UvA-DARE (Digital Academic Repository)
Wolbachia: intercellular manipulators of mite reproduction
Breeuwer, J.A.J.; Jacobs, G.
Published in:
Experimental and Applied Acarology
Link to publication
Citation for published version (APA):
Breeuwer, J. A. J., & Jacobs, G. (1996). Wolbachia: intercellular manipulators of mite reproduction. Experimental
and Applied Acarology, (20), 421-434.
General rights
It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s),
other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons).
Disclaimer/Complaints regulations
If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating
your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask
the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam,
The Netherlands. You will be contacted as soon as possible.
UvA-DARE is a service provided by the library of the University of Amsterdam (http://dare.uva.nl)
Download date: 16 Jun 2017
Experimental & Applied Acarology, 20 (1996) 421-434
421
Wolbachia" intracellular manipulators of mite
reproduction
J.A.J. Breeuwer* and G. Jacobs
Institute of Systematies and Population Biology, University of Amsterdam, Amsterdam
The Netherlands
ABSTRACT
Cytoplasmically transmitted Wolbachia (alpha-Proteobactefia) are a group of closely related
intracellular microorganisms that alter reproduction in arthropods. They are found in a few
isopods and are widespread in insects. Wolbaehia are implicated as the cause of parthenogenesis
in parasitic wasps, feminization in isopods and reproductive (cytoplasmic) incompatibility in
many insects. Here we report on the widespread occurrence of Wolbaehia in spider mites and
predatory mites based on a PCR assay for a 730 bp fragment of theftsZ gene with primers that are
specific for Wolbaehia. An additional PCR, using two primer pairs that amplify a 259 bp region of
the fisZ gene that are diagnostic for the two Wolbachia subdivisions A and B, showed that
infected mites only carried type B and not type A Wolbaehia. The fact that some species tested
negative for Wolbachia does not mean that the entire species is uninfected. We found that natural
populations of Tetranyehus urticae are polymorphic for the infection. The possible effects of
Wolbaehia on mite reproduction and post-zygotic reproductive isolation are discussed.
Key words: Wolbachia, reproduction, cytoplasmic incompatibility
INTRODUCTION
Wolbachia are a group of closely related intracellular bacteria that alter the
reproduction of their arthropod hosts in various ways. Wolbachia are typically
localized in the reproductive tissues of arthropods and are maternally
transmitted through the egg cytoplasm. They are widespread in insects,
occurring in 5-10% of species examined (O'Neill et aL, 1992; Rousset et al.,
1992; Werren et aL, 1995), but outside the insects they have only been reported
in a few isopods and a mite. So far, Wolbachia have been implicated as the
causative agent of post-zygotic reproductive incompatibility (or cytoplasmic
To whom correspondence should be addressed at: Kruislaan 320, 1098 SM Amsterdam, The
Netherlands
0168-8162 © 1996 Chapman & Hall
422
J.A.J.
B R E E U W E R A N D G. J A C O B S
incompatibility) in many insect species (Breeuwer et al., 1992; O'Neill et al.,
1992; Rousset et al., 1992; Werren and Jaenike, 1995; Giordano et al., 1995), an
isopod (Legrand et al., 1986; Rousset et al., 1992) and a predatory mite
(Johanowicz and Hoy, 1995), parthenogenesis in parasitic wasps (Stouthamer et
al., 1993; van Meer et al., 1995) and feminizing genetic males in isopods
(Legrand and Juchault, 1986; Rigaud et aL, 1991). In a few cases Wolbachia do
IIVJL IIO, V ~ O,I,L I.~ll~V~.iL ILJII I I O ~ L l~al-II~,.lIJ-LIt~LIOll ~ . . l l ~ L l . . I . a l l l , , / I~£ / , ~ . ,
177J9
IIUIIIIIalJL[I
allU
Turelli, 1995; Rousset and Solignac, 1995). Finally, many more insect hosts are
known to harbour Wolbachia, but their effect on host reproduction has not been
determined (O'Neill et al., 1992; Werren et al., 1995).
Wolbachia exploit two routes to increase in frequency in the host population
(i.e. proportion of infected hosts): (1) Conversion of genetic males into
phenotypic, functional females as in a number of isopods or female
parthenogenesis (thelytoky) in several parasitoid wasps. Everything else being
equal, this will immediately increase the frequency of the Wolbachiatransmitting sex, the females, in the host population. (2) Reducing or preventing
production of uninfected offspring. This phenomenon is called cytoplasmic
incompatibility and results in zygotic mortality in diploid species (Laven, 1957;
Hoffmann and Turelli, 1988; O'Neill and Karr, 1990) and male-biased or all
male offspring in haplodiploid hymenopteran species (Saul, 1961; Richardson et
al., 1987; Breeuwer and Werren, 1990).
Cytoplasmic incompatibility is typically observed in crosses between males
infected with Wolbachia and uninfected females and results in a reduced
number of surviving hybrid offspring, relative to all other, compatible, crossing
combinations between infected and uninfected individuals. This phenomenon
was first discovered in Culex mosquitoes (Laven, 1957; Yen and Barr, 1973) in
which certain crosses resulted in complete or partial embryonic death of
offspring. In subsequent introgression experiments, Laven (1957) demonstrated
that the factor causing incompatibility was cytoplasmically inherited. Cytological observation and antibiotic treatment showed that cytoplasmic incompatibility is caused by intracellular bacteria in reproductive tissues (Yen and Barr,
1973). These bacteria were described earlier in Culex pipiens and named
Wolbachia pipientis by Hertig (1936).
