A Survey of the Bacteriophage WO in the Endosymbiotic Bacteria Wolbachia
Laurent Gavotte,*1 Hélène Henri,* Richard Stouthamer, Delphine Charif,* Sylvain Charlat,à
Michel Boulétreau,* and Fabrice Vavre*
*Laboratoire de Biométrie et Biologie Evolutive (UMR 5558), CNRS, IFR 41, University Lyon 1, Villeurbanne, France; Department
of Entomology, University of California, Riverside; and àBiology Department, University College London, London, United Kingdom
Bacteriophages are common viruses infecting prokaryotes. In addition to their deadly effect, phages are also involved in
several evolutionary processes of bacteria, such as coding functional proteins potentially beneficial to them, or favoring
horizontal gene transfer through transduction. The particular lifestyle of obligatory intracellular bacteria usually protects
them from phage infection. However, Wolbachia, an intracellular alpha-proteobacterium, infecting diverse arthropod and
nematode species and best known for the reproductive alterations it induces, harbors a phage named WO, which has
recently been proven to be lytic. Here, phage infection was checked in 31 Wolbachia strains, which induce 5 different
effects in their hosts and infect 25 insect species and 3 nematodes. Only the Wolbachia infecting nematodes and Trichogramma were found devoid of phage infection. All the 25 detected phages were characterized by the DNA sequence of
a minor capsid protein gene. Based on all data currently available, phylogenetic analyses show a lack of congruency
between Wolbachia or insect and phage WO phylogenies, indicating numerous horizontal transfers of phage among
the different Wolbachia strains. The absence of relation between phage phylogeny and the effects induced by Wolbachia
suggests that WO is not directly involved in these effects. Implications on phage WO evolution are discussed.
Introduction
Wolbachia are maternally inherited obligatory intracellular symbionts, which infect a wide range of arthropods
and filarial nematodes (Bourtzis and Miller 2003). They infect more than 17% of insect species (Bourtzis and Miller
2003) and nearly all filarial nematodes (Bandi et al. 2001).
The success of Wolbachia is best explained by the variety of
phenotypes they induce, which ranges from mutualism in
nematodes to various reproductive alterations in arthropods, such as cytoplasmic incompatibility (CI) (O’Neill
and Karr 1990), parthenogenesis induction (PI) (Stouthamer
et al. 1990), feminization of genetic males (Bouchon et al.
1998), male killing (MK) (Jiggins et al. 2001), and oogenesis completion in one hymenopteran species (Dedeine
et al. 2001). In addition, some strains do not induce any
apparent reproductive effect (Vavre et al. 2002). The molecular targets used by Wolbachia and the mechanisms involved in their effects are not known, and the absence of
any correlation between Wolbachia phylogeny and the effects they induce in hosts have led several authors to speculate on the evolution of Wolbachia-induced phenotypes
(Reviewed in Stouthamer et al. 1999). Three hypotheses
have been posed: 1) the effects are determined by the host’s
genome, 2) transition from one effect to another is easy and
is a frequent mutational event, and 3) effects are encoded by
extrachromosomal elements harbored by the bacterium. No
plasmid was detected in Wolbachia, but the presence of
a phage, named WO, suspected for a long time (Wright et al.
1978), was confirmed recently (Masui et al. 2000).
Phages are widespread viruses infecting bacteria that
use the host cell molecular systems for replicating their own
nucleic acid and for synthesizing their proteins. At the end
of the phage infection cycle, the accumulation of phage particles in bacterial cytoplasm induces cell lysis and bacterial
1
Present address: Department of Entomology, S225 Agricultural
Science Center Building—North Lexington.
Key words: Wolbachia, phage WO, insect, phylogeny.
E-mail: [email protected].
Mol. Biol. Evol. 24(2):427–435. 2007
doi:10.1093/molbev/msl171
Advance Access publication November 9, 2006
Ó The Author 2006. Published by Oxford University Press on behalf of
the Society for Molecular Biology and Evolution. All rights reserved.
