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. 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