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Sperm competition risk generates
phenotypic plasticity in ovum
fertilizability
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Renée C. Firman and Leigh W. Simmons
Centre for Evolutionary Biology, School of Animal Biology (M092), University of Western Australia,
Nedlands 6009, Australia
Research
Cite this article: Firman RC, Simmons LW.
2013 Sperm competition risk generates
phenotypic plasticity in ovum fertilizability.
Proc R Soc B 280: 20132097.
http://dx.doi.org/10.1098/rspb.2013.2097
Received: 13 August 2013
Accepted: 19 September 2013
Subject Areas:
evolution, behaviour
Keywords:
sexual conflict, cryptic female choice,
polyspermy, house mice, cumulus oophorus,
zona pellucida
Author for correspondence:
Renée C. Firman
e-mail: [email protected]
Electronic supplementary material is available
at http://dx.doi.org/10.1098/rspb.2013.2097 or
via http://rspb.royalsocietypublishing.org.
Theory predicts that sperm competition will generate sexual conflict that
favours increased ovum defences against polyspermy. A recent study on
house mice has shown that ovum resistance to fertilization coevolves in
response to increased sperm fertilizing capacity. However, the capacity for
the female gamete to adjust its fertilizability as a strategic response to
sperm competition risk has never, to our knowledge, been studied. We
sourced house mice (Mus domesticus) from natural populations that differ
in the level of sperm competition and sperm fertilizing capacity, and
manipulated the social experience of females during their sexual development to simulate conditions of either a future ‘risk’ or ‘no risk’ of sperm
competition. Consistent with coevolutionary predictions, we found lower fertilization rates in ova produced by females from a high sperm competition
population compared with ova from a low sperm competition population,
indicating that these populations are divergent in the fertilizability of their
ova. More importantly, females exposed to a ‘risk’ of sperm competition produced ova that had greater resistance to fertilization than ova produced by
females reared in an environment with ‘no risk’. Consequently, we show
that variation in sperm competition risk during development generates phenotypic plasticity in ova fertilizability, which allows females to prepare for
prevailing conditions during their reproductive life.
1. Introduction
Studies of postcopulatory sexual selection have focused on the evolutionary
and facultative responses of the ejaculate to sperm competition, and mechanisms by which females influence the transfer and storage of sperm [1,2].
Much less attention has been given to the evolutionary consequences of
postcopulatory sexual selection for ovum form and function.
Selection upon the ejaculate occurs when the sperm of rival males are forced
to compete for fertilizations [3]. Male evolutionary adaptations to sperm competition have been well documented, and studies of experimental evolution
have shown that sperm competition selects for ejaculates with high sperm densities and enhanced competitive ability [4 –6]. Indeed, competitive conditions
are expected to favour sperm that rapidly penetrate the ovum. However, a detrimental outcome of increased sperm fertilizing capacity is an elevation in the
frequency of polyspermy [7]. Although the frequency of polyspermy in natural
fertilizations has received little attention, it has been shown to be present even
when sperm are limited [8,9]. As a response to this fatal threat, ova are expected
to counteradapt by decreasing their fertilizability (ova defensiveness) [10], leading to a coevolutionary arms race between the gametes [11]. Evolutionary
responses in ovum resistance to fertilization have been explored both among
and within Mus species. A comparative study of three species of Mus that
had evolved under different levels of sperm competition reported asymmetries
in interspecific fertilization rates and provided evidence that sexual conflict at
the gametic level has the potential to promote prezygotic reproductive barriers
[12]. Indeed, ovum defences could account for faster conspecific fertilization
rates, compared with heterospecific rates, in the naturally hybridizing species
& 2013 The Author(s) Published by the Royal Society. All rights reserved.
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2. Material and methods
(a) Experimental animals
Sexually mature male (20) and female (20) house mice were
trapped on Whitlock Island (308190 S, 1148590 E) and Rat Island
(288420 S, 1138470 E) located off the coast of Western Australia,
and brought to the University of Western Australia. We selected
these two populations because they are known to differ in the
frequency of multiple paternity [37]. In the Whitlock Island
population, 17% of litters are of mixed paternity, whereas in the
Rat Island population 71% of litters are of mixed paternity. Associated with this difference in the level of sperm competition, males
on Rat Island have relatively larger testes than males on Whitlock
Island [37]. The mice were bred under common-environment
conditions for four generations. Mice were maintained in a
constant temperature room (CTR; 248C) on a reversed light–
dark cycle, and food and water were provided ad libitum.
