Table Of ContentBiol.Lett.(2012)8,412–415 [4]. The sex dependence of death in this system
doi:10.1098/rsbl.2011.1114 suggests that wSca interferes with the sex-specific
Publishedonline4January2012 gene expression or physiology of the host. Indeed,
Evolutionary biology the occurrence of sexual mosaic individuals with an
exclusively male genotype upon incomplete elimin-
ation of wSca with antibiotics or heat treatment
A male-killing Wolbachia
strongly suggests that wSca has the ability to feminize
carries a feminizing factor genetic males [4–6]. Until now, however, molecular
interactions between the symbiont and host have not
and is associated with been investigated in this unique system.
In addition to Wolbachia, diverse bacteria from the
degradation of the generasuchasSpiroplasma,RickettsiaandArsenophonus
are known to cause male-specific death in their hosts
sex-determining system
[3]. Although the mechanism of male killing has been
studied in detail only in a few bacterium–host systems
of its host
[7–9], these studies suggested that mechanisms of
male killing might be diverse: dosage compensation is
Takafumi N. Sugimoto and Yukio Ishikawa* suggested to be involved in Spiroplasma-induced male
GraduateSchoolofAgriculturalandLifeSciences,Departmentof killing in Drosophila [7], whereas Arsenophonus is
AgricultureandEnvironmentalBiology,TheUniversityofTokyo, reported to target maternally inherited centrosomes to
Tokyo113-8657,Japan
killmalesinNasonia[9].
*Authorforcorrespondence([email protected]).
Inthepresentstudy,togaininsightintothemechan-
Endosymbiotic bacteria of the genus Wolbachia
ism of male killing by Wolbachia, we focused on a gene
induce diverse reproductive alterations in their
working at the bottom of the sex-determination cas-
insect hosts. Wolbachia (wSca) infecting the
cade, doublesex (dsx), which is transcribed into either a
moth Ostrinia scapulalis causes unusual male
male or female isoform by sex-specific splicing and
killing,in which males (genotype: ZZ) selectively
servesasafinalregulatorofsex-specificgeneexpression
die during embryonic and larval development,
whereas females (genotype: ZW), in turn, selec- in somatic cells of insects [10,11]. We investigated the
tively die when cured of infection. To gain developmental changes in phenotypic and genetic sex
insight into the interaction between wSca and ratios in the broods of normal, wSca-infected and
the host, we analysed phenotypic and genetic infected-and-cured O. scapulalis by diagnosing the iso-
sexes of the embryos and larvae of normal, wSca- forms of a dsx homologue (female-specific OsdsxFL or
infected, and infected-and-cured O. scapulalis by male-specific OsdsxM [12]), and sex chromatin in
diagnosing the sex-specifically spliced transcripts
interphase nuclei, respectively.
of Osdsx—a homologue of the sex-determining
gene doublesex—and sex chromatin in interphase
nuclei, respectively. It was observed that the 2. MATERIAL AND METHODS
female-type Osdsx was expressed in the infected
(a) Insects
male (ZZ) progenies destined to die, whereas the
Adult moths of O. scapulalis (Lepidoptera: Crambidae) were cap-
male-type Osdsx was expressed in the cured tured at Matsudo, Japan (35.88N, 139.98E) in the summer of
female(ZW)progeniesdestinedtodie.Thesefind- 2008–2009. Females infected with Wolbachia, which produce
ings suggest that (i) wSca, a male killer, carries all-female offspring, were screened by diagnostic polymerase chain
a genetic factor that feminizes the male host, reaction (PCR; see §2b), and maintained as matrilines through
crosseswithuninfectednormalmales[4].ThreeWolbachia-infected
(ii)thesex-determiningsystemofthehostisdegra-
matrilines and three uninfected cultures of O. scapulalis were used
ded, and (iii) a mismatch between the genetic and
forthepresentstudy.Insectswererearedat23+18Cwithaphoto-
phenotypicsexesunderliesthesex-specificdeath. period of 16L:8D. The larvae were reared on an artificial diet
(Silkmate2M,NosanCorp.)[4].
