to
c, P
9, A
stal
ity, P
Journal of Insect Physiology 56 (2010) 1755–1762
m st
es h
ces
Contents lists available at ScienceDirect
Journal of Insec
journa l homepage: www.e ls1. Introduction
Copulation is a key step toward reproduction for both male and
female insects, and is also an arena where the success of each sex is
intimately linked to actions of the other. Although copula duration
and insemination are important for both sexes, much greater
attention has been paid to male influences, especially the effects of
male condition (Parker and Simmons, 2000; reviewed in Simmons,
2001). However, there is now growing awareness ofmechanisms by
which females can exert substantial influence over termination of
matings (and hence copula duration), sperm transport and sperm
storage patterns (Eberhard, 1996; Simmons et al., 1999;Hosken and
Ward, 2000; Andre´s and Cordero Rivera, 2000; Simmons, 2001;
Bangham et al., 2003; Arnqvist and Rowe, 2005; Arthur et al., 2008).
For example, females can promote termination of copulations
through physical resistance such as kicking, shaking and abdomen
curling (Thornhill, 1976; Crean and Gilburn, 1998; Blanckenhorn
et al., 2007;Mazzi et al., 2009). In the dung fly, Sepsis cynipsea, larger
females exert control over the initiation of mating, but males may
influence fertilization by internally injuring their mates (Ding and
Blanckenhorn, 2002). In other species, females that matemore than
once may bias paternity in favour of preferred males by differential
sperm storage and usage (Eberhard, 1996; BlochQazi et al., 1996;
Hellriegel and Bernasconi, 2000; Fedina and Lewis, 2004). Through
controlled contractions of the genital tract, females may influence
the storage, ejection and utilization of sperm (Rodrı´guez, 1994;
Co´rdoba-Aguilar, 1999; Hosken and Ward, 2000; Bangham et al.,
2003; Pizzari, 2004). In addition to the transfer of sperm, prolonged
copulations can have diverse additional functions such as mate
guarding and receptivity reduction (Alcock, 1994).
Males and females may disagree about the optimal duration of
pairing. Which sex ‘‘wins’’ conflicts over copula duration ultimate-
ly depends on each sex’s ability to influence the outcome. Female
decisions on whether to stay in copula or disengage can depend on
a number of factors such as predation risk, lost time to find
additional mates, foraging or oviposition opportunities, as well as
the potential for paternity manipulation. In the present study, we
investigate the role of female Queensland fruit flies (Bactrocera
tryoni [Froggatt], or ‘Q-fly’) in copula termination and sperm
storage, a species for which we have good prior knowledge of
reproductive processes such as sperm and male accessory gland
product transfer.
Keywords:
Mating termination
Copulation
Tephritidae
Bactrocera tryoni
Ventral receptacle
Spermathecae
Repeatability
Copula control
Sperm ejection
females that were intact, or had been incapacitated through decapitation or abdomen isolation. We
found that copulations were far longer when females had been incapacitated, indicating that constraints
imposed on copula duration by intact females had been relaxed. Repeatability of copula duration for
males was very low regardless of female treatment, and this is also consistent with strong female
influence. Number of sperm in the spermathecae was not influenced by female treatment, suggesting
that female abdominal ganglia control the transport of sperm to these long-term storage organs.
However, more sperm were found in the ventral receptacles of incapacitated females compared to intact
females. Overall, results implicate cephalic ganglia in regulation of copula duration and short-term
sperm storage in the ventral receptacle and abdominal ganglia in regulation of long-term sperm storage
in the spermathecae.
 2010 Elsevier Ltd. All rights reserved.
* Corresponding author at: INBIOTECA, Universidad Veracruzana, Apartado
Postal 250, C.P. 91090, Xalapa, Ver., Mexico. Tel.: +52 228 842 27 73;
fax: +52 228 842 27 73.
E-mail addresses: diperez@uv.mx, dianuxperez@gmail.com (D. Pe´rez-Staples).