The precise mechanism of cytoplasmic incompatibility is not known. In the
parasitic wasp Nasonia, cytoplasmic incompatibility results in improper
condensation and fragmentation of the paternal chromosomes in the first
mitotic division of the fertilized egg (Ryan and Saul, 1968; Breeuwer and
Werren, 1990; Reed and Werren, 1995). Similar abnormal mitosis has been
observed in Drosophila simulans (O'Neill and Karr, 1990; Callaini et al., 1994)
and C. pipiens (Jost, 1970), indicating that the same mechanism may be
operating in diverse species. Apparently, paternal chromosome loss restores the
haploid status of fertilized eggs in haplodiploid Hymenoptera, because they
develop into normal males. In diploids, chromosome loss results in aberrant
development eventually leading to embryonic death.
WOLBACHIA: 1NTRACELLULAR MANIPULATORS OF MITE REPRODUCTION
423
Recently, Wolbachia have been reported in the predatory mite Galendromus
oceidentalis (Johanowics and Hoy, 1995). In mites, two observations suggest
that Wolbachia causing cytoplasmic incompatibility may be present: cytological
observations and crossing experiments. Several cytological studies in mites
reported on the presence of intracellular microbes in reproductive tissues (e.g.
Tetranychus urticae and G. occidentalis; see Table 1). Sometimes these
obligate intracellular microorganisms transmitted via arthropods (Krieg and
Holt, 1984). In most cases, these microbes are pathogenic to mammals, and the
arthropod host, mainly ticks and their allies, is the vector. In the case of the
parasitic mites of the genus Leptotrombidium, infection is possibly associated
with female-biased sex ratios (Roberts et al., 1977; Hastriter et al., 1987). In all
other instances, the microbes do not appear to have an obvious effect on their
arthropod host or the effects have not been studied. Recent molecular
phylogenetic studies using the 16S ribosomal DNA sequences showed that
RLOs are a group of obligate intracellular bacteria that belong to the 0~Proteobacteria and include the genus Wolbachia (Weisburg et al., 1991). The
Wolbachia from various arthropod hosts form a monophyletic group and are
closely related (more than 95% sequence similarity), despite their occurrence in
distantly related hosts (e.g. arthropod phyla and insect orders) and their different
phenotypic effects on host reproduction (O'Neill et al., 1992; Rousset et al.,
1992; Stouthamer et al., 1993; Werren et al., 1995).
In spider mites as well as in predatory mites, there are several reports on
embryo mortality and male-biased sex ratios of F1 offspring in crosses between
populations in several species (e.g.T. urticae (Helle and Pieterse, 1965; de Boer
and Veerman, 1983; Young et al., 1985; Gotoh et al., 1993), Tetranychus
neocaledonicus (Gutierrez and van Zon, 1973), Tetranychus telarius-bimaculatus complex (Dillon, 1958); Panonyehus ulmi (Gotoh and Noguchi, 1990),
Panonychus mori (Osakabe, 1993)). Note that because they have haplodiploid
sex determination, only females are hybrids. Recently, Gotoh et al. (1995a,b)
demonstrated almost complete non-reciprocal embryo mortality and malebiased sex ratios between northern and southern Japanese populations of
Tetranyehus quercivorus in a detailed crossing study. In predatory mites, Croft
(1970) and Hoy and Cave (1988) reported partial to complete F1 mortality,
shrivelled eggs and developmental mortality and males-biased sex ratios in
crosses between geographic strains of the predatory mite G. oceidentalis. Hoy
and Cave (1988) suggested the possibility that unidirectional incompatibility
might be mediated by microorganisms, similar to cytoplasmic incompatibility in
insects, based on earlier observations of rickettsia-like microorganisms in the
latter species (Hess and Hoy, 1982).
With a polymerase chain reaction (PCR) assay, detection and identification of
Wolbachia can now be reliably and rapidly performed on individual hosts
(Hoffmann and Turelli, 1995; Werren et aL, 1995). Here we report on a survey
for Wolbachia in various spider mite (Tetranychidae) and predatory mite
Rickettsia tsutsugamushi
R. tsutsugamushi
Parasitic mite
Leptotrombidium fletcheri
Leptotrombidium arenicola
Spider Mite
Tetranychus urticae
Phytoseiulus persimilis
Bacterium (digestive tract)
Type A (digestive tract)
Type B (reproductive tract)
Rickettsiella phytoseiuli
Rickettsia-like
Rickettsia sp.
Wolbachia persica
Tick
Ornithodorus savignyi
several species
Argus persicus
Predatory Mite
Typhlodromus (= Metaseiulus) occidentalis
Microbes
Host
Microbes reported in mites based on cytological studies
TABLE 1
Unknown
Unknown
Unknown
Unknown
Possible male killer
Possible male killer
Unknown
Vertebrate pathogen
No effect
Effect
Sologic and Rodriguez
(1971)
Su~t,~kovfiand ROttgen (1978)
Hess and Hoy (1982)
Hastriter et al. (1987)
Roberts et al. (1977)
Krieg and Holt (1984)
Reference
t,J
4~
WOLBACHIA: INTRACELLULAR MANIPULATORS OF MITE REPRODUCTION
425
(Phytoseiidae) species using a molecular assay for the ftsZ gene based on PCR
amplification of this gene with a primer pair that is specific for Wolbachia
(Holden et al., 1993; Werren et al., 1995) and discuss possible implications of
Wolbachia infection with respect to reproductive isolation and the sex ratio.
