Sperm Storage Patterns in Doubly Mated Female Anastrepha suspensa (Diptera: Tephritidae)

Sperm Storage Patterns in Doubly Mated Female Anastrepha suspensa (Diptera: Tephritidae) Abstract We examined sperm storage patterns of doubly mated females in a species of tephritid fly (Anastrepha suspensa (Loew) [Diptera: Tephritidae]) with four sperm storage organs and an unusually complex reproductive system to elucidate possible reproductive arenas in which sexual selection can or has played a role in sperm competition and cryptic female choice and to predict effects on paternity outcomes. The duration of copulation with each male of doubly mated female flies was recorded and the sperm storage organs were dissected to determine the location and identity of each male’s sperm (by microsatellite polymerase chain reaction) and its relative quantity. Short copulations with the first male generally resulted in little transfer of sperm and predicted the length of time spent in second copulations, resulting in disproportionate sperm storage from second copulations. For doubly mated females, most storage organs contained sperm from second males only, although the fertilization chamber often contained sperm from both males (allowing for possible sperm competition). Long-term storage organs (spermathecae) always contained sperm from single males exclusively possibly limiting opportunities for sperm competition. Our data provide a mechanism for second male precedence without invoking sperm competition, and caution that paternity patterns must be critically interpreted with inclusion of information on sperm storage patterns. Multiple mating in insects is well-documented (Ridley 1988, Arnqvist and Nilsson 2000), setting up circumstances where postcopulatory sexual selection may act through sperm competition (Parker 1970, 1979) and cryptic female choice (Thornhill 1983, Eberhard 1996). Evolutionary theory predicts male and female postcopulatory sexual selection is based on the disparity in investment by each sex; females usually invest more in the production of ova than males that invest in the development of spermatozoa (Bateman 1948, Parker 1970). Thus, female fitness is limited by the production of ova, whereas male fitness is limited by the number of ova fertilized (Parker 1970). However, it has become increasingly apparent from studies on a number of different insects that copulation per se does not guarantee paternity for a particular male (Eberhard 1996), and the cost of ejaculate and fluid production for males is high (Simmons 2001). In addition, within the Insecta, a temporal separation often occurs between copulation and ova fertilization, generating a dynamic timeframe for sexual selection to act at the level of reproductive tract morphology and gametes (Parker 1970, Eberhard 1996, Simmons 2001), especially when females are utilizing patchy resources for oviposition (Prokopy 1980, Burke 1981). In this study, we examine sperm storage patterns of doubly mated females in a species with four sperm storage organs and an unusually complex reproductive system to assess whether sexual selection plays a role in sperm competition and cryptic female choice. This study is based on the understanding that the temporal disjunction between copulation and fertilization may influence both male (sperm competition) and female (cryptic female choice) and that these interests are subject to evolutionary forces when females mate multiply and ejaculates from different sires are stored for long periods of time prior to ova fertilization (Parker 1970, Eberhard 1996, Simmons 2001). Multiple sperm storage organs and multiple mating provide the potential opportunity for the differential storage and use of sperm from two or more males and subsequently a unique chance to determine the importance of possible sperm competition and cryptic female choice. Until recently, with the advent of genetic lines with fluorescently labeled sperm, studies on sexual selection have been hampered by the inability to discern specific sperm storage patterns and other reproductive system dynamics in multiply mated females. These studies have generally tried to understand paternity outcomes, sperm storage, and sperm use indirectly by examining offspring only. We used a protocol developed by Fritz et al. (2010) in which the sperm storage organs are dissected and cleared of maternal cells, and the DNA is extracted, amplified (microsatellite PCR), and assigned to individual males. Our study species, the Caribbean fruit fly, Anastrepha suspensa (Loew), is a New World member of the invasive fruit fly family Tephritidae and is an agricultural pest in the Antilles, Puerto Rico and Florida (Aluja 1994, Aluja and Norrbom 2000, El-Sayed et al. 2009). As with many tephritid invasive generalists, female A. suspensa mate multiply (Sivinski and Heath 1988, Sivinski and Burk 1989) and possess a highly complex reproductive tract for storing and using sperm (Fritz and Turner 2002). In A. suspensa females, spermatozoa are stored in four different specialized organs (three spermathecae and a ventral receptacle; Fritz and Turner 2002). The spermathecae are thought to be sites for long-term storage to replenish the female’s ventral receptacle, the short-term storage organ where ova fertilization occurs, as described in other tephritids, such as Ceratitis capitata (Wiedemann) and Bactrocera tryoni (Froggatt) (Twig and Yuval 2005, Pérez-Staples et al. 2007). Fritz and Turner (2002) detailed the unusual complexity of the spermathecae and ventral receptacle in A. suspensa, which suggest strong selection for the maintenance, compartmentalization, and mobilization of sperm. The spermathecae have highly infolded sculptured interiors, where sperm are lodged in contact with exit pores layered with secretory cells. Spermathecal capsules also have valves that control the entry and release of sperm, and each spermathecae has its own separate duct surrounded by musculature. The ventral receptacle is surrounded by oblique musculature and embedded in a thick layer of mitochondrial-rich cells with numerous canuli, which may allow for the rapid evacuation of fluid from the ventral receptacle. The ventral receptacle also includes a mass of cuticular infoldings and a vacuole filled with secretory substances and surrounded by approximately 200 alveoli in which small numbers of sperm can reside (Fritz and Turner 2002). Thus, A. suspensa offers a rich morphological and physiological arena in which postcopulatory sexual selection may act. Sperm storage patterns in A. suspensa have been described by Fritz (2004) for singly mated flies, but the dynamics of sperm storage have not been examined in doubly mated females. Although sperm storage patterns have been examined in tephritid flies in a number of studies with single and doubly mated females (Yuval et al. 1996, Fritz 2004, Bertin et al 2010, Scolari et al. 2014), the present study represents the first to describe both the identity and location of sperm stored from two different males in all of the sperm storage organs. Materials and Methods Study Organisms Flies were obtained as pupae from the Biological Control Mass Rearing Facility at the Department of Agriculture Division of Plant Industry in Florida and reared in a quarantine insectary maintained under a photoperiod of 14:10 (L:D) h, a temperature of 25 ± 2°C and 