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Animal Behaviour 89 (2014) 131e139

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Animal Behaviour
journal homepage: www.elsevier.com/locate/anbehav

Sperm dynamics and cryptic male choice in tephritid ies


D. Prez-Staples a, *, G. Crdova-Garca b, M. Aluja b
a
b

INBIOTECA, Universidad Veracruzana, Av de las Culturas Veracruzanas, Xalapa, Veracruz, Mexico


Instituto de Ecologa, A.C., Xalapa, Veracruz, Mexico

a r t i c l e i n f o
Article history:
Received 19 June 2013
Initial acceptance 16 July 2013
Final acceptance 21 November 2013
Available online 1 February 2014
MS. number: A13-00523R2
Keywords:
copula duration
cryptic choice
diet
Diptera
host use
life history
size
Tephritidae

Variance in female quality can result in males discriminating females either through precopulatory or via
postcopulatory choice. Male cryptic choice can be exhibited through copulation duration and strategic
ejaculation. Female quality can also affect sperm storage distribution. Here, we studied sperm allocation
in three tephritid ies with contrasting life histories and multiple sperm storage organs, Anastrepha
ludens (MX ies), Anastrepha obliqua (WI ies) and Anastrepha spatulata (AS ies). In addition, for MX
ies, we assessed the effect of female potential fecundity, size and diet on sperm allocation and the effect
of female age on sperm storage distribution. Sperm distribution during copulation differed between
species, which may be explained by their oviposition strategies. MX males mating with more fecund and
younger females had shorter copulation durations. Female quality also inuenced sperm storage patterns, as well-fed females stored more sperm in long-term sperm storage organs such as the ventral
receptacle compared to malnourished females. Detailed studies on how female quality affects sperm
transfer and sperm storage asymmetry will further our understanding on cryptic male choice.
2014 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

It is now widely acknowledged that females are not the only sex
that is choosy. Male choice has now been demonstrated in a variety
of animal species (Droney & Thaker, 2006; Engqvist & Suaer, 2001).
Males can choose females during both pre- and postcopulatory
processes (Edward & Chapman, 2011). In species where there is no
paternal care, ejaculate costs in spermatogenesis (Dewsbury, 1982;
Wedell, Gage, & Parker, 2002) and seminal uid production
(Hayward & Gillooly, 2011) are thought to favour cryptic male
choice. Thus, males are expected not only to discriminate among
females as mates, but also to modulate investments in copula
duration and ejaculates, depending on female quality
(Bonduriansky, 2001; Engqvist & Sauer, 2003; Galvani & Johnstone,
1998; Janicke, Kesselring, & Schrer, 2012; Kelly & Jennions, 2011;
Simmons, 2001).
Copula duration and sperm transfer can vary with male quality
parameters such as size, diet, previous mating experience and age
(e.g. Taylor & Yuval 1999; Teng & Zhang, 2009). Female traits such as
nutrition, age, size, mating status and fecundity can also modulate
copula duration and sperm transfer (Kelly & Jennions, 2011).
Indeed, recent studies have highlighted the importance of female

* Correspondence: D. Prez-Staples, INBIOTECA, Universidad Veracruzana, Av de


las Culturas Veracruzanas 101, Col. E. Zapata, C.P. 91090, Xalapa, Veracruz, Mexico.
E-mail address: diperez@uv.mx (D. Prez-Staples).

effects in understanding sperm storage patterns (Lpold et al.,


2013).
Resources allocated during copulation, such as sperm, are expected to vary if males can gauge female quality (Janicke et al.,
2012; Reinhold, Kurtz, & Engqvist, 2002). For example, Drosophila
melanogaster males deliver signicantly more sperm to mated,
large or young females than they do to virgins, small or old females
(Lpold, Manier, Ala-Honkola, Belote, & Pitnick, 2011). Other qualities that males could be assessing are female fecundity (e.g. Teng &
Zhang, 2009). Male evaluation of these parameters could continue
during copulation, inuencing the duration of the copula and
sperm allocation patterns.
Furthermore, for species with multiple and different types of
sperm storage organs, there can also be plasticity in how sperm
are distributed and stored within the female (Curril & LaMunyon,
2006; Nakahara & Tsubaki, 2007; Pitnick, Markow, & Spicer,
1999). Sperm allocation patterns could help females bias paternity and utilize sperm efciently (Fedina & Lewis, 2004;
Hellriegel & Bernasconi, 2000; Otronen, Reguera, & Ward,
1997), while in multiply mated females, sperm precedence may
depend on the interaction between male and female genotype
(reviewed in Pai & Bernasconi, 2008). However, an understanding of how female quality inuences plasticity in copulation
duration and sperm storage is still incipient and remains poorly
understood.

