56(7), 2002, pp. 1331–1339
WITHIN- AND BETWEEN-POPULATION VARIATION FOR WOLBACHIA
REPRODUCTIVE INCOMPATIBILITY IN A HAPLODIPLOID MITE
F. VALA,1,2 A. WEEKS,3 D. CLAESSEN,4 J. A. J. BREEUWER,5 AND M. W. SABELIS6
Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, P.O. Box 94084,
1090 GB Amsterdam, The Netherlands
is a bacterium that induces cytoplasmic incompatibility (CI), the phenomenon in which
infected males are reproductively incompatible with uninfected females. CI spreads in a population of hosts becauseit reduces the fitness of uninfected females relative to infected females. CI encompasses two steps: modification (mod)of sperm of infected males and rescuing (resc) of these chromosomes by Wolbachia
in the egg. Infections associatedwith CI have mod1resc1 phenotypes. However, mod2resc1 phenotypes also exist; these do not result in CI. Assumingmod/resc phenotypes are properties of the symbiont, theory predicts that mod2resc1 infections can only spread in ahost population where a mod1resc1 infection already occurs. A mod2resc1 infection spreads if the cost it imposes onthe infected females is lower than the cost inflicted by the resident (mod1resc1) infection. Furthermore, introductionof a mod2 Wolbachia
eventually drives infection to extinction. The uninfected population that results can be recolonizedby a CI-causing Wolbachia
. Here, we investigated whether variability for induction of CI was present in two Tetranychusurticae
populations. In one population all isofemale lines tested were mod2. In the other, mod1resc1 and mod2resc1isofemale lines coexisted. We found no evidence for a cost difference to females expressing either type (mod1/2).
Infections in the two populations could not be distinguished based on sequences of two Wolbachia
genes. We considerthe possibility that mod2 is a host effect through a population dynamics model. A mod2 host allele leads to infectionextinction in the absence of fecundity differences. Furthermore, the uninfected population that results is immune toreestablishment of the (same) CI-causing Wolbachia
Cytoplasmic incompatibility, modification and rescue, resistance, spider mite, theoretical model, Wol-
Received August 13, 2001.
Accepted March 11, 2002.
is a cytoplasmically transmitted bac-
so haplodized eggs develop as males. Depending on the de-
terium that infects several arthropod and nematode hosts. In
gree of aneuploidy, eggs may: develop as a male, if eggs
the two-spotted spider mite, Tetranychus urticae
Koch, a phy-
revert to the haploid state; die, if haplodization is not com-
tophagous haplodiploid arthropod, Wolbachia
plete but insufficient to develop as a female; or develop as
both cytoplasmic incompatibility (CI; Breeuwer 1997; Vala
an aneuploid female (see Breeuwer 1997; Vala et al. 2000).
et al. 2000) and hybrid breakdown (Vala et al. 2000).
Mortality of aneuploid embryos would explain why, in hap-
CI is expressed in crosses between uninfected (U) females
lodiploids, CI is expressed as a bias of F1 sex ratio toward
and infected (W) males (reviewed by Hoffmann and Turelli
males associated with an increase in mortality (cf. Breeuwer
1997; Stouthamer et al. 1999). CI is not induced if the same
1997; Vala et al. 2000; Vavre et al. 2000).
strain that was present in the male is also presentin the fertilized egg (i.e., /W 3 ?W). CI reduces the fitness
The Modification and Rescuing Model
of uninfected females (which are incompatible with W males)relative to infected females (which are compatible with both
Although the molecular details of CI are not known, the
W and U males). As a result, the frequency of infected fe-
phenomenon is thought to involve a Wolbachia
males (and the CI trait) increases in the host population.
in and antitoxin (Hurst 1991; Rousset and Raymond 1991)
Cytological studies of CI in Nasonia
wasps (Reed and Wer-
or, similarly, Wolbachia
-mediated modification and rescuing
ren 1995) and Drosophila simulans
(Callaini et al. 1997)
steps (Hoffmann and Turelli 1997; Werren 1997). Chromo-
showed that in /U 3 ?W crosses the paternal set of chro-
somes from infected males are modified by Wolbachia
mosomes does not segregate properly in mitotic divisions
become unable to respond properly to cell-cycle cues in un-
early in embryonic development. This results in haploid or
infected eggs. If the fertilized egg is infected with the same
aneuploid embryos. In diploid species haploid and aneuploid
bacterial strain that was present in the father, paternal chro-
embryos abort, thus CI is expressed as increased F1 mortality.
mosomes are rescued, that is, they segregate properly during
In haplodiploids, females are diploid and males are haploid,
An infection in which Wolbachia
induces CI is denoted
2 Present address: Department of Biology, University College
mod1resc1 (Werren 1997). However, if CI is not induced,
London, Wolfson House, 4 Stephenson Way, London NW1 2HE,
the infection phenotype may be either mod2resc2, an infec-
tion that can neither modify sperm nor rescue modified chro-
3 Present address: Department of Entomology, University of Cal-
ifornia, Riverside, California 92521; E-mail: [email protected]
mosomes, or mod2resc1, an infection that cannot modify
sperm but can rescue modified chromosomes. The test to
q 2002 The Society for the Study of Evolution. All rights reserved.