55% relative humidity, according to the protocols described by Fritz (2004). Sexually mature adult flies used in mating crosses were sorted by sex as teneral adults. After separating males and females, females were individually marked with nontoxic paint dots according to a standardized combination of paint colors and locations on the dorsum (Dhakal 2008). Mating trials The age of flies in mating trials was standardized by using only post-teneral flies (9 d after emergence) and for 6 d thereafter (Kendra et al. 2005). All mating trials were run between 12:00 and 16:00 h, a time when flies are sexually active (Burk 1983). Sexually mature males emit pheromones and thus “call” for females (Nation 1972). Individual couples’ copulations were timed. Timing began when the male inserted his aedeagus into the female’s genital opening and ended when copulation was terminated and couples fully separated (e.g., observation of the complete withdrawal of the aedeagus). Cohorts of approximately 130 marked females were placed into identical mating cages (1.5 liter plastic jar with a cloth stockinette access sleeve over the opening) containing 200 males each; all flies were the same age and had the same emergence date. When copulation ensued, a pair was removed from the mating cage by allowing the female to walk forward upon a wooden applicator stick and enter a 75-ml plastic vial. Vials were capped with foam plugs and monitored until cessation of copulation. After each mating, the mated pair was held in absolute darkness for 2 h to allow sufficient time for sperm storage within the female and to prevent females from repeatedly copulating with the same male, following Fritz (2004). Subsequently, individual males were placed in an eppendorf tube, labeled with the female identification number and frozen at −65°C. Females were transferred to a new cage lacking oviposition substrate, provided with water and food ad libidum and were given the opportunity to mate the following day with virgin males. After second copulations ensued, flies were handled and subsequently frozen according to the protocols mentioned above. To examine the relative duration of copulation of females with their first male mate versus their second male mate, three mating-time categories were established. These categories included: females that mated from 1 to 15 min, females that mated 16 to 25 min, and females that mated ≥26 min. The first and second categories were established because prior literature indicated female A. suspensa mating <15 min often did not store sperm (Fritz 2004), while a mating time of 16–25 min corresponds to a range including the mean copulation durations noted by previous studies (Fritz 2004, Wallace 2005). Molecular Protocol The presence, origin (from which male), relative quantity, and location of sperm in the four sperm storage organs of doubly mated females were investigated using microsatellite primers and PCR. Microsatellite primers have been developed for A. suspensa (Fritz and Schable 2004), and Fritz et al. (2010) described the protocols for isolating, amplifying, and quantifying sperm DNA in A. suspensa. The protocols can be summarized in five major steps: 1) isolation of DNA from adult flies and sperm storage organs in females (three spermathecae and a ventral receptacle) by microdissection, 2) removal of the maternal cells from sperm storage organs through sonication (to prevent maternal contamination of DNA during PCR), 3) isolation of sperm DNA from sperm storage organs, 4) PCR amplification of DNA with microsatellite loci, and 5) sperm relative quantification. Of 180 females that copulated, 93 mated again with a second male. All doubly mated females (N = 93) and their males (N = 186) were genotyped for three polymorphic microsatellite loci (837 PCR reactions) using primers 1H, 3B, and 5E (Fritz and Schable 2004) and previously used to study sperm storage (Fritz et al. 2010). Doubly mated females along with their two male sires were subsequently chosen for further analysis if all three individuals differed genetically for at least two loci; 52 females and their 104 males fell into this category. These 52 females were then dissected for their sperm storage organs and visually inspected for the presence of sperm using 4',6-diamidino- 2-phenylindole (DAPI) staining and fluorescent microscopy. Of these 52 females, individuals with few (<10) sperm or no sperm in all their storage organs were eliminated from further analysis. Thus, our sample size of doubly mated females storing >10 sperm from at least 1 male was 36 females. If no sperm was visualized in a sperm storage organ or produced amplicons after PCR, then this organ was abbreviated as NV (Table 1). Missing data were abbreviated as MD (Table 1). Each spermatheca in the doublet (the paired spermathecae) was not individually distinguishable from its pair by position or any morphological marker. Thus, the assignment of sperm to one or the other spermatheca of the doublet was arbitrary in Table 1. Table 1. The Relative Percentage of Spermatozoa Contributed by First and Second Sires to Four Female Sperm storage Organs for 36 Doubly Mated Females No. of females (N = 36)  Sire  CD (min)  SP  DBL  VR  1  First sire  13  0%  NV/NV  0%  Second sire  45  100%  NV/NV  100%  2  First sire  50  NV  100%/0  100%  Second sire  20  NV  0%/NV  0%  3  First sire  11  NV  0%/NV  0%  Second sire  9  NV  100%/NV  100%  4  First sire  5  NV  0%/NV  0  Second sire  8  NV  100%/NV  100%  5  First sire  19  0%  NV/100%  38.2%  Second sire  17  100%  NV/0  61.8%  6  First sire  16  NV  100%/0%  46.2%  Second sire  15  NV  0%/100%  53.82%  7  First sire  15  NV  100%/NV  100%  Second sire  10  NV  0%/NV  0%  8  First sire  10  0%  0%/NV  0%  Second sire  38  100%  100%/NV  100%  9  First sire  7  NV  0%/0%  0%  Second sire  35  NV  100%/100%  100%  10  First sire  14  NV  0%/NV  0%  Second sire  30  NV  100%/NV  100%  11  First sire  12  0%  0%/0%  0%  Second sire  32  100%  100%/100%  100%  12  First sire  10  0%  0%/0%  0 %  Second sire  16  100%  100%/100%  100%  13  First sire  20  NV  100%/NV  100%  Second sire  7  NV  0%/NV  0%  14  First sire  9  NV  0%/NV  0%  Second sire  10  NV  100%/NV  100%  15  First sire  16  0%  100%/0%  50.3%  Second sire  21  100%%  0%/100%  49.7%  16  First sire  10  NV  0%/NV  0%  Second sire  20  NV  100%/NV  100%  17  First sire  21  0%  0%/0%  0%  Second sire  17  100%  100%/100%  100%  18  First sire  35  100%  100%/100%  100%  Second sire  11  0%  0%/0%  0%  19  First sire  40  NV  100%/NV  100%  Second sire  10  NV  0%/NV  0%  20  First sire  17  NV  100%/0%  73.9%  Second sire  16  NV  0%/100%  26.1%  21  First sire  16  NV  100%/0%  46.8%  Second sire  20  NV  0%/100%  53.2%  22  First sire  16  100%  100%/0%  28.7%  Second sire  17  0%  0%/100%  71.3%  23  First sire  55  0%  100%/0%  50.7%  Second sire  14  100%  0%/100%  49.3%  24  First sire  35  0%  0%/0%  0%    Second sire  11  100%  100%/100%  100%  25  First sire  13  0%  100%/0%  59.4%  Second sire  31  100%  0%/100%  40.6%  26  First sire  17  NV  100%/0%  67.8%  Second sire  24  NV  0%/100%  32.2%  27  First sire  19  0%  100%/0%  53.8%  Second sire  22  100%%  0%/100%  46.2%  28  First sire  20  NV  100%/0%  36.7%  Second sire  17  NV  0%/100%  63.3%  29  First sire  9  NV  0%/NV  0%  Second sire  35  NV  100%/NV  100%  30  First sire  31  NV  100%/0%  