Journal of Insect Physiology 57 (2011) 1501–1509
Contents lists available at SciVerse ScienceDirect
Journal of Insect Physiology
journal homepage: www.elsevier.com/locate/jinsphys
Effects of male nutrition on sperm storage and remating behavior in wild
and laboratory Anastrepha fraterculus (Diptera: Tephritidae) females
Solana Abraham a,b,⇑, Lucía Goane a,b, Jorge Cladera c, M. Teresa Vera a,b,1
a
Sección Zoología Agrícola, Estación Experimental Agroindustrial Obispo Colombres, Tucumán, Argentina
CONICET, Av. Rivadavia 1917 C1033AAJ CABA, Argentina
c
Instituto de Genética ‘‘E.A. Favret’’, INTA Castelar, Buenos Aires, Argentina
b
a r t i c l e
i n f o
Article history:
Received 17 June 2011
Received in revised form 31 July 2011
Accepted 2 August 2011
Available online 12 August 2011
Keywords:
Diet
Sperm storage
Remating
Refractory period
Tephritidae
a b s t r a c t
Male physiological condition can affect his ability to modulate female sexual receptivity. Thus, studying
this aspect can have biological and practical implications. Here, we examine how male nutritional status
affected the amount of sperm stored, remating rate and refractory period of the tephritid fruit fly Anastrepha fraterculus (Wiedemann) females. Both wild and laboratory flies were evaluated. We also examine
female sperm storage patterns. Experiments were carried out by manipulating male adult diet and exposing these males to virgin females. Females mated with differently treated males were either dissected to
count the amount of sperm stored or exposed to virgin males to determine remating rate and the length
of the refractory period. We found that male nutritional status affected the amount of the sperm stored
and the renewal of sexual receptivity in wild flies. For laboratory flies, male nutritional status affected the
length of the refractory period but not the amount of sperm stored by females. In addition, we report that
the ventral receptacle is not an important organ of sperm storage in this species. We conclude that male
nutritional condition influences the ability to modulate female sexual receptivity, possibly through a
combination of the quantity and quality of the ejaculate. From an applied perspective, providing males
with an enriched diet will likely result in increased efficacy of the sterile insect technique.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
One major challenge for male insects is that sperm transferred
during mating is stored in significant numbers and used to sire female offspring. In this respect, males may benefit if female receptivity is reduced after copulation. Many mechanisms are involved
in the reduction of female receptivity, and two of them are related
to male ejaculate. One effect associated with this physiological
change is triggered by the amount of sperm stored in female
spermathecae (Cunningham et al., 1971; Nakagawa et al., 1971;
Klowden, 2001). In such a case, female sexual receptivity returns
only when sperm stores are depleted (Whittier and Shelly, 1993;
Blay and Yuval, 1999; Gromko and Markow, 1993; Sakurai, 1998,
but see Steiner et al., 2008). The other is the effect produced by
the receptivity-inhibiting proteins secreted by the males’ accessory
glands (AGPs). They are transferred from the males to the females
during copulation (reviewed by Gillot, 2003 but also see Kuba and
⇑ Corresponding author. Present address: Cátedra de Terapéutica Vegetal,
Departamento de Sanidad Vegetal, Facultad de Agronomía y Zootecnia de la
Universidad Nacional de Tucumán, Florentino Ameghino s/n, El Manantial 4105,
Tucumán, Argentina. Tel.: +54 0381 4390040.
E-mail address: solanaabraham@yahoo.com.ar (S. Abraham).
1
Present address: Cátedra de Terapéutica Vegetal, Departamento de Sanidad
Vegetal de la Facultad de Agronomía y Zootecnia de la UNT, Tucumán, Argentina.
0022-1910/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jinsphys.2011.08.004
Ito, 1993; Fernández and Klowden, 1995; Harmer et al., 2006;
Radhakrishnan and Taylor, 2007; Himuro and Fujisaki, 2008;
Pérez-Staples et al., 2008a; Steiner et al., 2008; Yamane et al.,
2008a,b; Radhakrishnan et al., 2009; Sirot et al., 2008, 2009).
Receptivity renewal in this case, may not be linked to sperm reserves. Both of these mechanisms are likely to be affected by male
condition. If the quality and volume of the ejaculate is correlated
with the nutritional status of the male, it is expected that males
in good nutritional condition will better succeed in delaying the renewal of female receptivity in comparison with poorly nourished
males.
Fruit flies within the Tephritidae have emerged during the last
decades as good model systems in which to study male reproductive strategies in general and conditions that affect renewal of female receptivity in particular. This is mainly due to the wide use
of the sterile insect technique (SIT) (Knipling, 1955) to control
many fruit pest species within this family. To ensure the success
of the SIT, the sterile male must be able to mate with wild females,
transfer a suitable ejaculate in quantity and composition and inhibit the receptivity of their mates. A high remating rate and a fast
renewal of female receptivity can adversely affect the use of the
SIT, as a female could remate with a fertile male and sire viable offspring (Kraaijeveld and Chapman, 2004). In addition, laboratory
adaptation of wild strains, mass-rearing conditions, and the
1502
S. Abraham et al. / Journal of Insect Physiology 57 (2011) 1501–1509
number of generations a strain has been held in colonization may
produce certain detrimental changes associated with male sexual
competitiveness and related traits (Moreno et al., 1991; Lux
et al., 2002; Rull et al., 2005). Consequently, increasing our knowledge on factors that enhance male reproductive success will surely
contribute to a wider use of this environmentally friendly pest control technique.
The impact of protein feeding during the adult stage in male
reproductive parameters has widely been recognized among
several Tephritidae species. In Ceratitis capitata (Wiedemann),
males fed with protein achieve greater mating success (Shelly
and Kennelly, 2002), greater ability to court and copulate (Kaspi
and Yuval, 1999), greater probability of sperm transfer and storage
(Taylor and Yuval, 1999), and grater ability to inhibit female receptivity (Blay and Yuval, 1997; Yuval et al., 2002; Gavriel et al., 2009).
In Bactrocera tryoni (Froggatt), protein diet enhances male mating
success and copula duration (Prabhu et al., 2008; Pérez-Staples
et al., 2009), the probability of sperm storage, the amount of sperm
stored, and reduces the probability of female remating (Pérez-Staples et al., 2008b). In a study comprising four Anastrepha species,
Anastrepha obliqua (Macquart), Anastrepha serpentina (Wiedemann) and Anastrepha striata (Schiner) protein-fed males achieved
higher mating success than males fed sucrose, bird feces or an open
fruit (Aluja et al., 2001) while for Anastrepha ludens (Loew) adult
male diet showed no effect on male mating success (Aluja et al.,
2001, but see Aluja et al., 2008). Additionally, A. obliqua proteinfed males exhibited longer copula durations and induced longer
refractory periods in their mates than protein-deprived males
(Pérez-Staples et al., 2008a; Aluja et al., 2009). Although for Ragholetis pomonella (Walsh), food quality does not affect mating
behavior or the production of male gametes (Hendrichs et al.,
1992), overall evidence suggests that ingestion of protein at the
adult stage within tephritid fruit flies increases male sexual
competitiveness in parameters related to its mating and postcopulatory success. Yet the impact of different protein sources
and/or quality remains unexplored.