Keywords: Ostrinia scapulalis; male killing;
feminization; masculinization; doublesex; Wolbachia (b) DiagnosticPCR
DNAwasextractedfromtheovariesoffemalemothsusingaDNeasy
TissueKit (Qiagen),and PCRwasperformed usingExTaqDNA
polymerase (Takara Bio Inc.) and wsp gene-specific primers, wsp-
81Fandwsp-691R[13].Theprimersusedforamplificationofthe
1. INTRODUCTION actingene,asapositivecontrol,wereactin-Fandactin-R[12].
Wolbachia, a group of endosymbiotic bacteria har-
boured by a wide range of insects [1], is known for (c) Tetracyclinetreatment
Wolbachia was eliminated from the infected matrilines by rearing
variousmanipulationsofhostreproductiontoexpedite
larvae on an artificial diet containing tetracycline hydrochloride
theirownpropagation[2].Amongthemanipulationsis (0.06%, w/w) throughout the entire larval stage. Only female
male killing, in which males selectively die during adults were obtained in the generation treated. The absence of
embryonic and larval development, giving rise to all- Wolbachia in the femaleswasconfirmed by diagnostic PCR. These
Wolbachia-eliminated females, when crossed with normal males,
female progeny [3]. Despite the conspicuous effect of
produceall-maleprogeny[4,12].
the infection, the molecular interactions between Wol-
bachia and its hosts that may mediate male-specific (d) Observationofthesexchromatinforgeneticsexing
Thegeneticsexofindividualscanbedeterminedfromthepresence
death have remained unexplored. Wolbachia (wSca)
(female)orabsence(male)ofsexchromatinininterphasenucleiin
infectingtheadzukibeanborermoth(Ostriniascapula- the cells of Malpighian tubules or silk glands [4,12]. Malpighian
lis)causesmalekilling,however,themalekillinginthis tubules were dissected out from larvae or adults in sterile saline,
Wolbachia–host system is unusual in that females, in and fixed with methanol:acetic acid (3:1) for approximately
1min.Thepreparationswere stained withlacticacetic orcein, and
turn, selectively died when wSca was eliminated by
examinedunderalightmicroscope.Inthecaseofembryos,embryos
antibiotic treatment, giving rise to all-male progeny (pre-hatched larvae) were taken out from the eggs, and tissues
Received14November2011
Accepted9December2011 412 Thisjournalisq2012TheRoyalSociety
Wolbachia affects host sex determination T. N. Sugimoto & Y. Ishikawa 413
(a) 1.0 (i) (ii) ZZ ZW
(n = 23) (n = 22)
s 0.8 Osdsx
n ofmale 0.6 type -
oportioetic fe 0.4 type -
pren
g 0.2
actin
0
(b) 1.0 * * ZZ ZW
*
(n = 27) (n = 26)
0.8 Osdsx
s
oportion ofetic female 00..64 ttyyppee --
pren
g 0.2
actin
0
(c) 1.0 ZZ ZW
(n = 26) (n = 24)
s 0.8 Osdsx
n ofmale 0.6 type -
oportioetic fe 0.4 type -
pren
g 0.2
* actin
* *
0
5 12 17 40
embryos larvae larvae adults
age (days)
Figure 1. Changes in the genetic sex ratio associated with the development of Ostrinia scapulalis ((i) n¼23–92), and the
expression of male and female-type Osdsx transcripts in 5-day-old embryos ((ii) only examples are shown). Age refers to
days after oviposition. Genetic sex was determined by the presence/absence of sex chromatin in the cells [4]. The embryos
(pre-hatched larvae) were checked for sex chromatin and the type of Osdsx (OsdsxM or OsdsxFL, see the text). The actin
gene was used as a reference. ZZ, male genotype; ZW, female genotype. Asterisks represent significantly different from 0.5
byFisher’sexacttest (p,0.01).Wolbachia:(a)uninfected; (b)infected and (c)eliminated.
including the Malpighian tubules and silk glands were isolated by conversely, all genetic females freed from infection died
pulling the abdominal tip with fine forceps. The remainder of the at the early larval stage (figure 1c). Diagnoses of the
embryowasusedforRNAextraction.