MATERIALS AND METHODS
Mite species
The list of species and strains of spider mites, the origin, and the year of
collection are listed in Table 2. Spider mites were raised on detached common
bean leaves (Phaseolus vulgaris) in climate rooms (18 or 23°C, L:D = 16:8, RH
60%). The strains of predatory mites had been in the laboratory for almost 20
years. Predatory mites were raised on pollen (Vicia faba) or on detached lima
bean leaves (Phaseolus lunatus) infested with the two spotted spider mite T.
urticae (see Table 3).
DNA extraction
From each culture five egg-laying females or ten freshly laid eggs were
collected and crushed in 50 #1 sterile 5% Chelex solution (weight:volume =
Chelex resin:ddH20) and 1 #1 filter sterilized proteinase-K (20 mg
ml-1)(Walsh et al., 1991). Prior to egg DNA isolation, eggs were surface
sterilized by washing eggs in 70% EtOH and rinsed twice with sterile ddH20 to
eliminate external contamination. The solution was vortexed for 10 s and
incubated at 37°C for 30 rain, vortexed again and centrifuged (14 000 r.p.m., for
2 min) to pellet the tissue material and chelex. Subsequently 10/~1 of the solution
was used in a 25 #1 PCR. Care was taken to avoid bacterial contamination and
control DNA samples were prepared from known infected and uninfected strains
ofD. simulans (Diptera) (respectively, Riverside and Watsonville from Dr. G. de
Jong, University of Utrecht, The Netherlands; Hoffrnan et al. 1996)) and infected
Muscidifurax uniraptor (Hymenoptera) and uninfected Muscidifurax raptor
(Hymenoptera) (obtained from Dr. R. Stouthamer, Agricultural University
Wageningen, The Netherlands; Stouthamer et aL, 1993).
PCR amplification
Wolbachia fisZ DNA was amplified in a 25/tl PCR reaction volume (10/~1
sample DNA, 2.5 #1 10X buffer; 0.2 #1 nucleotide mix (10 mM each), 0.3/~1
20 mM of each primer, 0.1 #1 superTaq (HT Biotechnology Ltd.); and 11.4 #1
ddH20). The PCR master mix was prepared in one batch and then added to each
DNA sample. The PCR was run on a Hybaid thermal cycler and cycle
conditions were one cycle (1 min at 94°C, 1 rain at 55°C, 3 rain at 72°C)
followed by 35 cycles (15 s at 94°C, 1 min at 55°C, and 2 min at 72°C). After
PCR, 4 #1 of amplified reaction product was run on a 0.7% agarose gel and
ur6cae (60)
kanzawai
neocaledonicus
turkestani (6)
yusti
ludeni
mcdanieli
piercei
Eutetranychus
banksi
orientalis
Oligonychus
bessardi
biharensis
Tetranychus
gloveri
collyerae
lambi
lombardinii (4)
Species
1995
1991
1991
1977
1977
1971, (2), 1986 ( I )
1991
1987
1976
1991
1977
1967
1988
1992
1994
1994
1967
1995
1994
Ricinus sp.
Thunbergia sp.
Florida, USA
N e w Zealand
N e w Caledonia
Indonesia (1)
Kenya (3)
New Zealand
France
Indonesia
Florida, USA
Indonesia
Louisiana, USA
Greece
Poland
NL imported
Coastal field populations, NL
Italy
Greenhouses, NL
Coastal field populations, NL
Mersea island, England
1968
Unknown
Oxalis sp.
Unknown
Madagascar
Bangladesh
Uknown
Cassava (Manihot esculenta)
Cassava (M. esculenta)
Thunbergia sp.
Unknown
Unknown
Strawberry (Fragaria sp.)
Cassava (M. esculenta)
Unknown
Trifolium sp.
Blackberry (Rubus sp.)
Banana (Musa sp.)
Crataegus sp., Trifolium sp.
Unknown
Tomato, cucumber, rose
Polygonatum sp., Lonicera sp.,
Crataegus sp., Sambucus nigra
Althaea rosea, Malva sp.
1967
1979
Year of collection
Unknown
Unknown
Host plant
Florida, USA
Egypt
Origin
l
1
0
1
1
1
1
3
1
1
1
1
1
1
1
0
37
-
-
Negative
flsZ
1
1
6
0
0
1
1
1
1
2
0
5
11
-
-
Positive
species, several strains from different geographic locations were tested (numbers in parentheses). From all other species only a single strain was available. NL, The
Netherlands.
Wolbachia occurrence in different strains of different spider mites species based on PCR assay for the 730 bp fragment o f the Wolbachia fisZ gene. From a few
TABLE 2
to
WOLBACHIA:INTRACELLULARMANIPULATORS OF MITE REPRODUCTION
427
TABLE 3
t¥olbachia occurrence in predatory mites based on PCR assay for the 730 bp fragment of the WolbachiaftsZ.
Underlined species were raised on spider mites, others were raised on pollen. Generic names according to de
Moraes et al. (1986).
Species
~yFr~u~l
Year of collection
ftsZ
~.~VLULL~Ula
• JO
--
Colombia
The Netherlands
Commerciala
The Netherlands
USA
The Netherlands
Madagascar
USA
New Zealand
Marocco
1987
1972
Unknown
1972
Unknown
1989
Unknown
Unknown
1986
1982
Origin
vlr~r~.~
iJ~Lul~**~
Amblyseius herbicolus
.4mblyseius andersoni
Phytoseiulus persimilis
Typhlodromus pyri
Galendromus occidentalis
Neoseiulus barkeri
Neoseiulus bibens
Neoseiulus californicus
Neoseiulus cucumeris
lphiseius degenerans
Z.s~L.U~Lr~
ga.