32.9%  Second sire  15  NV  0%/100%  67.1%  31  First sire  10  NV  100%/0%  100%  Second sire  5  NV  0%/NV  0%  32  First sire  14  0%  100%/0%  74.3%  Second sire  38  100%  0%/100%  25.7%  33  First sire  12  NV  0%/0%  0%  Second sire  12  NV  100%/100%  100%  34  First sire  12  NV  100%/0%  MD    Second sire  39  NV  0%/100%  MD  35  First sire  NV  0%  100%  MD    Second sire  NV  100%  0%  MD  36  First sire  NV  100%  0  MD    Second sire  NV  0%  100%  MD  No. of females (N = 36)  Sire  CD (min)  SP  DBL  VR  1  First sire  13  0%  NV/NV  0%  Second sire  45  100%  NV/NV  100%  2  First sire  50  NV  100%/0  100%  Second sire  20  NV  0%/NV  0%  3  First sire  11  NV  0%/NV  0%  Second sire  9  NV  100%/NV  100%  4  First sire  5  NV  0%/NV  0  Second sire  8  NV  100%/NV  100%  5  First sire  19  0%  NV/100%  38.2%  Second sire  17  100%  NV/0  61.8%  6  First sire  16  NV  100%/0%  46.2%  Second sire  15  NV  0%/100%  53.82%  7  First sire  15  NV  100%/NV  100%  Second sire  10  NV  0%/NV  0%  8  First sire  10  0%  0%/NV  0%  Second sire  38  100%  100%/NV  100%  9  First sire  7  NV  0%/0%  0%  Second sire  35  NV  100%/100%  100%  10  First sire  14  NV  0%/NV  0%  Second sire  30  NV  100%/NV  100%  11  First sire  12  0%  0%/0%  0%  Second sire  32  100%  100%/100%  100%  12  First sire  10  0%  0%/0%  0 %  Second sire  16  100%  100%/100%  100%  13  First sire  20  NV  100%/NV  100%  Second sire  7  NV  0%/NV  0%  14  First sire  9  NV  0%/NV  0%  Second sire  10  NV  100%/NV  100%  15  First sire  16  0%  100%/0%  50.3%  Second sire  21  100%%  0%/100%  49.7%  16  First sire  10  NV  0%/NV  0%  Second sire  20  NV  100%/NV  100%  17  First sire  21  0%  0%/0%  0%  Second sire  17  100%  100%/100%  100%  18  First sire  35  100%  100%/100%  100%  Second sire  11  0%  0%/0%  0%  19  First sire  40  NV  100%/NV  100%  Second sire  10  NV  0%/NV  0%  20  First sire  17  NV  100%/0%  73.9%  Second sire  16  NV  0%/100%  26.1%  21  First sire  16  NV  100%/0%  46.8%  Second sire  20  NV  0%/100%  53.2%  22  First sire  16  100%  100%/0%  28.7%  Second sire  17  0%  0%/100%  71.3%  23  First sire  55  0%  100%/0%  50.7%  Second sire  14  100%  0%/100%  49.3%  24  First sire  35  0%  0%/0%  0%    Second sire  11  100%  100%/100%  100%  25  First sire  13  0%  100%/0%  59.4%  Second sire  31  100%  0%/100%  40.6%  26  First sire  17  NV  100%/0%  67.8%  Second sire  24  NV  0%/100%  32.2%  27  First sire  19  0%  100%/0%  53.8%  Second sire  22  100%%  0%/100%  46.2%  28  First sire  20  NV  100%/0%  36.7%  Second sire  17  NV  0%/100%  63.3%  29  First sire  9  NV  0%/NV  0%  Second sire  35  NV  100%/NV  100%  30  First sire  31  NV  100%/0%  32.9%  Second sire  15  NV  0%/100%  67.1%  31  First sire  10  NV  100%/0%  100%  Second sire  5  NV  0%/NV  0%  32  First sire  14  0%  100%/0%  74.3%  Second sire  38  100%  0%/100%  25.7%  33  First sire  12  NV  0%/0%  0%  Second sire  12  NV  100%/100%  100%  34  First sire  12  NV  100%/0%  MD    Second sire  39  NV  0%/100%  MD  35  First sire  NV  0%  100%  MD    Second sire  NV  100%  0%  MD  36  First sire  NV  100%  0  MD    Second sire  NV  0%  100%  MD  CD, copulation duration; DBL, doublet spermathecae; SP, singlet spermatheca; VR, ventral receptacle. Bold numbers are ventral receptacles with sperm from two males. View Large Amplicon peak areas generated by microsatellite PCR output on a DNA sequencer can be used to estimate the relative proportions of sperm contributed by each male storing sperm in a particular sperm storage organ (Fritz et al. 2010). Since male sperm is haploid, the ratio of the peak area of one allele (of one male) to that of another male provides a relative measure of sperm quantity. If a sperm storage organ contained the sperm from two different males (as was the case for the ventral receptacles) then the ratio of amplicon peak areas was used as a relative quantification of sperm contributed by each male (Fritz et al. 2010). PCR products were resolved on a Beckman Coulter (Fullerton, CA) CEQ 2000XL Sequence Analyser using the Beckman Coulter CEQ DNA Size Standard-400 and the CEQ Fragment Analysis Module Program (Beckman Coulter). Statistical Analysis One-way analysis of variance (ANOVA) was used to estimate the effect of the amount of time spent copulating with the first male on the amount of time spent mating with a second male (for three copulation duration categories: 1–15, 16–25, and >25 min). Tukey’s post hoc test was used for multiple comparisons of the means of the duration of copulation of female flies. Paired t-tests were used to compare the mean durations of copulation with first males with the mean time spent copulating with the second males. Homogeneity chi-square was used to test for frequency differences in the number of females copulating with their first male for a particular period of time and their propensity to mate with a second male. This statistic was also used to test for differences (for doubly mated females) in the frequency of females storing sperm from one versus two males at the three different time periods of copulation, and frequencies of sperm storage in the three spermathecae. Pearson’s ‘r’ correlation was used to examine the relationship between the durations of first and second copulations of doubly mated females. Data were analyzed using the software package R 3.0.2 (R Core Team 2013). Results Copulation Duration In total, 180 female flies copulated with at least one male. Approximately half (93) of these females subsequently mated again within 24 h. The possible effect of the duration of copulation on the propensity of females to mate again was assessed for all 180 females in this study in three first-copulation time categories (Fig. 1). The percent of females falling into all three copulation time categories showed an inverse relationship between singly mated versus doubly mated female flies (Fig. 1). Females that copulated with a second male had significantly shorter mean copulation times (t = 2.89, df = 165, P = 0.004) with their first sire (18.64 ± 10.09 min) than did females mating only once (23.43 ± 11.23 min). Almost half (44/93) of all females that mated twice had their first copulation last ≤15 min compared with about a quarter of females that only mated once (21/87; Fig. 1). Females whose first mating was 15 min or less were significantly more likely to mate a second time compared with females that had mated for 16–25 min (Fig. 1; homogeneity chi-square, χ2 = 5.75, df = 1, P < 0.02) or mated ≥26 min (homogeneity chi-square, χ2 = 8.46, df = 1, P < 0.01). Alternatively, females that had mated 16–25 min versus females mating ≥26 min with their first male did not differ significantly in their propensity to mate with a second male (homogeneity chi-square, χ2 = 0.42, df = 1, P = 0.52). Fig. 1. View largeDownload slide Percent female A. suspensa copulating once (open) or twice (stippled) grouped in three time periods according to the duration of the first copulation. Numbers within columns are sample sizes. Fig. 1. View largeDownload slide Percent female A. suspensa copulating once (open) or twice (stippled) grouped in three time periods according to the duration of the first copulation. Numbers within columns are sample sizes. Of 180 female flies, 93 copulated with a second male within the following 24 h and were, therefore, assessed