For permissions, please e-mail: [email protected]
death. However, some bacteriophages can establish a not
immediately lethal association with bacteria when they enter lysogenic cycles (Lwoff 1953). In that case, a phage coding for advantageous proteins such as antibiotic resistance
or toxins can be a valuable auxiliary for bacteria (Miao and
Miller 1999). Phages are also implicated in transduction,
a mechanism allowing genetic transfer between bacterial
cells (Miller 2001).
The lytic activity of the phage WO is well documented
(Masui et al. 2001; Fujii et al. 2004; Gavotte et al. 2004),
making its prolonged persistence in Wolbachia strains a
puzzling observation. Indeed, strong selection pressures
acting on endosymbiotic prokaryotes tend to eliminate parasitic DNA such as repeated DNA or phages (Andersson JO
and Andersson SG 1999). WO is one of the rare reported
cases of bacteriophage infection in an intracellular bacterium (Storey et al. 1989), suggesting that WO might provide some factors of importance to Wolbachia, for example,
by contributing to the reproductive alterations they induce
in their hosts.
Like numerous intracellular symbionts, Wolbachia
cannot be cultured outside of insect cells, rendering the
study of WO particularly difficult and making polymerase
chain reaction (PCR) surveys and sequence analyses useful
methods for studying Wolbachia–phage interactions. Based
on such PCR surveys, Bordenstein and Wernegreen (2004)
estimated that WO infects 90% of Wolbachia strains. This
estimate included 39 Wolbachia strains from A and B clade
and inducing various reproductive alterations. However, for
most of these phages, no sequence is available, especially
for those inducing effects other than CI (Masui et al. 2000;
Bordenstein and Wernegreen 2004; Gavotte et al. 2004;
Sanogo and Dobson 2004; but for phage sequences from
Wolbachia-inducing feminization, see Braquart-Varnier
et al. 2005 and for one involved in host oogenesis, see
Gavotte et al. 2004).
Analysis of the diversity and the evolutionary dynamics of WO–Wolbachia associations is the first step to better
understand the possible implication of WO in Wolbachia
dynamics and effects on hosts, as was already proposed
by some authors (Masui et al. 2000; Fujii et al. 2004). In
the present study, phage WO infection was characterized
428 Gavotte et al.
using PCR survey and sequencing of a minor capsid protein
in 31 new insect Wolbachia strains and 3 strains infecting
nematodes. Together with data already published (Masui
et al. 2000; Bordenstein and Wernegreen 2004; Gavotte
et al. 2004; Sanogo and Dobson 2004; BraquartVarnier et al. 2005), our data set contains 40 phage types
harbored by 52 Wolbachia strains covering the whole range
of Wolbachia effects (CI, PI, MK, feminization, commensalisms, and mutualism). This study allows drawing
new conclusions on the coevolutionary history of WO
and Wolbachia.
Materials and Methods
Biological Material
Table 1 reports all insect species used in this study
together with the other species for which phage sequences were available in the literature (Masui et al.
2000; Bordenstein and Wernegreen 2004; Gavotte
et al. 2004; Sanogo and Dobson 2004; Braquart-Varnier
et al. 2005).
DNA Extraction and PCR
DNA extraction was carried out using Chelex resin as
described in Vavre et al. (1999). PCR was performed in
a 25-ll final volume reaction containing 200 lM of each
dNTP, 200 nM of each primer, 0.5 units Taq DNA polymerase, and 2 ll of DNA solution. The PCR conditions
were 1 min at 95 °C and then 35 cycles of 30 s at 92 °C,
40 s at specific hybridization temperature (table 2), and
1 min 15 s at 72 °C. After the cycles, a 10-min elongation
time at 72 °C was realized. Amplified products were visualized under UV after 30 min migration under 100 volts
current in 1% agarose gels.
Enzymatic Digestion
Potential number of phage types was estimated by
screening all orf7 PCR products with 2 restriction enzymes.
Enzymes were selected to discriminate different phage
types in Asobara tabida and Leptopilina heterotoma
(Gavotte et al. 2004) and proved also efficient for other
bacteriophage types.