F. VALA ET AL.
distinguish between these alternatives is to mate females har-
and PCR were as in Breeuwer (1997). The R and C strains
boring a mod2resc? infection to males with a mod1resc1 Wol-
are the same as those described in Vala et al. (2000).
(Bourtzis et al. 1998; Merc¸ot and Poinsot 1998a). IfCI is observed, then the infection (in the female) is
mod2resc2. Of course, this test is valid only if females andmales are reproductively compatible in the absence of Wol-
Isofemale lines of the two strains were created by taking
. Note that this test does not indicate whether mod2
virgin females from the infected base populations and per-
is a property of the symbiont or of the host.
forming mother 3 son matings for four consecutive gener-
Mod2 is typically assumed to be a property of the sym-
ations; for arrenotokous haplodiploid organisms this gives
biont. Theory predicts that in a population where a mod1resc1
an expected inbreeding coefficient of 0.98 (Hartl and Clark
is fixed, a mod2resc1 infection increases when
1997). From each inbred isofemale line an uninfected coun-
rare, if it inflicts a cost to the infected female that is lower
terpart was created either by tetracycline curing as described
than the cost imposed by the resident type of infection (Prout
by Breeuwer (1997) or by heat treatment as described by Van
1994; Turelli 1994; Hurst and McVean 1996). However, if
Opijnen and Breeuwer (1999; whatever method worked first).
there are uninfected individuals in the population (e.g., be-
For tetracycline treatment, 20–30 females were placed on
cause not all progeny of an infected mother is infected),
arenas and fed an antibiotic solution (described by Breeuwer
mod2resc1 infections cannot spread or persist without the
1997). For heat treatment, 20–30 females were placed at 328C
mod1resc1 type (Hurst and McVean 1996). Therefore,
(Van Opijnen and Breeuwer 1999). Mites were reared as a
mod2resc1 phenotype is expected to coexist with a mod1resc1
culture at this temperature for eight to nine generations to
maximize the number of uninfected females at the end of
The mod2resc1 Wolbachia
type (initially) increases in fre-
treatment. Isofemale lines cured by tetracycline are labeled
quency within the infected subpopulation because it reduces
TET, and isofemale lines cured by heat treatment are labeled
host fecundity less than the resident mod1resc1 Wolbachia
HT. For one isofemale line (R3), two uninfected sublines
However, mod2resc1 Wolbachia
rely on the sterilizing effect
were established, one by curing with tetracycline the other
of the mod1resc1 infection to reduce the fitness of uninfectedindividuals. As mod2resc1 Wolbachia
increase within the in-
by curing with heat treatment. The TET and HT R3 sublines
fected subpopulation, the mod1resc1 phenotype decreases in
were compared to control for treatment effects. To establish
frequency and, consequently, the frequency of uninfected
the uninfected sublines, 10–15 mated females per isofemale
hosts increases. Thus, spread of a mod2 Wolbachia
line were placed alone on leaf discs to oviposit for three days
leads to infection extinction (Hurst and McVean 1996). Both
and subsequently collected for PCR. For each isofemale line,
mod2 and mod1 infection types disappear, and the population
offspring from females that did not give amplification prod-
returns to the uninfected state. Because potentially any un-
ucts on a PCR with Wolbachia
-specific primers were kept.
infected population may be (re)colonized by a CI-causing
Offspring from females positive in the PCR was discarded.
and again revert to the uninfected state, in a sense,
This process was repeated twice. Finally, F3 females were
reversible or cyclic evolution is possible (Hurst and McVean
pooled to establish the uninfected sublines. PCR assays with
-specific primers and DNA isolation were as in
Mod2resc1 infection phenotypes have been described in
(Bourtzis et al. 1998; Merc¸ot and Poinsot 1998a).
Because lines were inbred prior to curing and thus are
However, it was not investigated whether these mod2 infec-
expected to be nearly homozygous, differences between the
tions coexist with mod1resc1 infections as predicted by the-
infected and uninfected sublines of each isofemale line are
ory. In the present study we investigate whether variability
most likely due to presence or absence of Wolbachia
for mod and resc phenotypes is present between and/or within
suming nuclear genetic variation in the base population, dif-
two spider mite populations.
ferences between isofemale lines are likely to be due to ge-netic differences at the nuclear level (the genetic similarity
MATERIALS AND METHODS
in the two populations is discussed below).