Other aspects that condition male reproductive success is the
pattern in which sperm is stored. Many insects have several sperm
storage organs and in particular, tephritid fruit flies have two or
three spermathecae and one ventral receptacle. This morphological
feature provides the conditions for sperm to be stored asymmetrically (i.e. not in evenly distributed numbers in each sperm storage
organ) and could thus serve to discriminate among ejaculates of
different males leading to cryptic female choice (Eberhard, 1996).
Asymmetry in sperm storage has been documented in all studies
that evaluated patterns of sperm storage in the Tephritidae (Yuval
et al., 1996; Taylor and Yuval, 1999; Taylor et al., 2000, 2001; Fritz,
2004; Harmer et al., 2006; Pérez-Staples and Aluja, 2006; PérezStaples et al., 2007, 2010), where one spermatheca exhibits higher
number of sperm than the other/s spermatheca/e. The ventral
receptacle, also known as the fertilization chamber, has been mentioned as a transit point of sperm which is replenished by sperm
from the spermathecae; and has been proposed as the site where
egg fertilization occurs (Twig and Yuval, 2005). Although this
makes this structure less relevant as a morphological feature that
allow cryptic female choice to occur, it is also mentioned as a storage organ of sperm whose relative importance seems to vary
according to the species within the Tephritidae (Fritz, 2004; Twig
and Yuval, 2005; Pérez-Staples and Aluja, 2006; Pérez-Staples
et al., 2007).
The South American fruit fly, Anastrepha fraterculus (Wiedemann), is a major pest of fruit trees (Norrbom, 2004) and is susceptible to being controlled by the SIT (Ortíz, 1999). In this species,
females can remate up to eight times throughout their life
(De Lima et al., 1994) and during the first month after sexual
maturation almost 50% of the females will remate with a refractory
period of approximately 15 days (Abraham et al., 2011a). In turn,
when females are not allowed to remate by mating interruption,
female fertility drops, suggesting that females could be looking
for an additional copulation to replenish their sperm supply (Abraham et al., 2011a). A. fraterculus females have three spermathecae
with an oval pear like form and an elongated ventral receptacle
with lobular papillae (Bartolucci et al., 2006). There are no records
on sperm storage patterns for this species, and little is known on
female post-copulatory behavior and factors that modulate them
such as the impact of adult diet on sperm storage and renewal of
female receptivity.
Here, we aimed to determine the impact of male nutritional status on the amount of sperm stored and the renewal of female
receptivity of A. fraterculus. Given that in previous studies fly rearing history affected remating behavior (Abraham et al., 2011a) we
tested wild and laboratory females. Additionally, we determined
the patterns of sperm storage and the importance of the ventral
receptacle as a sperm storage organ. Under the hypothesis that
the inclusion of protein in the adult male diet improves its sexual
performance, we predicted that females mated with sugar-fed
males would have shorter copula durations, exhibit higher remating rates, shorter refractory periods, and would store lower
amounts of sperm when compared to females mated with males
fed an enriched diet.
2. Methods
2.1. Insects and culture
Laboratory A. fraterculus adult flies were obtained from a colony
established at the Agricultural Zoology laboratories of the Estación
Experimental Agroindustrial Obispo Colombres, Tucumán, Argentina. This colony was initiated in 1997 with pupae obtained from
infested guavas, collected in the vicinity of Tafi Viejo, Tucumán
province (north-western Argentina). Rearing followed methods described by Jaldo et al. (2001) and Vera et al. (2007). Wild flies were
recovered from infested guavas collected at Horco Molle, Tucumán,
close to Tafí Viejo. Fruits were taken to the laboratory and placed in
15 L plastic trays with sand. Larvae migrated from the fruit to the
sand where they entered the pupal stage. After 10 days, the sand
was sieved and recovered pupae were placed in 10 L containers
at 25 ± 1 °C and 70 ± 10 RH until adult emergence.
On the day of emergence, flies were sorted by sex and were
transferred to 750 mL plastic containers in groups of 25 adults,
with water and food provided ad libitum. Females were fed with
a standard adult diet consisting of sugar (57.9%) (Ledesma S.A., Jujuy, Argentina), hydrolyzed yeast (14.5%) (Yeast Hydrolyzated
Enzymatic, MP BiomedicalsÒ), hydrolyzed corn (27.3%) (Gluten
Meal, ARCORÒ, Tucumán, Argentina), and vitamin E (0.3%) (ParafarmÒ, Buenos Aires, Argentina) (w/w) (Jaldo et al., 2001). Males
were fed with one of the following four diets: (1) sugar, (2) low
quality protein (sugar and brewer’s yeast, CALSAÒ, Tucumán,
Argentina, 3:1 ratio), (3) high quality protein (sugar and MP BiomedicalÒ hydrolyzed yeast, 3:1 ratio) or (4) the standard adult diet.
Both laboratory and wild flies were evaluated. In the case of wild
flies, diet (3) was not administered.
Laboratory flies were tested 14–16 days after adult emergence
and wild flies at the age of 21 days. This ensured that all individuals were sexually mature (Jaldo, 2001; Petit-Marty et al., 2004;
Jaldo et al., 2007).
2.2. Experimental procedures
On the day of testing, 80 males and 40 virgin females were
released into a plastic cage (12 L) with an artificial plastic branch
S. Abraham et al. / Journal of Insect Physiology 57 (2011) 1501–1509
inside, at 7:30–8:00 h. Released males were from a given origin
(laboratory males laboratory females; wild males wild females) and diet and therefore, couples were obtained under no
choice conditions. Cages were checked for copulating pairs at 10min intervals for 2 h after releasing the flies. This time was chosen
given that Argentine populations of A. fraterculus exhibit a narrow
period of mating activity early in the morning (Petit-Marty et al.,
2004; Vera et al., 2006). Copulating pairs were carefully coaxed
into test tubes (20 mL) which were then plugged and numbered.
The time at which the copulation was detected was recorded. Couples were checked every 5 min until copulation finished and this
time was recorded. After the end of copulation, one set of females
was randomly used to determine female remating rate and refractory period, another set was dissected for sperm counts.
Females taken to determine remating rate and duration of the
refractory period were kept singly in 750 mL plastic containers
with water and the standard adult diet. Despite the fact that the
use of small containers (750 mL) may have favored higher remating rates than those prevalent under natural conditions, our main
goal was to compare the effect of male nutritional condition after
the first mating on remating propensity. Two days after, they were
offered two sexually mature virgin males of the same strain as the
female, fed with the standard adult diet, for a period of 2 h at dawn.
For those females that remated, the unsuccessful male was removed from the container to prevent disturbance of the copulating
pair. The couples were allowed to complete copulation freely, and
once they separated, the male and the female were discarded from
the experiment. If no copulation occurred after the 2-h period, the
two males were removed from the container. This procedure was
repeated every 48 h. Females that did not remate were offered an
oviposition substrate consisting in an agar (30 g of agar in
500 mL of water) cylindrical slice (3 cm diameter and 2 cm high)
wrapped in parafilmÒ that was replaced every 48 h. For each fly
origin and male nutritional status three cages were set up. The trial
lasted 32 days with a total of 16 observation dates.
The other set of females was dissected between 2 and 10 h from
the end of copulation under a dissecting microscope (Leica MZ 95)
using a 60 magnification, following Taylor et al. (2000) and Twig
and Yuval (2005). Reproductive tracts were removed and placed
over a slide with a 50 lL drop of sterilized water. Spermathecae
were dissected and placed separately on slides with 7 lL of sterilized water containing 0.1% of soap (TritonÒ). Each of the three
spermathecae was broken apart with fine forceps and a 3 lL drop
of acetic orcein was added to allow the staining of spermatozoa.
