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56(10), 2002, pp. 1976–1981
INTERACTIONS THAT DETERMINE
Department of Ecology and Evolutionary Biology, Brown University, Box G-W, Providence, Rhode Island 02912
We have recently described a mutualistic symbiosis in which Wolbachia
bacteria were shown to improve
the fitness of some Drosophila melanogaster
did not extend longevity in all Drosophila
genotypes,even though 16s rDNA sequences indicated that our Drosophila
stocks were infected with the same Wolbachia
Here, we use reciprocal hybrid crosses between two Drosophila
strains, one that lived longer with Wolbachia
(Z53)and one that did not (Z2), to investigate the inheritance of the survival phenotype and its dependence on the hostgenotype, sex, and mating conditions. Wolbachia
’s positive effects were more apparent in hybrid flies than in parentalflies, ruling out exclusive maternal inheritance or the dependence of the survival phenotype on Wolbachia
straindifferences. The Wolbachia
survival effects were more apparent in single-sex cages, where courtship and mating werenot permitted. In these cages, nearly all flies with Wolbachia
lived longer than uninfected flies, even though strainZ2 showed no Wolbachia
effect in mixed-sex mating cages. We used comparisons between single- and mixed-sexcages to estimate the cost of reproduction for both sexes. Our data suggest that Wolbachia
infection may increase theinferred cost of reproduction, particularly in males. Wolbachia
can even produce a positive survival effect almost aslarge as the negative survival effect associated with reproduction. We discuss the implications of our experiments forthe study of insect symbioses.
Cost of reproduction, hybrid, mortality, survival, symbiosis.
is in transition from parasitic to
thought to infect millions of insect species (Werren et al.
mutualistic (A. J. Fry, M. R. Palmer, and D. M. Rand, unpubl.
1995a; Jeyaprakash and Hoy 2000; Werren and Windsor
ms.). To better understand these survival effects, we used
2000; Jiggins et al. 2001a). Their widespread distribution
reciprocal hybrid crosses between two Drosophila
among insects makes them one of the most common infec-
one that lived longer with Wolbachia
(Z53) and one strain
tious microorganisms. These bacteria inhabit the reproductive
(Z2) that did not show a significant Wolbachia
tissues of their hosts (Dobson et al. 1999), where they induce
design allowed us to compare the survival effects of the Wol-
a number of reproductive modifications intended to enhance
strains found in Z53 and Z2 as each hybrid had a
their transmission through females. Their effects include the
strain. We also investigated Wolbachia
induction of parthenogenesis (Stouthamer et al. 1993), fem-
effects in both inbred (Z53 and Z2) and outbred (hybrid)
inization of genetic males (Rousset et al. 1992), male-killing
genotypes and we manipulated the mating conditions to un-
(Jiggins et al. 1998; Hurst et al. 1999), and cytoplasmic in-
’s interaction with reproduction. This was
compatibility (CI; Caspari and Watson 1959; Yen and Barr
done because courtship and mating are known to affect Dro-
1971; Fine 1978; Hoffmann et al. 1986, 1990). Wolbachia
survival (e.g., Fowler and Partridge 1989; Partridge
does not induce strong reproductive modifications in Dro-
and Fowler 1990; Chapman et al. 1995) and could interact
, and this has led to the suggestion that
’s effects on survival.
may benefit D. melanogaster
fitness (Hoffmann etal. 1994, 1998; Solignac et al. 1994). Attempts to identify
these benefits revealed that, in the field, Wolbachia
couldimprove survival, but the positive survival effects depended
on the population background and the location of the field
The Z53 and Z2 D. melanogaster
stocks were originally
site (Olsen et al. 2001). In the laboratory, we found that
collected in Zimbabwe, Africa. These laboratory strains were
could significantly improve both survival and fe-
started as isofemale lines, have been in laboratory culture for
male fecundity, and these effects depended on the host ge-
several hundred generations, and have been maintained at
notype (A. J. Fry, M. R. Palmer, and D. M. Rand, unpubl.
moderate population size (100–200 pairs) in the laboratory
ms.). DNA sequences from 16s rDNA (O’Neill et al. 1992)
of D. M. Rand. They also carry the endosymbiotic bacterium
and the fts
Z cell cycle gene (Holden et al. 1993; Werren et
. Marc Tatar (Brown University) provided Ri-
al. 1995b) suggested that our Wolbachia
strains were iden-
RedE, an uninfected control strain. We created paired Wol-
tical. However, recent reports of recombination between dif-
-infected (W) and tetracycline-treated (T) experimen-
strains preclude exact strain identification
tal lines with a standard protocol (Hoffmann et al. 1994;
unless a large number of relevant loci are compared (Werren
Poinsot and Mercot 1997). The uninfected fly lines are called
et al. 1995b; Werren and Bartos 2001; Jiggins et al. 2001b).