To detect variation in Wolbachia
-induced reproductive in-
compatibility, several isofemale lines from each population
Two populations of T. urticae
spider mites were established
were tested for CI. The test for aneuploidy of F
in the laboratory, one from mites collected from rose plants
hybrid breakdown test, is presented in a separate paper. To
(the R strain, hereafter) in a greenhouse at Aalsmeer, The
test for CI, all possible crosses between infected (W) and
Netherlands and another from mites collected from cucumberplants (the C strain, hereafter) obtained from the Institute for
uninfected (U) individuals were performed (/ 3 ?: W 3
Horticultural Plant Breeding in Wageningen, The Nether-
W, W 3 U, U 3 U, U 3 W). In haplodiploids, CI may result
lands. Since collection, spider mites have been reared on
in a male bias of F1 sex ratio associated with an increase in
detached leaves of Phaseolus vulgaris
‘Arena'. Cultures were
mortality in /U 3 ?W crosses compared to U 3 U crosses.
maintained, and experiments were performed in one climate
is present in the female and/or absent in the
room, at 238C, 60–80% relative humidity, and 16L:8D pho-
male (thus, / 3 ?: W 3 W and W 3 U), crosses should be
toperiod. Both strains were infected with Wolbachia
on a polymerase chain reaction (PCR) assay with Wolbachia
mod2resc1, crosses were performed between mod2resc? fe-
specific primers (Breeuwer and Jacobs 1996). DNA isolation
males and mod1resc1 males of another isofemale line.
Procedures for All Experiments
1262R, which amplify 730 base pairs (bp) of the cell-division gene (Holden et al. 1993), and the primer pairs
Twenty-five to 30 females of each line laid eggs on de-
81F and wsp
619R (Zhou et al. 1998), which amplify 590–
tached bean leaves (P. vulgaris
) placed on water-soaked cot-ton balls. These females were transferred at three-day inter-
632 bp of the wsp
gene, were used in separate PCR ampli-
vals to produce age cohorts. Offspring from these cohorts
fications. PCR reaction mixes and amplification conditions
were used in the experiments to ensure that all mites tested
were the same as described in Weeks and Breeuwer (2001).
were of the same age. All experiments were performed on
PCR products were then cleaned using Genecleant (BIO 101,
bean leaf discs (1.5 cm in diameter). Leaf discs were placed
Inc., Bingham, Nottingham, U.K.) and cloned into a pGEMt-
on water-soaked cotton sheets stretched on sponges. Sponges
T vector (Promega, Madison, WI). We extracted five vectors
were placed on plastic trays, and water was added regularly
from recombinant colonies for each gene from each strain
to prevent the leaf discs from drying. In all experiments,
using the alkaline-lysis method (Sambrook et al. 1989). After
crosses and spider mite lines were randomized across spong-
extraction, 1 mg of vector DNA was used as template for a
cycle sequencing reaction (Thermosequenase kit, Amersham/
For F1 analysis (test for CI), experimental females were
Pharmacia, Piscataway, NJ) using fluorescent-labeled primer
collected at the last molting stage from age cohorts (to ensure
(IRD 700/800, Biolegio, Malden, The Netherlands) and sub-
they were virgin) and placed in groups of five females and
sequently run on an NEN Global IR2 DNA analyzer (LI-
three males for 48 h to mate. Subsequently, females were
COR, Lincoln, NE).
transferred individually to fresh leaf discs for oviposition. Intotal six days of oviposition were scored, and females were
transferred to a fresh leaf disc after three days. Offspring (F1female, F1 male, unhatched eggs, and dead individuals) were
Eight isofemale lines of two spotted spider mites were
counted 10–12 days later and used to compute clutch size
established through mother 3 son mating. One reason why
(CS 5 number unhatched eggs 1 number dead 1 number
it may be easy to establish these lines is haplodiplody: Re-
F1 females 1 number F1 males), F1 sex ratio (SR 5 number
cessive deleterious mutations are mostly eliminated through
F1 males/[number F1 females 1 number F1 males]), and F1
male mortality (Crozier 1985). Thus, in a sense, mothers
mortality (mortality 5 [number unhatched eggs 1 number
usually mated to good sons.
Lines established from PCR-negative females in an assay
-specific primers remained negative without
further treatment. For isofemale line 3, two cured sublines
Effect of factors was analyzed by MANOVAs on derived
were established by curing with tetracycline (TET) and by
variables (i.e., variables computed from what was actually
curing with heat treatment (HT). No differences were found
measured in the experiments: clutch size, sex ratio, and mor-
in crosses with mites cured by one or the other method (Table
tality) because these variables will generally not be inde-
1, R3). Therefore, for assessment of reproductive compati-
pendent. We report the MANOVA Wilk's l test statistic.
bility, we conclude that method of curing had no effect other
Normality of data was tested graphically and significance was
than removal of the symbiont.
examined using the Shapiro-Wilk test. Homocedasticity(equality of group variances) was tested using Levine's test.