0003-3472/$38.00 2014 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.anbehav.2013.12.016

132

D. Prez-Staples et al. / Animal Behaviour 89 (2014) 131e139

Here, we sought to elucidate the dynamics of sperm storage in


tephritid ies by asking the following three questions. (1) What is
the relationship between copulation duration and sperm storage in
species with short or relatively long copula durations and contrasting life histories? (2) Can males adjust the amount of sperm
transferred according to female potential fecundity? (3) How do
female age, size and diet affect copulation duration and sperm
storage? For the rst question, we took advantage of a truly unique
opportunity to compare sperm dynamics in three species with
contrasting life histories and copulation durations, the Mexican
fruit y, Anastrepha ludens (hereafter MX ies), the West Indies fruit
y, Anastrepha obliqua (hereafter WI ies) and Anastrepha spatulata
(hereafter AS ies) (Table 1). WI ies are egg limited, meaning females cannot produce enough eggs to exploit all available hosts
(Daz-Fleischer & Aluja, 2003). In addition, they can be characterized as having a short life span, a very concentrated reproductive
window, high daily egg production (Liedo, Carey, Celedonio, &
Guillen, 1992), host-dependent oocyte maturation (Aluja, DazFleischer, Papaj, Lagunes, & Sivinski, 2001), a numerous but shortlived native ancestral host and a relatively long female sexual refractory period (Aluja, Rull, Sivinski, Trujillo, & Prez-Staples, 2009).
Thus, as this species needs to oviposit quickly, we predicted that
proportionally more sperm would be stored in short-term storage
organs such as the ventral receptacle. MX ies in contrast, have a
longer life span, a longer reproductive window and lower daily egg
production (Liedo et al., 1992), and oocyte maturation does not
depend on host presence (Aluja et al., 2001). Their native ancestral
hosts are available for longer periods and the female sexual refractory period is shorter (Aluja, Rull, Sivinski, et al., 2009; DazFleischer & Aluja, 2003). Thus, we expected proportionally more
sperm stored in long-term sperm storage organs such as the
spermathecae. For AS ies, we expected similar patterns to WI ies,
as their native hosts are only present for short periods. To address
male adjustment in the ejaculate and copulation duration according to female fecundity, we contrasted a polyphagous species (MX
ies) with a monophagous species (AS ies). We predicted that in
polyphagous species with many hosts available, males would
ejaculate more sperm compared to the monophagous species. In
addition, in MX ies, we evaluated the effect of female quality
parameters, such as age, size and diet on copula duration and
sperm storage. We predicted that more sperm would be transferred
to high-quality females. We expected malnourished females to
have fewer sperm than well-fed females immediately after copulation as a result of cryptic male choice discriminating against
malnourished females. Twelve hours after copulation ended, we
expected differential sperm storage patterns between females to be
more the result of female inuences than of male inuences. We
also predicted well-fed females to store proportionally more sperm
in the ventral receptacle (VR), where it would be readily available
for oviposition, compared to malnourished females, which may not
have energy reserves available to oviposit.
METHODS
Study Species
Copula duration varies widely among species of tephritid fruit
ies (Aluja, Piero, Jcome, Daz-Fleischer, & Sivinski, 2000). We
chose the WI, MX and AS fruit ies with a range of average copulation durations from 47 to 320 min and varying life history characteristics (Table 1). WI and AS ies are similar in their oviposition
strategies compared to MX ies. Even though WI is currently a
polyphagous species, its native and ancestral hosts are fruits of
Spondias (Anacardiaceae trees) that mature quickly and synchronously (Daz-Fleischer & Aluja, 2003). AS ies oviposit in

Table 1
Life history characteristics of three tephritid ies*
Natural history

Anastrepha
ludens
(MX y)

Anastrepha obliqua
(WI y)

Anastrepha
spatulata
(AS y)

Female sexual
maturation

Does not
depend on
host presence
10e15

Depends on host
presence

7e13

70

73.46.6

47.010.9

20553y

20

20

12

17

? (probably
monandrous)
?

1e40
Long
Stable
(2e3 months)
Polyphagous
51.72.2\,
71.96.6_
More in SP

1
Short
Highly ephemeral
(2e3 weeks)
Polyphagous
39.922.4\,
38.521.7_
More in VR

Sexual maturation
period (days)
Mean (SE) copulation
duration
Daily male mating
frequency
% Females remating
Average sexual
refractory period
(days)
Clutch size (eggs)
Reproductive period
Native host availability
Host breadth
Life expectancy
Predicted sperm
storage patterns

1
Shortz
Ephemeral
(1 month)z
Monophagous
?
More in VR

?: data not available. SP: spermathecae; VR: ventral receptacle.


* After Aluja, Herrera, et al. (2000); Aluja, Piero, et al. (2000); Aluja, Ordano, et al.
(2009); Aluja, Rull, Sivinski, et al. (2009) and Liedo et al. (1992) unless otherwise
noted.
y
Present study.
z
Lpez-Ortega et al. (2013).