During breeding, the females were paired with a single male
until they were seen to be pregnant upon which males were
removed. Mice of the fifth generation were used as focal individuals
in this experiment.
(b) Social environment manipulation
House mice can recognize and identify conspecifics via individual scent signals [38]. Specifically, male scent has been shown to
influence female reproductive development [39] and function
[40,41]. Thus, we exposed developing females to the soiled
chaff of one to 10 males and created variation in their perception
of male density, and thus the sperm competition environment.
From the time that they were weaned (three weeks old) until
they reached sexual maturity, ‘risk’ females were maintained in
a CTR that also housed caged males (see the electronic supplementary material, figure S1). These females received soiled
chaff from multiple males and ‘encountered’ sexually mature
males. Thus, each week ‘risk’ females received approximately
15 g of a mixture of soiled chaff that consisted of 30 g of bedding
from each of the boxes of 10 sexually mature males. ‘Encounters’
were conducted once a fortnight. The focal females were placed
in large, opaque plastic tubs (49 74 41 cm) containing two
individually caged mature males. For 30 min, the females
roamed freely inside the tub, which allowed them to receive
olfactory, auditory and visual (but not tactile) stimuli from the
encounter animals. Each time the focal females encountered
two different males.
By contrast, ‘no risk’ females were maintained in a CTR that
contained only females. Focal females received soiled chaff from
a single male and they ‘encountered’ sexually mature females.
2
Proc R Soc B 280: 20132097
with conspecifics, and thus created local environments in
which females perceived either a ‘risk’ or ‘no risk’ of sperm
competition. We retrieved ova of females from the low and
high sperm competition populations that had matured
under either a ‘risk’ or ‘no risk’ of sperm competition, and
quantified ovum resistance to fertilization by using in vitro
fertilization (IVF) techniques. Our IVF assays produced a pattern of fertilization rates among females from the different
populations that is consistent with the theoretical prediction
that sperm competition should generate divergence in
ovum defensiveness via sexual conflict at the gametic level
[11]. More importantly, we found that females exposed to a
‘risk’ of sperm competition produced more defensive ova
than females that were reared under ‘no risk’ of sperm competition. Thus, our investigation provides evidence of
phenotypic plasticity in a female trait in response to the
risk of sperm competition.
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Mus domesticus and Mus musculus [13]. More recently, a study
of experimental evolution has shown that female house mice
(M. domesticus) that had evolved under a polyandrous mating
regime produced ova that were more resistant to fertilization
compared with females that had evolved under a monogamous regime [14]. These investigations have provided
compelling evidence for the evolution of ovum resistance to
fertilization in response to sperm competition.
Soliciting copulations from multiple males can also be
beneficial to females by allowing them to ‘choose’ between
males in the postcopulatory arena; ‘cryptic female choice’
[1]. Although isolating the subtle processes of prezygotic
sperm choice from sperm competition is difficult [15],
sperm selection is expected to generate a fitness advantage
by enabling females to skew paternity toward males of high
genetic quality [1,15]. Consequently, females may use mechanisms of ovum defence to filter male quality and ensure that
only the highest quality sperm penetrate the outer coat of the
ovum, the zona pellucida (Zp), and fuse with the nucleus.