The drop was stirred quickly with entomological pins for 1 min.
A 18 18 mm coverslip was then placed on top of each of the storage organs and secured on each corner with a drop of transparent
nail polish. After 5 days spermatozoa were counted under a 400
magnification lens with a microscope (Leitz Laborlux 11). The
whole slide was covered by counting all spermatozoids in 250 randomly selected fields (10 rows by 25 columns), which corresponds
to 10.2% of the total area. To obtain the total amount of sperm for
each storage organ, a conversion factor of 9.82 was applied to the
sperm counted for each storage organ (Pérez-Staples and Aluja,
2006).
A separate set of laboratory females was used to determine the
amount of sperm stored in the ventral receptacle. Fifty couples of
laboratory flies fed with standard adult diet were allowed to mate
and females were dissected at each of the following different time
periods either before or after the end of copulation: 10–30 min
after the beginning of the copulation (interrupted), 0–30 min after
the end of copulation, 2–3 h after the end of copulation, 6–8 h after
the end of copulation and 24 h after the end of copulation. Each
storage organ (the ventral receptacle and the three spermathecae)
were dissected following the methodology described above and
the total amount of sperm stored was estimated.
1503
2.3. Statistical analysis
From the set of females used to determine remating rate, only
those females that remated or survived until the end of the trial
were considered for the analysis. Those that died during this period
but did not remate were not included. Remating rate was estimated as the percentage of females that copulated twice. We estimated a ‘‘short-term’’ remating rate at 48 h after the first
copulation and a ‘‘long-term’’ remating rate at the end of the trial
(32 days after the first copulation). The refractory period was estimated as the time, in days, between the first and the second copulation. The proportion of females with empty spermathecae was
estimated as the percentage of females that did not store sperm
in any of their spermathecae. Total amount of sperm stored was
estimated by adding the number of spermatozoids found in each
spermathecae.
Remating rate was compared within each fly strain by means of
v2-tests of independence. For a comparison among more than two
treatments, the sequential Bonferroni method was applied after
the v2-tests. Copula duration, refractory period, the relationship
between copula duration and refractory period, and amount of
sperm stored were compared within each fly strain by means of
a one-way ANOVA, where male diet was the independent variable.
Differences among means were determined by means of a Tukey
test. Non-parametric Kruskal–Wallis was applied when ANOVA
assumptions were not met and multiple comparisons were carried
out by means of Dunn’s test. The relationship between amount of
sperm stored and copula duration was determined with a Pearson’s correlation analysis. The proportion of females with empty
spermathecae was tested with a v2 of Homogeneity. Bias in the
quantity of sperm stored in the spermathecae was tested by v2
of Goodness of Fit. The expected ratio of sperm in each spermathecae was 1:1:1 under the assumption that the spermathecae are
filled with spermatozoa independently of one another. We interpreted this distribution as ‘‘one instance for the spermatozoa to
choose’’ the way toward any of the three spermathecae, hereafter
named as ‘‘one step’’. Another possibility is that sperm has two instances to ‘‘choose’’, where the sperm is equally divided twice: first
between the single spermatheca and the paired spermathecae
(which may share a common opening in the vagina) and then between the two paired spermathecae (when this common opening
divides); this 2:1:1 (single:double:double) expected proportion
will be referred as ‘‘two steps’’. A significant deviation from these
ratios would indicate differential filling among the three spermahecae and thus non-random distribution (Fritz, 2004). All analyses were done with InfoStat (2009).
3. Results
3.1. Wild flies
3.1.1. Copula duration
Male diet affected copula duration (Kruskal Wallis test, H = 6.55,
df = 2, 169, P = 0.037). Copulation was shorter for pairs involving
sugar-fed males than for those involving males fed an enriched
diet, irrespective its quality (Table 1).
3.1.2. Remating rate
Male diet also affected the number of females that remated in
the short-term (v2-tests, v2 = 24.75, df = 2, P < 0.0001) and the
long-term (v2 = 5.96, df = 2, P = 0.050). In the short-term, females
mated with sugar-fed males (N = 53) remated in higher proportions. In the long-term, females mated with sugar-fed males presented a similar remating rate than females mated with brewer’s
yeast fed males (N = 55) (P = 0.477 after Bonferroni’s correction)
S. Abraham et al. / Journal of Insect Physiology 57 (2011) 1501–1509
Table 1
Copula duration (hh:mm) (median with upper and lower quartiles) of wild and
laboratory Anastrepha fraterculus pairs involving males fed with: sugar, low quality
protein (BY), standard diet (SD) or high quality protein (MP). N = sample size.
Male diet
Copula duration
Wild pairs
Sugar
BY
SD
MP
Laboratory pairs
25%
Med.
75%
N
25%
Med.
75%
N
00:16
00:24
00:33
–
00:33 a
00:47 b
00:40 b
00:51
00:56
01:00
53
55
61
00:20
00:41
00:30
00:31
00:34 a
00:59 c
00:46 b
00:51 b
00:50
01:10
00:57
00:59
83
60
77
46
Within each column, medians followed by a different letter were statistically different (Dunn’s test, P < 0.05).
Refractory period duration (days)
1504
20
b
b
16
46
46
12
a
8
49
4
0
Brewer’s yeast
Standard diet
Male diet
100
Fig. 2. Refractory period for wild Anastrepha fraterculus females mated with males
fed with different diets (mean + SE). Same letters over bars show no significant
differences (ANOVA analysis, followed by Tukey test, P > 0.05). Numbers inside bars
indicate sample size.
80
60
40
20
0
2
4
6
8 10 12 14 16 18 20 22 24 26 28 30
Days after first copulation
Fig. 1. Cumulative remating curves for wild Anastrepha fraterculus females mated
with sugar-fed males (h), mated with brewer’s yeast fed males (d) and mated with
standard diet fed males (j).
Refractory period duration (days)
Cumulative percent of remated females
Sugar
and a higher remating rate than females mated with standard diet
fed males (N = 61) (P = 0.045 after Bonferroni’s correction). Remating curves are shown in Fig. 1.
3.1.4. Proportion of females with empty spermathecae
Male diet affected the proportion of females with empty spermathecae (v2-tests, v2 = 9.98, df = 2, P = 0.007). Eight out of 25 females (32%) mated with sugar-fed males and three of 24 females
(12.5%) mated with brewer’s yeast fed males had no spermatozoa
in any of their spermathecae. There were no copulations in which
the spermathecae were found to be empty for females mated with
standard diet fed males. Multiple comparisons revealed differences
between females mated with sugar-fed males versus standard diet
fed males (v2-tests, v2 = 9.18, df = 1, P = 0.007).
3.1.5. Amount of sperm stored
The amount of sperm stored was strongly affected by male diet.
(Kruskal Wallis test, H = 27.55, df = 2, 73, P < 0.001) (Fig. 4). There
was no correlation between copula duration and amount of sperm
stored by females (Pearson’s r = 0.16, P = 0.170).