Orf7 PCR products (5 ll) were digested over night using 2 restriction enzymes: DraI, and MboII. The restriction
reaction was performed at 37 °C in 4-CORE B buffer (Tris–
HCl: 6 mM; MgCl2: 6 mM; NaCl: 50 mM; dithiothreitol:
50 mM; pH 7.5 at 37 °C) (PROMEGA, Charbonnières,
France). Restriction profiles were observed under UV light
after 1 h 30 min migration at 100 volts current on 3.5%
agarose gel containing Ethidium Bromide.
Cloning and Sequencing
PCR products with digestion profile corresponding to
a known unique sequence were sequenced directly; all
others were cloned before sequencing. PCR products were
cloned using the TOPO-TA Cloning Kit (Invitrogen,
Abingdon, UK) after purification by cartridge method on
Concert Rapid PCR Purification System kit (Life Technologies). Purified products were inserted into a plasmid pCR
2.1-TOPO containing ampicillin resistance gene. This construction was transferred into competent Escherichia coli of
TOP10. Colonies containing plasmid were selected on
Luria broth (LB) medium plates (Trypton 1%, Yeast extract
0.5%, NaCl 1%, and agarose 1%) with ampicillin (25 lg/
ml), and those containing plasmids with the PCR product
were discriminated by a white/blue screening. Selected
clones were incubated in LB liquid medium with ampicillin
(25 lg/ml). Plasmids were purified by alkaline extraction
(Birnboim and Doly 1979). A specific PCR with WOF/
WOR primers was performed to select clones containing
the right insert. Clones were selected using the same restriction method, and 4–10 clones were sequenced for each profile. In all cases, within a Wolbachia strain, clones sharing
the same restriction profile gave all the same sequence.
DNA sequences are available on GenBank under the following accession numbers: orf7: DQ380533 to DQ380547, orf2:
DQ380528 to DQ380532, wsp: DQ380525 to DQ380527.
Alignments and Phylogeny
DNA sequences were first translated and peptide
sequences aligned with MUSCLE software (Edgar 2004)
with parameter by default. Using RevTrans (Wernersson
and Pedersen 2003), peptide alignments were then used
as a scaffold for constructing the corresponding DNA multiple alignment. GBLOCKS program (Castresana 2000)
was used to select reliable wsp regions. This left 390 sites,
that is, 59% of the original alignment. The orf7 informative
regions were selected manually. This left 281 sites, that is,
62% of the original alignment.
For both genes, maximum likelihood (ML) trees were
computed using phyml (Guindon and Gascuel 2003). Substitution models were determined with MrAIC.pl (Nylander
2004). For wsp, the optimal model was GTR 1 I 1 gamma
(4), that is, general time reversible with a proportion of invariant sites and 4 categories of substitution rate to estimate
the alpha parameter of the gamma law, whereas the best
model for orf7 was the model GTR 1 gamma (4). Five hundred replicates of Bootstraps were made with SEQBOOT
from the PHYLIP package (Felsenstein 1989).
Test for Bacteria–Phage Coevolution
In order to test the significance of the global hypothesis of coevolution between the phages WO and the Wolbachia strains, we used the ParaFit test (Legendre et al.
2002). This test is the function of the 2 matrices of phylogenetic distances (B and C) and of the matrix of host–
parasite association links (A). These 3 matrices can be combined in a fourth one: D (D 5 CA#B), which describes the
host–parasite association. To test the congruence between
the 2 phylogenies, a global statistic, called ParaFitGlobal, is
derived from D [ParaFitGlobal 5 trace(D#D)]. By permuting at random the row of the matrix A, a distribution of the
ParaFitGlobal values can be obtained. The probability for
the ParaFitGlobal value obtained from the data to be larger
than or equal to most of the ParaFitGlobal values obtained
under permutation can then be calculated. The ParaFit test
is robust to the existence of several parasites per host
(Legendre et al. 2002).
A Bacteriophage WO Survey in Wolbachia 429
Table 1
Infection of Phage WO in the Wolbachia Strains Studied
Insect host
Wolbachia
strain
Clade
Effecta
Phage type
number
Strain origin
No.
tested
No.
positive
5
34
10
10
10
5
20
8
?
?
?
25
6
?
?
?
?
?