(T) because they were treated with the antibiotic tetracycline
The positive fitness effects we found could have important
for two generations to remove Wolbachia
(0.25 mg/ml tet-
implications for the maintenance of Wolbachia
racycline in water added to Carolina dry food in a 1:1 mix).
and may indicate that the symbiosis between
-infected (W) lines received identical food and
᭧ 2002 The Society for the Study of Evolution. All rights reserved.
TABLE 1. Proportional hazards analysis of females from fly strains
TABLE 2. Risk ratios of Wolbachia
-infected (W) to tetracycline-treat-
Z53, Z2, and their hybrids. Mating environments were mixed- or sin-
ed (T ) lines from two different mating environments, mixed- and sin-
refers to Wolbachia
-infected or tetracycline-treated
gle-sex, left columns. Ratios greater than one indicate W lived longer
flies. Likelihood-ratio (LR) chi-squared values and associated proba-
than T and measure Wolbachia
’s survival benefits. The two right col-
umns are risk ratios of survival in single-sex to mixed-sex cages fordifferent infection states (T or W) and are a rough measure of the
benefits to survival obtained by not reproducing. W and T or mixed-and single-sex cages were compared with log-rank tests and signifi-
Ͻ 0.001) is indicated by an asterisk.
environment, except tetracycline was not added. The infec-
tion status of all lines was confirmed with Wolbachia
16S rRNA polymerase chain reaction primers (O’Neill et al.
1992). To insure that differences between T and W lines were
not due to tetracycline treatment, we used two controls. First,
we treated an uninfected fly strain, RiRedE, with tetracyclineto see if treatment affected survival. Second, we held ex-perimental flies for two generations on standard corn-meal
together in one cage. Single-sex environments were con-
laboratory food after treatment, but before the experimental
structed with 100 virgin flies of only one sex per cage. There
crosses, to minimize maternal effects of treatment.
were three replicate cages for each cross, sex, and mating
Twenty-five pairs of parental flies from the T and W lines
environment combination. Our treatment control, RiRedE,
were placed into food bottles and held until approximately
was reared in the mixed-sex environment only.
120–150 eggs were deposited. We controlled egg density
In total, we monitored 78 demography cages and scored
because W females from Z53 can lay more eggs than T fe-
the survival of more than 10,000 flies. Statistical analyses
males (A. J. Fry, M. R. Palmer, and D. M. Rand, unpubl.
were performed using the JMP statistical package (SAS In-
ms.), which could influence larval density and in turn affect
stitute 1995) and a semiparametric proportional hazards sta-
development time (Gonzalez-Candelas et al.
tistical model (Cox 1972). In our model, the dependent var-
1990), survival (Buck et al. 1993), and Wolbachia
iable was time of death measured to the nearest 48 h, with
levels (Hoffmann et al. 1998). Eggs were allowed to develop
host genotype (four crosses), infection status (T or W), mat-
and virgin adults collected and paired into one of four cross
ing environment (mixed- or single-sex), and their interactions
types: two hybrid crosses, Z53 male ϫ Z2 female and Z2
male ϫ Z53 female, and two parental crosses, Z53 ϫ Z53and Z2 ϫ Z2. To avoid confounding effects of cytoplasmic
incompatibility on survival, all crosses were made with flies
The survival of female flies from Z53, Z2, and their hybrids
of the same infection status; that is, we did not cross T and
depended on interactions between Wolbachia
’s effects on sur-
W flies. Twenty-five pairs of adults from each cross were
vival, the host genotype, and whether courtship and mating
placed into food bottles until 120–150 eggs were deposited.
occurred (Table 1). A proportional hazards analysis of male
Virgin F1 adults from these crosses were collected over a
survival was nearly identical except the three-way interaction
24-h period and used to initiate demography cages.
was not significant (likelihood-ratio 2 ϭ 5.82, df ϭ 3, P
ϭ0.12). Due to the large number of pairwise comparisons be-
tween T and W flies, we show only some of the more im-
The demography cages were constructed from quart-serv-
portant comparisons. The complete dataset is summarized in
ing plastic containers with a screened lid; a side coupling the
same dimension as a standard food vial; and a double-walled,
The survival curves from the four crosses were signifi-
rubber side entrance, made from bicycle inner tube. The de-
cantly heterogeneous for females and males (Fig. 1), regard-
mography cages were kept in a walk-in incubator on a 12L:
less of Wolbachia
infection status, in both mixed- and single-
12D photoperiod at 25ЊC and 40% relative humidity. Fresh
sex cages. Hybrid flies survived longer than flies from either
food vials we added to the cages every other day, when dead
parental strain (Fig. 1). Wolbachia
had a pronounced survival
flies were removed with an aspirator, sexed, and counted.