3.1.6. Asymmetry in sperm storage
The ratio of sperm in the three spermathecae did not fit in
any of the two randomly expected distributions (i.e. 1:1:1 or
24
b
a
a
0-15
16-30
b
b
31-45
46-60
16
8
0
>61
Intervals of mating duration (min)
Fig. 3. Female refractory period at different intervals of mating duration for wild
Anastrepha fraterculus females (median with upper and lower quartiles). Same
letters on each boxes show no significant differences (Kruskal–Wallis analysis,
followed by Dunn’s test, P > 0.05). Numbers inside boxes indicate sample size.
1600
Number of sperm stored
3.1.3. Refractory period
The refractory period was affected by male diet (ANOVA,
F = 10.72, df = 2, 138, P < 0.001) (Fig. 2). The refractory period of females significantly increased as mating duration increased beyond
30-min (Fig. 3; Kruskal–Wallis test, P < 0.001).
32
c
1200
b
800
24
400
a
24
0
25
Sugar
Brewer’s yeast
Standard diet
Male diet
Fig. 4. Amount of sperm stored in the three spermathecae for wild Anastrepha
fraterculus females mated with males fed with different diets (median with upper
and lower quartiles). Same letters over boxes show no significant differences
(Kruskal–Wallis analysis, followed by Dunn’s test, P > 0.05). Numbers inside boxes
indicate sample size.
1505
S. Abraham et al. / Journal of Insect Physiology 57 (2011) 1501–1509
Table 2
Total amount of sperm stored (median with upper and lower quartiles) and amount of sperm stored in each spermathecae (in percentage) in wild
Anastrepha fraterculus females mated with male fed with: sugar, low quality protein (BY) or standard diet (SD). N = sample size.
Male diet
Total amount of sperm stored
Double spermathecae
Double spermathecae
Med.
75%
N
%
%
%
0
58.9
176.7
19.6
191.5
402.6
98.2
422.2
726.7
25
24
24
18.7
27.5
35.8
42.0
47.6
48.2
7.3
12.4
16
3.2. Laboratory flies
1000
3.2.1. Copula duration
Copula duration was affected by male diet (Kruskal Wallis test,
H = 29.52; df = 3, 266, P < 0.001). Copulation was shorter for pairs
involving sugar-fed males than for those pairs involving males
fed an enriched diet (Table 1).
800
600
200
0
2 4 6
8 10 12 14 16 18 20 22 24 26 28 30
Days after first copulation
Fig. 5. Cumulative remating curves for laboratory Anastrepha fraterculus females
mated with sugar-fed males (h), brewer’s yeast fed males (d), MP hydrolyzed yeast
fed males (s) and standard diet fed males (j).
Refractory period duration (days)
2:1:1 relationships) (v2 test, P < 0.001 in all the cases). There was a
bias for storing more sperm in one of the two paired spermathecae
over sperm stored in the other paired spermatheca or in the single
one (Table 2). Interestingly, in those females that mated with sugar-fed males and brewer’s yeast fed males, the amount of sperm
stored among the three spermathecae adjusted to a 1:2:1 relationship (v2-tests, v2 = 1.05, P = 0.591 and v2 = 5.28, P = 0.071, for females mated with sugar-fed and brewer’s yeast fed males
respectively). In both cases, one of the paired spermatheca had half
of the total sperm stored and the other two had one quarter each.
Refractory period duration (days)
400
32
c
ab
a
ab
bc
0-15
16-30
31-45
46-60
24
16
8
0
>61
Intervals of mating duration (min)
Fig. 7. Female refractory period at different intervals of mating duration for
laboratory Anastrepha fraterculus females (median with upper and lower quartiles).
Same letters on each boxes show no significant differences (Kruskal–Wallis
analysis, followed by Dunn’s test, P > 0.05). Numbers inside boxes indicate sample
size.
2000
b
20
b
46
16
ab
a
12
63
37
70
8
Number of sperm stored
Cumulative percent of remated females
Sugar
BY
SD
Single spermathecae
25%
1500
1000
24
500
24
23
4
0
0
Sugar
Sugar
Brewer’s
yeast
MP
hydrolyzed
yeast
Standard
diet
Male diet
Fig. 6. Refractory period for laboratory Anastrepha fraterculus females mated with
males fed with different diets (mean + SE). Same letters over bars show no
significant differences (ANOVA analysis, followed by Tukey test, P > 0.05). Numbers
inside bars indicate sample size.
24
MP
Standard
Brewer’s
diet
yeast hydrolyzed
yeast
Male diet
Fig. 8. Amount of sperm stored in the three spermathecae for laboratory Anastrepha
fraterculus females mated with males fed with different diets (median with upper
and lower quartiles). Same letters over boxes show no significant differences
(Kruskal–Wallis analysis, followed by Dunn’s test, P > 0.05). Numbers inside boxes
indicate sample size.
1506
S. Abraham et al. / Journal of Insect Physiology 57 (2011) 1501–1509
Table 3
Total amount of sperm stored (median with upper and lower quartiles) and the amount of sperm stored in each spermathecae (in percentage) in
laboratory Anastrepha fraterculus females mated with male fed with: sugar, low quality protein (BY), high quality protein (MP) or standard diet (SD).
N = sample size.
Male diet
Sugar
BY
SD
MP
Number of sperm stored
1600
Single spermathecae
Double spermathecae
Double spermathecae
25%
Total amount of sperm stored
Med.
75%
N
%
%
%
29.5
98.2
29.5
196.4
93.3
373.2
589.2
525.4
628.5
491.0
923.1
962.4
24
23
24
24
25.2
25.2
23.5
34.3
54.5
49.6
52.9
42.6
11.9
16.6
15.3
14.6
3.2.6. Asymmetry in sperm storage
The ratio of sperm in the three spermathecae did not fit any of
the two expected random distributions (i.e. 1:1:1 or 2:1:1 relationships) (v2-tests, P < 0.001 in all the cases).There was a bias for storing more spermatozoa in one paired spermatheca over that stored
in the other paired spermatheca and in the single one (Table 3). For
those females that mated with brewer’s yeast fed males, the
amount of sperm stored in the three spermathecae fitted a 1:2:1
relationship (v2-tests, v2 = 5.96, df = 3, P = 0.051).
Spermathecae
Ventral receptacle
1200
800
400
3.3. Amount of sperm stored in the spermathecae and the ventral
receptacle of laboratory females
0
Interrupted 0-30 min
2-3 h
6-8 h
24 h
Time after copulation
Fig. 9. Amount of sperm stored in the three spermathecae and ventral receptacle
for laboratory Anastrepha fraterculus females at different dissection times (median
with upper and lower quartiles). Same letters on each boxes show no significant
differences (Kruskal–Wallis analysis, followed by Dunn’s test, P > 0.05).
3.2.2. Remating rate
Remating rate was not affected by male diet. Remating rates of
females mated with sugar-fed males (N = 83), brewer’s yeast fed
males (N = 60), MP hydrolyzed yeast fed males (N = 77) and standard diet fed males (N = 46) were similar at the beginning and at
the end of the trial (v2-tests, v2 = 1.74, df = 3, P = 0.627 and
v2 = 1.38, df = 3, P = 0.710, in the short-term and in the long-term,
respectively). Remating curves are shown in Fig. 5.
3.2.3. Refractory period
Refractory period was affected by male diet (ANOVA, F = 7.88,
df = 3, 212; P < 0.001). Females first mated with sugar-fed males
displayed a shorter refractory period than those first mated with
brewer’s yeast or standard diet fed males (Fig. 6). The refractory
period of females significantly increased as mating duration increased beyond 45-min (Fig. 7; Kruskal–Wallis test, P = 0.001).