The Effect of Wolbachia on Cytoplasmic Incompatibility
In MANOVAs, equality of covariance matrices was testedusing the Box's test. Nonparametric tests (Kruskal-Wallis)
Variability for Wolbachia
-induced reproductive incompat-
were used when assumptions of normality and homocedas-
ibility was found among isofemale lines of the rose strain.
ticity were violated (provided they could not be solved by
Presence of Wolbachia
in males of isofemale line R1 and R2
transformation). When (M)ANOVA were performed, sex ra-
resulted in induction of CI when mated with R1 and R2
tio and mortality were arcsine-square-root transformed. Sta-
uninfected females, respectively. CI was expressed as in-
tistic analysis was performed using SPSS (Chicago, IL).
creased F1 mortality and a sex-ratio bias toward males (Table
MANOVAs were followed by a series of univariate ANO-
1). However, in crosses of infected R3 males with uninfected
VAs. The significance a-level of these ANOVAs (P
R3 females CI was not observed (Table 1). PCR with Wol-
was adjusted following the Bonferroni procedure to correct
primers confirmed that this result was not due to a
for multiple analysis (Field 2000). Pairwise comparisons
change in infection status of the uninfected sublines (or of
were performed using Tukey post hoc tests.
the infected one). Finally, absence of CI was stable over time;the same result was obtained in later experiments (cf. Table
Cloning and Sequencing of Wolbachia ftsZ and wsp Genes
) from both the cu-
Although in isofemale line C3 higher F1 mortality was
cumber and rose populations of T. urticae
were cloned and
observed in U 3 W crosses (Table 2), this effects was not
sequenced to determine the relatedness of their Wolbachia
statistically significant. Thus, the cucumber isofemale lines
strains. Ten individual female mites from each mass-bred
tested did not show CI associated with presence of Wolbachia
population of cucumber and rose were pooled separately.
in males—all infections in C lines were associated with mod2
DNA was extracted using the CTAB method adapted for
phenotypes. In C5 a significant effect in sex ratio was found:
mites from Breeuwer (1997). The primers ftsZ
Infected females produced more female-biased sex ratios. A
F. VALA ET AL.
TABLE 1. Test for induction of cytoplasmic incompatibility: crosses within isofemale lines from the Rose population. W, Wolbachia infected;U, uninfected (cured); TET, cured by tetracycline; HT, cured by heat treatment; variables significant (after Bonferroni correction) according tounivariate ANOVAs are marked with an asterisk; identical superscripts (a, b, c, d) within columns indicate nonsignificant differences betweencrosses at the 5% level (Tukey test).
5 29.09, Wilk's l 5 0.21, P
5 3.70, P
5 4.13, P
5 37.06, Wilk's l 5 0.22, P
5 3.70, P
5 4.13, P
Rose 3 (HT and TET )
5 3.96, Wilk's l 5 0.81, P
5 5.64, P
5 3.70, P
5 2.54, P
sex-ratio shift toward females provides a spreading mecha-
Wsp and ftsZ Sequence Variation for the Two Strains of
nism for Wolbachia
(Egas et al. 2002) and is consistent with
results obtained previously in the base population (Vala et
No differences were found in either of the ftsZ
al. 2000, 2002).
sequences within or between the cucumber and rose mass-bred populations of T. urticae
. All 10 inserts sequenced (five
from cucumber and five from the rose populations) for both
To test whether infected R3 females retained the property
were identical (Genbank accession numbers are
of rescuing modified sperm, despite the fact that sperm from
-AF404763 and wsp
-AF404765 for cucumber and ftsZ
infected R3 males is not modified, R3 and R1 mites were
AF404764 and wsp
-AF404766 for rose).
crossed. Results are presented in Table 3. First, as for pre-vious experiments (Tables 1, 2), infected R1 males induced
CI in uninfected R1 females, but infected R3 males did not
This is the first report of within-population variation for
induce CI in uninfected R3 females. Second, infected R1
-induced phenotypes. The variation reported here
males induced CI in uninfected R3 females, whereas infected
is likely to reflect genetic differences because environmental
R3 males did not induce CI in uninfected R1 females. Third,
conditions were constant throughout experiments. The ques-
presence of Wolbachia
in R3 females eliminated incompat-
tion is whether these differences are due to genetic variation
ibility in crosses with infected R1 males. In other words,
at the host, at the symbiont level, or both.
mortality and sex ratio of /R3W 3 ?R1W was not signif-icantly different from mortality and sex ratio of /R3U 3
2 a Property of Wolbachia or of the Host?
R1U crosses, whereas both differed from /R3U 3 ?R1W
crosses. Thus, we conclude that the phenotype of the Wol-
Three genes are commonly used to infer Wolbachia
-host association in isofemale line R3 is of type
logeny, 16S rDNA, wsp,
The latter two evolve faster
is the most variable and informative (Zhou et al.
TABLE 2. Test for induction of cytoplasmic incompatibility: crosses within isofemale lines from the cucumber population (for table legend,see Table 1).