±VZU.IIIa
(Chant)
(Chant)
Athias-Henriot
Scheuten
(Nesbitt)
Hughes
(Blommers)
(McGregor)
(Oudemans)
(Berlese)
I
+
+
+
+
-
a Commercial strain obtained from Koppert BV, Berkel en Rodenrijs, The Netherlands.
stained with ethidium bromide to determine the presence and size of amplified
DNA.
The same primers were used that Holden et al. (1993) used to amplify 730
base pairs (bp) of the 3'-end of the WolbachiaflsZ gene (forward primer: 5'-gTT
gTC gCA AAT ACC gAT gC-3' and reverse primer, 5'-CTT AAg TAA gCT
ggT ATA TC-3'). Holden et al. (1993) showed that these primers specifically
amplify the 600 bp of the 3r end oftheftsZgene plus 130 bp into the 3' flanking
region of Wolbachia and not of closely related members of the ~-proteobacteria.
In addition, we further tested the specificity ofthefisZ Wolbachia primer pair on
Escherichia coli (strain JM 101) and unidentified gut microbes that are routinely
isolated from spider mites and grown on LB medium (Breeuwer, unpublished
results). Both bacteria did not yield any PCR product, confirming specificity of
ftsZ primers for Wolbachia.
Subsequently, the Wolbachia type was determined in two PCRs using fresh
DNA extracts of all the mite strains that had yielded a 730 bp DNA fragment. Presently, two types of Wolbachia can be distinguished based upon
both the 16S rDNA genes (Breeuwer et al., 1992; Stouthamer et al., 1993) and
thefisZ gene (Werren et aL, 1995). Werren et al. (1995) developed two primer
pairs which specifically amplify either Wolbachia type A (forward primer: 5'CTC AAg CAC TAg AAA AgT Cg-3' and reverse primer: 5'-TTA gCT CCT
TCg CTT ACC Tg-3') or Wolbachia type B (forward primer, 5'-TTC ggC Cgg
ATT TTA CAC AA-3' and reverse primer, 5'-TAg ggA TTA gCT TAg gCT Tg3'). These primers amplify a 259 bp region of the 3'-end of the ftsZ gene. The
specificity and reliability of these two primer sets were confirmed using infected
and uninfected strains ofD. simulans and infected M. uniraptor (Hymenoptera)
428
J . A . J . BREEUWER AND G. JACOBS
and uninfected M. raptor (Hymenoptera). Infected D. simulans from Riverside
is known to be infected with type A Wolbachia only, whereas M. uniraptor is
infected only with type B Wolbachia (Stouthamer et al. 1993; Werren et al,
1995). Our primer sets correctly detected and identified Wolbachia in these
insects.
RESULTS
Wolbachia were found in both predatory mites and spider mites based on the
presence of a 730 bp fragment of the fisZ gene in PCR assays. The chelex
(without mites) and PCR water control did not yield a 730 bp fragment, thus
contamination can be ruled out. In spider mites, six out of 18 species that are
maintained in our laboratory were infected (Table 2). In addition, in the two
spotted spider mite species, T. urticae, infection with Wolbachia was
polymorphic: 22 strains were infected and 37 were not.
Predatory mites that tested positive in the PCR assay were all fed with spider
mites (Table 3). It is possible that we amplified Wolbachia from ingested prey,
because of the presence of Wolbachia in T. urticae. To rule out this possibility, a
PCR was also performed with the total DNA isolates of surface sterilized eggs
of the predatory mites. All species that tested positive using adults were also
positive for Wolbachia using eggs. This indicated that PCR detection of
Wolbachia in predatory mites was not due to contamination by infected prey in
the gut.
Wolbachia in both spider mites and predatory mites belonged to Wolbachia
type B; PCR amplification with the Wolbachia type B primer set yielded the
expected 259 bp DNA fragment in all infected mite strains and species as
determined earlier in this study based on the presence of a 730 bp fragment of
thefisZ gene. None of the infected mites yielded any PCR product with primers
for Wolbachia type A. This primer set, however, was able to amplify a
Wolbachia type A f i s Z gene from a positive control, M. uniraptor wasps known
to be infected with type A only (Stouthamer et al., 1993; Werren et aL, 1995).
DISCUSSION
Wolbachia are widespread among insects. Approximately 5-10% of insect
species that have been surveyed appear to be infected (O'Neill et al., 1992;
Werren et al., 1995). Outside the insects Wolbachia have only been reported in a
few isopod species (Rigaud et al., 1991; Rousset et al., 1992). This is the first
report on the widespread occurrence of Wolbachia infections in the Acari class.
This suggests that more species of Acari are likely to harbour Wolbachia and its
distribution among mite species may very well parallel the situation in insects.