further in this study. Singly mated flies were not examined in this study since sperm storage patterns for singly mated females has already been described by Fritz (2004). For doubly mated females, the duration of copulation from 3 to 55 min and the mean (± SD) for all copulation durations was 19.20 ± 9.90 min (N = 93). The duration of copulation was, however, significantly different between first versus second copulations (e.g., mean 20.90 ± 9.69 min vs 17.49 ± 9.86 min, respectively; t = 2.03, df = 92, P = 0.04). The mean copulation durations with the second males, for females in the three time categories mentioned above, were 1–15 min = 25.02 ± 11.69, 16–25 min = 19.97 ± 3.51 and ≥26 min = 12 ± 4.06 min, respectively (Fig. 2). One-way ANOVA showed an overall significant difference in mean copulation durations with the second males among the three time categories (F = 14.14, df = 2, P < 0.01). Post-ANOVA pair-wise comparison (Tukey’s HSD test) showed that the mean copulation duration of the first time category was not significantly different from the mean copulation duration of the second time category (P > 0.05). But, the mean copulation duration of first and second time categories were significantly different from the third time category (P < 0.01). Fig. 2. View largeDownload slide Mean (±SD) copulation duration with first sires (open) versus second sires (stippled) of doubly mated females grouped in three time periods according to the females’ first copulation duration (N = 93). Fig. 2. View largeDownload slide Mean (±SD) copulation duration with first sires (open) versus second sires (stippled) of doubly mated females grouped in three time periods according to the females’ first copulation duration (N = 93). For the 93 females that copulated twice and were categorized in the first and second time categories (1–15 min and 16–25 min), the mean duration of copulation with first males was shorter than the mean time spent copulating with second males and this pattern was reversed in the third time category (≥26 min). This apparent inverse relationship between the time females spent in copulation with first versus second males was congruent with correlation analysis (Pearson correlation r = −0.37691, P < 0.01, N = 93, Fig. 3). Fig. 3. View largeDownload slide Pearson correlation between females’ first copulation duration versus females’ second copulation duration (r = −0.37691, P = 0.00001, N = 93). Fig. 3. View largeDownload slide Pearson correlation between females’ first copulation duration versus females’ second copulation duration (r = −0.37691, P = 0.00001, N = 93). Microsatellite Genotypes of Males and Females The genotypes of 93 doubly mated females and the 186 males with which they mated were determined for three polymorphic microsatellite loci. Only 52 of these females (along with their 104 sires), however, were genotypically distinguishable allowing for the genetic discrimination of sperm from both males in a particular female’s sperm storage organs. For inclusion in this study, sperm had to be distinguishable between males and from the female genotype by a minimum of two microsatellite loci. The four sperm storage organs of each of the 52 females (N = 208 sperm storage organs) mentioned above were dissected and visually inspected for the presence of sperm before proceeding with the isolation of DNA using DAPI stain. Although all copulations of these 52 females lasted for at least 3 min, 16 females had no stored sperm or had an insignificant quantity (<10). Thus, 36 females (69.23% of the 52 doubly mated females) of the original 180 mated flies were found suitable for discerning the pattern of sperm storage by molecular analysis (Table 1). Sperm Storage and Copulation Duration Ninety-two percent of females that mated twice (N = 36) and copulated relatively short periods of time with the first sire (1–15 min) stored sperm from one male only (Table 1, Fig. 4): this sperm came from the second sire almost exclusively (12 out of 13 females). This pattern was reversed for females that mated with their first sire for ≥15 min (Table 1, Fig. 4); when copulation time with the first sire was greater than 15 min and the female fly stored the sperm from only one male, then it was almost exclusively sperm from the first male (5/6 = 83%; Fig. 4). In summary, short copulations with the first sire generally led to storage of sperm from a single male, the second sire. Longer copulations were less likely to lead to females storing sperm from single males (only 20%, Fig. 4), but if they did, the sperm was almost always from the first male. The percent of females storing sperm from both males was significantly different for short copulations (1–15 min) versus those lasting 16–25 min (homogeneity chi-square, χ2 = 9.31, df = 1, P < 0.01). Although inconclusive due to sample size (N = 5), 60% of females that initially copulated for ≥26 min stored the sperm from a single male (Fig. 4). Fig. 4. View largeDownload slide Number of doubly mated females (N = 36) storing sperm from one or both males’ with respect to the duration of their copulation with the first male (N = 36). Hatched columns = number of females storing sperm from the second male only; open bars = number of females storing sperm from the first male only; stippled bars = females storing sperm from both males. Fig. 4. View largeDownload slide Number of doubly mated females (N = 36) storing sperm from one or both males’ with respect to the duration of their copulation with the first male (N = 36). Hatched columns = number of females storing sperm from the second male only; open bars = number of females storing sperm from the first male only; stippled bars = females storing sperm from both males. Storage Patterns in the Ventral Receptacle and Spermathecae All 36 doubly mated females were analyzed for sperm storage patterns, all females examined stored sperm in their ventral receptacle and in at least one of the three spermathecae (Table 1). Doubly mated females that stored the sperm of only a single male, however, were more likely to have one or two empty sperm storage organs than females storing sperm from two males. Almost all females (12/13) storing sperm from two males in their ventral receptacle also had both spermathecae of the doublet (the paired spermathecae) with sperm (and from separate males). No spermathecae were observed to contain the sperm of more than one male, but 39% of all females had sperm from both males in their ventral receptacle (Table 1). If the sperm from only one male was present in the ventral receptacle of a female fly then sperm stored in any of her spermathecae were from the same male. In addition, when the sperm from a single male was stored in the ventral receptacle, they were typically from the second sire (14/20). Among the 36 doubly mated females, 61% (22/36) had no sperm in the singlet (the single spermathecae that occurs on one side of the reproductive tract) spermatheca (Table 1). If there was sperm in the singlet spermatheca then this sperm was almost always from the second male (12/14 individuals) whether the female had stored sperm from one or both males she mated with (Table 1). Considering just those 20 females with only one male’s sperm in their ventral receptacle, seven also had sperm in the singlet spermatheca; of these singlet spermathecae, all except one had the sperm of the