2
12
19
10
10
10
15
?
?
?
13
?
9
1
1
?
?
20
?
2
2
14
9
?
1
1
14
?
10
10
28
10
10
20
5
34
10
10
10
4 (?)
20
8
?
?
?
25
6
?
?
?
?
?
0
12
19
10
10
10
15
?
?
?
7
?
3
1
1
?
?
20
?
2
2
5
8
?
1
1
14 (?)
?
0
0
0
0
0
20
10
10
10
10
40
10
10
?
1
1
1
0
0
0
0
0
0
0
0
0
0
0
Asobara rufescens
Asobara tabida
wAruf
wAtab1
wAtab2
wAtab3
A
A
CI
CI
CI
oog
1
1
?
3
Drosophila bifasciata
Drosophila melanogaster
wDbif
wDmel
A
A
MK
CI
Drosophila recens
Drosophila simulans
wDres
wRI
A
A
CI
CI
?
1
1
2
0
1
wCof
wAu
wNo
wHa
wEkue
wEcauA
wBol2
wLgui
wLhet1
wLhet2
wLhet3
A
A
B
A
A
A
A
A
A
A
A
None
CI
CI
CI
CI
CI
Unk.
CI
CI
CI
CI
1
1
5
1
1
4
0
1
1
1
1
Muscidifurax uniraptor
Nasonia giraulti
Nasonia logicornis
Nasonia vitripennis
Pachycrepoideus dubius
Protocalliphora siala
Trichopria drosophilae sp.
Armadillidium vulgare
Adalia bipunctata
Culex pipiens pipiens
wMuni
wNgir
wNlon
wNvitA
wPdub
wPsia
wTdro
wAvul
wAbipY
wCpip
A
A
A
A
A
A
A
B
B
B
PI
CI
CI
CI
None
CI
CI
Fem.
MK
CI
2
1
3
4
2
1
1
2
1
3
Encarsia formosa
Ephestia cautella
Hypolimnas bolina
wEfor
wEcauB
wBol1
B
B
B
PI
CI
MK
1
2
2
Leptopilina clavipes
Leptopilina victoriae
Nasonia vitripennis
Porcellio dilatatus petiti
Porcelliunides pruinosus
Telenomus nawai
Teleogryllus taiwanemma
Trichogramma brevicapillum
Trichogramma cordubensis
Trichogramma deion
Trichogramma embryophagum
Trichogramma evanescens
Trichogramma kaykai
wLcla
wLvic
wNvitB
wPpet
wPprui
wTnaw
wTtai
wTbre
wTcor
wTdei
wTemb
wTeva
wTkay1
wTkay2
wTmin
wTnub
wTole
wTpla
wTpre
wTsem
wTsib
wBmal
wDimm
wLsig
Uninfected
B
B
B
B
B
B
B
B
B
B
B
B
B
A
B
B
B
B
B
B
B
D
C
D
nd
PI
CI
CI
Fem.
Fem.
PI
CI
PI
PI
PI
PI
PI
PI
Unk.
PI
PI
PI
PI
PI
PI
PI
Mut.
Mut.
Mut.
nd
2
1
1
1
1
?
2
0
0
0
0
0
1
France
France
USA
Greece
Canada
nd
France
Portugal
China
USA
USA
France
Australia
Australia
Noumea
Hawaı̈
Japan
Japan
Tubai-FP
South Africa
France
The Netherlands
Spain
Tunisia
USA
USA
USA
USA
France
nd
France
nd
Russia
USA
France
The Netherlands
Japan
Tahiti–FP
Moorea–FP
The Netherlands
Maurice Island
USA
nd
nd
Japan
nd
USA
Portugal
USA
Iran
France
USA
0
0
0
0
0
0
0
0
0
0
0
USA
Canada
Yugoslavia
USA
Uruguay
France
Canada
nd
France
France
France
Ephestia kuehniella
Ephestia cautella
Hypolimnas bolina
Leptopilina guineaensis
Leptopilina heterotoma
Trichogramma nr minutum
Trichogramma nubilale
Trichogramma oleae
Trichogramma platneri
Trichogramma pretiosum
Trichogramma semblidis
Trichogramma sibercum
Brugia malayi
Dirofilaria immitis
Litosomoides sigmodontis
Setaria equine
Referencesb
1
1
1
1
1
2
3
4
3
3
3
4
4
1
1
1
3
3
3
3
5
6
4
3
5
5
4
7
NOTE.—Data from this study are in bold. FP, French Polynesia.