Phenotypic plasticity is defined as the capacity of a single
genotype to exhibit variable phenotypes in different environments [16]. Phenotypic plasticity is ubiquitous in nature and
has been demonstrated to occur in a large number of traits
across a diverse range of taxa (reviewed in [17,18]). Anticipatory plastic responses have been shown to be stimulated by an
individual’s interaction with cues that reflect future environmental or social conditions [18]. Plastic responses are usually
highly adaptive and may influence an individual’s survival
[19 –21] and/or reproductive success [22 –25]. For example,
social cues that are indicative of an elevated risk of sperm
competition have been shown to generate adaptive responses
in the ejaculate. When presented with a risk of sperm competition, males have been shown to make both immediate
adjustments in the number of ejaculated sperm [22,26–29],
as well as long-term, developmental changes in sperm production [24,30 –32]. There is also evidence of phenotypic
plasticity in sperm morphology. Thus, studies have shown
that sperm size can be adjusted under different social scenarios [33– 35]. The influence of the social environment on
the ovum phenotype has not before been investigated. In
this study, we addressed whether female house mice
(M. domesticus) can phenotypically adjust the fertilizability of
their ova to variation in the sperm competition environment.
Population density varies considerably in natural house
mouse populations and has been shown to be predictive of
the level of sperm competition as measured by the incidence
of multiply sired litters [36,37]. We sourced house mice from
two island populations that have rates of multiply sired litters
that represent a low (17% multiple paternity) and a high
(71% multiple paternity) level of sperm competition [37]. As
predicted by sperm competition theory, males from the
high sperm competition population produce more sperm
and better quality sperm compared with males from the
low sperm competition population [32]. Moreover, males
from these populations were found to increase the fertilizing
potential of their sperm when reared under a risk of sperm
competition [32]. Here, we exposed maturing females to
different sperm competition environments by creating variation in their perception of male density. Olfaction is the
primary sensory modality in house mice, and conspecifics
are recognized by individually distinct scent signals [38].
We therefore manipulated the social experience of developing
females via exposure to male odours and direct encounters
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s.d.
assay ID
0.523
0.723
fixed
effects
estimate
s.e.
t12
(intercept)
0.994
0.359
22.628
0.009
population
treatment
20.870
21.610
0.416
0.416
2.092
3.872
0.036
,0.001
proportion of ova fertilized
variance
0.8
0.6
0.4
0.2
0
p
Once every two weeks, females in the ‘no risk’ treatment received
soiled chaff (15 g) from the same mature male that was housed in
a different CTR. Every other week ‘no risk’ female chaff (15 g)
was transferred from the back to the front of their own box.
During encounters, ‘no risk’ females were exposed to two
mature females. The ‘risk’ and ‘no risk’ individuals were
switched between CTRs after four weeks of social manipulation.
The IVF assays commenced after eight weeks of social
manipulation. One replicate assay for each of the four treatment
groups was performed each week for four weeks. Consequently,
the age of the females at the time of ova donation ranged from
11 to 14 weeks. The females received the social manipulation
regime until they were sacrificed for ova donation. Ova were
mixed with the sperm of males from the same population that
had been reared under standard colony conditions. A total of
16 IVF assays were performed.
(c) In vitro fertilization
The protocol for the IVF assays has been described previously
[12,42]. Briefly, adult females were induced to superovulate
with 5IU pregnant mare’s serum gonadotropin and 5IU human
chorionic gonadotropin that were administered 48-h apart. The
female reproductive tracts were removed and cumulus –oocyte
complexes (COCs) were released from the ampullae. Epididymides were removed from males and placed in a 1 ml drop of
human tubal fluid (HTF) and incubated to allow sperm to disperse into the medium [12,42]. Following an initial 10 min
incubation, sperm concentration and sperm motility was quantified using a CEROS computer-assisted sperm analysis system
(v. 10, Hamilton and Thorne Research) using standard mouse
parameters. On average, the sperm traits did not differ significantly among the small sample of males used in the IVF assays
(see the electronic supplementary material, tables S1 and S2).
The IVF assays were performed in 500 ml drops of HTF under
mineral oil. The COCs from four females of the population were
pooled in a single assay. Previous IVF experiments both within
and between Mus species produced higher fertilization rates of
conspecific ova– sperm combinations compared with heterospecific combinations [12], and trial assays performed on wildderived, laboratory populations of mice at sperm concentrations
of 2.0 106 motile sperm ml21 provided evidence of a ceiling
effect with fertilization rates close to 100%. Thus, to ensure that
higher fertilization rates across the different treatments would
be detectable, the IVF assays were mixed with a final concentration of 1.5 106 motile sperm ml21. The gametes were
coincubated for 4 h at 378C under 5% CO2/air, which allowed
the sperm to penetrate the ova, fuse with the oolema, decondense
3
risk
no risk
low SC population
risk
no risk
high SC population
Figure 1. Phenotypic plasticity in ova fertilizability. Fertilization rates of ova
donated by females from a low-level or high-level sperm competition (SC)
population that matured under a ‘risk’ or ‘no risk’ of sperm competition.