guayabillo Schoepa schreberi that are available only for approximately 1 month during the year (Lpez-Ortega et al., 2013). Both WI
and AS ies need to oviposit quickly into their native host plants,
because they are available only for a brief time (Aluja, Herrera,
Lopez, & Sivinski, 2000; Daz-Fleischer & Aluja, 2003; LpezOrtega et al., 2013). In comparison, MX ies have more time to
oviposit into their native hosts, yellow chapote, Casimiroa greggii, as
the fruiting period is more prolonged (Thomas, 2012). MX females
produce eggs continuously, dumping eggs if no hosts are available
(Aluja, Birke, Guilln, Daz-Fleischer, & Nestel, 2011).
Sperm competition is limited in all three species. Both MX and
WI females have long sexual refractory periods after mating during
which females oviposit (Aluja, Ordano, et al., 2009; Table 1). For AS
ies, remating is unknown. All three species are synspermatogenic
(Boivin, Jacob, & Damiens, 2005), as males produce sperm
throughout their lives. Females have three spermathecae (one
doublet and a singlet) and a ventral receptacle (VR), which is the
site for short-term sperm storage and is also the fertilization
chamber (Fritz, 2004; Twig & Yuval, 2005). For MX ies, previous
studies have shown that females prefer to mate with older, sexually
experienced males (Prez-Staples, Martnez-Hernndez, & Aluja,
2010), while copulation duration varies with the degree of genetic relatedness (Aluja, Rull, Prez-Staples, Daz-Fleischer, &
Sivinski, 2009). In a related tephritid, females have considerable
control over copulation termination (Prez-Staples, Weldon,
Radhakrishnan, Prenter, & Taylor, 2010). Thus, we predicted that
parameters of female quality could further inuence copulation
duration and sperm storage.
Experimental Procedures
Wild MX ies were collected from infested Citrus auriantum
(cultivar Cucha) from Martinez de la Torre and Misantla in the State
of Veracruz, Mexico. Wild WI ies were collected from Spondias

D. Prez-Staples et al. / Animal Behaviour 89 (2014) 131e139

purpurea from the surroundings of Xalapa, Veracruz. Wild AS ies


were collected from infested Schoepa schreberi from Llano Grande,
Tejera and Vado de Ixtla, Veracruz. Infested fruit was placed in
containers with vermiculite. Pupae were collected from the sieved
vermiculite and placed in plastic containers with humid vermiculite until adults emerged. All adults were separated by sex and
species, placed in Plexiglas cages (30  30  30 cm) and fed strips
of paper soaked in a 1:3 solution of hydrolysed yeast protein (Yeast
Hydrolysate Enzymatic; ICN Biomedicals, Aurora, OH, U.S.A.) and
sucrose (J. T. Baker, Mallinckrodt Baker S.A. de C.V., Xalostoc, Estado
de Mexico, Mexico), unless otherwise noted. Water was provided as
soaked cotton balls.
All observations took place in Plexiglas cages (30  30  30 cm)
with food and water. Foliage was placed inside a glass bottle
plugged with cotton to provide leaves where individuals could
court. For all experiments, three sexually mature males and females
were placed inside cages, except for AS ies, where ve pairs were
allowed to mate, as this species does not readily mate in laboratory
cages with few individuals (G. Crdova-Garca, personal observation). Flies of the different species were used at ages corresponding
to when they mature sexually: MX pairs were 15e18 days old, WI
pairs were 15e20 days old and AS pairs were 71e97 days old, unless otherwise noted. Observations began according to the sexual
activity period for each species (Aluja, Piero, et al., 2000). MX y
observations began at 1500 hours, WI ies at 0900 hours and AS
ies at 1700 hours. Females were dissected immediately after
copulation except when otherwise noted. Mated females were
anaesthetized with ethyl acetate and dissected with a drop of
modied tris buffer (after Taylor, Kaspi, & Yuval, 2000) under a
stereomicroscope (Olympus SZX12). The VR and three spermathecae were placed in separate drops, crushed and covered with a
coverslip. Coverslips were secured with a clear drop of nail polish
and left to dry. Sperm were counted in 36 elds of vision for each
storage structure under a phase-contrast microscope (Nikon
Eclipse 50i). To estimate total sperm numbers, each sperm storage
structure was multiplied by 9.78 as a conversion factor (PrezStaples & Aluja, 2006). Males and females were digitally photographed under the stereomicroscope. Images were measured using
Image-Pro Plus software. As indicators of overall body size, we
measured head width, thorax length and hindtibia length.
Timing of sperm transfer
For all three species, we analysed the relationship between
sperm transfer and copulation duration. Pairs were placed in cages
as described above. Once copulation started, randomly chosen pairs
were interrupted by spraying them with ethyl chloride (Traumazol
R, Aguascalientes, Mexico). This quickly froze individuals that were
sacriced. For MX ies, pairs were randomly separated at 3, 20 or
40 min, or left to separate naturally. For WI ies, pairs were
randomly interrupted at 2, 10, 30 or 40 min, or left to separate
naturally. For AS ies, pairs were randomly separated at 3, 10, 20,
40, 76, 114 or 150 min, or left to separate naturally. As only a few
matings were observed for AS ies at 71 days of age, nonmating
pairs were given the opportunity to mate again from 86 to 97 days
of age. Observations were repeated until 15e25 females per
treatment were obtained. Females were frozen with ethyl chloride
and dissected immediately after copulations were interrupted or
ended naturally.
Sperm transfer and potential fecundity
For MX and AS ies whose copulations ended naturally (see
above), we also analysed the relationship between copula duration,
sperm transfer, oocyte quantity (potential fecundity) and female
and male size. Oocyte numbers of virgin females were also counted.