a
indicates bacterial effect: none, no reproductive effect detected; oog., effect on oogenesis; Unk., effect unknown; Fem., feminization; Mut., mutualism; ?, indicates
unknown data; nd., not determined.
b
Results previously presented in (1) Gavotte et al. 2004; (2) Wu et al. 2004; (3) Bordenstein and Wernegreen 2004; (4) Masui et al. 2000; (5) Braquart-Varnier et al. 2005;
(6) Sanogo and Dobson 2004; (7) by Blast on complete genome http://tools.neb.com/wolbachia/search.html.
430 Gavotte et al.
Table 2
Primers Used in the Present Work
Organism
Phage WO
Gene
orf7
WD0633
orf2
Wolbachia
ftsZ
wsp
Insect
a
ITS2
Primer
Primer sequence
WOF
WOR
WD0633F
WD0633R
ORF2F
ORF2R
F2
R2
81F
691R
ITS2F
ITS2R
5#-CCC ACA TGA GCC AAT GAC GTC TG-3#
5#-CGT TCG CTC TGC AAG TAA CTC CAT TAA AAC-3#
5#-TGG GTA TCT CTT AGA TGC AAA AG-3#
5#-AAG AGC AAG GCT TTT ACA TTA GG-3#
5#-GCA GGG CTA TAT TTT GGC GAG AA-3#
5#-AAC TCC ATT AAA ACT TCC CTG GC-3#
5#-TTG CAG AGC TTG GAC TTG AA-3#
5#-CAT ATC TCC GCC ACC AGT AA-3#
5#-TGG TCC AAT AAG TGA TGA AGA AAC-3#
5#-AAA AAT TAA ACG CTA CTC CA-3#
5#-TGT GAA CTG CAG GAC ACA TG-3#
5#-AAT GCT TAA ATT TAG GGG GTA-3#
Annealing
temperature
Referencesa
57 °C
1
56 °C
56 °C
55 °C
2
52 °C
3
55 °C
4
References: (1) Masui et al. 2000; (2) Holden et al. 1993; (3) Braig et al. 1998; (4) Campbell et al. 1993.
Results
Phage Typing
Phage presence was detected by specific PCR on the
orf7 marker, a gene coding for a minor capsid protein used
to detect WO (Masui et al. 2000). PCR specificity was
tested on individuals cured from Wolbachia by antibiotic
treatment, and WO was never detected in Wolbachia-free
individuals (data not shown). Individuals showing Wolbachia infection but no signal on orf7 were tested using 2
other phage genes, the orf2 and the wd0634, and congruent
results were always obtained on the 3 markers. The quality
of DNA extracts tested negative for phage and bacteria was
assessed by PCR using primers for the internal transcribed
spaces 2 (ITS2). Samples, where we failed to amplify the
ITS2, were excluded from the analysis.
To determine the diversity of phage infection within
each bacterial strain, the nucleotide sequence of orf7 was
used. A phage type was defined by grouping all phages
sharing more than 99% similarity on orf7 DNA sequences
based upon pure clones sequencing repeats. Usefulness of
orf7 as a phage phylogenetic marker was assessed by comparing the phylogenies obtained with orf7 and orf2 markers
as in Bordenstein and Wernegreen (2004). Unfortunately,
our sequences do not locate in the same gene region of
the orf2, making it impossible to directly compare the 2
studies. Phylogenies were established with ML method,
HKY model for orf2, and GTR for orf7 with 500 bootstrap
replicates. Congruency between orf7 and orf2 phylogenies
is good (fig. 1) as has been reported by Bordenstein and
Wernegreen (2004). Moreover, for 3 different Wolbachia
strains (wRi, wDmel, wLhet1) showing the same orf7
and orf2 sequences, a part of wd0634 gene that codes
for a resolvase was also sequenced. The 3 sequences obtained were also completely identical. Thus, even though
recombination can happen in phages (Bordenstein and
Wernegreen 2004), the orf7 seems to be a good phylogenetic marker.
insects (wTkay1 and wTkay2) or with weak orf7 PCR
signal (wTnaw and wDbip), phage infection has not been
resolved (table 1).