3.2.4. Proportion of females with empty spermathecae
Male diet did not affect the proportion of females with empty
spermathecae (v2-tests, v2 = 0.405, df = 3, P = 0.939). Of the seven
out of 95 females that had no spermatozoa in any of their spermathecae, two mated with sugar-fed males, two mated with MP
hydrolyzed yeast fed males, two mated with standard diet fed
males and one mated with brewer’s yeast fed males.
3.2.5. Amount of sperm stored
Similarly, the amount of sperm stored was not affected by male
diet (Kruskal Wallis test, H = 5.18; df = 3, 95; P = 0.158) (Fig. 8).
There was a weak positive correlation between copula duration
and the amount of sperm stored by the females (Pearson’s
r = 0.20, P = 0.050).
Only one female out of 50 had no sperm in their spermathecae
or the ventral receptacle. Of those that stored sperm, 18 had no
sperm in the ventral receptacle. There were no cases where sperm
was found in the ventral receptacle but not in the spermathecae.
The amount of sperm stored in the three spermathecae and the
ventral receptacle was constant at the different dissection times
(Kruskal Wallis, H = 4.10, df = 4, 50; P = 0.392 for spermathecae
and H = 2.96, df = 4; P = 0.533 for ventral receptacle). The amount
of sperm stored in the ventral receptacle represented 10.1% of
the total sperm (max: 16.8% at 2–3 h from the end of copulation
and min: 2.2% at 6–8 h from the end of copulation) (Fig. 9).
4. Discussion
Our study found an effect of male nutritional status on the
amount and distribution of sperm stored and on the renewal of
sexual receptivity for wild and laboratory A. fraterculus females.
Wild females mated with sugar-fed males engaged in shorter copulations, exhibited higher remating rates, shorter refractory periods, higher proportions of females without sperm and lower
amounts of sperm stored in the spermathecae than females mated
with males fed with an enriched diets. Laboratory females mated
with sugar-fed males engaged in shorter copulations, exhibited
shorter refractory periods than females mated with males fed an
enriched diet. Taken together these results clearly demonstrate
the enhancing effect of protein and other nutrients on male sexual
performance. This is consistent with previous studies on other
tephritids (Blay and Yuval, 1997; Taylor and Yuval, 1999; Aluja
et al., 2008, 2009; Pérez-Staples et al., 2008b, 2009; Prabhu et al.,
2008; Gavriel et al., 2009). In addition, we found that sperm is
stored in the spermathecae in a nonrandom pattern with one of
the paired spermathecae storing always more than 30% of the total
sperm stored compared to the other spermatheca of the pair. Finally, we report for this species that the ventral receptacle is not used
to store large amounts of sperm.
Copulas involving males of poorer nutritional condition were
shorter in wild and laboratory flies. Similarly for B. tryoni, Sugarfed males have significantly shorter copula durations than males
fed sugar and protein (Pérez-Staples et al., 2007). Two alternative
explanations can be proposed. Sugar-fed males may have not been
S. Abraham et al. / Journal of Insect Physiology 57 (2011) 1501–1509
able to copulate as long as males fed an enriched diet or alternatively, females may have been able to discriminate among males’
of different nutritional status and cease copulation earlier. The second explanation seems more plausible in view of the recent evidence that, at least for B. tryoni, females exert control over
copula duration (Pérez-Staples et al., 2010).
The finding that male nutrition affected both the amount of
sperm stored and the renewal of female receptivity in wild flies
supports the hypothesis that male nutrition impacts male sexual
competitiveness. Females mated with sugar-fed males stored less
sperm and remated in higher numbers than females mated to
males fed an enriched diet. Sugar-fed males may have failed to inhibit female receptivity at least for two reasons: (i) the amount of
sperm transferred to females was low, which resulted in a lower
amount of sperm being stored and/or (ii) the amount or efficacy
of the AGPs was reduced (Fernández and Klowden, 1995; Abraham
et al., in 2011b). Our results indirectly support the former alternative given that 32% of the females that mated with sugar-fed males
had no sperm stored in their spermathecae and at the same time
34% of females mated with sugar-fed males remated 48 h after
the first mating (i.e. in the first opportunity they were given to remate). Mossinson and Yuval (2003) suggested that sperm transfer
and male AGPs act in succession to inhibit female sexual receptivity with sperm acting in the short-term and AGPs in the long-term.
The fact that the number of females remating at the end of the
observation period (30 days) was marginally affected by male diet
is another indirect evidence that remating tendency is affected by
sperm numbers soon after the first copulation and that other factors may contribute to inhibit female receptivity in the long-term.
Although this study did not address issues such as the effect of the
concentration and quality of the AGPs, Abraham et al. (2011b) by
directly injecting AGPs in females, found that sugar-fed males were
less capable of inhibiting the receptivity of their mates.
For laboratory flies, the amount of sperm stored and the number of females that remated were not affected by male diet. The
lack of differences in remating rate among male dietary treatments, either in the short and long-term is in concordance with
the hypothesis that sperm (or any feature correlating with the
amount of sperm stored) modulates female receptivity, at least in
the short-term. However, the fact that the refractory periods varied
between diet treatments, suggests that another factor, not related
with the number of sperm is involved (see below for discussion of
refractory periods in laboratory flies). Alternatively, Weldon and
Taylor (2011) found that the ejaculatory apodeme (erectile and
pumping organ) of wild male B. tryoni fed only sugar was very
poorly developed whereas laboratory males had developed ejaculatory apodemes regardless of adult diet.
Male nutrition also had an impact on the female sexual refractory period. In this case, females mated with sugar-fed males
exhibited shorter refractory periods both in wild and laboratory
flies, evidencing that sugar-fed males had a lower ability to inhibit
female sexual receptivity irrespective of their rearing history. As
described above, adult male diet did not affect the amount of
sperm stored by females in laboratory flies. Therefore, under the
hypothesis that sperm is the only factor modulating female postcopulatory behavior, similar refractory periods among treatments
involving laboratory insects should be expected. However, this
was not the case and male diet did affect refractory period. This
fact suggests that besides the amount of sperm, another ejaculate
component, most likely male AGPs’, is involved in the length of
the refractory period. This has been shown to occur, for example,
in Togo hemipterus (Scott) (Himuro and Fujisaki, 2008). The time
in which each factor impacts on female behavior may not be the
same (see Figs. 1 and 5), emphasizing the need to evaluate not only
if females remate (i.e. remating rate) but also when does this occur
in order to draw conclusions on male factors that affect female
1507
behavior. However, our experimental design does not allow to
determine the timing of expression of different mechanisms.
The differences between wild and laboratory females in the effect of adult male diet on the amount of sperm stored might be
attributed to differences in the nutritional quality of the larval
medium in which both developed. Wild flies were obtained from
guavas with high levels of infestation and probably strong competition for the resources, while laboratory insects were obtained
from a larval diet rich in carbohydrates, lipids and proteins. Many
empirical studies in insects support this hypothesis, where the
nutritional deficiency as larvae decreases sperm production (Gage
and Cook, 1994), male remating behavior, fecundity of their mates
(Delisle and Bouchard, 1995) and adult traits related with their
reproductive success (Engels and Sauer, 2007). Within tephritids,
C. capitata larvae that developed in a rich medium emerged as
adults with more nutritional reserves (e.g., increased lipids) and
matured sexually earlier than adults stemming from a protein-deprived diet (Kaspi et al., 2002). These authors suggest that flies
need to reach a nutritional threshold to mature sexually, and that
this nutritional level may result from a combination of nutrients
from the larval and adult diet. The effects of larval host of lesser
nutritional quality coupled with a protein-deprived adult diet
could explain the low sperm numbers stored by wild females mated with sugar-fed males observed in our study. Interestingly, it
seems that an enriched adult diet is sufficient to overcome any detrimental impact carried over from the larval stage.