(Online version in colour.)
and form a pronucleus [12,42]. After the incubation period, the
ova were washed in 100 ml drops of HTF to remove remaining
cumulus cells and/or attached sperm. The oocytes where then
stained with a DNA fluorochrome (Hoechst 33342) and viewed
under a Zeiss Axio Imager.A1 fluorescent microscope. Fertilization rate was determined as the number of ova with stained
pronuclei over the total number of mature ova. The number of
ova scored and the proportion of ova fertilized in each IVF
assay are presented in the electronic supplementary material,
table S3.
3. Results
We analysed IVF rates among the treatment groups using a
generalized linear mixed model (GLMM) fit by the Laplace
approximation using the ‘lme4’ library in the R-statistical
analysis package [43], which is one of the recommended
maximum-likelihood estimation methods for generalized
mixed-effects models [44]. Fixed effects in the GLMM were
population, treatment and their interaction, while assay ID
was modelled as a random effect. The interaction term was
non-significant ( p ¼ 0.976), and therefore removed from the
model (table 1). We found that females which had matured
under a perceived risk of sperm competition had increased
ova defensiveness (38% of ova fertilized) compared with
females that had matured in a social environment with no
risk of sperm competition (73% of ova fertilized; table 1
and figure 1). Our statistical analysis also revealed that
there was a significant difference between populations in
fertilization rate (table 1 and figure 1). Under identical fertilization conditions, we found reduced IVF rates in ova derived
from the high sperm competition population (44% of ova fertilized) compared with the low sperm competition population
(67% of ova fertilized).
4. Discussion
Phenotypic plasticity is widespread in nature, and plastic
responses have been shown to include changes in behavioural,
physiological, morphological and life-history traits [17,18].
Individuals may be sensitive to cues or stimuli that reflect
future social conditions, and thus make ontogenic adjustments
Proc R Soc B 280: 20132097
random
effects
1.0
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Table 1. Fertilization rates of ova of female house mice from two
populations that matured under a ‘risk’ or ‘no risk’ of sperm competition.
(A GLMM fit by the Laplace approximation (R) revealed that both
population and sperm competition risk (treatment) had a significant effect
on IVF rates in house mice.)
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4
Proc R Soc B 280: 20132097
polyspermy [48]. For mice (M. musculus), it is known that
polyspermy does occur under natural mating conditions
(approx. 2%) [49], however the relationship between the
number/quality of naturally inseminated sperm and the
rate of polyspermy is yet to be determined. Although IVF
conditions are likely to produce inflated rates of polyspermy
compared with natural conditions, it has been shown that the
in vitro rate of polyspermy increases as sperm density
increases [50]. Consequently, we might expect females to
adjust the fertilizability of their ova in accordance with
expected sperm densities at the site of fertilization.
We documented variation in ova fertilizability between
females that were exposed to different perceived male
densities during development and for the first time, to our
knowledge, provide evidence of phenotypic plasticity in
a female trait in response to the sperm competition environment. It could be argued that the observed result is
attributable to sperm limitation in the ‘no risk’ treatment
and the production of more fertilizable ova. However, it is
important to note that the IVF rates in the ‘no risk’ treatment
were comparable to those that were obtained during trial
assays when animals were reared under standard colony
conditions (approx. 80%) [14]. Consequently, the rates of fertilization in the ‘risk’ treatment are representative of a reduction
in fertilization success and show that female house mice
respond to an elevated risk of sperm competition by increasing
their ova resistance to fertilization. The response of increased ovum defences among females parallels our finding
that males from these populations increase their fertilization
efficiency when reared under a perceived risk of sperm competition [32]. Thus, in house mice we have found that both male
(sperm) and female (ova) responses to sperm competition risk
align with the sexual conflict hypothesis [11].