Osdsx isoforms in the same individuals showed that the
(e) AnalysisofthetypeofOsdsxexpressedinembryos female-type OsdsxFL was expressed in all individuals
TotalRNAwasisolatedfromembryos,larvaeandadultsofWolbachia- infected with wSca irrespective of genetic sex (ZZ and
infected, infected-and-cured and uninfected O. scapulalis using
ZW; figure 1b), indicating that wSca feminized genetic
RNAiso (Takara Bio Inc.), and treated with RNase-free DNase I
(Qiagen). First-strand cDNA was synthesized from the total RNA males (ZZ). By contrast, the male-type OsdsxM was
using a PrimeScript first-strand cDNA Synthesis Kit (Takara Bio expressedinallindividualsfreedfrominfectionirrespec-
Inc.).PCRamplificationwasperformedusingKODFXDNApoly- tive of the genetic sex (figure 1c), indicating that
merase (Toyobo) with the primers exon 1-F and exon 5-R under
elimination of wSca brought about the masculinization
thefollowingconditions:988Cfor2min,30cyclesof988Cfor15s,
608C for 10s, 688C for 60s and a final extension at 728C for of genetic females (ZW). Inviability of embryos/larvae
10min[12].ThesizesofthePCRproductswiththeaboveprimers was observed when the genetic sex and phenotypic sex
were468bpforOsdsxMand725bpforOsdsxFL.
differed(table1).
3. RESULTS
InanuninfectednormalstrainofO.scapulalis,thesexratio 4. DISCUSSION
of individuals did not deviate significantly from 1:1 Inthesilkmoth(Bombyxmori),thepresenceofasingle
(proportion of females¼0.5) throughout development W chromosome ensures female development, whereas
(figure 1a), and the type of dsx homologue expressed in its absence male development takes place [14].
wasinaccordancewiththegeneticsex,thatis,themale- Thus, the W chromosome is believed to carry an
type OsdsxM was expressed in individuals with the ZZ epistatic female-determining factor (referred to here-
genotype,andthefemale-typeOsdsxFLwasexpressedin after as the F factor), although its molecular nature
individuals with the ZW genotype (figure 1a). We con- as well as the transcriptional cascade leading to the
firmed that all genetic males in the wSca-infected strain female-specific splicing of dsx is largely unknown
eventually died during the larval stage (figure 1b), and (reviewed in earlier studies [11,15,16]).
Biol.Lett.(2012)
414 T. N. Sugimoto & Y. Ishikawa Wolbachia affects host sex determination
Table 1. Viability of progenies produced by normal (uninfected), Wolbachia-infected and infected-and-cured Ostrinia
scapulalis female moths with reference to the genotypic and phenotypic sexes of progenies. (W* indicates a W chromosome
suggestedtohaveadysfunctionalfemale-determiningfactor.)
infectionstateofmother Wolbachiaa genotypeb sexualphenotypec viability
uninfected 2 ZW female viable
ZZ male viable
infected þ ZW* female viable
ZZ female inviable
curedd 2 ZW* male inviable
ZZ male viable
aMinusandplusindicateabsenceandpresenceofWolbachiainprogenies,respectively.
bThe genotype of an individual was determined by the presence/absence of sex chromatin in interphase nuclei, which is a condensed
heterochromatinformedfromtheWchromosome.
cThe sexual phenotype of an individual was determined by the sex-specific isoforms of Osdsx, a homologue of the sex-determining gene
doublesex(seethetextformoredetails).
dCuredofWolbachiainfectionbytetracyclinetreatment.
