Heredity 81 (1998) 254–260 Received 6 August 1997, accepted 2 January 1998Distribution and reproductive effects of Wolbachia in stalk-eyed flies (Diptera: Diopsidae)
AHMAD R. HARIRI†§, JOHN H. WERREN‡ & GERALD S. WILKINSON*†
†Department of Biology, University of Maryland, College Park, MD 20742, USA and ‡Department of Biology,University of Rochester, Rochester, NY 14627, USAWolbachia are cytoplasmically inherited bacteria capable of altering the reproductive biology oftheir hosts in a manner which increases their spread within a population. These microbes cancause cytoplasmic incompatibility, parthenogenesis and feminization of genetic males. BecauseWolbachia have been associated with female-biased sex ratio distortion, we used a PCR assayto examine 17 species of stalk-eyed flies (Diptera: Diopsidae), two of which exhibit female-biased sex ratios, for the presence of these microbes. Type A Wolbachia was detected in fourdiopsid species, three from the genus Sphyracephala, none of which exhibit biased progeny sexratios. The reproductive effects of the microbe were examined in one of those species, S. bec-carii, by conducting reciprocal crosses between infected and uninfected strains. In this species,Wolbachia do not cause detectable cytoplasmic incompatibility or reduce host fecundity. Incontrast, our results are consistent with an association between the microbes and enhancedmale fertility. Possible explanations for the pattern of distribution and effects on male fertilityinclude a predisposition for acquiring Type A Wolbachia by these flies and accommodation bythe host genome to bacterial presence. Keywords: cytoplasmic bacteria, Diopsidae, Sphyracephala, stalk-eyed flies. Introduction
promotes the spread of the microorganisms. Theeffects of Wolbachia infection include unidirectional
Wolbachia are a monophyletic group of proteobac-
and bidirectional cytoplasmic incompatibility (CI),
teria with two major divisions, Type A and B, which
parthenogenesis, and feminization of genetic males
have been associated with a wide range of repro-
(see Werren, 1997 for review). Wolbachia can have
ductive changes in arthropods (Werren et al.,
either positive effects on host fecundity (Girin &
1995a). They are primarily found within the cells of
Bouletreau, 1995; Stolk & Stouthamer, 1996;
the gonadal tissues of infected individuals (O’Neill,
Poinsot & Mercot, 1997) and fertility (Wade &
1995) and are typically inherited by vertical trans-
Chang, 1995) or negative effects on host fecundity
mission through the maternal cytoplasm (Hoffmann
(Hoffmann et al., 1990). The positive or negative
& Turelli, 1988). Horizontal transmission, especially
consequences of infection may be related to differ-
of Type A Wolbachia, is believed to play an import-
ences in the mode of microbe transmission and
ant role in spreading the infection between species
maintenance in different groups of organisms.
and maintaining phylogenetically similar strains in
Models of symbiont transmission predict that nega-
diverse Orders of arthropods (Werren et al., 1995b).
tive effects on host reproduction will be prevalent in
Wolbachia have not been found extracellularly and
systems where symbiont populations are maintained
their existence and proliferation appear to be inti-
by horizontal transmission, and positive effects will
mately linked with that of their arthropod hosts
be more common when vertical transmission domi-
Wolbachia typically alter the reproductive biology
Recently, female-biased sex ratio distortion has been
of their hosts in a manner which ultimately
reported in two species of stalk-eyed flies (Diptera:Diopsidae), Cyrtodiopsis dalmanni and C. whitei
*Correspondence. E-mail: email@example.com§Present address: Brain Research Institute, UCLA, Los Angeles,
(Burkhardt & De La Motte, 1983), and ascribed to X
chromosome meiotic drive (Presgraves et al., 1997).
1998 The Genetical Society of Great Britain. WOLBACHIA IN STALK-EYED FLIES 255
Because Wolbachia have been associated with
was confirmed by measuring product length from an
female-biased sex ratio distortion, either through
agarose gel. All solutions were filter-sterilized
parthenogenesis in haplodiploids (Stouthamer et al.,
(0.22 m pore diameter) to reduce potential
1993) or feminization in diploids (Rousset et al.,
bacterial contamination. Control DNA samples were
1992), we decided to determine whether or not these
prepared from pupae of known infected and unin-
microbes are present in C. dalmanni and C. whitei.
fected strains of Nasonia vitripennis and compared
In order to determine the potential distribution of
with amplified products from each stalk-eyed fly
Wolbachia infection in stalk-eyed flies an additional
species to determine the presence of Wolbachia.