Cucumber 1 (TET )
5 1.85, Wilk's l 5 0.89, ns
Cucumber 2 (TET )
5 2.61, Wilk's l 5 0.81, P
5 3.70, P
5 4.13, P
5 6.49, Wilk's l 5 0.68, P
5 16.75, P
5 5.75, P
Cucumber 4 (TET )
5 3.79, Wilk's l 5 0.77, P
5 4.54, P
5 4.54, P
5 4.74, Wilk's l 5 0.78, P
5 4.17, P
5 7.70, P
1998; Jiggins et al. 2001). In our study, wsp
genes through recombination (Jiggins et al. 2001; Werren
quences did not correlate with infection phenotype. Sequenc-
and Bartos 2001). Alternatively, the phenotypes associated
es were identical in the rose and cucumber strains, and re-
with these infections may be influenced by host effects.
productive incompatibility was induced in isofemale lines of
Crosses within isofemale lines of the cucumber strain re-
the first, but not of the second strain. Fialho and Stevens
vealed that infection phenotype was mod2resc? in all C iso-
(2000) report a similar result. In Tribolium madens Wolbachia
female lines (Tables 2). The same result is observed in crosses
is associated with male killing and in T. confusum
within the cucumber base population (Vala et al. 2002). Pre-
tion results in CI. However, based on ftsZ
vious results suggest that C infections can, to some extent,
the two bacteria cannot be distinguished (Fialho and Stevens
rescue R-modified sperm (cf. Vala et al. 2000). However, the
2000). Of course, it is possible that the Wolbachia
two strains are reproductively isolated even in the absence
in these pairs of strains and species differs in genes other
(Vala et al. 2000). Thus, it is difficult to con-
than the two we have sequenced. The two strains of Wol-
clusively determine whether infected C females can rescue
may have acquired similar copies of wsp
sperm from infected R males. In any case, how does an in-
F. VALA ET AL.
TABLE 3. Crosses between R1 and R3 isofemale lines for resc2 or
in R3. If we repeat these calculations for sex ratio, then R1
resc1 status (for table legend, see Table 1).
has the least female-biased sex ratio (0.48), whereas R2 andR3 have similar sex ratios (0.27 and 0.26). Thus, infections
in R1 or R2 do not appear more costly to females than the
infection in R3. Absence of a difference in cost to infected
Does R1W induce CI in R1U? Yes.
R1, R2, and R3 females is expected if the Wolbachia
these lines is the same. This hypothesis cannot be refuted
based on our sequence data. The phenotypic differences ob-
Does R1W induce CI in R3U? Yes.
served may be due to genetic differences between hosts.
Like a symbiont mod2 allele, a host mod2 allele cannot
invade a population unless there is a resident mod1. CI pro-
Can R3W rescue R1W? Yes.
vides a spreading mechanism to the symbiont, but for nuclear
host genes CI means that not all crosses between infected
Does R3W induce CI in R3U? No.
and uninfected individuals will produce viable offspring. In
a population with infected and uninfected hosts, males that
possess a nuclear allele conferring resistance to sperm mod-
Does R3W induce CI in R1U? No.
ification are compatible with all females in the population.
Therefore, such a host allele invades (Turelli 1994), even if
the mod2 trait is not associated with a lower cost to the
5 18.74, Wilk's l 5 0.48, P
infected (mod2) host. The allele spreads in the absence of
5 23.24, P
, 0.001 F
5 21.11, P
other fitness differences because, being a nuclear allele, it istransmitted by both sexes. If uninfected individuals are notpresent in the population, like in our laboratory population,
fection that does not induce CI maintain itself in the host
a mod2 host allele is, like a symbiont mod2 allele, a neutral
population? A separate study suggests that C-Wolbachia
duces a sex-ratio bias toward females (Vala et al. 2002).
To summarize, in a field situation, where uninfected in-
Mathematical analysis shows that such an effect provides a
dividuals are likely to be present, a mod2 host allele invades
spreading mechanism for the bacteria (Egas et al. 2002).
if there is a mod1 allele present. Under the same circum-
Mod1resc1 and mod2resc1 infection phenotypes were iso-
stances a symbiont allele can only invade if it entails a lower
lated from the rose strain. Crosses within isofemale line R3
cost to infected females than the resident mod1. Our results
did not show induction of CI (Table 1), whereas CI was
do not support the hypothesis that mod2 is less costly to
induced in crosses within isofemale lines R1 and R2. Ad-
infected females than mod1. Thus, if mod2 were a symbiont
ditional crossing experiments demonstrated that the infection
trait, it would be rare and essentially neutral in the field.
in R3 exhibits a mod2resc1 phenotype (Table 3). Thus,
Consequently, in the absence of a cost effect to infected fe-
mod1resc1 and mod2resc1 phenotypes co-exist within a sin-
males, the probability of picking up a host mod2 in the field
gle host population.
seems higher. Once in the laboratory, where uninfected in-
Theory predicts that if a resident mod1resc1 Wolbachia
dividuals are not present, a mod2 host allele is a neutral trait.
present and the mod2resc1 type is more deleterious than the
The probability of it drifting to fixation or to extinction de-
resident mod1, then mod2 will be excluded by selection. If
pends on its original frequencies. If introduced at interme-
the mod2 and mod1 infections are equally harmful, then mod2
diate frequencies it could be maintained.