0022-1910/$ – see front matter  2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jinsphys.2010.07.005Control of copula duration and sperm s
Diana Pe´rez-Staples a,b,*, Christopher W. Weldon a,
John Prenter a, Phillip W. Taylor a
aDepartment of Brain, Behaviour & Evolution, Macquarie University, Sydney, NSW 210
b Instituto de Biotecnologı´a y Ecologı´a Aplicada, Universidad Veracruzana, Apartado Po
cCentre for Invasion Biology, Department of Botany and Zoology, Stellenbosch Univers
dDepartment of Biology, University of Central Florida, Orlando, FL 32816, USA
A R T I C L E I N F O
Article history:
Received 26 May 2010
Received in revised form 12 July 2010
Accepted 14 July 2010
A B S T R A C T
Copula duration and sper
insects. Although both sex
investigate female influenrage by female Queensland fruit flies
reethi Radhakrishnan a,d,
ustralia
250, C.P. 91090, Xalapa, Ver., Mexico
rivate Bag X1, Matieland 7602, South Africa
orage patterns can directly or indirectly affect fitness of male and female
ave an interest in the outcome, research has tended to focus on males. To
, we compared copula duration and sperm storage of Queensland fruit fly
t Physiology
evier .com/ locate / j insphys

D. Pe´rez-Staples et al. / Journal of Insect Physiology 56 (2010) 1755–17621756Tephritid fruit flies have proven popular subjects for studies of
factors influencing copula duration, including male nutritional
status (Blay and Yuval, 1997; Taylor and Yuval, 1999; Field et al.,
1999;Taylor et al., 2000;Alujaet al., 2001; ShellyandKennely,2002;
Pe´rez-Staples and Aluja, 2004; Pe´rez-Staples et al., 2007a, 2008a),
male mating history (Pe´rez-Staples et al., 2008b; Radhakrishnan
etal., 2009),presenceof rivalsandhosts (Alonso-Pimentel andPapaj,
1996, 1999), source of population (Aluja et al., 2000), genetic
similarity (Aluja et al., 2009) and sperm transfer (Tsubaki and Sokei,
1988; Kuba and Ito, 1993; Pe´rez-Staples and Aluja, 2006). Yet,
despite the sizeable literatureontephritidmatingbehaviour,westill
know surprisingly little about the involvement of each sex in copula
termination and sperm storage.
Q-fly copulations are of highly variable duration, lasting on
average 1–2 h but sometimes lasting as long as 7 h (Pe´rez-Staples
et al., 2007b). Copula duration is longer for males fed on diets
including autolyzed yeast as a protein source in addition to sucrose
(Pe´rez-Staples et al., 2007a, 2008a), but female diet, male size and
female size do not influence copula duration (Pe´rez-Staples et al.,
2007a). Mated females and females injected with fluids extracted
from male accessory glands tend to have shorter copula durations
than virgin females (Radhakrishnan and Taylor, 2007, 2008),
suggesting either thatmales are able to detect femalemating status
and limit their copulations with mated females or that previously
mated females tend to terminate copulations sooner than virgins.
Once fully engaged in copulation, Q-flies remain still throughout
most of itsduration.However, toward the endof a copulation female
Q-flies commonly become quite active, walking about, while males
usually dismount soon after females become active. Females can
oviposit eggs the day after mating and 30–40% may remate the
followingnight (Harmeretal., 2006). Ina related tephritidBactrocera
cacuminata 23% of wild females have been reported to fertilize
offspring with the sperm of more than one male (Song et al., 2007).
Probability of a female remating is not influenced by sperm reserves
(Harmer et al., 2006; Radhakrishnan et al., 2009), thus there is ample
opportunity for sperm competition.
In addition to copula duration, there is also potential for direct
female involvement in sperm storage. Q-fly females store sperm in
two spermathecae and a ventral receptacle (Pe´rez-Staples et al.,
2007b). During tephritid copulation the distal portion of themale’s
intromittent organ (aedeagus) reaches the distal end of the
female’s copulatory bursa, but is unable to reach the spermathecae
(Eberhard and Pereira, 1995; Eberhard, 2005). Themale’s aedeagus
has three gonopores (openings) fromwhich sperm are ejected, one
opens into the ventral receptacle (=fertilization chamber, the site
of egg fertilization), while two openings face the spermathecal
ducts (Marchini et al., 2001). Thus after ejaculation, sperm still
needs to travel through the long spermathecal ducts to the
spermathecae, suggesting ample opportunity for femalemediation
of sperm storage. As in theMediterranean fruit fly, Ceratitis capitata
(Twig and Yuval, 2005), Q-fly spermathecae serve as long-term
sperm storage sites whereas sperm in the ventral receptacle are
stored for shorter periods (Pe´rez-Staples et al., 2007b). The ventral
receptacle and the spermathecae have contractile capabilities, and
the ventral receptacle is surrounded by layers of muscle fibers and
nerve synaptic boutons (Fritz, 2002). In a related tephritid, the
Caribbean fruit fly Anastrepha suspensa, a single abdominal
ganglion innervates the spermathecae and their ducts suggesting
female mediation of sperm transport (Fritz, 2002; Fritz and Turner,
2002). In a recent study, Fritz (2009) reported that matings of
decapitated A. suspensa females were longer than matings of intact
females but had similar sperm storage patterns, suggesting that
while cephalic control may be involved in copula termination of
this tephritid it is not required for regulation of sperm storage.