effect in hybrid flies and flies reared in the single-sex cages
Because courtship and mating can affect Drosophila
(Table 2). We found that Wolbachia
affected the survival of
and could interact with Wolbachia
’s effects on survival
hybrid flies more than parental flies. Hybrids that carry Wol-
(Fowler and Partridge 1989; Partridge and Fowler 1990;
survived significantly longer than T hybrids, even
Chapman et al. 1995), we scored survival in two different
though all hybrids had at least one parent (Z2) that did not
mating environments. Mixed-sex environments were con-
show a significant Wolbachia
survival effect (Fig. 2). The
structed with 100 virgin F1 males and 100 virgin females
exception involves male hybrids with a Z2 father. Here, the
Survival curves from females (A) and males (B) from
parental crosses and hybrids. Data are from Wolbachia
-infected (W)flies reared in mixed-sex mating cages. Symbols: Z53 ϫ Z53, filledtriangle; Z53 ϫ Z2, square; Z2 ϫ Z53, inverted triangle; Z2 ϫ Z2,diamond. (A) Females are significantly heterogeneous by log-rank(LR) test (2 ϭ 254, df ϭ 3, P
Ͻ 10Ϫ3), (B) as are males (LR 2
ϭ 270, df ϭ 3, P
survival curves for T and W flies were not different in mixed-sex cages (Table 2). However, in single-sex cages, W flieslived longer than T flies, even if W and T flies were notdifferent in the mixed-sex cages as was the case for Z53males and both sexes of Z2 (Fig. 3). There were no significant
Comparison of survival curves from Wolbachia
survival effects associated with tetracycline (T) treatment in
(W, closed circles) and tetracycline-treated (T, open circles) flies
our treatment control, RiRedE (P
Ͼ 0.05 for both sexes by
from four genotypes. Data are from females reared in mixed-sex
log-rank test), indicating that tetracycline treatment was not
cages. (A) LR 2 ϭ 52.28, df ϭ 1, P
Ͻ 10Ϫ3; (B) LR 2 ϭ 0.05,
directly responsible for the survival effects we observed.
ϭ 0.89; (C) LR 2 ϭ 69.96, P
Ͻ 10Ϫ3; (D) LR 2 ϭ 15.59, P
survival depended on strong interactions be-
infection, host genotype, and the matingconditions experienced by both male and female flies (Table1). The parental strains Z53 and Z2 have been in laboratoryculture for several hundred generations, they are inbred andhave probably accumulated mutations affecting survival. Notsurprisingly, hybrid flies were longer-lived than their parentsin both mixed- (Fig. 1) and single-sex mating environments.
With one exception, the hybrids also showed a significantpositive Wolbachia
effect, even though each hybrid had atleast one parent (Z2) that did not. The presence of a Wol-bachia
effect in nearly all hybrids suggests that there maybe a relationship between inbreeding and the expression ofthe Wolbachia
survival effect. Other Wolbachia
phenotypiceffects such as the expression of cytoplasmic incompatibilityand the fidelity of maternal transmission are more pronouncedin laboratory stocks than in field populations (Hoffmann etal. 1990; Turelli and Hoffmann 1995). Drosophila
fitness canbe adversely affected by inbreeding (e.g., Miller et al. 1993;Fry et al. 1998; Aspi 2000). As Wolbachia
infection spreadsthroughout a host population, genetic variation at other cy-toplasmically inherited molecular markers can be reduced,such as mitochondrial DNA variation in D. simulans
(e.g.,Ballard 2000). Thus, the interaction between Wolbachia
andinbreeding could be important for predicting the equilibrium
Effects of Wolbachia
in mixed- and single-sex mating cag-
es. Comparisons between Wolbachia
-infected (W, closed circles)
frequency and spread of Wolbachia
infection in natural pop-
and tetracycline-treated (T, open circles) flies. (A) LR 2 ϭ 3.61,
df ϭ 1, P
ϭ 0.05; (B) LR 2 ϭ 35.40, df ϭ 1, P
Ͻ 10Ϫ3; (C) LR
There is little evidence to suggest that the positive survival
2 ϭ 0.05, df ϭ 1, P
ϭ 0.89; (D) LR 2 ϭ 48.83, df ϭ 1, P
effects associated with Wolbachia
are uniparentally inherited.
TABLE 3. Mortality model analysis from Z53, Z2, and their hybrids
TABLE 4. Mortality model analysis from Z53, Z2, and their hybrids
from the mixed-sex mating cages. See Discussion for explanation of
from the single-sex mating cages. See Discussion for explanation of
models and symbols. Values give the proportional contribution of that
models and symbols. Values give the proportional contribution of that
parameter to the total difference in mortality between tetracycline-
parameter to the total difference in mortality between tetracycline-
treated (T ) and Wolbachia
-infected (W) flies.
treated (T ) and Wolbachia
-infected (W) flies.