133

Oocytes were xed in sodium chloride (Solucin CS, Pisa S.A. De


C.V., Guadalajara, Mexico) for later quantication.
Sperm allocation according to female diet and size
For MX ies, we tested whether female diet affected sperm
transfer. Female diet was varied by providing females with either
continuous access to protein and sugar, consisting of 16 days of
sugar and hydrolysed yeast in a 1:3 ratio (well-fed females), or
limited access to protein, consisting of sugar and hydrolysed yeast
for the rst 8 days of age after emergence and the following 8 days
with sugar only (malnourished females). The latter treatment
assured that females could reach sexual maturity with developed
ovaries (Aluja et al., 2001) but were of a lower physiological condition compared to females with continuous access to hydrolysed
yeast. Males were given sugar and hydrolysed yeast in a 1:3 ratio ad
libitum. Three males were placed with either three well-fed females or three malnourished females. No food was placed in mating cages. Mating pairs were chosen randomly. Mated females were
anaesthetized with ethyl acetate and dissected immediately after
copulation. Mated males and females were also anaesthetized,
photographed and measured.
Sperm use through time
Female diet was manipulated as above, except that after copulation ended, females were returned to their respective precopulation diet (well-fed or malnourished). The following morning
(approximately 12 h later), pairs were anaesthetized in a sealed
container with ethyl acetate. Females were dissected to quantify
sperm numbers. Mated males and females were photographed and
measured.
Sperm storage according to female age
For MX ies, we further tested the effect of female age and diet
on sperm storage 12 h after mating. Young females of 13 days of age
and old females of 35 days of age were placed separately in
observation cages with 20-day-old males. Mating pairs were
randomly selected for each treatment. Copula duration was recorded (ending at night). The following morning, mated females were
anaesthetized with ethyl acetate and dissected. Sperm numbers
were counted as described above.
Statistical Analysis
Sperm storage asymmetry was calculated as the proportion of
sperm stored in the VR relative to the total sperm stored. To assess
how sperm were stored throughout copulation, linear regressions
were tted on copulation duration and total sperm stored or sperm
storage asymmetry for the three species. Species size and total
amount of sperm were analysed by ANOVA.
The effect of mating on potential fecundity (oocyte counts) was
analysed in an ANOVA by species. For mated females, the effects of
copulation duration and total sperm stored were analysed by
ANOVA on potential fecundity. The effect of female diet was analysed separately for copulation duration, total sperm transferred
and proportion of sperm stored in the VR by ANCOVA with female
and male tibia length and thorax used as independent variables.
Copula duration, sperm storage and the proportion of sperm in the
VR after 12 h were also each analysed by ANCOVA. Total sperm
stored was ln transformed, and the proportion of sperm stored in
the VR was arcsine transformed to meet normality assumptions.
Two outliers were deleted from the proportion of sperm stored in
the VR (one from the well-fed group and one from the malnourished group). Backtransformed least square means  SE are presented after controlling for other factors in the model. Age effects
on copulation duration and sperm storage were analysed by

134

D. Prez-Staples et al. / Animal Behaviour 89 (2014) 131e139

ManneWhitney U tests, because data did not conform to assumptions of parametric models. Predictors are presented as medians and percentiles.
RESULTS
Sperm Transfer and Copulation Duration
There was a positive linear relationship between copulation
duration and total sperm transfer for each of the three species (MX
ies: R2 0.64, F1,98 175.57, N 100, P < 0.001; WI ies:
R2 0.34, F1,116 59.45, N 118, P < 0.001; AS ies: R2 0.45,
F1,118 96.67, N 120, P < 0.001; Fig. 1). For MX ies, 10e577
sperm were found in 40% (N 10) of females 3 min after the
initiation of mating, mainly in the VR. At 10 min and beyond, all
other matings resulted in successful sperm transfer regardless of
whether they were interrupted or not. Similarly, for WI, at 3 min,
more than one sperm was counted in 45.83% (N 11) of females,
and one female had up to 2308 sperm, which were found almost
exclusively in the VR. At 10 min and beyond, all matings resulted in
sperm transfer and only two more females (interrupted at 10 min)
had no sperm. For AS ies, sperm was not observed for pairs
interrupted at 3 or 10 min, except for one female interrupted at
10 min, which had 29 sperm. At 20 min, sperm was not found in
33% (N 5) of females. At this time, the maximum amount of
sperm found was 1095, with approximately 60% already transferred
to the three spermathecae. At 40 min, an additional three females
had no sperm, and one full-length copulation, which lasted
229 min, also resulted in no sperm transferred.
Despite the fact that MX ies are bigger than WI ies and AS ies
are the smallest (e.g. female head size: F2,335 88.37, P < 0.001;
male head size: F2,335 42.92, P < 0.001), MX males did not
transfer more sperm to females compared to WI ies. AS ies had
the least amount of sperm stored (F2,60 5.62, P 0.006). Post hoc
Tukey HSD tests revealed signicant differences only in total
amount of sperm stored between AS ies and the other two species.
The rate of sperm transfer (mean sperm stored/mean full-length

A. ludens
A. obliqua

Potential Fecundity

10000
8000
6000
4000
2000
50

100
150
200
250
Copula duration (min)

300

350

Figure 1. Total sperm stored and copulation duration for three tephritid ies. Copulas
were interrupted at 3, 20 or 40 min for Anastrepha ludens (MX y; open circles, solid
line), at 2, 10 30 or 40 min for A. obliqua (WI y; crosses, intermittent line), at 3, 10, 20,
40, 76, 114 or 150 min for A. spatulata (AS y; lled circles, dotted line), or left to
separate naturally.

