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Animal Behaviour
journal homepage: www.elsevier.com/locate/anbehav
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
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
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
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
133
134
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.
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
18000
16000
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.
90
80
135
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
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.
Proportion in the VR
136
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
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138
139
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Ward, P. I. (1993). Females inuence sperm storage and use in the yellow dung
y Scathophaga stercoraria (L.). Behavioral Ecology and Sociobiology, 32, 313e
319.
Wedell, N., Gage, M. J. G., & Parker, G. A. (2002). Sperm competition, male prudence
and sperm-limited females. Trends in Ecology & Evolution, 17, 313e320.