second male (Table 1). Sperm Storage Bias If each of the three spermathecae are used equally in the storage of sperm from males, then each of the three spermathecae should have an equal frequency of sperm presence. This assumption was tested by chi-square analysis. As described previously, the three spermathecae of A. suspensa occur as a single organ (singlet spermathecae) on one side of the fly’s abdomen, whereas the other two spermathecae (doublet) occur as a pair on the opposite side of the abdomen; neither of the latter can be distinguished from one another. Thus, the null hypothesis = the singlet spermatheca accounts for 1/3 of all the spermathecae with stored sperm and the doublet accounts for 2/3 of stored sperm. Females storing sperm from single males were less likely to have sperm in the singlet spermatheca than in the doublet spermathecae (N = 36, χ2 = 11.4, df = 1, P < 0.01). Similarly, chi-square analysis of the observed and expected number of empty singlet and empty doublet spermathecae for females storing both males’ sperm showed a significantly higher number of empty singlet spermathecae (χ2 = 14.46, df = 1, P < 0.01). Although the number of females storing only one male’s sperm did have a higher frequency of empty singlet spermathecae than females with sperm from two males, this difference was not significant (χ2 = 2.68, df =1, P = 0.1). Finally, of all spermathecae storing sperm in doubly mated females, almost 2/3 stored the sperm of the second male exclusively (43/67). Relative Quantification of Sperm The only sperm storage organ that contained the sperm of more than one male was the ventral receptacle (Table 1). There were no instances where a doubly mated female was devoid of sperm in the ventral receptacle, whereas this was not the case for any of the spermathecae (Table 1). There were only 13 females, though, that stored the sperm of both males in their ventral receptacle. The sperm DNA was amplified by PCR in these 13 samples using the aforementioned microsatellite primers to generate relative peak areas of the amplicons from both males. Although the relative quantity of sperm stored within a ventral receptacle was sometimes skewed to one sire or the other, there was no discernable pattern of relative sperm quantity in the ventral receptacles of these 13 females (Table 1). Furthermore, the average relative percent of sperm in the ventral receptacle was very similar for both males—50.51% for first sires and 55.02% for second sires (Table 1). Discussion Copulation Duration Copulation durations for doubly mated A. suspensa females sampled in this study (N = 93) ranged from 3 to 55 min, consistent with copulation durations found in previous studies for singly mated females (Fritz 2001, 2004, Wallace 2005). The overall mean copulation time was consistent with the mean duration of copulation reported in previous studies for A. suspensa (Aluja et al. 2000, Fritz 2001, 2004, Wallace 2005). Previous studies on A. suspensa, however, did not consider or include the duration of copulation for doubly mated females. In this study, a significant difference was found in the amount of time females spent copulating with the first male versus the second male; more time was spent in copulation with second males. Sperm Storage Patterns Females typically use their hind legs and other body movements to dislodge males in copula as reported by Sivinski (2000), Wallace (2005), and Fritz (2009). Since females in first and second copulations were the same individuals, but the sires were different, the change in the duration of copulation suggests females are affecting this difference rather than males. The possible reason for this difference may be the significant percent of females storing little to no sperm during their first copulation and, as a consequence, mating for longer periods of time during their second copulation. Evidence in support of this hypothesis includes a study by Fritz (2004) in which sperm storage patterns in females that had copulated once were examined. Fritz (2004) reported that short copulations (e.g., <15 min) often led to insignificant storage of sperm. Furthermore, the duration of copulation correlated positively with the amount of sperm females stored (Fritz 2004). In this study, the duration of the first copulation correlated negatively with the duration of the second copulation for doubly mated females (Fig. 3, N = 93) and probably reflects a similar relationship to the quantity of sperm stored as observed by Fritz (2004). Over 30% of all females that had copulated twice, and whose sperm storage organs were visually scanned, had little or no detectable sperm in their sperm storage organs, and the majority of these females had copulated for less than 15 min. These data agree with results obtained for A. suspensa by Mazomenos et al. (1977) who reported similarly high rates of females storing few to any sperm after copulation. The molecular analysis of sperm storage patterns in 36 doubly mated females provided some insight and plausible explanations for the relationship between sperm storage and copulation duration discussed above. Over one-third (13/36) of all 36 females stored sperm from only the second male (Table 1). Moreover, most of these females were individuals that had copulated with the first male for less than 15 min. Thus, a pattern emerges whereby females that initially mate for a short period of time are more likely to have little if any sperm stored (Fritz 2004), but more likely to mate with a second male and for a longer period of time, which results in the storage of sperm primarily from the second male (Figs. 1–4). This outcome is somewhat reversed for females that had longer copulations with the first male. Females that copulated with the first male for 16–25 min copulated with the second male for only a slightly longer period of time (Fig. 2), whereas females that copulated with the first male for more than 26 min had a much shorter copulation time with the second male (Fig. 2). If these females (copulating >15 min) stored sperm from only one male, it was most likely to be sperm from the first male (Fig. 4). Finally, females copulating with the first male for more than 15 min were more likely to store sperm from both males than females copulating initially for less than 15 min; the latter generally had sperm from only one male, typically the second male. Among the spermathecae, sperm was stored significantly more often in the doublet spermathecae. The doublet spermathecae are two independent sperm storage organs held together by a muscular sheath enclosing their individual ducts for approximately one-third of their length (Fritz and Turner 2002). While previous studies on A. suspensa have documented the differential use of the paired spermathecae in singly mated females (Fritz 2004, Wallace 2005), this study is the first to document patterns of storage specific to each male in doubly mated females. For example, whereas one male’s sperm may be stored in one of the spermathecae of the doublet, the second male’s sperm may exclusively occupy the remaining doublet spermatheca. This phenomenon highlights the independent use of these two paired spermathecae and provides a unique storage site for each mate whose sperm is stored. Sexual selection theory advances two hypotheses for storage of sperm. The first, sperm competition, suggests