Although male diet affected copula duration and the amount of
sperm stored, both variables were not correlated for wild flies. That
is, a longer copulation did not imply a greater amount of sperm
being stored. This was also demonstrated for C. capitata, B. tryoni
and A. obliqua (Taylor et al., 2000; Harmer et al., 2006; Pérez-Staples and Aluja, 2006), strengthening the hypothesis that in tephritids longer copulations do not necessarily translate into higher
reproductive success of the males. For laboratory females there
was a weak positive correlation between copula duration and the
total amount of sperm stored, as in Anastrepha suspensa (Fritz,
2004). Nevertheless, this tendency may be negligible considering
that when copulations were interrupted within 10–30 min, females had similar amounts of sperm stored in their storage organs
compared to females that were not interrupted and mated freely.
This reinforces the idea that extra time in copulation is not necessarily used to transfer more sperm.
Longer copulations implied a longer female refractory period.
Therefore, after sperm transfer, additional time in copula could
be used by males to transfer more AGPs (Pérez-Staples and Aluja,
2006), complete female stimulation, and/or exercise a form of mate
guarding (Mossinson and Yuval, 2003; Vera et al., 2003). The later
hypothesis makes sense for A. fraterculus due to the fact that the
window of mating activity during the day is relatively short (2 h)
(Petit-Marty et al., 2004; Vera et al., 2006) and a long copulation
(1 h) can prevent remating on the same day.
We found a marked asymmetry in sperm storage among A. fraterculus spermathecae, as one of the paired spermatheca stored
more sperm than the other paired spermatheca or the single one,
as described for A. suspensa by Fritz (2004). Asymmetry in sperm
storage has been reported in all studies evaluating patterns of
sperm storage in the Tephritidae (Yuval et al., 1996; Taylor and Yuval, 1999; Taylor et al., 2000, 2001; Fritz, 2004; Harmer et al., 2006;
Pérez-Staples and Aluja, 2006; Pérez-Staples et al., 2007, 2010), but
the way the sperm is distributed within those organs differs among
species (Fritz, 2004; Pérez-Staples and Aluja, 2006). The presence
of multiple organs of sperm storage and asymmetric storage in females are considered as ground for both cryptic female choice and
sperm competition to occur (Ward, 1993; Eberhard, 1996). However, in A. fraterculus the overlap of sperm from rival males in time
and space seems unlikely as the time between the first and second
1508
S. Abraham et al. / Journal of Insect Physiology 57 (2011) 1501–1509
mating is long. We would therefore not expect sperm competition
sensu stricto to be a common phenomenon as postulated for A. obliqua (Pérez-Staples and Aluja, 2006). This hypothesis is supported
by recent studies where we determined that A. fraterculus females
accept a new copulation when they need to restore their sperm
supplies (Abraham et al., 2011a). It is interesting to note that A.
fraterculus females in our study stored less than half the mean
number of spermatozoids than those reported for C. capitata,
A. obliqua and B. tryoni (Twig and Yuval, 2005; Pérez-Staples and
Aluja, 2006; Pérez-Staples et al., 2007) with our values being more
similar to those reported for A. suspensa (Fritz, 2004). It would be
interesting to understand the underlying causes of such
differences.
Sperm storage in the ventral receptacle for A. fraterculus was
closer to that reported for C. capitata than for other species. In B.
tryoni females stores only 3% of the total sperm (Pérez-Staples
et al., 2007) in the ventral receptacle, in C. capitata females stores
15% (Twig and Yuval, 2005), in A. suspensa approximately 37%
(Fritz, 2004) and in A. obliqua approximately 50% (Pérez-Staples
and Aluja, 2006). In A. fraterculus, the ventral receptacle stored
approximately 10% of total sperm and this amount remained constant at least 24 h after the end of copulation. In C. capitata the
amount of sperm in the ventral receptacle also remains constant
from the third day and up to 18 days after copulation, but this does
not occur with the amount of sperm in the spermathecae (Twig
and Yuval, 2005), leading the authors to suggest that the ventral
receptacle is replenished with sperm from the spermathecae. Unlike A. suspensa and A. obliqua, the ventral receptacle in A. fraterculus does not seem to be used to store large amounts of sperm.
These differences could be due to different egg laying strategies
where use of sperm could be more efficient or occur at smaller
or slower rates in A. fraterculus.
Finally, within the context of the SIT, the present study indicates that the incorporation of an enriched adult male diet before
release will have a positive impact in delaying the renewal of female sexual receptivity. Moreover, laboratory males fed with
brewer’s yeast, a locally available and cheaper protein, were as
effective as those males fed with the high-quality yet more expensive protein. Thus, the local protein could be used as an alternative
in the pre-release male diet, decreasing costs of mass-production.
Overall, our findings provide relevant information on how diet enhances male sexual competitiveness and female post-copulatory
behavior. The effect of irradiation on the amount of sperm stored
and the ability of sterile males to inhibit female receptivity remains to be assessed.
Acknowledgments
This work was supported by FAO/IAEA Research Contracts
11894 and 14111 to M.T.V. We thank Compañía Argentina de Levaduras S.A. (CALSAÒ) for providing brewer’s yeast and ARCORÒ S.A.
for providing corn protein for the diets. We thank D. Pérez-Staples,
J.A. Rull, F. Pennacchio and two anonymous reviewers for their
comments on an earlier draft of the manuscript.
References
Abraham, S., Goane, L., Rull, J., Cladera, J., Willink, E., Vera, M.T., 2011a. Multiple
mating in Anastrepha fraterculus females and its relationship with fecundity and
fertility. Entomologia Experimentalis et Applicata. doi:10.1111/j.15707458.2011.01160.x.
Abraham, S., Cladera, J., Goane, L., Vera, M.T., 2011b. Factors affecting Anastrepha
fraterculus female receptivity modulation by accessory gland products. Journal
of Insect Physiology. doi:10.1016/j.Jingphys.2011.08.007.
Aluja, M., Jácome, I., Macías-Ordóñez, R., 2001. Effects of adult nutrition on male
sexual performance in four neotropical fruit fly species of the genus Anastrepha
(Diptera: Tephritidae). Journal of Insect Behavior 14, 759–775.
Aluja, M., Pérez-Staples, D., Sivinski, J., Sánchez, A., Piñero, J., 2008. Effects of male
condition on fitness in two tropical tephritid flies with contrasting life histories.
Animal Behavior 76, 1997–2009.
Aluja, M., Rull, J., Sivinsky, J., Trujillo, G., Pérez-Staples, D., 2009. Male and female
condition influence mating performance and sexual receptivity in two tropical
fruit flies (Diptera: Tephritidae) with contrasting life histories. Journal of Insect
Physiology 55, 1091–1098.