is essentially neutral (Hurst and McVean 1996). Its frequencymay drift around the level at which it was introduced and,
Population Dynamics Consequences of a Host mod
assuming the mutation that causes the shift in mod is notvery common, disappear. Thus, the probability of finding a
Hosts may mutate or otherwise protect the target sites of
neutral mod2 symbiont seems low. A mod2resc1 Wolbachia
modification by Wolbachia
, or bacterial growth in males may
increases in frequency when rare if it entails a lower cost to
be prevented (Bressac and Rousset 1993; Poinsot et al. 1998;
infected females than the resident mod1 (Prout 1994; Turelli
McGraw et al. 2001). Two examples suggest mod2 host al-
1994; Hurst and McVean 1996). If uninfected individuals are
leles may occur in Drosophila
hosts. First, a Wolbachia
present in the population, spread of the mod2resc1 type leads
that does not induce CI in D. melanogaster
, because it is
both infections to extinction (Hurst and McVean 1996). If
excluded from sperm cysts, induces CI when present D. si-
uninfected individuals are not present, like in our infected
males (McGraw et al. 2001). Second, the observation
laboratory populations, a less costly mod2 symbiont type
that wAu, a symbiont type occurring in D. simulans
, fails to
would spread to fixation.
induce CI in flies from Australia (Hoffmann et al. 1996;
Our results do not indicate any correlated differences be-
Merc¸ot and Poinsot 1998b), but induces CI in some isofemale
tween fecundity costs to infected females and CI induction.
lines of flies collected in Florida (Ballard et al. 1996).
Infected R1 and R3 females generally produce similar clutch
It is therefore interesting to ask what happens after invasion
sizes (Table 1). Average F1 mortality in W 3 W and W 3
by a mod2 host allele. It is conceivable that spread of a
U crosses is 0.25 for R1, 0.16 for R2, and 0.21 for R3.
nuclear mod2 would result in conditions for reestablishment
Therefore, the mod1resc1 infection in R2 incurs lower mor-
of uninfected individuals, analogous to spread of a Wolbachia
tality in broods of W females than the mod2resc1 infection
mod2 (Hurst and McVean 1996). Simulations presented in
infected with wAu in Australia constitute an example of hostallele–mediated dynamics such as those simulated here.
To conclude, we found within-population variation for
mod. The possibility that the differences we observed are dueto host effects cannot be excluded. Simple mathematical anal-ysis of the dynamics of a host mod2 allele suggests interestingevolutionary implications and novel interpretations of exist-ing data. Clearly, more effort should be made to distinguishbetween Wolbachia
and host mod2 alleles.
F. Vala was supported by Fundac¸a˜o para a Ciencia e Tec-
nologia (scholarship reference: Praxis XXI/BD/9678/96).
Ballard, J. W. O., J. Hatzidakis, T. L. Karr, and M. Kreitman. 1996.
Reduced variation in Drosophila simulans
Bourtzis, K., S. L. Dobson, H. R. Braig, and S. L. O'Neill. 1998.
have been overlooked. Nature 391:852–853.
Breeuwer, J. A. J. 1997. Wolbachia and cytoplasmic incompatibility
in the spider mite Tetranychus urticae
and T. turkestani
Breeuwer, J. A. J., and G. Jacobs. 1996. Wolbachia
The effect of a host allele resistant to modification by
manipulators of mite reproduction. Exp. Appl. Acarol. 20:
on the population dynamics of the infection (see Ap-
pendix and Table A1 for details). The dynamics depicted here are
Bressac, C., and F. Rousset. 1993. The reproductive incompatibility
based on m 5 0.9, F
5 0.9, H
5 0.1. With these parameters and
system in Drosophila simulans
: DAPI staining analysis of the
in the absence of mod2 (p
0), there is an unstable equilib-
symbionts in sperm cysts. J. Invertebr. Pathol. 61:
rium at p
0.765. With initial conditions ps
0, the dynamics quickly converge to the
Callaini, G., R. Dallai, and M. G. Riparbelli. 1997. Wolbachia
0.014. After one generation, a mod2
induced delay of paternal chromatin condensation does not pre-
allele was introduced (p
1025), thus mimicking a mutation in a
vent maternal chromosomes from entering anaphase in incom-
single infected host. (a) Frequency of types susceptible to modi-
patible crosses of Drosophila simulans
. J. Cell Sci. 110:271–280.
fication by Wolbachia
(dashed) and qs
(solid). (b) Fre-
Crozier, R. H. 1985. Adaptive consequences of male-haploidy. Pp.
quency of types resistant to modification by Wolbachia
W. Helle and M. W. Sabelis, ed. Spider mites: their
(dashed) and qr
biology, natural enemies and control. Elsevier, Amsterdam.
De Boer, R. 1985. Reproductive barriers. Pp. 193–200 in
and M. W. Sabelis, ed. Spider mites: their biology, natural en-
Figure 1 show that this is indeed the case (see the Appendix
emies and control. Elsevier, Amsterdam.
for details). However, invasion by a host allele conferring
Egas, M., F. Vala, and J. A. J. Breeuwer. 2002. On the evolution
resistance to sperm modification by Wolbachia
leads to es-
of cytoplasmic incompatibility in haplodiploids. Evolution 56:1101–1109.
tablishment of resistant uninfecteds. Spread of a host mod2
Fialho, R. F., and L. Stevens. 2000. Male-killing Wolbachia
allele differs from spread of a mod2 Wolbachia
flour beetle. Proc. R. Soc. Lond. B 267:1469–1474.
uninfected population that results can only be recolonized by
Field, A. 2000. Complex ANOVA (GLM2). Pp. 294–322 in
using a novel modification site (i.e., a new in-
Wright, ed. Discovering statistics using SPSS for Windows.
compatibility type). This could provide selective pressure for
SAGE Publications, London.