The fact that some species in Tables 2 and 3 tested negative for Wolbaehia
does not necessarily mean that the entire species is uninfected. Some strains
WOLBACHIA: INTRACELLULAR MANIPULATORS OF MITE REPRODUCTION
429
have been kept under laboratory conditions for many years and could have lost
the infection during that time. In addition, individual species can be
polymorphic for Wolbachia infection in natural situations. For example, natural
populations of Drosophila melanogaster in Australia (Hoffmann et al., 1994;
Solignac et al., 1994) and D. simulans (Hoffmann et aL, 1986; Turelli and
Hoffmann, 1991; Hoffmann and Turelli, 1995) differ in their Wolbachia
infection level. Similarly, the parasitoid wasp Trichogramma (Stouthamer et al.,
1990) and the isopod Armadillidium vulgare (Rigaud et al., 1991) are
polymorphic for Wolbachia inducing respectively, parthenogenesis or feminization. In addition, T. urticae spider mites collected in the dunes along the coast of
The Netherlands were polymorphic for the infection (Table 2). Polymorphism
for cytoplasmic incompatibility Wolbachia may be transient and Wolbachia are
expected to spread to fixation in the population (Caspari and Watson, 1959).
However, polymorphism is predicted under a variety of conditions, e.g.
evolution of resistance, reduced fitness of infected individuals and stochastic
loss of bacteria within infected individuals (Wade and Stevens, 1985; Rousset et
al., 1991; Stevens and Wicklow, 1992; Stouthamer and Luck, 1993; Turelli,
1995; Wade and Chang, 1995). Such conditions do occur in natural
populations. For example, in natural D. simulans populations, Hoffmann and
Turelli (1995) showed that transmission fidelity from mother to daughter is not
perfect and incompatibility, i.e. the hatching rate increases with male age.
Finally, it is possible that the absence of an amplification product in a given
species is an artefact due to sequence variation of the primer sites in the ftsZ
gene in different Wolbachia from different hosts or inappropriate DNA
isolation. The primer set we used may not amplify every member of the genus
Wolbachia. However, this is unlikely, because this particular primer set is
capable of detecting both Wolbachia types (A and B) from many different
arthropod hosts.
At this point little is known about the effects of Wolbachia in mites. Postfertilization cytoplasmic incompatibility due to Wolbachia typically results in
paternal chromosome loss and renders the egg haploid. In haplodiploids such
eggs develop into males, but are inviable in diploids. The paternal chromosome
loss may not be complete and give rise to aneuploid zygotes (Ryan et al., 1987).
In haplodiploids, this will lead to zygotic mortality in addition to a male-biased
sex ratio (Breeuwer and Werren, 1993). Thus, male-biased sex ratios and/or
high zygotic mortality are often indications that Wolbachia are involved. Such
incompatibilities have been reported in both spider mites and predatory mites.
Indeed, many mite species that tested positive for Wolbachia in our assay have
also been reported to show incompatibility in crosses between geographic
strains. Although this correlation may be circumstantial, it strongly suggests that
Wolbachia might play a role in earlier observed incompatibilities in these
species.
J. A. J. Breeuwer (unpublished data) showed that Wolbachia in a strain of the
two spotted spider mite T. urticae causes cytoplasmic incompatibility between
430
J . A . J . BREEUWER AND G. JACOBS
uninfected females and infected males. Such crosses produced high numbers of
eggs that did not hatch. The numbers of adult males were normal, but the
numbers of adult females were greatly reduced. This suggests that mortality
occurred only in fertilized eggs, which normally give rise to females and not in
unfertilized eggs, which develop into males. As in insects, Wolbachia induced
cytoplasmic incompatibility probably results in paternal genome loss in spider
egg and subsequent development into a (haploid) male. Probably, paternal
chromosome elimination is not complete and produces aneuploid inviable
embryos as has been reported in the wasp Nasonia (Breeuwer and Werren,
1993).
Gotoh et al. (1995a,b) reported on the unidirectional incompatibility between
northern and southern populations of T. quercivorus in Japan. Gotoh et al.
(1995b) dismissed the possibility of microbial involvement, because antibiotic
and heat treatment of individual mites did not alter the compatibility patterns.
There are several reasons for the fact that these treatments did not alter the
incompatibility. Wolbachia induced unidirectional incompatibility is typically
observed between uninfected females and infected males. Thus, because
incompatibility occurs in the cross between northern Sapporo females and
southern Tsukuba males, the Sapporo strain is expected to be uninfected and the
Tsukuba strain to be infected. Therefore, the antibiotic and heat treatment of the
'uninfected' Sapporo females is not expected to alter their incompatibility with
'infected' Tsukuba males. This is exactly what they found, but this cannot rule
out microbial involvement. Treatment of infected males during development
could, in principle, render them uninfected at the time of mating and compatible
with uninfected females. However, treatment of infected males may not be very
effective, because the compatibility type of the males (their sperm) is probably
determined very early in their development (Breeuwer and Werren, 1993). This
may explain why, after antibiotic treatment, infected Tsukuba males remained
incompatible with uninfected Sapporo females (Gotoh et al., 1995b). In order to
establish microbial involvement, the infected strain is usually treated with
antibiotics (tetracycline or rifampicin) for a few generations. The treated strain
is then crossed to the original infected strain and the incompatible uninfected
strain. If microorganisms are involved, the treated strain is now expected to be
incompatible with the original infected strain, but compatible with the
uninfected strain.