The expression of the female-type OsdsxFL in genetic feminization [2]. It is intriguing that a feminizing
males (ZZ) of the wSca-infected O. scapulalis clearly effect underlies the male killing, because feminized
indicates that wSca, a male killer, carries a feminizing individualsareviablewhenproducedbya‘true’femin-
factor that interferes with upstream sex-determination izer in the butterfly, Eurema hecabe [19]. Comparison
processes, or possibly the sex-specific splicing of of the male killer in O. scapulalis and the feminizer in
Osdsx itself. Meanwhile, the masculinization of genetic E.hecabemightshedlightonhowWolbachiadeveloped
females(ZW)freedfrominfectionindicatesthatafactor their repertoire of reproductive manipulations.
in the female-determining cascade is degraded in the
wSca-infected strains. Here, it should be noted that We thank Drs T. Matsuo, S. Hoshizaki, D. Kageyama,
T. Kayukawa, E. Sunamura, H. Sakamoto and T. Fujii
wSca-infectedstrains,whichcomprisefemalesonly,have
foradvice.
been maintained by crossing with males from normal
strains. Given that the degraded factor has been stably 1 Hilgenboecker, K., Hammerstein, P., Schlattmann, P.,
transmittedfrommother todaughter,itismostlikelyto Telschow, A. & Werren, J. H. 2008 How many species
belocatedontherecombinationallyisolatedWchromo- are infected with Wolbachia? A statistical analysis of cur-
some,anditispossiblethattheFfactoritselfisdegraded. rentdata.FEMSMicrobiol.Lett.281, 215–220. (doi:10.
Although discordanceof the genetic and phenotypic 1111/j.1574-6968.2008.01110.x)
sexes is suggested to underlie the sex-specific death 2 Werren,J.H.,Baldo,L.&Clark,M.E.2008Wolbachia:
master manipulators of invertebrate biology. Nat. Rev.
(table 1), the mechanism of death is unresolved. In
Microbiol. 6, 741–751. (doi:10.1038/nrmicro1969)
the male killing, caused by wSca, (i) the growth of
3 Hurst,G.D.D.&Jiggins,F.M.2000Male-killingbacteria
embryo/larvae destined to die was generally retarded,
ininsects:mechanisms,incidence,andimplications.Emerg.
(ii) no discernible abnormalities were found in their
Infect.Dis.6,329–336.(doi:10.3201/eid0604.000402)
morphology, and (iii) the occurrence of death was irre-
4 Kageyama, D. & Traut, W. 2004 Opposite sex-specific
gular.Therefore,themechanismofdeathisnotlikelyto effects of Wolbachia and inference with the sex determi-
be linked to a specific developmental process or event. nation of its host Ostrinia scapulalis. Proc. R. Soc. Lond.
One plausible explanation for growth retardation and B271,251–258. (doi:10.1098/rspb.2003.2604)
eventual death may be discordance in dosage compen- 5 Kageyama, D., Ohno, S., Hoshizaki, S. & Ishikawa, Y.
sation, i.e. sex-dependent adjustment of the expression 2003 Sexual mosaics induced by tetracycline treatment
levels of genes on the sex chromosomes. Indeed, invol- intheWolbachia-infectedadzukibeanborer,Ostriniasca-
pulalis. Genome46,983–989. (doi:10.1139/g03-082)
vement of dosage compensation is suggested in the
6 Sakamoto,H.,Kageyama,D.,Hoshizaki,S.&Ishikawa,Y.
Spiroplasma-induced male killing in Drosophila [7]. In
2008 Heat treatment of the Adzuki bean borer, Ostrinia
moths, however, the presence of dosage compensation
scapulalis infected with Wolbachia gives rise to sexually
is itself currently under discussion [17,18]. Studies on
mosaic offspring. J. Insect Sci. 8, 1–5. (doi:10.1673/031.
theexpressionlevelsofZ-linkedgenesinwSca-infected 008.6701)
and infected-and-cured individuals may shed light on 7 Veneti, Z., Bentley, J. K., Koana, T., Braig, H. R. &
the dosage compensation in moths. Hurst,G.D.D.2005Afunctionaldosagecompensation
Our recent identification of sex-specific isoforms of complex required for male killing in Drosophila. Science
Osdsx [12] paved the way for the simultaneous analysis 307,1461–1463. (doi:10.1126/science.1107182)
of genetic and phenotypic sexes.It was hitherto difficult 8 Bentley, J. K., Veneti, Z., Heraty, J. & Hurst, G. D. D.