Bartolucci, A., Vera, M.T., Yusef, V., Oviedo, A., 2006. Morphological characterization
of the reproductive system of irradiated Anastrepha fraterculus. Proceedings of
the 7th International Symposium on Fruit Flies of Economic Importance, 45–52.
Blay, S., Yuval, B., 1997. Nutritional correlates of reproductive success of male
Mediterranean fruit flies (Diptera: Tephritidae). Animal Behavior 54, 59–66.
Blay, S., Yuval, B., 1999. Oviposition and fertility in the Mediterranean fruit fly
(Diptera: Tephritidae): effects of male and female body size and the availability
of sperm. Annals of the Entomological Society of America 92, 278–284.
Cunningham, R.T., Farias, G.H., Nakagawa, S., Chambers, D.L., 1971. Reproduction in
the Mediterranean fruit fly: depletion of stored sperm in females. Annals of the
Entomological Society of America 64, 312–313.
De Lima, I.S., Howse, P.E., Salles, L.A.B., 1994. Reproductive behavior of the South
American fruit fly Anastrepha fraterculus (Diptera: Tephritidae): laboratory and
field studies. Physiological Entomology 19, 271–277.
Delisle, J., Bouchard, A., 1995. Male larval nutrition in Choristoneura rosaceana
(Lepidoptera: Tortricidae): an important factor in reproductive success.
Oecologia 104, 508–517.
Eberhard, W.G., 1996. Female Control: Sexual Selection by Cryptic Female Choice.
Princeton University Press, Princeton.
Engels, S., Sauer, K.P., 2007. Energy beyond the pupal stage: larval nutrition and its
long-time consequences for male mating performance in a scorpionfly. Journal
of Insect Physiology 53, 633–638.
Fernández, N.M., Klowden, M.J., 1995. Male accessory gland substances modify the
host-seeking behavior of gravid Aedes aegypti mosquitoes. Journal of Insect
Physiology 41, 965–970.
Fritz, A.H., 2004. Sperm storage patterns in singly mated female of the Caribbean
fruit fly, Anastrepha suspensa (Diptera: Tephritidae). Annals of the
Entomological Society of America 97, 1328–1335.
Gage, M.J.G., Cook, P.A., 1994. Sperm size or numbers? Effects of nutritional stress
upon eupyrene and apyrene sperm production strategies in the moth Plodia
interpunctella (Lepidoptera: Pyralidae). Functional Ecology 8, 594–599.
Gavriel, S., Gazit, Y., Yuval, B., 2009. Remating by female Mediterranean fruit flies
(Ceratitis capitata, Diptera: Tephritidae): temporal patterns and modulation by
male condition. Journal of Insect Physiology 55, 637–642.
Gillot, C., 2003. Male accessory gland secretions: modulators of female reproductive
physiology and behavior. Annual Review of Entomology 48, 163–184.
Gromko, M.H., Markow, T., 1993. Courtship and remating in field populations of
Drosophila. Animal Behavior 45, 253–262.
Harmer, A.M.T., Radhakrishnan, P., Taylor, P.W., 2006. Remating inhibition in female
Queensland fruit flies: effects and correlates of sperm storage. Journal of Insect
Physiology 52, 179–186.
Himuro, C., Fujisaki, K., 2008. Males of the seed bug Togo hemipterus (Heteroptera:
Lygaeidae) use accessory gland substances to inhibit remating by females.
Journal of Insect Physiology 54, 1538–1542.
Hendrichs, J., Cooley, S.S., Prokopy, R.J., 1992. Post-feeding bubbling behavior in
fluid feeding Diptera: concentration of crop contents by oral evaporation.
Physiological Entomology 17, 153–161.
InfoStat, 2009. InfoStat, versión 2009. Manual del Usuario. Grupo InfoStat, FCA,
Universidad Nacional de Córdoba, 1st edition. Editorial Brujas, Argentina.
Jaldo, H.E., 2001. Estudios biológicos y poblacionales de Anastrepha fraterculus
(Wiedemann) (Diptera: Tephritidae). Ph.D. Thesis, Universidad Nacional de
Tucumán, Argentina.
Jaldo, H.E., Gramajo, M.C., Willink, E., 2001. Mass rearing of Anastrepha fraterculus
(Diptera: Tephritidae): a preliminary strategy. Florida Entomologist 84, 716–
718.
Jaldo, H.E., Willink, E., Liedo, P., 2007. Demograhic analysis of mass-reared
Anastrepha fraterculus (Diptera: Tephritidae) in Tucumán, Argentina. Revista
Industrial y Agrícola de Tucumán 84, 15–20.
Kaspi, R., Mossinson, S., Drezner, T., Kamensky, B., Yuval, B., 2002. Effects of larval
diet on development rates and reproductive maturation of male and female
Mediterranean fruit flies. Physiological Entomology 27, 29–38.
Kaspi, R., Yuval, B., 1999. Lek site selection by male Mediterranean fruit flies. Jounal
of Insect Behavior 12, 267–276.
Klowden, M.J., 2001. Sexual receptivity in Anopheles gambiae mosquitoes: absence
of control by male accessory gland substances. Journal of Insect Physiology 47,
661–666.
Knipling, G.F., 1955. Possibilities of insect control or eradication through the use of
sexually sterile males. Journal of Economical Entomology 48, 459–462.
Kraaijeveld, K., Chapman, T., 2004. Effects of male sterility on female remating in
the Mediterranean fruitfly, Ceratitis capitata. Philosophical Transactions of the
Royal Society of London, Series B: Biological Sciences 271, 209–211.
Kuba, H., Ito, J., 1993. Remating inhibition in the melon fly: Bactrocera cucurbitae,
copulation with spermless males inhibits female remating. Journal of Ethology
11, 23–28.
Lux, S.A., Vilardi, J.C., Liedo, P., Gaggl, K., Calagno, G.E., Munyiri, F.N., Vera, M.T.,
Manso, F., 2002. Effects of irradiation on the courtship behavior of medfly mass
reared for the sterile insect technique. Florida Entomologist 85, 102–112.
S. Abraham et al. / Journal of Insect Physiology 57 (2011) 1501–1509
Moreno, D.S., Sanchez, M., Robacker, D.C., Worley, J., 1991. Mating competitiveness
of irradiated Mexican fruit fly (Diptera: Tephritidae). Journal of Economical
Entomology 8, 1227–1234.
Mossinson, S., Yuval, B., 2003. Regulation of sexual receptivity of female
Mediterranean fruit flies: old hypotheses revisited and new synthesis
proposed. Journal of Insect Physiology 49, 561–567.
Nakagawa, S., Farias, G.J., Suda, D., Cunningham, R.T., Chambers, D.L., 1971.
Reproduction of the Mediterranean fruit fly: frequency of mating in the
laboratory. Annals of the Entomological Society of America 64, 949–950.
Norrbom, A.L., 2004. Host plant database for Anastrepha 428 and Toxotripana
(Diptera: Tephritidae: Toxotripanini) <http://www.sel.barc.usda.gov/diptera/
tephriti/tephriti.htm>.
Ortíz, G., 1999. Potential use of the sterile insect technique against the South
American fruit fly. In: International Atomic Energy Agency (Ed.), The South
American Fruit Fly, Anastrepha fraterculus (Wied.): Advances in Artificial
Rearing, Taxonomic Status and Biological Studies. IAEA TEC-DOC 1064.
Vienna, Austria, 121–130.