Phage WO was never detected in the nematode species
studied (Litosomoides sigmodontis and Dirofilaria immitis), and no related sequences were found on the complete
genome of Brugia malayi symbiont by Blast (http://tools.
neb.com/wolbachia/search.html, Foster et al. 2005). Because of their particular phylogenetic position compared
with Wolbachia from arthropods, these strains are not included in subsequent analyses. Wolbachia from Trichogramma were also found to be devoid of phage. The only
exception is the Wolbachia infecting Trichogramma kaykai. However, a second Wolbachia strain wTkay2, different
from the strains inducing PI, is also present in T. kaykai
(Van Meer et al. 1999). Based on different genetic markers,
this strain is identical to the Wolbachia infecting Ephestia
kuehniella and could have been transferred horizontally
because E. kuehniella is the host used for Trichogramma
rearing.
Phage Distribution
Together with previously published data (Masui et al.
2000; Bordenstein and Wernegreen 2004; Gavotte et al.
Phage Diversity
The phage infection status (including presence of WO
and sequence on orf7) of 27 new Wolbachia strains was
established. For 4 other strains, present in multiply infected
FIG. 1.—Comparison between orf2 and orf7 phylogenies for 5
Wolbachia strains.
A Bacteriophage WO Survey in Wolbachia 431
FIG. 2.—Distribution of the number of phage types found per Wolbachia strain. The high proportion of uninfected strains is due principally
to Trichogramma symbionts. Data from Masui et al. (2000); Gavotte et al.
(2004); Bordenstein and Wernegreen (2004); and Braquart-Varnier et al.
(2005) are included.
2004; Braquart-Varnier et al. 2005), the phage infection status of 48 bacterial strains is known (fig. 2). Among these
strains, 30% are devoid of phage, but this percentage is
probably overestimated because most uninfected strains
(12 out of 14) are from Trichogramma hosts that we extensively surveyed. Most phage-infected Wolbachia strains
display low numbers of phages, 85% (28 of 34) showing
only 1 or 2 different phage types.
Most Wolbachia strains display identical and complete
infection for all individuals tested, but 4 proved polymorphic for phage infection: wPdub infecting Pachycrepoideus
dubius (7 Wolbachia-infected individuals showing phage
infection out of 13 tested), wTdro infecting Trichopria
drosophilae sp. (3 of 9), wLcla infecting Leptopilina clavipes (5 of 14), and wLvic infecting Leptopilina victoriae
(9 of 11).
Multiple Phage Infection
Multiple phage infection, where a Wolbachia strain
displays more than one phage type, has been observed in
12 bacterial strains: wEcauA displays 5 phage types, wNvitA 4 phage types, wNlonA, wAtab3, and wTtai 3 phage
types each, and wMuni, wPdub, wAvul, wEcauB, wLcla,
and wBol1 phage types each (table 1).
In order to test whether phages infecting a given Wolbachia strain are more closely related to each other than to
phages chosen at random, we rebuilt the observed distribution of multiple infection (1 strain with 5 phages, 1 with 4
phages, 4 with 3 phages, and 6 with 2 phages) by randomly
drawing phages. Monte Carlo simulation (Raeside 1976)
was done by 5000 random drawings. Between each drawing,
all phages were put back because some multiply infected
bacteria display common phage types. For each drawing,
we calculated the average of tree branch length realized
by ML method between coinfecting phages. The sampling
distribution of genetic distances is normal (Kolmogorov–
Smirnov test, D 5 0.0147; P 5 0.2138) with an average
of 0.261 and a standard deviation of 0.029 (fig. 3). The
FIG. 3.—Distribution of the average genetic distance between phages
present in a single Wolbachia strain, obtained by Monte Carlo simulation.
observed average is 0.252 with a standard deviation of
0.032. Thus, phage types coinfecting a Wolbachia strain
are not more related than expected by chance (P 5 0.628).