Here we carried out four experiments to investigate whether
female Q-flies play a role in copula termination and sperm storage.We limited female control in two ways: (1) by decapitating the
female, we severed all 6 pairs of cephalic ganglia (Armati, 1975) and
(2) by removing both head and thorax, we eliminated female
influences mediated by the thoracic ganglia (Schmitt, 1962). We
compared the copula duration and sperm storage patterns of intact
pairs with pairs in which female nervous control from either the
head (decapitated) or head and thorax (isolated abdomen) had been
surgically eliminated. If females influence copula duration we
predicted that copula duration would be longer in pairs where
females have been incapacitated either through abdomen isolation
ordecapitation. Ifneural regionsof theheador thoraxare involved in
long-term sperm storage, we predicted that transfer of sperm to the
spermathecaewill bedisrupted or reduced in incapacitated females.
We calculated repeatability of copula duration in males mated
to several different virgin intact females, decapitated females, or
isolated abdomens. If females are involved in mediation of copula
duration and males have characteristic duration targets con-
strained by females from reaching them, we expected longer
copulations and higher repeatability inmale copula durationwhen
males were mated with incapacitated females. Alternatively
stated, we expected shorter copula durations and lower repeat-
ability in intact pairs for which females would be able to constrain
male behaviour. If males alone control copula duration and have
characteristic duration targets, then we expected no differences in
copula duration among the treatments and high repeatability for
all. Finally, if females alone control copula duration then we
expected longer copulations when females are incapacitated and
low repeatability of male copula duration in all treatments.
2. Methods
2.1. General methods
Q-flies were obtained as pupae from the Fruit Fly Production
Facility located at Industry & Investment New South Wales’
Elizabeth Macarthur Agricultural Institute, Menangle, Australia.
Adult flies emerged in a laboratory at Macquarie University,
Sydney, Australia, andwere initially housed in 5 L cages containing
c. 200 flies. All cages were suppliedwithwater-soaked cottonwool
as well as separate dishes of dry granular sucrose and dry
hydrolyzed yeast enzymatic (MP Biomedicals, Aurora, OH, USA) as
food. All cages were maintained at 24–26 8C and 65–75% relative
humidity. A light:dark 12:12 photocycle was maintained in the
laboratory and flies also experienced a simulated dawn and dusk in
which a set of full spectrum incandescent lights sequentially
turned on and off over 1 h, respectively. Adult flies were separated
according to sex using an aspirator within 3 days after emerging.
No calling, courting, or mating was observed in cages prior to
separating the sexes. Flies from this source begin mating at
approximately 5 days of age, withmaximummating frequency not
observed until around 10 days of age (Pe´rez-Staples et al., 2008a).
2.2. Experiment 1: female mediation of copula duration
When flies were 14 days old, 90 pairs of virgin females and
males were placed in 70 mL clear plastic jars with mesh lids. Once
copulation had begun (the males’ aedeagus was inserted into the
females’ ovipositor sheath and he was not immediately dislodged),
each femalewas randomly assigned to one of three groups; ‘intact’,
‘decapitated’, or ‘isolated abdomen.’ While pairs were in copula,
and as soon as intromission of the male’s aedeagus into the
female’s ovipositor sheath was observed, we surgically excised the
head of females in the decapitated treatment and the abdomen of
females in the isolated abdomen treatment using a number 24
carbon steel ultra sharp surgical blade (Livingstone International
Pty, Ltd., Rosebery, NSW, Australia) attached to a metal rod, taking