The typical route of Wolbachia transmission is via egg cytoplasm, from
mother to offspring, i.e. vertical transmission. However, recent molecular
phylogenetic studies indicated that horizontal transmission may occur as well,
even between different insect orders (Rousset et al., 1992; Werren and Jaenike,
1995; Werren et al., 1995). In particular, type B Wolbachia appear to have
spread relatively recently to new insect species. The mechanisms of horizontal
transmission are unknown, but Werren et al. (1995) proposed that transmission
may take place within ecological associations between the host and parasitoid
WOLBACHIA: INTRACELLULARMANIPULATORSOF MITEREPRODUCTION
431
insects. Predator--prey relationships, e.g. predatory mites feeding on spider mite
eggs and insects, represent another group of ecological associations that provide
a route for the horizontal transmission of WoIbachia. Although the phylogeny of
mite Wolbachia has yet to be determined, only type/3 Wolbachia have been
found in mites so far. In addition, many mites are parasitic, not only on mites
but also on insects. This provides yet another route for horizontal transfer of
Wolbachia across taxa. Further analysis of ecological and phylogenefic patterns
of associations may provide insights into the mode of horizontal transmission of
Wolbachia and effects on Wolbachia distribution across taxa (Werren and
Jaenike, 1995).
Finally, the apparent widespread occurrence of Wolbachia in mites has
several implications for taxonomic and population biology/genetic research in
mites. The absence of hybrids or embryo mortality in crosses between mites that
are morphologically difficult to distinguish is frequently used to determine
whether or not they belong to the same species. However, Wolbachia-induced
cytoplasmic incompatibility can give similar results. In the latter case, it does
not necessarily indicate that the two populations belong to different taxonomic
groups. Therefore, care should be taken to use crossing experiments as a key for
species identification. Within species, the variation in Wolbachia infection may
greatly affect the population dynamics of its host by influencing the sex ratio,
offspring production and population structure.
NOTE ADDEDIN PROOF
Recently, the phylogeny of Wolbachia from mites was determined. Based on
16S rDNA andfis2 DNA sequences, they cluster together with Wolbachia found
in other arthropods (Johanowicz and Hoy, 1996; Tsagkarakou et al., 1996).
ACKNOWLEDGEMENTS
These results were presented at the Third International Symposium on the
Population Dynamics of Plant-inhabiting Mites, Gilleleje, Denmark, June 1995.
We would like to thank Hans Bolland for maintaining the stocks and identifying
mite species. This research has been funded by the Royal Netherlands Academy
of Arts and Sciences.
REFERENCES
Breeuwer, J.A.J. and Werren, J.H. 1990. Microorganisms associated with chromosome
destruction and reproductive isolation between two insect species. Nature 346: 558--560.
Breeuwer, J.A.J. and Werren, J.H. 1993. Cytoplasmic incompatibility and bacterial density in
Nasonia vitripennis. Genetics 135: 565-574.
Breeuwer,J.A.J., Stouthamer,R., Barns, S.M., Pelletier,D.A.,Weisburg,W.G. and Werren,J.H.
1992. Phylogenyof cytoplasmicincompatibilitymicroorganismsin the parasitoidwasp genus
432
J.A.J. BREEUWERAND G. JACOBS
Nasonia (Hymenoptera: Pteromalidae) based on 16S ribosomal DNA sequences. Insect Mol.
Biol. 1: 25-36.
Callaini, G., Riparbelli, M.G. and Dallai, R. 1994. The distribution of cytoplasmic bacteria in the
early Drosophila embryo is mediated by astral microtubules. J. Cell Sci. 107: 673~582.
Caspari, E. and Watson, G.S. 1959. On the evolutionary importance of cytoplasmic sterility in
mosquitoes. Evolution 13: 568-570.
Croft, B.A. 1970. Comparative studies on four strains of Typhlodromus occidentalis (Acarina:
Phytoseiidae). I. Hybridization and reproductive isolation studies. Ann. Entomol. Soc. Am. 63:
1558--563.
de Boer, R. and Veerman, A. 1983. A case of hybrid inviability in the two-spotted spider mite,
Tetranychus urticae. Entomol. Exp. Appl. 34: 127-128.
de Moraes, G.J., McMurty, J.A. and Denmark, H.A. 1986. A Catalog of the Mite Family
Phytoseiidae: Reference to Taxonomy, Distribution and Habitat, EMBRA-PA, DF.
Dillon, L.S. 1958. Reproductive isolation among certain spider mites of the Tetranychus urticae
complex with preliminary notes. Ann. Entomol. Soc. Am. 51: 441-448.
Giordano, R., O'Neill, S.O. and Robertson, H.M. 1995. Wolbachia infections and the expression
of cytoplasmic incompatibility in Drosophila sechellia and D. mauritiana. Genetics 140:
1307-1317.
Gotoh, T. and Noguchi, O. 1990. Developmental success and reproductive incompatibility among
populations of the European red mite, Panonychus ulmi (Acari: Tetranychidae). Exp. Appl.
Acarol. 10: 157-165.
Gotob, T., Bruin, J., Sabelis, M.W. and Menken, S.B.J. 1993. Host race information in
Tetranychus urticae: genetic differentiation, host plant preference, and mate choice in a tomato
and cucumber strain. Entomol. Exp. Appl. 68: 171-178.
Gotoh, T., Abe, T., Kurihara, A. and Suzuki, M. 1995a. Genetic incompatibility in local
populations of the spider mite, Tetranychus quercivorus Ehara et Gotoh (Acari:
Tetranychidae). Appl. Entomol. Zool. 30: 361-368.
Gotoh, T., Oku, H., Moriya, K. and Odawara, M. 1995b. Nucleus-cytoplasm interactions causing
reproductive incompatibility between populations of Tetranychus quercivorus Ehara et Gotoh
(Acari: Tetranychidae). Heredity 74: 405-414.
Gutierrez, J. and van Zon, A.Q. 1973. A comparative study of several strains of the Tetranychus
neocaledonicus complex and sterilization tests of males by X-rays. Entomol. Exp. Appl. 16,
123-134.