2007 The pathology of embryo death caused by the
to determine the phenotypic sex of embryos and young
male-killingSpiroplasmabacteriuminDrosophilanebulosa.
larvae, because they do not show any sexual difference
BMCBiol. 5, 9. (doi:10.1186/1741-7007-5-9)
in morphology. Discordance between the genetic and
9 Ferree, P. M., Avery, A., Azpurua, J., Wilkes, T. &
phenotypic sexes in embryos/larvae destined to die was
Werren,J.H.2008Abacteriumtargetsmaternallyinher-
uncovered for the first time, to our knowledge, by this
itedcentrosomestokillmales inNasonia.Curr.Biol. 18,
simultaneous analysis. 1409–1414. (doi:10.1016/j.cub.2008.07.093)
In addition to male killing, reproductive altera- 10 Hoshijima, K., Inoue, K., Higuchi, I., Sakamoto, H. &
tions performed by Wolbachia include, among others, Shimura,Y.1991Controlofdoublesexalternativesplicing
Biol.Lett.(2012)
Wolbachia affects host sex determination T. N. Sugimoto & Y. Ishikawa 415
bytransformerandtransformer-2inDrosophila.Science252, 16 Fujii, T. & Shimada, T. 2007 Sex determination in the
833–836. (doi:10.1126/science.1902987) silkworm, Bombyx mori: a female determinant on the W
11 Shukla, J. N. & Nagaraju, J. 2010 Doublesex: a conserved chromosome and the sex-determining gene cascade.
downstreamgenecontrolledbydiverseupstreamregulators. Semin. Cell Dev. Biol. 18, 379–388. (doi:10.1016/j.
J.Genet.89,341–356.(doi:10.1007/s12041-010-0046-6) semcdb.2007.02.008)
12 Sugimoto,T.N.,Fujii,T.,Kayukawa,T.,Sakamoto,H.& 17 Suzuki, M. G., Shimada, T. & Kobayashi, M. 1998
Ishikawa,Y.2010Expressionofadoublesexhomologueis Absence of dosage compensation at the transcription
altered in sexual mosaics of Ostrinia scapulalis moths level of a sex-linked gene in a female heterogametic
infected with Wolbachia. Insect Biochem. Mol. Biol. 40, insect, Bombyx mori. Heredity 81, 275–283. (doi:10.
847–854.(doi:10.1016/j.ibmb.2010.08.004) 1046/j.1365-2540.1998.00356.x)
13 Zhou,W.,Rousset,F.&O’Neill,S.1998Phylogenyand 18 Walters, J. R. & Hardcastle, T. J. 2011 Getting a full
PCR-based classification of Wolbachia strains using wsp dose? Reconsidering sex chromosome dosage compen-
gene sequences. Proc. R. Soc. Lond. B 265, 509–515. sation in the silkworm, Bombyx mori. Genome Biol. Evol.
(doi:10.1098/rspb.1998.0324) 3,491–504. (doi:10.1093/gbe/evr036)
14 Tazima, Y. 1964 The genetics of the silkworm. London, 19 Hiroki,M.,Kato,Y.,Kamito,T.&Miura,K.2002Fem-
UK: Academic Press. inization of genetic males by a symbiotic bacterium in a
15 Traut, W., Sahara, K. & Marec, F. 2007 Sex chromo- butterfly, Eurema hecabe (Lepidoptera: Pieridae). Natur-
somes and sex determination in Lepidoptera. Sex. Dev. wissenschaften 89, 167–170. (doi:10.1007/s00114-002-
1,332–346. (doi:10.1159/000111765) 0303-5)
Biol.Lett.(2012)