Phage Phylogeny
Phage phylogeny, based on orf7, and Wolbachia phylogeny, based on wsp (Braig et al. 1998), were compared for
33 bacterial strains infecting insects (only a few strains infecting Trichogramma, devoid of phages are included,
others were not included for readability) and 55 phage types
(fig. 4).
No congruence was found between phage and bacterial phylogenies (ParaFit test, P 5 0.1319). Thus, it appears
that phages do not cospeciate often enough with their Wolbachia host to create strong phylogenetic signal, similar to
the lack of congruence between the phylogenies of Wolbachia and their insect hosts. Comparisons between phage
and insect host phylogenies are also not congruent (data
not shown).
However, a few correlations can be observed: the 2
pairs of Wolbachia wPdub/wMuni and wEkue/wTkay2
are potentially originating from bacterial horizontal transfers (Van Meer et al. 1999; Vavre et al. 1999) and display
the same wsp, 16S RNA, and ftsZ sequences and also carry
the same phage infection. This bacteriophage WO–Wolbachia combination was probably transferred between different insect hosts. However, absence of correlation between
phage and bacterial phylogenies indicates that the bacteriophage WO can be transferred horizontally without its bacterial host between different Wolbachia strains or insects
and can infect new Wolbachia hosts, as previously suggested based on smaller data sets (Masui et al. 2000;
Bordenstein and Wernegreen 2004).
432 Gavotte et al.
FIG. 4.—Comparison between bacterial phylogeny on the left side (based on wsp sequence) and phage phylogeny on the right side (based on orf7
sequence) infecting these bacteria. Wolbachia strains uninfected by phage were not included for a better readability.
Phage and Wolbachia Effects
Comparing the phage phylogeny with the effects induced by their Wolbachia hosts (fig. 5) does not suggest
any evident correlation between the phage phylogeny
and the effect of the Wolbachia strain it infects. Indeed,
the CI, MK, and PI phenotypes are scattered throughout
the phylogeny, and the phages associated with these effects
form paraphyletic groups. Moreover, a number of phageinfected bacteria (e.g., wPdub) do not induce any reproductive effects, whereas some uninfected Wolbachia strains are
able to induce reproductive effects: all Trichogramma symbionts induce PI and wDres induces CI (Stouthamer and
Kazmer 1994; Werren and Jaenike 1995).
Discussion
Intracellular bacteria are usually not prone to bacteriophage infection, owing both to selection for a reduction of
their genome size and limited exposure to infection associated with their isolated lifestyle (Franck et al. 2002). However, we observed that infection with the bacteriophage WO
is a common feature in Wolbachia because it was detected
in 70% of the strains and this proportion is probably an underestimation due to the high representation of Trichogramma species in the data set.
The general absence of congruence between phages
and Wolbachia phylogenies demonstrates that WO is able
to successfully transfer itself horizontally between different insects, with or without its bacterial host, as already
suggested by some studies on fewer Wolbachia–phage associations (Masui et al. 2000; Bordenstein and Wernegreen
2004). Random mixing of phages is also suggested by
the fact that phages sharing the same Wolbachia are not
more related to each other than expected by chance. Three
hypotheses can be drawn for these transfers: 1) Bacteriophage WO particles can be released, under some conditions, in the proximity of insect cells infected by
Wolbachia, and they can pass through the eukaryote cell
wall and then initiate new infections. Parasitoid infection
represents a potential route for such horizontal transfer.
Whatever the result of parasitism (success or failure), the
Wolbachia strain present in the ‘‘winner’’ insect comes into
contact with new potential phage infection. 2) Bacteriophage WO is able to infect other bacteria than Wolbachia.