Proportion of sperm in ventral receptacle

12000
Total sperm

The proportion of sperm stored in the VR for full-length copulations varied signicantly with species (F2,58 17.90, P < 0.001)
but not with copula duration (F1,58 0.20, P 0.65). For copulations that were not interrupted, more sperm were stored in the VR
of WI ies (mean  SE 0.64  0.05, N 23), less so for AS ies
(0.40  0.07, N 14), and even less for MX ies (0.26  0.04,
N 25). For WI and AS ies, there was no signicant relationship
between the proportion of sperm stored in the VR and copulation
duration (WI ies: R2 0.001, F1,101 0.12, N 103, P 0.73; AS
ies: R2 0.005, F1,80 0.38, N 82, P > 0.54), whereas for MX
ies, there was a signicant negative relationship between the
proportion of sperm in the VR and copulation duration (R2 0.24,
F1,83 26.66, N 85, P < 0.001). That is, for MX females, sperm
were initially stored in the VR. As the copulation progressed, sperm
were increasingly stored in the spermathecae, until eventually
there were few sperm left in the VR at the end of the copulation.
This relationship did not hold for the other two species (Fig. 2).

A. spatulata

14000

0
0

Asymmetry in Sperm Storage

There were signicant differences in the potential fecundity


(oocyte counts) for MX and AS ies (F1,58 5.16, P 0.027). MX
females had more oocytes than AS ies (least square means  SE:

18000
16000

copulation duration) was faster in MX ies, 135.55 sperm were


transferred/min. WI ies stored 124.30 sperm/min, while AS ies
had only 16.53 sperm/min, consistent with a longer copulation
duration. For MX and WI females, the sperm transfer rate was
constant except for the early minutes of copulation (MX ies:
F2,68 40.87, P < 0.001; WI ies: F3,87 7.77, P < 0.001). For both
species, post hoc Tukey HSD tests revealed signicant differences
only between the rst 3 min and the remainder of copulation. No
differences were found in the sperm transfer rate during the
remainder of copulation for both species. For AS ies, the sperm
transfer rate also differed between interruptions (F6,94 20.34,
P < 0.001). Tukey post hoc tests revealed differences between the
rst interruptions (3, 10 and 20 min) and the rest of the copulation
but no differences between the interruptions after the rst 20 min
of copulation (i.e. 40, 76, 114 and 150 min).

1
A. ludens
A. obliqua
A. spatulata

0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0

50

100
150
200
250
Copula duration (min)

300

350

Figure 2. Proportion of sperm stored in the ventral receptacle of three tephritid ies
according to copulation duration. Copulas were interrupted at 3, 20 or 40 min for
Anastrepha ludens (MX y; open circles, solid line), at 2, 10, 30 or 40 min for A. obliqua
(WI y; crosses, intermittent line), at 3, 10, 20, 40, 76, 114 or 150 min for A. spatulata
(AS y; lled circles, dotted line), or left to separate naturally.

D. Prez-Staples et al. / Animal Behaviour 89 (2014) 131e139

56.90  7.05 versus 34.23  7.05, respectively). The intraspecic


variance in oocyte counts ranged from 0 to 134 oocytes for MX ies
and from 0 to 113 for AS ies.
Differences between species in potential fecundity were also
evident according to their mating status. For AS ies, mated females
had more oocytes than did virgin females (F1,28 6.75, P 0.015;
least square mean  SE: mated females: 46.13  6.47; virgin females: 22.33  6.47). There was no signicant difference in oocyte
amount for mated or virgin MX females (F1,28 0.15, P 0.70).
For mated MX females, there was a signicant relationship between potential fecundity and copulation duration (R2 0.30,
F1,12 5.02, P 0.04). Females with more oocytes had shorter
copulations (Fig. 3), yet these females did not store more sperm
(F1,12 1.27, P 0.28). Also, for these females, there was no relationship between potential fecundity and size (female head:
F1,11 0.01, P 0.92; thorax: F1,11 0.06, P 0.94; hindtibia length:
F1,11 0.52, P 0.48). For AS ies, there was no signicant relationship between potential fecundity and copulation duration
(R2 0.16, F1,12 1.45, P 0.25) or sperm storage (F1,12 0.99,
P 0.34). Also, there was no relationship between fecundity and
female size (female head: F1,11 0.33, P 0.57; thorax: F1,11 1.92,
P 0.19; hindtibia length: F1,11 0.66, P 0.43). There was no
relationship between potential fecundity and proportion of sperm
in the VR for either species (MX ies: R2 0.02, F1,13 0.24,
P 0.63; AS ies: R2 < 0.001, F1,13 0.002, P 0.96).
Female Diet
For MX ies, there was no signicant effect of female diet on
copula duration (F1,22 0.43, P 0.52). No other parameter of female or male size or their interaction had a signicant effect on
copula duration (female thorax: F1,22 0.27, P 0.61; female
hindtibia: F1,22 0.21, P 0.65; male thorax: F1,22 1.89, P 0.18;
male hindtibia: F1,22 0.06, P 0.81; female  male thorax:
F1,22 1.18, P 0.29; female  male tibia: F1,22 0.46, P 0.50).
Female diet did not inuence the amount of sperm transferred and
stored immediately after copulation (F1,22 0.07, P 0.81). No
other parameter of female or male size or their interaction had a
signicant effect on total sperm transferred (female thorax:
F1,22 0.57, P 0.45; female hindtibia: F1,22 0.13, P 0.72; male