An alternative though not mutually exclusive interpretation of our data, is that females may moderate ova
fertilizability under different risks of sperm competition as
a mechanism of cryptic female choice [1]. When male density
is high, and females have the opportunity to mate with multiple partners, they may benefit from elevating ova resistance
to fertilization to ensure that only the best quality sperm,
with the greatest fertilizing potential, are able to penetrate
the Zp. We are unable to separate processes of sperm selection from sperm competition in our study. Future research
will endeavour to determine whether cryptic female choice
at the gametic level selects for enhanced offspring fitness.
Regardless of the process(es) accounting for the observed pattern of fertilization, we have shown that females are sensitive
to cues of sperm competition risk and have the ability to
adjust the degree of ovum resistance to fertilization accordingly. Indeed, phenotypic plasticity in ova defensiveness
during development would allow females to prepare for conditions during their reproductive life and potentially reduce
the likelihood of polyspermy in an environment where the
risk of sperm competition is high.
The plastic responses we have documented in ova fertilizability could be explained by physical, biochemical and/or
molecular mechanisms of the ovum and its associated extracellular matrix. The cumulus oophorus is a group of closely
associated granulosa cumulus cells that support the maturing
oocyte prior to ovulation [51]. Following ovulation, the
cumulus oophorus surrounds the oocyte as a cloud of cumulus cells, which facilitates the transfer of the oocyte into the
oviduct [52] and participates in the passage of sperm to the
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to enhance specific traits that will be adaptive during their
reproductive life [18]. Phenotypic plasticity in a female trait
in response to sperm competition has not before, to our knowledge, been studied. Here, we used IVF to explore whether
females had the capacity to respond to increased fertilization
effort among males by increasing their resistance to fertilization. We sourced house mice from two natural populations
that differed in the strength of selection via sperm competition
and tested whether there were adjustments in ovum fertilizability
in response to cues that corresponded to different sperm competition environments. We found that females exposed to a ‘risk’ of
sperm competition produced more defensive ova compared with
the ova of females that were reared under ‘no risk’ of sperm
competition. We thereby provide, to our knowledge, the first
demonstration of phenotypic plasticity in the female gamete in
response to the sperm competition environment.
Sexual conflict over the postmating interests of males and
females is widespread in nature [45] and arises at the gametic
level when the sperm of rival males compete for fertilizations
[11]. Selection for increased sperm fertilizing potential is predicted to generate an elevation in the frequency of the fatal
condition of polyspermy [7]. Sexual conflict theory proposes
that an increase in male sperm competitiveness will provoke
a counteradaptation in females of increased ova resistance to
fertilization [11]. Until recently, empirical support for this
sexual conflict paradigm was limited to just two studies.
A comparative study on free-spawning invertebrates reported
a correlation between sperm density (a proxy for the strength
of sperm competition) and ova resistance to fertilization [10].
Similarly, ova fertilizability was found to be correlated with
relative testes size among species of Mus [12]. The first
direct line of evidence that sperm competition can generate
sexual conflict at the gametic level, and create asymmetries
in fertilization rates among populations, comes from a study
of experimental evolution on house mice (M. domesticus)
[14]. Females that had evolved with sperm competition were
found to produce ova with increased resistance to fertilization
compared with the ova of females that had evolved under a
monogamous regime [14]. The natural mouse populations
that we used in this study are likely to differ in many respects
other than the level of sperm competition. However, it is interesting to note that the pattern of divergence in IVF rates
observed between these two populations of mice is consistent
with the theoretical prediction that sperm competition should
generate sexual conflict at the gametic level, leading to
an evolutionary divergence in ova defensiveness among
populations and species [11].
A number of theoretical models have been developed to
predict how males will strategically adjust the ejaculate in
response to sperm competition risk [46,47], and empirical
studies of rodents have provided evidence for phenotypic
plasticity in both sperm production [24,32] and expenditure
[28]. Previously, we found that males from our high sperm
competition population produced more sperm and better
quality sperm than did males from the low sperm competition population [32]. Moreover, males reared under a
perceived risk of sperm competition produced more aggressive sperm compared with males reared under no risk [32].