Hastriter, M.W., Kelly, D.J., Chan, T.C., Phang, O.W. and Lewis Jr., G.E., Jr. 1987. Evaluation of
Leptotrombidium (Leptotrombidium)fletcheri (Acari: Trombiculidae) as a potential vector of
Ehrlichia sennetsu. J. Med. Eutomol. 24: 542-546.
Helle, W. and Pieterse, A.H. 1965. Genetic affinities between adjacent populations of spider mites
(Tetranychus urticae Koch). Entomol. Exp. Appl. 8: 305-308.
Hertig, M. 1936. The Rickettsia, Wolbachiapipientis and associated inclusions of the mosquito
Culex pipiens. Parasitology 28: 453--490.
Hess, R.T. and Hoy, M.A. 1982. Microorganisms associated with the spidermite predator
Metaseiulus ( = Typhlodromus) occidentalis: electron microscope observations. J. Invert.
Pathol. 40: 98--106.
Hoffmann, A.A. and Turelli, M. 1988. Unidirectional incompatibility between populations of
Drosophila simulans: inheritance, geographic variation and fitness effects. Genetics 119: 435444.
Hoffmann, A.A. and Turelli, M. 1995. Cytoplasmic incompatibility in Drosophila simulans:
dynamics and parameter estimates from natural populations. Genetics 140: 1319--1338.
Hoffmann, A.A., Claney, D.J. and Merton, E. 1994. Cytoplasmic incompatibility in Australian
populations of Drosophila melanogaster. Genetics 136: 993-999.
Hoffmann, A.A., Turelli, M. and Simmons, G.M.., 1986. Unidirectional incompatibility between
populations of Drosophila simulans. Evolution 40: 692-701.
WOLBACHIA:INTRACELLULARMANIPULATORSOF MITEREPRODUCTION
433
Holden, P.E., Brookfield, J.F. and Jones, P. 1993. Cloning and characterization of an fisZ
homologue from a bacterial symbiont of Drosophila melanogaster. Mol. Gen. Genet. 240:
213--220.
Hoy, M.A. and Cave, F.E. 1988. Premating and postmating isolation among populations of
Metaseiulus occidentaIis (Nesbitt) (Aearina: Phytoseiidae). Hilgardia 56: 1-20.
Johanowicz, D.L. and Hoy, M.A. 1995. Molecular evidence for a Wolbachia endocytobiont in the
predatory mite Metaseiulus occidentalis. J. Cell. Biochem. 21A: 198.
Johanowicz, D.L. and Hoy, M.A. 1996. Wolbachia in a predator-prey system: 16S ribosomal
DNA analysis of two phytoseiids (Acari: Phytoseiidae) and their prey (Acari: Tetranychidae).
Ann. Entomol. Soc. Am. 89: 435-441.
Jost, E. 1970. Untersuchungen zur Kreuzungssterilit~it im Culex pipiens Komplex. Wilhelm Roux
Arch. Entwickl. Org. 166: 173-188.
Krieg, N.R. and Holt, J.H. 1984. Bergey's Manual of Systematic Bacteriology. The Williams and
Wilkins Co., Baltimore.
Laven, H. 1957. Vererbnng durch Kerngene und das Problem der ausserkaryotischen Vererbung
bei Culex pipiens. II. Ausserkaryotische Vererbung. Z. Indukt. Abstamm. Vererbungsl. 88:
478-516.
Legrand, J.J. and Juchault P., 1986. Rfle de bact6ries symbiotiques dans l'intersexualit6, la
monog6nie et la sp6ciation chez Crustac6s Oniscoides. Bull. Zool. 53: 161-172.
Legrand, J.J., Juchault, P., Moraga, D. and Legrand-Hamelin, E. 1986. Microorganismes
symbiotiques et sp6ciation. Bull. Soc. Zool. Fr. 111: 135-147.
O'Neill, S.L. and Karr, T.L. 1990. Bidirectional incompatibility between conspecific populations
of Drosophila simulans. Nature 348: 178-180.
O'Neill, S.L., Giordano, R., Colbert, A.M.E., Karr, T.L. and Robertson, H.M. 1992. 16S rRNA
phylogenetic analysis of the bacterial endosymbionts associated with cytoplasmic incompatibility in insects. Proc. Natl. Acad. Sci. USA 89: 2699--2702.
Osakabe, M. 1993. Divergence of the northern and southwestern populations ofPanonychus mori
(Yokoyama) (Acari: Tetranychidae) in Japan in host range and reproductive compatibility.
Appl. Entomol. Zool. 28: 189--197.
Reed, K. and Werren, J.H. 1995. Induction of paternal genome loss by the paternal sex ratio
chromosome and cytoplasmic incompatibility bacteria (Wolbachia): a comparison of early
embryonic events. Mol. Reproduct. Devel. 40: 408-418.
Richardson, P.M., Holmes, W.P. and Saul, G.B., II. 1987. The effects of tetracycline on
nonreciprocal incompatibility in Mormoniella [ =Nasonia] vitripennis. J. Invert. Pathol. 50:
176--183.
Rigaud, T., Souty-Grosset, C., Raimond, R., Mocquard, J. and Juchault, P. 1991. Feminizing
endocytobiosis in the terrestrial crustacean Armadillidium vulgare Latr. (Isopoda): recent
acquisitions. Endocyto. Cell. Res. 7: 259-273.
