryonic lethality occurs.
doi:10.1371/journal.ppat.1005048.g006
How Does Wolbachia Kill Male Moths Selectively?
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previously [14]. Ostrinia Masc was identified from assembled contigs by BLAST using the B.
mori Masc amino acid sequence as a query.
Because extensive synteny conservation is observed among several lepidopteran insects
including Ostrinia [22–24], we identified putative corresponding chromosomes for Ostrinia
RNA-seq derived contigs by BLAST using 13,789 B.mori gene models (putative protein-coding
genes whose chromosomal locations are identified). Transcript abundance in each contig was
quantified as described previously [14–15]. RNA-seq data of B.mori Masc RNAi experiments
(GFP and Masc RNAi embryos of each sex, 72 h post-injection, 4 data sets) [14] were used as a
control data set.
RT-PCR
Total RNA and genomic DNA were prepared from Ostrinia embryos (25–50 embryos at each
time point) using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. Total
RNA was subjected to reverse transcription using avian myeloblastosis virus (AMV) reverse
transcriptase with an oligo-dT primer (TaKaRa). PCR was carried out with KOD FX-neo DNA
polymerase (TOYOBO). Sex-specific splicing of Ostrinia dsx by RT-PCR and Wolbachia detec-
tion by wsp PCR were performed with primers reported previously [6]. RT-qPCR analyses
were performed using a KAPA
TM
SYBR FAST qPCR kit (Kapa Biosystems) and specific prim-
ers. The expression levels of rps3 were used to normalize transcript levels. Primers used for RT-
qPCR are listed below:
OstriniaMasc_F: 5'-TTTGCCGCATTCATTCGCAG-3'
OstriniaMasc_R: 5'-TGGTTTTGGTGCAAGCAATTCG-3'
OstriniaRPS3_F: 5'-TGGCCACCAGAACTCAAAGC-3'
OstriniaRPS3_R: 5'-GAAACGCTTCTGGACTACGGA-3'
Transfection of Ostrinia Masc cDNA in BmN4 cells
Ostrinia Masc cDNA was cloned into the pIZ/V5-His vector (Invitrogen). Transfection experi-
ments were performed as described previously [14]. In brief, BmN4 cells were transfected with
plasmid DNAs using X-tremeGENE HP (Roche). Cells were collected at 72 h after transfection,
and RT-PCR for Bmdsx and RT-qPCR for BmIMP were performed. B.mori Masc was used as a
positive control for this experiment. BmIMP mRNA level was normalized to that of rp49. Prim-
ers used for RT-qPCR are listed below:
Bmdsx_F: 5'-AACCATGCCACCACTGATACCAAC-3'
Bmdsx_R: 5'-GCACAACGAATACTGCTGCAATCG-3'
rp49_F: 5'-CCCAACATTGGTTACGGTTC-3'
rp49_R: 5'-GCTCTTTCCACGATCAGCTT-3'
BmIMP_F: 5'-ATGCGGGAAGAAGGTTTTATG-3'
BmIMP_R: 5'-TCATCCCGCCTCAGACGATTG-3'
How Does Wolbachia Kill Male Moths Selectively?
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cRNA injection
The DNA template for cRNA synthesis was amplified by PCR using the pIZ/V5-His vector
containing Ostrinia Masc (described above) or GFP (control) cDNA. Primers used for PCR are
listed below:
pIZ-F-T7: 5'-TAATACGACTCACTATAGGGAGACAGTTGAACAGCATCTGTTC-3'
pIZ-R: 5'-GACAATACAAACTAAGATTTAGTCAG-3'
Capped, poly(A)-tailed cRNA was synthesized using mMESSAGE mMACHINE T7 Ultra
Kit (Ambion). cRNA injection was performed as described previously [14] with some modifi-
cations. We injected 1–2nlofMasc or GFP cRNA solution (1 μg/μl in 100 mM potassium ace-
tate, 2 mM magnesium acetate, 30 mM HEPES-KOH; pH7.4) into Wolbachia-infected
Ostrinia embryos within 4 h after oviposition. The hatched larvae were collected and molecu-
larly sexed by qPCR.
Phylogenetic analysis
Phylogenetic analysis of Ostrinia Masc protein was performed as described previously [14].
The amino acid sequences of proteins in the NCBI database and those deduced form the RNA-
seq data obtained in this study with significant homology (E-value of <1×10
−9
) to residues
51–122 of Masc were identified using the BLAST program. A neighbor-joining tree was con-
structed using 43 sequences [including 3 Ostrinia sequences (Ostrinia Masc, c66984_g1_i4,
and c66984_g1_i3)] and the reliability of the tree was tested by bootstrap analysis with 1000
replications.
Sequence deposition
The nucleotide sequence of Ostrinia Masc has been submitted to the DDBJ/EMBL/GenBank
data bank under the accession number LC028928. Deep sequencing data obtained in this study
are available under the accession number DRA003038 (DDBJ).
Supporting Information
S1 Fig. Characterization of a male-killing Wolbachia found in O.furnacalis.(A) Establish-
ment of the molecular sexing method for O.furnacalis. Molecular sexing of Wolbachia-unin-
fected O.furnacalis moths was performed by qPCR using two Z-linked genes Tpi and kettin.
The autosomal gene EF-1αwas used for normalization. Moth sexes were judged by external
morphology. The number indicates the sample size of each group. (B) Molecular sexing of
Wolbachia-infected O.furnacalis moths. The number indicates the sample size of each group.
(C) Molecular sexing of Wolbachia-infected embryos (just prior to hatching, left panel) or
hatched larvae (right panel). The number indicates the sample size of each group. (D) Brood
sex ratios in a Wolbachia-eliminated matriline by tetracycline treatment. The female:male ratio
of two independent mating is shown. (E) Splicing patterns of Ostrinia dsx in Wolbachia-free
embryos at 60 hpo. The female-type splicing variants were clearly detected from the same RNA
pool used in Fig 1C.
(EPS)
S2 Fig. Identification of Ostrinia Masc protein. Alignment of B.mori and Ostrinia Masc pro-
teins was performed by the Clustal W program. The locations of putative zinc finger domains
are indicated by red lines.
(EPS)
How Does Wolbachia Kill Male Moths Selectively?
PLOS Pathogens | DOI:10.1371/journal.ppat.1005048 July 14, 2015 12 / 14
S3 Fig. MA plots of RNA-seq data of Wolbachia-infected and uninfected Ostrinia during
embryogenesis. The Ostrinia contigs that were assigned to B.mori gene models were used for
analysis. Z-linked genes are indicated by red dots.
(EPS)
Acknowledgments
We thank Takeshi Fujii for pheromone analysis and Shizuo George Kamita for critical reading
of the manuscript.
Author Contributions
Conceived and designed the experiments: SK TK. Performed the experiments: TF MK KS TK
SK. Analyzed the data: TF MK KS TK SS TS YS SK. Wrote the paper: SK.
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