selection has acted to promote conditions where male gametes would be stored together to provide an arena for male–male gametic competition (Parker 1970, Simmons 2001). The second, cryptic female choice, suggests that female anatomy and physiology have been selected to maintain control of different males’ sperm to set the stage for differential use in ova fertilization (Eberhard 1996, Simmons 2001). The storage patterns exhibited in the three spermathecae of A. suspensa suggest the latter as a possible hypothesis, since different male gametes are held in separate spermathecae ruling out an arena for male–male competition; in no instance were sperm from two different males found together in a spermatheca. The singlet spermathecae were less likely to house sperm than were the doublets, a finding consistent with earlier studies (Fritz 2004, Wallace 2005). The singlet spermatheca primarily stored the second males’ sperm regardless of the duration of copulation; thus, first male copulations rarely end in sperm deposition in this particular storage organ (Table 1). The spermathecae are thought to be long-term storage organs and are used to fill the ventral receptacle as it gets depleted of sperm (Twig and Yuval 2005, Pérez-Staples et al. 2007). Under-utilization of the singlet spermathecae is poorly understood and may be an artifact of the studies to date, which until now have documented single matings exclusively or may represent an anatomical or physiological difference not yet elucidated. The ventral receptacle not only disproportionately stored sperm more often than did the spermathecae (all doubly mated females had sperm in the ventral receptacle), but Fritz (2004) found this organ accounts for an average of about 45% of all sperm stored by singly mated females in A. suspensa. The ventral receptacle is considered to be the site of ova fertilization (Solinas and Nuzzaci 1984) and the site of short-term sperm storage (Twig and Yuval 2005). Scolari et al. (2014) reported that, for doubly mated females of C. capitata, sperm from two males occur in equal amounts in the ventral receptacle. Our study found that although relative quantities of sperm present in the ventral receptacle from both sires can vary, the overall average amount of sperm stored is about equal for first versus second sires. Scolari et al. (2014) investigated sperm storage patterns in the ventral receptacle of doubly mated C. capitata using fluorescently labeled sperm and reported sperm stratification in the ventral receptacle. Their data indicated a disproportionately greater number of offspring from second males, although the percent of offspring sired by the first male increased over time. Without knowledge of sperm storage patterns, this outcome might have been interpreted as a case of sperm precedence in male sperm competition. Scolari et al. (2014), however, describes sperm storage stratification in the ventral receptacle that alone can account for the preponderance of second male offspring without invoking sperm competition. Scolari et al. (2014), however, did not include spermathecae in their analysis and our sperm storage patterns provide another possible set of circumstances that can also lead to skewed paternity outcomes. For example, in our study the majority of doubly mated females storing sperm from one male represented the second sire. Furthermore, of all spermathecae storing sperm in doubly mated females (N = 36), two-third of them (43/67) stored sperm from the second sire only (Table 1). Thus, as the ventral receptacle gets depleted during oviposition, sperm remaining in the long-term storage organs (spermathecae) available to replenish the site of fertilization, are primarily of the second male. If sperm stratification occurs in the ventral receptacle of A. suspensa, as has been described for C. capitata (Scolari et al. 2014), then an overabundance of second male offspring during the initiation of oviposition would ensue simply due to the way sperm are stored. Therefore, we would further predict that as females continue to oviposit and deplete sperm from their ventral receptacle, the proportion of offspring sired by the second male would rise again, on average, as spermathecae replenished the site of fertilization, the ventral receptacle. In summary, the results of this study describe the copulatory and sperm storage outcomes when there are multiple sires and where there is no opportunity for females to oviposit (since mating trials were accomplished within 24 h). Short copulations with a male generally resulted in little transfer of sperm and predicted the length of time spent in second copulations, resulting in disproportionate sperm storage from second copulations. Sperm storage organs can fill independently with sperm from different males, but each spermatheca stores sperm from one sire only. Independent sperm storage from different males provides the possibility for females to control paternity outcomes and avoid sperm competition where plausible and possible. Thus, in the absence of the details of sperm storage patterns and reproductive tract dynamics in females, invocation of sperm competition or cryptic female choice as the explanation for particular paternity outcomes may be unwarranted; our results imply that paternity outcomes over the lifetime of a female fly may be more dynamic and complex than hitherto considered. Acknowledgments We thank S. Fraser for assistance on this project. This project was supported, in part, by the Eastern Illinois University Council on Faculty Research, Eastern Illinois University Department of Biological Sciences, and the National Research Initiative of the USDA Cooperative State Research Education and Extension Service, grant no. 2004-3502-17673. References Cited Aluja, M. 1994. Bionomics and management of Anastrepha. Ann. Rev. 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Polyandry in the medfly – shifts in paternity mediated by sperm stratification and mixing. BMC Genetics  15 ( Suppl 2): S10. http://www.biomedcentral.com/1471–2156/S1/S2/S10. Accessed 29 May 2015. Simmons, L. W. 2001. Sperm competition and its evolutionary consequences in insects . Princeton University Press, Princeton, NJ. Sivinski, J. M., and Heath R. R.. 1988. Effects of oviposition on remating, response to pheromones, and longevity in female Caribbean fruit fly, Anastrepha suspensa (Diptera: Tephritidae). Ann. Entomol. Soc. Am . 81: 1021– 1024. Google Scholar CrossRef Search ADS   Sivinski, J. M., and Burk T.. 1989. Reproductive and mating behavior, pp. 343– 351. In A. S. Robinson and G. Hooper, (eds.), Fruit flies: their biology, natural enemies and control . In W. Helle (ed.), World crop pests, vol. 3A. Elsevier Science Publishers, Amsterdam, The Netherlands. Sivinski, J. M., Aluja, G. Dodson, A. Freidberg, D. Headrick, K. Kaneshiro, and Landolt P.. 2000. Topics in the evolution of sexual behavior in the Tephritidae, pp. 751– 792. In M. Aluja, and A. L. Norrbom (eds.), Fruit flies (Tephritidae): phylogeny and evolution of behavior . CRC Press, Boca Raton, FL. Solinas, M., and Nuzzaci G.. 1984. Functional anatomy of Dacus oleae Gmel. Female genitalia in relation to insemination and fertilization processes. Entomologica . 19: 135– 165. Team, R. 2013. R Development Core Team. RA Lang. Environ. Stat. Comput . 55: 275– 286. Thornhill, R. 1983. Cryptic female choice and its implications in the in the Scorpionfly Hylobittacus nigriceps. Am. Nat . 6: 765– 788. Google Scholar CrossRef Search ADS   Twig, E., and Yuval B.. 2005. Function of multiple sperm storage organs in female Mediterranean fruit flies (Ceratitis capitata, Diptera: Tephritidae). J. Insect. Physiol . 51: 67– 74. Google Scholar CrossRef Search ADS PubMed  Yuval, B., Blay S., and Kaspi R.. 1996. Sperm transfer and storage in the Mediterranean fruit fly (Ceratitis capitata, Diptera: Tephritidae).). Ann. Entomol. Soc. Am . 86: 486– 492. Google Scholar CrossRef Search ADS   Wallace, H. J. 2005. Copulatory behaviors of male Caribbean fruit flies (Anastrepha suspensa) and female sperm storage patterns . MS thesis. Eastern Illinois University, Charleston. © The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of the Entomological Society of America Oxford University Press