15 diopsid species from six genera were also
Additional amplifications were performed using
examined. We also report on the reproductive
Type A and Type B specific primers for ftsZ
effects of infection in one of those species, Sphyr-acephala beccarii, which harbours Type A Wolbachia. Materials and methods
Flies were maintained in modified mouse cages with
a 12 L:12D photoperiodic cycle and 30 min simu-
We collected flies from six of the 11 diopsid genera
lated dawn and dusk periods (Lorch et al., 1993).
(Feijen, 1989): Teleopsis, Cyrtodiopsis, Diasemopsis,
Flies were allowed to feed ad libitum on a standard
Diopsis, Sphyracephala and Eurydiopsis. These six
genera contain over 96% of the described species
with a commercial mould inhibitor (Wilkinson,
and occupy the entire geographical range of diopsids
1993). For breeding purposes, :35–40 mL of
(Steyskal, 1972). All Teleopsis species (breviscopium,
medium was presented in small plastic cups and
rubicunda, quadriguttata) and Cyrtodiopsis species
changed biweekly. Newly eclosed flies were isolated
(dalmanni, whitei, quinqueguttata) were collected in
by sex and maintained as virgins until they were
January 1989 in peninsular Malaysia. All Diasemop-
reproductively mature and then utilized in reciprocal
sis (aethiopica, dubia, munroi, silvatica) and Diopsis
(apicalis and fumipennis), as well as Sphyracephala
In order to determine the optimum concentration
munroi, were collected in December 1994 in the
of antibiotic, which would maximize elimination of
Natal Province, South Africa. Sphyracephala brevi-
the infection and minimize the mortality caused by
cornis were collected in October 1994 in Maryland
the toxicity of the antibiotic, flies were bred on
and S. detrahens and Eurydiopsis subnotata in
media treated with 0.5 mg mL1, 1.0 mg mL1, or
January 1996 in peninsular Malaysia.
2.0 mg mL1 concentrations of aqueous tetracycline,
Sphyracephala beccarii were collected on 22
prepared by dissolving tetracycline hydrochloride
November 1993 along a stream near Sudwala Caves,
(Sigma T-3383) in water over low heat. Fecund
40 km west of Nelspruit, Transvaal Province, South
females (3 weeks post eclosion) from these treat-
Africa. Approximately 20 flies were used to establish
ment classes were then tested for the presence of
a laboratory population and were subsequently bred
infection using the PCR assay described above.
in the laboratory for 10 months prior to the start of
These analyses, in addition to the number of pupae
this study. We estimate that the flies used in the first
produced by five pairs of flies from each of the three
antibiotic treatment experiment (see below) were at
tetracycline concentrations, were used to identify the
least three generations removed from those
optimum tetracycline concentration for curing.
These results indicated that the optimum concen-
tration of tetracycline was 1.0 mg mL1. At thisconcentration, the infection was eliminated and the
average daily pupal production was 8.0<1.9 (SE)
The presence of Wolbachia was determined for each
per pair. At 0.5 mg mL1, average daily pupal
species by extracting DNA from pooled ovaries of
production was higher (10.0<1.5), but the infection
two to four fecund females and then using the poly-
was not eliminated. At 2.0 mg mL1, the infection
merase chain reaction (PCR) to amplify a Wolba-
was eliminated, but average daily pupal production
chia-specific bacterial cell-cycle gene, ftsZ, sequence
(Werren & Jaenike, 1995). All samples were
Bacteria were eliminated by allowing 20 females
screened with universal 28S primers as a positive
and 20 males to mate and oviposit ad libitum for
control for amplification ability (see Werren et al.,
48 h on 1.0 mg mL1 tetracycline-treated medium.