Pérez-Staples, D., Aluja, M., 2006. Sperm allocation and cost of mating in a tropical
tephritid fruit fly. Journal of Insect Physiology 52, 839–845.
Pérez-Staples, D., Harmer, A.M.T., Taylor, P.W., 2007. Sperm storage and utilization
in female Queensland fruit flies (Bactrocera tryoni). Physiological Entomology
32, 127–135.
Pérez-Staples, D., Aluja, M., Macías-Ordóñez, R., Sivinski, J., 2008a. Reproductive
trade-offs from mating with a successful male: the case of the tephritid fly
Anastrepha obliqua. Behavioral Ecology and Sociobiology 62, 1333–1340.
Pérez-Staples, D., Harmer, A.M.T., Collins, S.R., Taylor, P.W., 2008b. Potential for prerelease diet supplements to increase the sexual performance and longevity of
male Queensland fruit flies. Agricultural and Forest Entomology 10, 255–262.
Pérez-Staples, D., Weldon, C.W., Smallridge, C., Taylor, P.W., 2009. Pre-release
feeding on yeast hydrolysate enhances sexual competitiveness of sterile male
Queensland fruit flies in field cages. Entomologia Experimentalis et Applicata
131, 159–166.
Pérez-Staples, D., Weldon, C.W., Radhakrishnan, P., Prenter, J., Taylor, P.W., 2010.
Control of copula duration and sperm storage by female Queensland fruit flies.
Journal of Insect Phisiology 56, 1755–1762.
Petit-Marty, N., Vera, M.T., Calcagno, G., Cladera, J.L., Segura, D.F., Allinghi, A.,
Rodriguero, M., Gómez Cendra, P., Viscarret, M.M., Vilardi, J.C., 2004. Sexual
behavior and mating compatibility among four populations of Anastrepha
fraterculus (Diptera: Tephritidae) from Argentina. Annals of the Entomological
Society of America 97, 1320–1327.
Prabhu, V., Pérez-Staples, D., Taylor, P.W., 2008. Protein: carbohydrate ratios
promoting sexual activity and longevity of male Queensland fruit flies. Journal
of Applied Entomology. doi:10.1111/j.1439-0418.2007.01265.x.
Radhakrishnan, P., Taylor, P.W., 2007. Seminal fluids mediate sexual receptivity and
copula duration in Queensland fruit flies. Journal of Insect Physiology 53, 741–
745.
Radhakrishnan, P., Pérez-Staples, D., Weldon, C.W., Taylor, P.W., 2009. Multiple
mating and sperm depletion in male Queensland fruit flies: effects on female
remating behaviour. Animal Behavior 78, 839–846.
Rull, J., Brunel, O., Mendez, M.E., 2005. Mass rearing history negatively affects
mating success of male Anastrepha ludens (Diptera: Tephritidae) reared for the
sterile insect technique. Journal of Economical Entomology 98, 1510–1516.
Sirot, L.K., Poulson, R.L., McKenna, M.C., Girnary, H., Wolfner, M.F., Harrington, L.C.,
2008. Identity and transfer of male reproductive gland proteins of the dengue
vector mosquito, Aedes aegypti: potential tools for control of female feeding and
reproduction. Insect Biochemistry and Molecular Biology 38, 176–189.
1509
Sirot, L.K., LaFlamme, B.A., Sitnik, J.L., Rubinstein, C.D., Avila, F.W., Chow, C.Y.,
Wolfner, M.F., 2009. Molecular social interactions: Drosophila melanogaster
seminal fluid proteins as a case study. Advances in Genetics 68, 23–56.
Sakurai, T., 1998. Receptivity of female remating and sperm number in the sperm
storage organ in the bean bug, Riptortus clavatus (Heteroptera: Alydidae).
Researches on Population Ecology 40, 167–172.
Shelly, T.E., Kennelly, S., 2002. Influence of male diet on male mating success and
longevity and female remating in the Mediterranean fruit fly (Diptera:
Tephritidae) under laboratory condition. Florida Entomologist 85, 572–578.
Steiner, S., Henrich, N., Ruther, J., 2008. Mating with sperm-depleted males does not
increase female mating frequency in the parasitoid Lariophagus distinguendus.
Entomologia Experimentalis et Applicata 126, 131–137.
Taylor, P.W., Yuval, B., 1999. Postcopulatory sexual selection in Mediterranean fruit
flies, Ceratitis capitata: advantages for large and protein-fed males. Animal
Behavior 58, 247–254.
Taylor, P.W., Kaspi, R., Yuval, B., 2000. Copula duration and sperm storage in
Mediterranean fruit flies from a wild population. Physiological Entomology 25,
94–99.
Taylor, P.W., Kaspi, R., Mossinson, S., Yuval, B., 2001. Age-dependent insemination
success of sterile Mediterranean fruit flies. Entomological Experimentalis et
Applicata 98, 27–33.
Twig, E., Yuval, B., 2005. Function of multiple sperm organs in females
Mediterranean fruti flies (Ceratitis capitata, Diptera: Tephritidae). Journal of
Insect Physiology 51, 67–74.
Vera, M.T., Cladera, J.L., Calcagno, G., Vilardi, J.C., McInnis, D.O., 2003. Remating of
wild Ceratitis capitata (Diptera: Tephritidae) females in field cages. Annals of the
Entomological Society of America 96, 563–570.
Vera, M.T., Cáceres, C., Wornoayporn, V., Islam, A., Robinson, A.S., de la Vega, M.H.,
Hendrichs, J., Cayol, J.P., 2006. Mating incompatibility among populations of the
South American fruit fly Anastrepha fraterculus (Wied.) (Diptera: Tephritidae).
Annals of the Entomological Society of America 99, 387–397.
Vera, M.T., Abraham, S., Oviedo, A., Willink, E., 2007. Demographic and quality
control parameters of Anastrepha fraterculus (Diptera: Tephritidae) artificial
rearing. Florida Entomologist 90, 53–57.
Ward, P.I., 1993. Females influence sperm storage and use in the yellow dung fly
Scathophaga stercoraria (L.). Behavioral Ecology Sociobiology 32, 313–319.
Weldon, C.W., Taylor, P.W., 2011. Sexual development of wild and mass-reared
male Queensland fruit flies in response to natural food sources. Entomologia
Experimentalis et Applicata 139, 17–24.
Whittier, T.S., Shelly, T.E., 1993. Productivity of singly vs. multiply mated female
Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae). Journal of the
Kansas Entomological Society 66, 200–209.
Yamane, T., Miyatake, T., Kimura, Y., 2008a. Female mating receptivity after
injection of male-derived extracts in Callosobruchus maculatus. Journal of Insect
Physiology 54, 1522–1527.
Yamane, T., Kimura, Y., Katsuhara, M., Miyatake, T., 2008b. Female mating
receptivity inhibited by injection of male-derived extracts in Callosobruchus
chinensis. Journal of Insect Physiology 54, 501–507.
Yuval, B., Blay, S., Kaspi, R., 1996. Sperm transfer and storage in the Mediterranean
fruit fly (Diptera: Tephritidae). Annals of the Entomological Society of America
89, 486–492.
Yuval, B., Kaspi, R., Field, S.A., Blay, S., Taylor, P., 2002. Effects of post-teneral
nutrition on reproductive success of male Mediterranean fruit flies (Diptera:
Tephritidae). Florida Entomologist 85, 165–170.