Adaptive adjustments in the ejaculate would enhance male
fitness in competitive situations [24,29] but are also likely to
lead to an increased risk of polyspermic fertilizations [10].
In the rat Rattus norvegicus, increased numbers of sperm at
the fertilization site will generate elevated rates of
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This research was approved by the UWA animal ethics committee
(07/100/607).
Acknowledgement. We are grateful to E. Roldan and M. Gomendio from
the National Museum of Natural Sciences (Spain) for IVF training.
We also thank T. Friend, T. Button, S. Stankowski and C. McHarrie
for assistance with field collections; F. Simmons and S. Lobind for
assistance with the social manipulation regime and animal husbandry;
and B. Buzatto for statistical assistance.
Funding statement. This research was funded by the Australian Research
Council.
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5
Proc R Soc B 280: 20132097
mouse ova regulate the establishment of the membrane
block is largely unknown, but has been suggested to be the
culmination of multiple postfertilization events that together
modify the ovum’s receptivity to sperm and is established
in approximately the same time frame as the Zp block [61].
Certainly, responses in the cumulus oophorus, the Zp and
the plasma membrane that account for plasticity in ova fertilizability need not be mutually exclusive. In order to gain
insights into these potential mechanisms of ova defensiveness,
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genes and compare fertilization rates of cumulus-enclosed
versus cumulus-denuded oocytes among females that have
prepared for different sperm competition environments.
In conclusion, it is well known that males show phenotypic plasticity in ejaculate investment in response to their
perception of sperm competition risk, and it has long been
recognized that male responses to sperm competition can
be costly for females, for example, if overly aggressive ejaculates result in polyspermy and ova mortality. Here, we
provide, to our knowledge, the very first account of adaptive
plasticity in a female trait in response to the risk of sperm
competition. We suggest that strategic adjustments in the
physical microenvironment of the cumulus oophorus and/
or variation in the expression of glycoprotein/s associated
with the Zp genes could account for variation in ova fertilizability. Importantly, this research has shown that variation in
sperm competition risk during development generates plastic
responses in the ovum that would allow females to prepare
for prevailing conditions during their reproductive life.
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surface of the oocyte, the Zp [53]. Sperm are required to move
through the extracellular matrix of the cumulus oophorus to
achieve successful fertilization. It has been suggested that the
cumulus oophorus attracts sperm via chemotaxis [54] and
directs the few sperm that reach the ampulla to the immediate
vicinity of the oocyte [55]. Thus, the cumulus oophorus has
an important role in controlling the number of sperm that
interact with the Zp. Consequently, variation in the number
or density of cumulus cells around the oocyte may account
for the plastic response in ovum defences that we have
observed in house mice. A greater concentration of cumulus
cells, or more compacted cumuli, around the oocyte may
create a physical barrier that inhibits the sperms’ passage to
the oocyte. In this case, it could be that females exposed to
a risk of sperm competition made strategic adjustments in
the density of cumuli that surrounded the ooctyes, restricting
the ease at which the sperm reach the Zp, and thus increasing
ovum resistance to fertilization. Alternatively, considering
that the oophurus physiochemical environment controls the
number of sperm that are attracted to the Zp [54], it might
be that fewer cumulus cells around the oocytes resulted in
reduced fertilization rates of females reared under a risk of
sperm competition.
Following successful fertilization, cortical granules (membrane bound organelles located in the cortex of unfertilized
oocytes) undergo exocytosis to release their contents into the
perivitelline space [56]. The released cortical granule proteins
are responsible for blocking polyspermy by modifying the Zp
[57]. Although other biochemical and structural modifications
of the Zp following cortical granule exocytosis have been
inferred, only a change in a Zp glycoprotein (Zp2) has been
experimentally described [58]. The ‘zona hardening’ inhibits
additional sperm from binding to the Zp and prevents already
bound sperm from penetrating the ovum [59,60]. It is possible
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the Zp’s block. In this case, variation in the expression
of the Zp genes may account for differences in ova resistance
to fertilization, offering an alternative potential mechanism for
strategic adjustments in ovum defences of females reared in
different social environments. The mechanism/s by which
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