However, more crosses will be needed to determine the exactinheritance pattern. We reported previously that 16S rRNAsequences from our Wolbachia
strains were identical, whichimplicated the host genome in the differential expression of
can decrease sperm production in D. simulans
survival effects (A. J. Fry, M. R. Palmer,
It is unclear whether a similar mechanism increases the cost
and D. M. Rand, unpubl. ms.). However, we note that recent
of reproduction in our W males. Our data reject the notion
reports of recombination between some Wolbachia
’s positive survival effects are achieved by
(Jiggins et al. 2001b; Werren and Bartos 2001) make it nec-
decreasing the cost of reproduction. This is consistent with
essary to obtain sequences from a large number of Wolbachia
our earlier result that found Wolbachia
could improve sur-
loci to determine if the strains are identical by descent. With
vival without decreasing female fecundity. In fact, Wolbachia
genomes currently being sequenced
improved both survival and fecundity of female flies (A. J.
(e.g., Slatko et al. 1999), it should soon be possible to de-
Fry, M. R. Palmer, and D. M. Rand, unpubl. ms.). The final
termine the extent to which different Wolbachia
point to make about Table 2 is that by comparing Wolbachia
host genomes interact to produce the various Wolbachia
effects on survival with the inferred cost of reproduction, we
find that Wolbachia
’s positive effects can be substantial. For
survival effects in our experiment showed
example, the positive effect of Wolbachia
(25%) is close to
a strong dependence on the mating cage. W flies reared in
half the inferred cost of reproduction (58%) in Z53 females
single-sex cages lived longer than T flies, even if T and W
(Table 2). We suggest that Wolbachia
effects of this mag-
flies were the same in the mixed-sex cages. The benefits of
nitude are probably important determinants of life-history
in the two environments can be compared using
evolution in chronically infected host populations. Whether
risk ratios (Table 2). For example, neither females nor males
these positive and negative survival effects might be offset-
from Z2 show a significant Wolbachia
effect in the mixed-
ting in natural populations is uncertain and requires addi-
sex environment (1.01 and 1.06 risk ratios, respectively). In
the single-sex environment, however, there is a 21% and 19%
To understand how Wolbachia
affects survival, we used
benefit to Wolbachia
infection. Aside from Z2 and Z53 males,
maximum-likelihood analyses (Pletcher 1999) to compare es-
however, the effect of Wolbachia
on survival is about the
timated mortality models from W and T flies (Tables 3, 4).
same in the mixed- and single-sex cages. For example, in
In most cases, similar models described T and W mortality
Z53 females, Wolbachia
’s positive effect is about 25% under
curves from the same genotype and sex. The inferred models
were either two-parameter Gompertz models, which describe
The right two columns of Table 2 compare fly survival in
a simple exponential increase in mortality rate with age, or
mixed- and single-sex cages. If we consider these risk ratios
Gompertz-Makeham models (Vaupel and Yashin 1985) that
as a very rough index of the cost of reproduction, we can
include an additional parameter (C
) to describe age-indepen-
’s effects on fly survival with the effects
dent mortality. The proportional contribution of each param-
of courtship, mating, and egg laying on fly survival. Inspec-
eter to the total difference in mortality was determined using
tion of these ratios indicates several things. First, nearly all
the method of Pletcher et al. (2000). The results are shown
of the flies experience a significant decrease in survival when
in Table 3 for mixed-sex mating cages and Table 4 for single-
courtship and mating occur. This has been well documented
sex cages. Our results indicate that Wolbachia
and presumably represents a physiological cost
contribute to age-independent mortality. For example, 22.6%
to reproduction (e.g., Fowler and Partridge 1989; Partridge
of the total difference in mortality between T and W flies for
and Fowler 1990; Chapman et al. 1995). Second, there is a
Z53 females is due to an age-independent (C
) mortality con-
trend suggesting that Wolbachia
infections can contribute to
tribution. More commonly, though, Wolbachia
the cost of reproduction. This cost appears much greater for
the rate of aging (slope, ␤), and the initial mortality rate
the parental Z53 and Z2 males than for any female. Again,
(intercept, ␣). Our data suggest that Wolbachia
can affect the
this effect could be related to the level of inbreeding in pa-
mortality schedules of flies in ways that are difficult to pre-
rental males because hybrid males show no cost of repro-
dict. Clearly, Wolbachia
but must be controlled in experi-
duction, infected or not (Table 2). Snook et al. (2000) showed
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Dionicio Rhodes Siegel The University of Texas at Austin Department of Chemistry and Biochemistry Fax: (512) 471-0397 Norman Hackerman Building, 5.132 Education 2003 B.A. Chemistry Reed College, Portland, OR Advisors: Arthur Glasfeld and Patrick McDougal Activities and Awards 2013 College of Natural Sciences Outreach Excellence Award Camille and Henry Dreyfus Special
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