90

Copula duration (min)

80

135

thorax: F1,22 3.31, P 0.08; male hindtibia: F1,22 0.25, P 0.62;


female  male thorax: F1,22 0.39, P 0.54; female  male tibia:
F1,22 2.46, P 0.13). There was no relationship between female
diet and the proportion of sperm stored in the VR (F1,19 1.22,
P 0.28). No other female or male size parameter inuenced sperm
transferred to the VR (female thorax: F1,19 2.39, P 0.14; female
hindtibia: F1,19 0.00, P 1.00; male thorax: F1,19 2.42, P 0.14;
male hindtibia: F1,19 1.93, P 0.18; female  male thorax:
F1,19 0.10, P 0.75; female  male tibia: F1,19 0.05, P 0.83).
Sperm Use through Time
For MX ies dissected 12 h after mating, there was no signicant
effect of female diet on copulation duration (F1,52 1.82, P 0.18).
However, in this case, the interaction between female and male
head length (F1,52 12.25, P 0.001), female and male thorax size
(F1,52 5.69, P 0.02) and male head length (F1,52 5.19, P 0.03)
had a signicant positive effect on copulation duration. Male thorax
(F1,52 7.58, P 0.007) had a signicant negative relationship,
whereas female head length (F1,52 0.05, P 0.82) and female
thorax (F1,52 0.87, P 0.03) had no effect.
Irrespective of female dietary treatment, more sperm were
stored immediately after mating than 12 h later (F1,58 6.05,
P 0.02). Females stored a mean of 5801  568 immediately after
mating compared to 3825  568 stored 12 h later. Total sperm
stored (ln transformed) did vary with female diet 12 h after mating
was completed (Table 2). Well-fed females stored more sperm
(backtransformed least square mean  SE: 3545.54  10.95)
compared to malnourished females (1220.54  12.32). The proportion (arcsine transformed) of sperm stored in the VR also varied
with female diet (Table 2). Backtransformed least square means
revealed that well-fed females stored proportionally fewer sperm
in the VR (0.18  0.0001) compared to malnourished females
(0.30  0.004).
Female Age
For MX ies, copula duration was longer for older females
(median  25the75th interquartile range) (47  32e57 min)
compared to younger females (31  26e41 min) (ManneWhitney
U test: U 56, N1 N2 30, P 0.021). Young females stored a
median of 2445 (25the75th percentile: 1633e4274) sperm 12 h
after copulation, whereas older females stored a median of 3912
(2973e7687) sperm. However, this difference was not statistically
signicant (U 67, N1 N2 30, P 0.062). There were no signicant differences in the proportion of sperm stored in the VR by
females of different ages (U 69, N1 N2 30, P 0.074).

70
Table 2
Effects of female diet, female size and male size on total sperm stored (ln transformed) and proportion of sperm stored (arcsine transformed) in the ventral
receptacle (VR) of Anastrepha ludens (MX females)

60

50

Variable

Total sperm stored


F1,50

F1,48

Female diet
Female thorax
Male thorax
Female head
Male head
Femalemale thorax
Femalemale head

14.96
0.51
0.02
1.05
0.07
1.14
0.89

<0.003
0.48
0.88
0.31
0.79
0.35
0.66

5.44
0.27
0.001
1.08
0.96
0.05
1.07

0.024
0.61
0.97
0.30
0.33
0.82
0.31

40

20

40

60
80
No. oocytes

100

120

140

Figure 3. Oocyte load (number of oocytes) and copulation duration for Anastrepha
ludens (MX y) females.

Signicant values are shown in bold.