Hartl, D. L., and A. G. Clark, 1997. Population substructure. Pp.
new incompatibility types. For example, in D. simulans
D. L. Hartl and A. G. Clark, eds. Principles of pop-
strains have been identified (wRi, wHa, wAu, wKi,
ulation genetics. Sinauer Associates, Sunderland, MA.
wMa, and wNo), a classification that is consistent with se-
Hoffmann, A. A., and M. Turelli. 1997. Cytoplasmic incompatibility
quence data from wsp
and 16S rDNA (cf. James and Ballard
in insects. Pp. 42–80 in
S. L. O'Neill, A. A. Hoffmann, and J.
H. Werren, eds. Influential passengers. Oxford Univ. Press, Ox-
2000). These symbiont types correspond to four incompati-
bility groups. It would be interesting to test if D. simulans
Hoffmann, A. A., D. Clancy, and J. Duncan. 1996. Naturally oc-
hosts resist modification by other simulans Wolbachia
infection in Drosophila simulans
The theoretical result that a host mod2 allele may lead to
not cause cytoplasmic incompatibility. Heredity 76:1–8.
symbiont exclusion is also interesting. For example, infec-
Holden, P. R., J. F. Y. Brookfield, and P. Jones. 1993. Cloning and
characterization of an fstZ
homologue from a bacterial symbiont
tions by wAu in Australia occur at zero to low frequencies
of Drosophila melanogaster
. Mol. Gen. Genet. 240:213–220.
in different populations (Hoffmann et al. 1996). The effect
Hurst, L. D. 1991. The evolution of cytoplasmic incompatibility or
of these infections on reproductive incompatibility was tested
when spite can be successful. J. theor. Biol. 148:269–277.
on pooled samples from an Australian population and shown
Hurst, L. D., and G. T. McVean. 1996. Clade selection, reversible
evolution and the persistence of selfish elements: the evolution-
not to cause CI. However, when isofemale lines were created
ary dynamics of cytoplasmic incompatibility. Proc. R. Soc.
from Florida populations, CI was detected in some isofemale
Lond. B. 263:97–104.
lines (Ballard et al. 1996). It is possible that populations
James, A. C., and J. W. O. Ballard. 2000. Expression of cytoplasmic
F. VALA ET AL.
incompatibility in Drosophila simulans
and its impact on infec-
2000. Evidence for female mortality in Wolbachia
tion frequencies and distribution of Wolbachia pipientis
toplasmic incompatibility in haplodiploid insects: epidemiologic
and evolutionary consequences. Evolution 54:191–200.
Jiggins, F. M., J. H. G. von der Schulenburg, G. D. D. Hurst, and
Werren, J. H. 1997. Biology of Wolbachia
. Annu. Rev. Entomol.
M. E. N. Majerus. 2001. Recombination confounds interpreta-
tions of Wolbachia
evolution. Proc. R. Soc. Lond. B 268:
Werren, J. H., and J. D. Bartos. 2001. Recombination in Wolbachia
Curr. Biol. 11:431–435.
McGraw, E. A., D. J. Merrit, J. N. Droller, and S. L. O'Neill. 2001.
Zhou, W., F. Rousset, and S. O'Neill. 1998. Phylogeny and PCR-
mediated sperm modification is dependent on host
based classification of Wolbachia
strains using wsp
genotype in Drosophila
. Proc. R. Soc. Lond. B 268:2565–2570.
quences. Proc. R. Soc. Lond. B 265:509–515.
Merc¸ot, H., and D. Poinsot. 1998a. . and discovered on Mount
Kilimanjaro. Nature 391:853.
Corresponding Editor: M. Riley
———. 1998b. Wolbachia transmission in a naturally bi-infected
strain from New Caledonia. Entomol. Exp.
Poinsot, D., K. Bourtzis, G. Markakis, C. Savakis, and H. Merc¸ot.
We study the dynamics of allele frequencies after invasion of the
transfer from Drosophila melanogaster
mod2 host allele, with a population genetic model that follows
: host effects and cytoplasmic incompatibility relation-
Turelli (1994). The variables in our model are the frequency of
ships. Genetics 150:227–237.
infected hosts susceptible to modification by Wolbachia
Prout, T. 1994. Some evolutionary possibilities for a microbe that
frequency of infected hosts resistant to modification by Wolbachia
causes incompatibility in its host. Evolution 48:909–911.