Sperm Storage Patterns in Doubly Mated Female Anastrepha suspensa (Diptera: Tephritidae)

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Abstract

Abstract We examined sperm storage patterns of doubly mated females in a species of tephritid fly (Anastrepha suspensa (Loew) [Diptera: Tephritidae]) with four sperm storage organs and an unusually complex reproductive system to elucidate possible reproductive arenas in which sexual selection can or has played a role in sperm competition and cryptic female choice and to predict effects on paternity outcomes. The duration of copulation with each male of doubly mated female flies was recorded and the sperm storage organs were dissected to determine the location and identity of each male’s sperm (by microsatellite polymerase chain reaction) and its relative quantity. Short copulations with the first male generally resulted in little transfer of sperm and predicted the length of time spent in second copulations, resulting in disproportionate sperm storage from second copulations. For doubly mated females, most storage organs contained sperm from second males only, although the fertilization chamber often contained sperm from both males (allowing for possible sperm competition). Long-term storage organs (spermathecae) always contained sperm from single males exclusively possibly limiting opportunities for sperm competition. Our data provide a mechanism for second male precedence without invoking sperm competition, and caution that paternity patterns must be critically interpreted with inclusion of information on sperm storage patterns. Multiple mating in insects is well-documented (Ridley 1988, Arnqvist and Nilsson 2000), setting up circumstances where postcopulatory sexual selection may act through sperm competition (Parker 1970, 1979) and cryptic female choice (Thornhill 1983, Eberhard 1996). Evolutionary theory predicts male and female postcopulatory sexual selection is based on the disparity in investment by each sex; females usually invest more in the production of ova than males that invest in the development of spermatozoa (Bateman 1948, Parker 1970). Thus, female fitness is limited by the production of ova, whereas male fitness is limited by the number of ova fertilized (Parker 1970). However, it has become increasingly apparent from studies on a number of different insects that copulation per se does not guarantee paternity for a particular male (Eberhard 1996), and the cost of ejaculate and fluid production for males is high (Simmons 2001). In addition, within the Insecta, a temporal separation often occurs between copulation and ova fertilization, generating a dynamic timeframe for sexual selection to act at the level of reproductive tract morphology and gametes (Parker 1970, Eberhard 1996, Simmons 2001), especially when females are utilizing patchy resources for oviposition (Prokopy 1980, Burke 1981). In this study, we examine sperm storage patterns of doubly mated females in a species with four sperm storage organs and an unusually complex reproductive system to assess whether sexual selection plays a role in sperm competition and cryptic female choice. This study is based on the understanding that the temporal disjunction between copulation and fertilization may influence both male (sperm competition) and female (cryptic female choice) and that these interests are subject to evolutionary forces when females mate multiply and ejaculates from different sires are stored for long periods of time prior to ova fertilization (Parker 1970, Eberhard 1996, Simmons 2001). Multiple sperm storage organs and multiple mating provide the potential opportunity for the differential storage and use of sperm from two or more males and subsequently a unique chance to determine the importance of possible sperm competition and cryptic female choice. Until recently, with the advent of genetic lines with fluorescently labeled sperm, studies on sexual selection have been hampered by the inability to discern specific sperm storage patterns and other reproductive system dynamics in multiply mated females. These studies have generally tried to understand paternity outcomes, sperm storage, and sperm use indirectly by examining offspring only. We used a protocol developed by Fritz et al. (2010) in which the sperm storage organs are dissected and cleared of maternal cells, and the DNA is extracted, amplified (microsatellite PCR), and assigned to individual males. Our study species, the Caribbean fruit fly, Anastrepha suspensa (Loew), is a New World member of the invasive fruit fly family Tephritidae and is an agricultural pest in the Antilles, Puerto Rico and Florida (Aluja 1994, Aluja and Norrbom 2000, El-Sayed et al. 2009). As with many tephritid invasive generalists, female A. suspensa mate multiply (Sivinski and Heath 1988, Sivinski and Burk 1989) and possess a highly complex reproductive tract for storing and using sperm (Fritz and Turner 2002). In A. suspensa females, spermatozoa are stored in four different specialized organs (three spermathecae and a ventral receptacle; Fritz and Turner 2002). The spermathecae are thought to be sites for long-term storage to replenish the female’s ventral receptacle, the short-term storage organ where ova fertilization occurs, as described in other tephritids, such as Ceratitis capitata (Wiedemann) and Bactrocera tryoni (Froggatt) (Twig and Yuval 2005, Pérez-Staples et al. 2007). Fritz and Turner (2002) detailed the unusual complexity of the spermathecae and ventral receptacle in A. suspensa, which suggest strong selection for the maintenance, compartmentalization, and mobilization of sperm. The spermathecae have highly infolded sculptured interiors, where sperm are lodged in contact with exit pores layered with secretory cells. Spermathecal capsules also have valves that control the entry and release of sperm, and each spermathecae has its own separate duct surrounded by musculature. The ventral receptacle is surrounded by oblique musculature and embedded in a thick layer of mitochondrial-rich cells with numerous canuli, which may allow for the rapid evacuation of fluid from the ventral receptacle. The ventral receptacle also includes a mass of cuticular infoldings and a vacuole filled with secretory substances and surrounded by approximately 200 alveoli in which small numbers of sperm can reside (Fritz and Turner 2002). Thus, A. suspensa offers a rich morphological and physiological arena in which postcopulatory sexual selection may