Proportion in the VR

136

D. Prez-Staples et al. / Animal Behaviour 89 (2014) 131e139

DISCUSSION
A positive relationship between copulation duration and total
sperm was found for all three species. Even though AS ies had
much longer copulations, males transferred fewer sperm than
either MX or WI ies, consistent with the lower potential fecundity
of AS ies. The distribution and fate of the male ejaculate within the
females sperm storage organs also differed between species. We
suggest that differences in their life history parameters, and in their
oviposition strategies in particular, account for these interspecic
differences.
Sperm Storage and Copulation Duration
Differences in sperm transfer and storage appear to be related to
the contrasting life histories and copulation durations of the three
species. MX ies did not transfer more sperm than WI ies, despite
being bigger. Rather, their sperm transfer rate was faster. For both
MX and WI ies, sperm were found in the VR within 3 min of
mating. Sperm transfer rate for both interrupted and uninterrupted
copulations was constant after the initial 3 min for both MX and WI
ies, consistent with previous studies for MX ies (Novelo-Rincn,
Montoya, Hernndez-Ortz, Liedo, & Toledo, 2009). In contrast,
sperm transfer in AS ies was slower, as no sperm were found
within the rst 20 min of copulation, consistent with a longer
copulation, which can last more than 5 h. Sperm transfer rate for AS
ies was linear but only after the initial 20 min. Males may also
ejaculate other substances in the seminal uid, such as accessory
gland products (AGPs), even when no sperm transfer takes place,
and/or engage in copulatory courtship. The transfer rate of AGPs
remains to be studied. In AS ies, copulations were recorded with
no sperm storage, indicating that male infertility may be common,
as occurs in wild Anastrepha fraterculus and other tephritids
(Abraham, Goane, Cladera, & Vera, 2011; Prez-Staples, Shelly, &
Yuval, 2013). All males were virgin and sexually mature, and thus
could not have been sperm depleted.
For all species, during the rst minutes of copulation, most
sperm were found in the VR, suggesting that the VR is lled rst
and then the spermathecae (Fritz, 2004; Prez-Staples, Harmer, &
Taylor, 2007; Twig & Yuval, 2005). Likewise, in Drosophila melanogaster (but not D. simulans or D. mauritiana), the seminal receptacle is lled rst and then the spermathecae (Manier et al., 2013).
In MX ies, the deepest penetration of the male genitalia is to the
anterior end of the bursa, with the male ejaculating at the base of
the spermathecal ducts (Briceo, Orozco, Quintero, Hanson, &
Hernndez, 2011). Thus, sperm still need to migrate to the VR and
then the spermathecae, allowing the female to inuence sperm
storage patterns (Bangham, Chapman, Smith, & Partridge, 2003;
Bussire, Demont, Pemberton, Hall, & Ward, 2010; Eberhard,
1996; Fedina, 2007; Fritz, 2004; Hellriegel & Bernasconi, 2000;
Siva-Jothy & Hooper, 1996). Furthermore, sperm storage asymmetry differed between species. More sperm were found in the VR of
WI females than in the VR of females of the other two species (see
below). The VR seems to be an important sperm storage organ for
the cariby (Anastrepha suspensa), and the WI y, but less so for the
medy (Ceratitis capitata), the Q-y (Bactrocera tryoni) and the
South American y (A. fraterculus) (Abraham et al., 2011; Fritz,
2004; Prez-Staples et al., 2007; Prez-Staples & Aluja, 2006;
Twig & Yuval, 2005).
For MX females, there was a decrease in the proportion of sperm
stored in the VR during copulation as sperm migrated almost
entirely to the spermathecae by the end of copulation, whereas AS
and WI females had similar amounts of sperm stored in the VR
throughout copulation. This disparity in sperm storage asymmetry
between species can perhaps be explained by host use and

oviposition strategies. Both AS and WI ies deposit single eggs in


ephemeral native hosts (Schoepa schreberi and Spondias spp.,
respectively). AS ies have unlimited fruit available but only for a
brief period during the year (Lpez-Ortega et al., 2013). WI ies do
not have enough eggs to oviposit all available hosts (Daz-Fleischer
& Aluja, 2003), which suggests that they would need a readily
available source of sperm from the VR to oviposit quickly (PrezStaples & Aluja, 2006). The greater amount of sperm in the VR of
WI ies compared to AS ies could also reect this need to oviposit
quickly. AS ies are most abundant when there are 10 000e17 000
fruits available (Lpez-Ortega et al., 2013), and WI ies have
approximately 2500e20 000 fruits maturing simultaneously on a
single tree during 2e4 weeks. In contrast, the native hosts of MX
ies, Casimiroa greggii and Casimiroa edulis, are available for months
at a time (Daz-Fleischer & Aluja, 2003; Thomas, 2012). Thus, MX
females store more sperm in long-term storage organs, such as the
three spermathecae, whereas AS and WI ies would probably
benet from keeping a proportion of sperm stored in the VR ready
for fertilization.
Female Quality
The greater the variance in female quality, the more prudent
males should be in sperm allocation (Galvani & Johnstone, 1998;
Wedell et al., 2002). However, the strength of male cryptic choice
will be dependent on whether males can gauge differences in female condition (Parker & Pizzari, 2010). Here, copula duration was
shorter for males mating with more fecund females, yet males did
not allocate more sperm to more fecund, bigger or well-fed females.
We suggest that copula duration may be under considerable female
control, and males may not be able to distinguish between females
of different quality. Alternatively, males may not need to be prudent
in sperm allocation if they are not sperm limited (Prez-Staples &
Aluja, 2006; Radhakrishnan et al., 2009).
Fecundity
Higher sperm allocation is predicted for males mating with
more fecund females (Parker & Pizzari, 2010). Indeed, in many
species, males choose females based on phenotypic indicators of
fecundity, such as body size, that can be assessed directly through
visual or tactile mechanisms (e.g. Andersson, 1994; Bonduriansky,
2001; Jeswiet, Lee-Jenkins, & Godin, 2012). In other insects, the
width of the abdomen (or fatness) has been correlated with
ovarian load (e.g. Maxwell, Gallego, & Barry, 2010; Valera &
Zdkov, 2012). Certainly in tephritids, the complex internal and
external interaction between male and female genitalia as well as
leg rubbing and male proboscis contact with the female thorax
should offer the male ample opportunity to assess female phenotype (Briceo et al., 2011). However, contrary to predictions, it
seems that both MX and AS males did not adequately gauge female
potential fecundity, because they did not transfer more sperm to
more fecund females. Selection for characters that uctuate
throughout the females life such as fatness can be weak and, in
addition, there may be strong selection to conceal fecundity indicators (Bonduriansky, 2001). Likewise, if there is no large variance in fecundity between females, then there should be weak
selection for sperm allocation strategies (Reinhold et al., 2002). Yet,
for both species, there was large variance in potential fecundity, as
female oocyte counts ranged from 0 to more than 100. More oocytes were found in mated AS ies than in randomly selected virgin
females. Females that were prone to mate could have had more
oocytes, or alternatively, mating could have stimulated oogenesis
through AGPs transferred in the ejaculate (e.g. Heifetz, Lung,