If these bacteria are free living and potentially present in
various environments, the possibility for Wolbachia to acquire new phages is increased compared with transmission
being only possible between Wolbachia strains. It is interesting to note that the closest relative of the WO phage of
Wolbachia is a phage found in the plant pathogen Xylella
fastidiosa, which is transmitted by the Wolbachia-infected
Glassy-winged sharpshooter (Simpson et al. 2000). 3) Previous studies observed that Wolbachia could be transmitted
horizontally between insects naturally (Huigens et al. 2004)
or by artificial methods (Boyle et al. 1993). Because Wolbachia is present in numerous ecologically related species,
transfer of a Wolbachia strain to an insect already infected
by another Wolbachia strain will establish contact among
various bacterial strains with various phage types. Even
A Bacteriophage WO Survey in Wolbachia 433
FIG. 5.—Bacteriophage WO phylogeny based on orf7 sequences. Reproductive effects on Wolbachia host are reported. None, No reproductive
effects known; Fem., Feminization of genetic males; and Oog., effect on
oogenesis.
Could the widespread association of Wolbachia and
the bacteriophage WO mean that the bacteriophage WO
may be a beneficial auxiliary to Wolbachia, as is found
in various other phage/bacteria couples (Miao and Miller
1999)? Among the functions potentially encoded for by
the phage genome are the reproductive effects of Wolbachia
on their insect host (Stouthamer et al. 1999). However, 1)
no correlation was observed between phage presence or phylogeny and reproductive effects and 2) among PI-inducing
Wolbachia, some are infected by phages (wLcla, wMuni,
and wTnaw) but others are not (Trichogramma strains),
showing that the diverse effects of Wolbachia are not
due to a small number of specialized WO phages that move
from one Wolbachia strain to another. This does not rule out
a possible implication of WO in the effect of Wolbachia, as
suggested by recent finding in mosquito where ankyrin
genes within the phage genome might be involved in the
induction of CI (Sinkins et al. 2005). Different phages
might be involved in the induction of similar (on the basis
of their phenotype) effects. Moreover, molecular mechanisms involved in reproductive effect of Wolbachia probably involve Wolbachia 3 host interaction that the phage
might mediate. Finally, the phage WO could be the vector
of genes involved in Wolbachia’s effect, but the frequent
rearrangements occurring in the Wolbachia genomes could
rapidly lead to the transfer of these genes on the bacterial
chromosome itself, thereby breaking down the association
between the phage and the Wolbachia effect. In conclusion,
the present study suggests that the success of the bacteriophage WO mainly derives from its ability to be transferred
among bacterial hosts (using a currently unknown mechanism), rather than from any beneficial effect contributed to
its Wolbachia host. This situation parallels the relationship
between Wolbachia and its arthropod hosts (Hurst and
Mc Vean 1996).
Acknowledgments
if the bacteria transferred are eliminated later, which is
probably the most common case, new phage infection could
have been initiated.
Whatever the mechanism, all observations suggest that
the bacteriophage WO can spread among Wolbachia strains
through horizontal transfers of phage, indicating that WO
has important infectious capacities. However, phage transfer might not be as frequent as is suggested by the absence of
congruence between Wolbachia and phage phylogenies.
For instance, the 3 Wolbachia strains coinfecting each individual of the parasitoid L. heterotoma do not share phage
infections (Gavotte et al. 2004). Moreover, phages were not
detected in 2 specific groups of Wolbachia: those infecting
nematodes and Trichogramma species (Schilthuizen and
Stouthamer 1997; Bandi et al. 2001). Whether these groups
have lost previous infection or have never been infected is
unclear, but it does show that Wolbachia strains can remain
uninfected for long periods of time. The unusually high specialization of these 2 Wolbachia clades, both in terms of host
range and phenotypic effects, might limit the opportunity of
phage acquisition. Alternatively, lack of phage infection
might also explain the high specialization of these Wolbachia, by limiting genome plasticity and gene transfers.
We thank Stephen Dobson and Tim Vogel for suggestions for improving this paper. We thank Serge Morand,
Gilsang Jeong, Greg Hurst, Chris Jiggins and Bernard Pintureau to provide a part of insect and nematodes samples
used in this study. This study was partly supported by
EuWol project (EU) QLK3-2000-01079 and CNRS
(UMR 5558 and IFR 41).
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Aoife McLysaght, Associate Editor
Accepted November 3, 2006