1995a, for details). Bacterial ftsZ DNA amplification
Twenty pairs of progeny were then bred to produce
The Genetical Society of Great Britain, Heredity, 81, 254–260. 256 A. R. HARIRI ET AL.
a second generation of flies which had developed in
could potentially influence the reproductive biology
1.0 mg mL1 tetracycline-treated media. Fecund
females obtained after both one and two generations
We explored the potential effects of these factors
of tetracycline exposure were tested with the PCR
assay to determine if there were detectable levels of
(confirmed by PCR) in a subset of treated indi-
Wolbachia. Progeny derived from both levels of
viduals which were then used in reciprocal crosses
tetracycline exposure were then bred on standard,
with individuals that were untreated and known to
tetracycline-free maize medium for three genera-
be infected. A multiway contingency table analysis
tions (20 males and 20 females in each generation)
was then used to determine if there was an associa-
in order to reduce the possibility of decreased fecun-
tion between fertility, presence of Wolbachia and
dity resulting from maternal exposure to tetra-
infection status. If reproductive effects are the result
of any of these factors, then reproductive perform-ance should not change in this second set of crosses. If, however, the effects are associated with Wolba-chia, then the two sets of crosses should exhibit a
After three generations of maintenance on tetra-
cycline-free medium, uninfected individuals fromboth levels of tetracycline exposure were used in
reciprocal crosses with infected individuals. Crosseswere conducted by pairing single males with single
In fertile crosses between infected and uninfected
females. Mating and oviposition on the medium was
individuals, an effect of Wolbachia on egg produc-
allowed to occur for 120 h. For each of the four
tion or development would be indicated by inequal-
possible crosses between infected and uninfected
ities in mean daily pupal production between
individuals, 20 replicates were established for flies
reciprocal crosses. Analysis of variance was used to
derived from one generation of tetracycline expo-
determine if heterogeneity existed between crosses
sure, and 15 replicates for flies derived from two
in pupal production. Fisher’s Paired Least Signifi-
cant Difference test (PLSD) was used to identify
An effect of Wolbachia on fertility would be indi-
significant differences between crosses in pupal
cated by an association between the presence or
absence of pupae and infection. Therefore, a multi-way contingency table analysis was conducted on the
number of fertile and infertile pairs from each treat-ment using
SYSTAT v.5.2 (Wilkinson, 1989). Fertility
(pupae or no pupae) was used as the response vari-
The results of the PCR screen for the Wolbachia
able and the level of antibiotic exposure (one or two
ftsZ gene are presented in Table 1. Four positive
generations), female condition (infected or uninfec-
amplifications were detected. Three species from the
ted), and male condition (infected or uninfected)
genus Sphyracephala and Eurydiopsis subnotata were
were used as explanatory variables in a four-dimen-
found to be positive for Type A Wolbachia, but
sional analysis. The significance of each potential
negative for Type B Wolbachia. Amplification
interaction was determined by testing the difference
products of the appropriate length, 1035–1047
2, calculated by finding the difference between like-
bases, were present for all four species. Neither
lihood-ratio 2 statistics of two models differing in
Type A nor Type B Wolbachia was detected in C.dalmanni or C. whitei; thus, the microbes do not
Reproductive performance between crosses may
contribute to the observed female-biased sex ratio
be influenced by several factors other than Wolba-chia. First, tetracycline-treated media yielded rela-tively few offspring. Therefore, inbreeding may have
occurred for four generations prior to the reciprocalcrosses and could be responsible for fertility differ-
Comparison of the fertility of flies from reciprocal
ences between infected and uninfected individuals.
crosses between infected and uninfected strains
Secondly, the two treated lines may differ genetically
(Table 2) revealed a significant association between
because of stochastic effects of small population size
fertility and the presence of infection in males (diff.
following tetracycline exposure. Thirdly, antibiotic
21 = 11.53, P0.005), with 43% of 70 uninfected
toxicity has an effect on the survival of eggs and
males failing to produce pupae compared to only
The Genetical Society of Great Britain, Heredity, 81, 254–260. WOLBACHIA IN STALK-EYED FLIES 257 Table 1 PCR amplification of Wolbachia ftsZ gene in 17
17% of 70 infected males. No significant association
was detected between fertility and presence of theinfection in females (diff. 21 = 0.55, P0.10) or
fertility and number of generations of tetracyclineexposure (diff. 21 = 3.55, P0.05). Consequently,
results from the two levels of tetracycline exposure
were combined in subsequent analyses of fertility.
After recovery of Wolbachia in a subset of anti-
biotic-treated individuals, the number of infertile
males decreased from 43% to 18% (Table 3). A
multiway contingency table analysis revealed a signi-
ficant association between fertility and the presence
of Wolbachia in males (diff. 21 = 6.66, P0.01), but
not in females (diff. 21 = 4.09, P0.10). This analy-
sis also indicated that the fertility effect observed in
the first set of crosses was not present in the second
set where there was recovery of Wolbachia (diff.