D. Prez-Staples et al. / Animal Behaviour 89 (2014) 131e139

Frongillo, & Wolfner, 2000; Heifetz, Vandenberg, Cohn, & Wolfner,


2005).
Female Size
Female size is often a factor that determines strategic ejaculation (Rodgers, Reaka, & Hines, 2011; Ward, 1993; but see MagallnGayn, Briones-Fourzn, & Lozano-lvarez, 2011). Males will
benet from allocating more sperm to larger females if larger females are more fecund or have higher viability. In our study, female
size was not a consistent predictor of sperm allocation, storage or
copulation duration. Also, we found no relationship between female size and fecundity, although this may be due to small sample
sizes. In one experiment we found that the interaction between
female and male head and thorax size affected copula duration.
Male thorax length and face width have been identied as targets of
precopulatory sexual selection (Rodriguero, Vera, Rial, Cayol, &
Vilardi, 2002; Sciurano et al., 2007), whereas male and female
wing size have been correlated with sperm stores in medies and
Q-ies (Prez-Staples et al., 2007; Taylor & Yuval, 1999; but see
Aquino & Joachim-Bravo, 2013). Thus, the importance of male and
female size on male cryptic choice clearly needs to be investigated
further.
Female Diet
More sperm were found in well-fed females than in malnourished females 12 h after mating. However, as there were no differences in sperm stored between females of different diet regimes
immediately after mating, sperm storage patterns appeared to be
more the result of female manipulation of the ejaculate than of
males ejaculating more sperm into well-fed females. One possibility
is that malnourished females might oviposit eggs more quickly,
resulting in less sperm being stored. In MX ies, there is no egg
reabsorption and females dump eggs when no hosts are available
(Aluja et al., 2011). However, malnourished females have lower rates
of egg production and egg dumping than well-fed females (Aluja
et al., 2011). Thus, rather than more oviposition by malnourished
females, it seems more likely that the difference in sperm stored
between females of different dietary regimes could be attributed to
sperm ejection after copulation. Female D. melanogaster have the
ability to remove sperm (e.g. Radhakrishnan & Fedorka, 2012) and
can do so 2e5 h after mating (Lpold et al. 2013; Manier et al. 2010).
Well-fed females may dump nonfertile eggs but keep sperm reserves for when hosts become available. The inuence of female
quality on sperm dumping remains to be studied.
Contrary to what we expected, well-fed females also stored
proportionally more sperm in the spermathecae than did
malnourished females, which stored more sperm in the VR.
Malnourished females may need to use sperm stores immediately
instead of storing sperm in longer-term storage sites. Long-term
sperm storage may be advantageous, as it decreases the risks and
costs of nding and mating with further mates and may promote
sperm competition if sperm from different males can be maintained (Crudgington & Siva-Jothy, 2000; Roth & Reinhardt, 2003).
Female Age
Variance in female age can be an important determinant of male
investment in copulation and female inuence over sperm storage.
In MX ies, copulation duration was shorter for younger and more
fecund females, although there was no effect on sperm stored.
While copula duration may be the result of both male and female
interests, females have been shown to have considerable control
over the duration of the copula (Fritz, 2009; Prez-Staples, Weldon,

137

et al., 2010). Thus, females of better physiological condition may be


able to shorten copulation and dislodge the male, compared to
females of lower condition.
Males are expected to allocate more resources to older females if
the risk of sperm competition increases with female age. For
example, in the sepsid y, males copulate for longer and transfer
more sperm to older females (Martin & Hosken, 2002). However, in
tephritid les (present study) and in scorpionies, there is no evidence for increased sperm storage with female age (Engqvist &
Sauer, 2003). While female inuences cannot be ruled out, cryptic
parameters, such as age, may not be so easily assessed by males,
although, in D. melanogaster, males can gauge female age possibly
through cuticular hydrocarbons and tailor their ejaculate accordingly (Lpold et al., 2011).
CONCLUSION
This study furthers our understanding on pericopulation processes, which can vary widely between species according to their
life history characteristics. Thus, for example, oviposition strategies
should be taken into account when explaining sperm storage patterns. Our results highlight the degree to which males can effectively evaluate female condition and exert male cryptic choice, and
how female quality can inuence sperm storage distribution.
Further tailoring of the ejaculate, such as adjustment of the seminal
uid components, remains to be evaluated.
Acknowledgments
We thank Alberto Anzures Dadda and Filimn Hernndez Durn
for technical support. We thank Dinesh Rao, Francisco DazFleischer and two anonymous referees for comments on the
manuscript. Financial support was provided by the Mexican
Council for Science and Technology (Project CONACyT e SEP-2004C01-46846).
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