Roberts, L.W., Rapmund, G. and Cadigan, F.C., Jr. 1977. Sex ratios in Rickettsia tsutsugamushiinfected and noninfected colonies of Leptotrombidium (Acari: Trombiculidae). J. Med.
Entomol. 14: 89-92.
Rousset, F. and Solignac, M. 1995. Evolution of single and double Wolbachia symbionts during
speciation in the Drosophila simulans complex. Proc. Natl. Acad. Sci. USA. 93: 63896393.
Rousset, F., Raymond, M. and Kjellberg, F. 1991. Cytoplasmic incompatibilities in the mosquito
Culex pipiens: how to explain a cytotype polymorphism? J. Evol. Biol. 4: 69-81.
Rousset, F., Bouchon, D., Pintureau, B., Juchault, P. and Solignac, M., 1992. Wolbachia
endosymbionts responsible for various alterations of sexuality in arthropods. Proc. R. Soc.
Lond. B 250: 91-98.
Ryan, S.L. and Saul, G.B., II. 1968. Post-fertilization effect of incompatibility factors in
Mormoniella. Mol. Gen. Genet. 103: 29-36.
Ryan, S i . , Saul, G.B., II and Conner, G.W. 1987. Separation of factors containing R-locus genes
434
J.A.J. BREEUWERANDG. JACOBS
in Mormoniella stocks derived from aberrant segregation following incompatible crosses. J.
Hered. 78: 273-275.
Saul, G.B., II. 1961. An analysis of non-reciprocal cross incompatibility in Mormoniella
vitripennis (Walker). Z. Vererbungsl. 92: 28-33.
Sologic, H.D. and Rodriguez, J.G. 1971. Microorganisms associated with the two-spotted spider
mite Tetranychus urticae. J. Invert. Pathol. 17: 48-52.
Solignac, M., Vautrin, D. and Rousset, F. 1994. Widespread occurrence of the proteobacteria
Wolbachia an partial cytoplasmic incompatibility in Drosophila melanogaster. Compt. Rend.
Acad. Sci. Paris (Life Sci.) 317: 461-470.
Stevens, L. and Wicklow, D.T. 1992. Multispecies interactions affect cytoplasmic incompatibility
in Tribolium flour beetles. Am. Nat. 140: 642-653.
Stouthamer, R. and Luck, R.F. 1993. Influence of microbe-associated parthenogenesis on the
fecundity of Trichogramma deion and T. pretiosum. Entomol. Exp. Appl. 67: 183-192.
Stouthamer, R., Luck, R.F. and Hamilton, W.D. 1990. Antibiotics cause parthenogenetic
Trichogramma to revert to sex. Proc. Natl. Acad. Sci. USA. 87: 475-581.
Stouthamer, R., Breeuwer, J.A.J., Luck, R.F. and Werren, J.H. 1993. Molecular identification of
microorganisms associated with parthenogenesis. Nature 361: 66-68.
Sut~kov~, G. and Riittgen, F. 1978. Rickettsiella phytoseiuli and virus-like particles in
Phytoseiulus persimilis (Gamasoidea: Phytoseiidae) mites. Acta Virol. 22: 333-336.
Tsagkarakou, A., Guillemaud, T., Rousset, F. and Navajas, M. 1966. Molecular identification of a
Wolbachia endosymbiont in a Tetranychus urticae strain (Acari: Tetranychidae). Insect Mol.
Biol. 5: 217-221.
Turelli, M. 1995. Evolution of incompatibility-inducing microbes and their hosts. Evolution 48:
1500-1513.
Turelli, M. and Hoffmann, A.A. 1991. Rapid spread of an inherited incompatibility factor in
California Drosophila. Nature 353: 440-442.
van Meet, M.M.M., van Kan, F.P.J.M., Breeuwer, J.A.J. and Stouthamer, R. 1995. Identification
of symbionts associated with parthenogenesis in Encarsia formosa (Hymenoptera:
Aphelinidae) and Diplolepis rosae (Hymenoptera: Cynipidae). Proc. Exper. Appl. Entomol.,
NEV 6: 81-86.
Wade, M.J. and Stevens L., 1985. Microorganism mediated reproductive isolation in flour beetles.
Science 227: 527-528.
Wade, M.J. and Chang, N.W. 1995. Increased fertility in Tribolium confusum beetles after
infection with the intracellular parasite Wolbachia. Nature 373: 72-74.
Walsh, P.S., Metzger, D.A. and Higuchi, R. 1991. Chelex 100 as a medium for simple extraction
of DNA for PCR-based typing from forensic material. Biotechniques 10: 506-513.
Weisburg, W.G., Barnes, S.M., Pelletier, D.A. and Lane, D.J. 1991. 16S ribosomal DNA
amplification for phylogenetic study. J. Bacteriol. 173: 697-703.
Werren, J.H. and Jaenike, J. 1995. Wolbachia and cytoplasmic incompatibility in mycophagous
Drosophila and their relatives. Heredity 75: 320-326.
Werren, J.H., Zhang, W. and Guo, L.R. 1995. Evolution and phylogeny of Wolbachia:
reproductive parasites of arthropods. Proc. R. Soc. Lond. B 261: 55-71.
Yen, J.H. and Barr, A.R. 1973. The etiological agent of cytoplasmic incompatibility in Culex
pipiens L. J. Invert. Pathol. 22: 242-250.
Young, S.S.Y., Wrensch, D.L. and Kongchuesin, M. 1985. Geographic variations and combining
abilities in the two-spotted spider mite, Tetranychus urticae. Entomol. Exp. Appl. 39: 109113.