Analysis of variance failed to reveal any significantdifferences (F3,87 = 1.7, P = 0.17) in pupal production
*Positive amplifications are Type A.
between fertile reciprocal crosses (Table 4). However, there was a significant difference
Table 2 Fertility of reciprocal crosses between strains of Sphyracephala beccarii infected and uninfected with Wolbachia Table 3 Comparison of the fertility of reciprocal crosses between strains of Sphyracephala beccarii infected and uninfected with Wolbachia. Wolbachia (ǹ) strains treated with tetracycline had subsequent recovery of Wolbachia Wolbachia (), absent; (ǹ), present. I, infected (not treated with tetracycline);T, treated with tetracycline. Values are totals from Table 2.
The Genetical Society of Great Britain, Heredity, 81, 254–260. 258 A. R. HARIRI ET AL. Table 4 Mean daily pupal production (<SE) per pair for all fertile reciprocal crosses between strains of Sphyracephala beccarii infected and uninfected with Wolbachia
(F1,89 = 5.0, P = 0.03) in mean pupal production
divergence in these species is needed to differentiate
between lines receiving different levels of tetra-
between these two potential routes of infection.
cycline exposure. Mean daily pupal production per
Our results did not reveal any differences in pupal
pair was 11.3<1.3 (SE) in lines with one generation
production between fertile crosses. Specifically,
of tetracycline exposure and 8.0<0.5 in lines with
fertile crosses between uninfected females and
two generations of exposure. There was no inter-
infected males, which have significantly lower fecun-
action between cross type and level of tetracycline
dity in systems exhibiting CI, did not produce signifi-
exposure for pupal production (F1,3 = 0.8, P = 0.47).
cantly different levels of pupae from all other
PCR analysis of uninfected females used in all
crosses. Thus, we can conclude that Wolbachia in
crosses confirmed that these treated strains were
this species do not cause CI or that the level of CI is
sufficiently weak not to be detected by this assay. The absence of CI in this system is not surprising. For example, although some Wolbachia-infected
populations of Drosophila simulans exhibit CI, others
The pattern of Wolbachia infection in the 17 species
do not (Hoffmann et al., 1996).
of stalk-eyed flies screened, with three of the four
The observed reduction in fecundity in lines
positive amplifications occurring in the genus Sphyr-
receiving two generations of tetracycline exposure in
acephala, suggests the existence of a unique relation-
comparison to those receiving only one, is most
ship between the microbe and these species.
likely an artifact of the crosses being conducted at
Assuming the overall probability of infection in
two different times. Slight environmental differ-
diopsids is 23% (4/17), the probability that at least
ences, such as media quality or laboratory tempera-
three of four Sphyracephala harbour the infection by
ture, could translate into significant effects on both
chance is only 0.04. There are two possible scenarios
frequency of mating and survival of offspring.
which can explain this nonrandom pattern of infec-
Interestingly, the results of our crosses are consist-
tion. First, the Wolbachia may have been acquired
ent with an association between the microbes and
prior to the divergence of the Sphyracephala genus
enhanced male fertility. Approximately 43% of unin-
and then subsequently lost in S. detrahens. Secondly,
fected males exhibit infertility compared to only
given the high rate of horizontal transmission of
17% of infected males. This pattern is also evident
Type A Wolbachia, the flies in this genus may have a
in treated flies that did not lose Wolbachia compared
predisposition for horizontal acquisition of the
to those that did. Positive effects of Wolbachia on
microbe and have been infected independently of
host productivity have been reported for Trichog-
each other. Although flies in the genus Sphyrace-ramma bourarachae (Girin & Bouletreau, 1995),
phala can be found on several continents, all of the
Tribolium confusum (Wade & Chang, 1995), Nasonia
infected species are commonly found in dense aggre-
vitripennis (Stolk & Stouthamer, 1996) and D. simu-
gations near streams (Feijen, 1989). Possible sources
lans (Poinsot & Mercot, 1997).
of infection include predatory mites (Johanowicz &
The male fertility effect we found in S. beccarii is
Hoy, 1995) and parasitoid wasps (Feijen & Schulten,
unlikely to be caused by inbreeding depression, line
1981a,b). Determination of Wolbachia ftsZ sequence
divergence or antibiotic toxicity. A multiway contin-
The Genetical Society of Great Britain, Heredity, 81, 254–260. WOLBACHIA IN STALK-EYED FLIES 259
gency analysis revealed a significant association
between fertility and the presence of Wolbachia in
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