), the frequency of uninfected hosts possessing the allele for
Reed, K. M., and J. H. Werren, 1995. Induction of paternal genome
susceptibility to modification by Wolbachia
), and the frequency
loss by the paternal sex-ratio chromosome and cytoplasmic in-
of uninfected hosts possessing the resistant allele to modification
compatibility bacteria (Wolbachia
): a comparative study of early
). Other notation follows Turelli (1994); m is the
embryonic events. Mol. Reprod. Dev. 40:408–418.
proportion infected offspring produced by an infected mother, F
Rousset, F., and M. Raymond. 1991. Cytoplasmic incompatibility
the fecundity of infected females relative to uninfected females,
in insects: Why sterilize females? Trends Ecol. Evol. 6:54–57.
is the hatchability in incompatible crosses (H
5 0 implies
Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular clon-
that CI results in 100% F1 mortality). Note that m, F
, and H
ing a laboratory manual. Cold Spring Harbor Laboratory Press,
respond to a, 1 2 U
, and 1 2 k
, respectively, in Hurst and McVean
Cold Spring Harbor, NY.
Stouthamer, R., J. A. J. Breeuwer, and G. D. D. Hurst. 1999. Wol-
Table A1 lists all possible matings, the frequency at which they
: microbial manipulator of arthropod reproduc-
occur assuming random mating, and the expected distribution of
tion. Annu. Rev. Microbiol. 53:71–102.
offspring over the four different host categories. Assuming non-
Turelli, M. 1994. Evolution of incompatibility-inducing microbes
overlapping generations and haploid genetics for reasons of model
and their hosts. Evolution 48:1500–1513.
tractability, the dynamics of the population is described by four
Vala, F., J. A. J. Breeuwer, and M. W. Sabelis. 2000. Wolbachia-
difference equations. For example, the frequency of infected, sus-
induced ‘hybrid breakdown' in the two-spotted spider mite Te-
ceptible hosts in the next generation (p
) is obtained by summing
Koch. Proc. R. Soc. Lond. B 267:1931–1937.
all separate contributions to Ws individuals in Table A1, and di-
Vala, F., T. Van Opijnen, J. A. J. Breeuwer, and M. W. Sabelis.
viding by the sum of all contributions:
2002. Genetic conflicts over sex ratio: mite-endosymbiont in-
teractions. Am. Nat. In press
9 5 (p
1 ½p p
1 . . 1 ½p q
Van Opijnen, T., and J. A. J. Breeuwer. 1999. High temperatures
1 . . 1 q
, a cytoplasmic incompatibility inducing en-
dosymbiont, from the two-spotted spider mite. Exp. Appl. Acar-
Note that because p
1, one variable can be
eliminated, leaving three difference equations.
Vavre, F., F. Fleury, J. Varaldi, P. Fouillet, and M. Bouletreau.
The objective of this exercise is to investigate whether Hurst and
TABLE A1. List of possible crosses between host types, the frequency by which they occur, and the distribution of offspring over the fourtypes in a population that segregates a host allele resistant to modification by Wolbachia.
Host types are classified by infection status (W,infected; U, uninfected) and susceptibility to modification (s, susceptible or mod1; r, resistant or mod2). The columns Ws, Wr, Us, and Ur areexpressed in units of the clutch size of an uninfected female.
(1 2 m)F H
½ (1 2 m)F
½(1 2 m)F
½(1 2 m)F
½(1 2 m)F
½(1 2 m)F H
½(1 2 m)F H
½(1 2 m)F
½(1 2 m)F
McVean's (1996) predictions hold assuming mod2 is a property of
that m , 1 and that the two equilibria (ps
, 0, 0) exist (i.e., the
the host. In other words, do uninfected individuals reestablish fol-
unstable, or threshold, equilibrium and the high prevalence, or poly-
lowing invasion by mod2? Using the same parameter values as Hurst
morphic, internal equilibrium).
and McVean (1996, fig. 2), we find: (1) invasion of the mod2 host
Note that if there were a cost to mod2, the uninfected population
allele, followed by extinction of Wolbachia
(Fig. 1); (2) the resulting
would slowly return to the susceptible state. However, population
uninfected host population is immune to any Wolbachia
immunity is provided by any frequency q
0. Therefore, we argue
the same modification site; (3) results (1) and (2) hold provided
that reinvasion of Wolbachia
requires a new modification site.
CREENCIAS SOBRE LA DIABETES MELLITUS Y SU INFLUENCIA EN EL ACTO DE AMAMANTAR EN PUÉRPERAS CON DIABETES GESTACIONAL PREVIA UNIVERSIDAD DE CARABOBO FACULTAD DE CIENCIAS DE LA SALUD DIRECCION DE POSTGRADO SEDE CARABOBO MAESTRIA EN ENFERMERIA SALUD REPRODUCTIVA CREENCIAS SOBRE LA DIABETES MELLITUS Y SU
Veja nesta edição Dicionário Sefaradi de SobrenomesDictionary of Sephardic Surnames Judeus no Colégio Pedro II, RJ O alfaiate Aldo Campagnano, vítima italianado "Holocausto" O Libelo de Damasco J.L. Cardozo de Bethencourt Minhas Memórias de um Mundo Perdido Os Judeus de Itapicuru