act. Sperm storage patterns in A. suspensa have been described by Fritz (2004) for singly mated flies, but the dynamics of sperm storage have not been examined in doubly mated females. Although sperm storage patterns have been examined in tephritid flies in a number of studies with single and doubly mated females (Yuval et al. 1996, Fritz 2004, Bertin et al 2010, Scolari et al. 2014), the present study represents the first to describe both the identity and location of sperm stored from two different males in all of the sperm storage organs. Materials and Methods Study Organisms Flies were obtained as pupae from the Biological Control Mass Rearing Facility at the Department of Agriculture Division of Plant Industry in Florida and reared in a quarantine insectary maintained under a photoperiod of 14:10 (L:D) h, a temperature of 25 ± 2°C and 55% relative humidity, according to the protocols described by Fritz (2004). Sexually mature adult flies used in mating crosses were sorted by sex as teneral adults. After separating males and females, females were individually marked with nontoxic paint dots according to a standardized combination of paint colors and locations on the dorsum (Dhakal 2008). Mating trials The age of flies in mating trials was standardized by using only post-teneral flies (9 d after emergence) and for 6 d thereafter (Kendra et al. 2005). All mating trials were run between 12:00 and 16:00 h, a time when flies are sexually active (Burk 1983). Sexually mature males emit pheromones and thus “call” for females (Nation 1972). Individual couples’ copulations were timed. Timing began when the male inserted his aedeagus into the female’s genital opening and ended when copulation was terminated and couples fully separated (e.g., observation of the complete withdrawal of the aedeagus). Cohorts of approximately 130 marked females were placed into identical mating cages (1.5 liter plastic jar with a cloth stockinette access sleeve over the opening) containing 200 males each; all flies were the same age and had the same emergence date. When copulation ensued, a pair was removed from the mating cage by allowing the female to walk forward upon a wooden applicator stick and enter a 75-ml plastic vial. Vials were capped with foam plugs and monitored until cessation of copulation. After each mating, the mated pair was held in absolute darkness for 2 h to allow sufficient time for sperm storage within the female and to prevent females from repeatedly copulating with the same male, following Fritz (2004). Subsequently, individual males were placed in an eppendorf tube, labeled with the female identification number and frozen at −65°C. Females were transferred to a new cage lacking oviposition substrate, provided with water and food ad libidum and were given the opportunity to mate the following day with virgin males. After second copulations ensued, flies were handled and subsequently frozen according to the protocols mentioned above. To examine the relative duration of copulation of females with their first male mate versus their second male mate, three mating-time categories were established. These categories included: females that mated from 1 to 15 min, females that mated 16 to 25 min, and females that mated ≥26 min. The first and second categories were established because prior literature indicated female A. suspensa mating <15 min often did not store sperm (Fritz 2004), while a mating time of 16–25 min corresponds to a range including the mean copulation durations noted by previous studies (Fritz 2004, Wallace 2005). Molecular Protocol The presence, origin (from which male), relative quantity, and location of sperm in the four sperm storage organs of doubly mated females were investigated using microsatellite primers and PCR. Microsatellite primers have been developed for A. suspensa (Fritz and Schable 2004), and Fritz et al. (2010) described the protocols for isolating, amplifying, and quantifying sperm DNA in A. suspensa. The protocols can be summarized in five major steps: 1) isolation of DNA from adult flies and sperm storage organs in females (three spermathecae and a ventral receptacle) by microdissection, 2) removal of the maternal cells from sperm storage organs through sonication (to prevent maternal contamination of DNA during PCR), 3) isolation of sperm DNA from sperm storage organs, 4) PCR amplification of DNA with microsatellite loci, and 5) sperm relative quantification. Of 180 females that copulated, 93 mated again with a second male. All doubly mated females (N = 93) and their males (N = 186) were genotyped for three polymorphic microsatellite loci (837 PCR reactions) using primers 1H, 3B, and 5E (Fritz and Schable 2004) and previously used to study sperm storage (Fritz et al. 2010). Doubly mated females along with their two male sires were subsequently chosen for further analysis if all three individuals differed genetically for at least two loci; 52 females and their 104 males fell into this category. These 52 females were then dissected for their sperm storage organs and visually inspected for the presence of sperm using 4',6-diamidino- 2-phenylindole (DAPI) staining and fluorescent microscopy. Of these 52 females, individuals with few (<10) sperm or no sperm in all their storage organs were eliminated from further analysis. Thus, our sample size of doubly mated females storing >10 sperm from at least 1 male was 36 females. If no sperm was visualized in a sperm storage organ or produced amplicons after PCR, then this organ was abbreviated as NV (Table 1). Missing data were abbreviated as MD (Table 1). Each spermatheca in the doublet (the paired spermathecae) was not individually distinguishable from its pair by position or any morphological marker. Thus, the assignment of sperm to one or the other spermatheca of the doublet was arbitrary in Table 1. Table 1. The Relative Percentage of Spermatozoa Contributed by First and Second Sires to Four Female Sperm storage Organs for 36 Doubly Mated Females No. of females (N = 36)  Sire  CD (min)  SP  DBL  VR  1  First sire  13  0%  NV/NV  0%  Second sire  45  100%  NV/NV  100%  2  First sire  50  NV  100%/0  100%  Second sire  20  NV  0%/NV  0%  3  First sire  11  NV  0%/NV  0%  Second sire  9  NV  100%/NV  100%  4  First sire  5  NV  0%/NV  0  Second sire  8  NV  100%/NV  100%  5  First sire  19  0%  NV/100%  38.2%  Second sire  17  100%  NV/0  61.8%  6  First sire  16  NV  100%/0%  46.2%  Second sire  15  NV  0%/100%  53.82%  7  First sire  15  NV  100%/NV  100%  Second sire  10  NV  0%/NV  0%  8  First sire  10  0%  0%/NV  0%  Second sire  38  100%  100%/NV  100%  9  First sire  7  NV  0%/0%  0%  Second sire  35  NV  100%/100%  100%  10  First sire  14  NV  0%/NV  0%  Second sire  30  NV  100%/NV  100%  11  First sire  12  0%  0%/0%  0%  Second sire  32  100%  100%/100%  100%  12  First sire  10  0%  0%/0%  0 %  Second sire  16  100%  100%/100%  100%  13  First sire  20  NV  100%/NV  100%  Second sire  7  NV  0%/NV  0%  14  First sire  9  NV  0%/NV  0%  Second sire  10  NV  100%/NV  100%  15  First sire  16  0%  100%/0%  50.3%  Second sire  21  100%%  0%/100%  49.7%  16  First sire  10  NV  0%/NV  0%  Second sire  20  NV  100%/NV  100%  17  First sire  21  0%  0%/0%  0%  Second sire  17  100%  100%/100%  100%  18  First sire  35  100%  100%/100%  100%  Second sire  11  0%  0%/0%  0%  19  First sire  40  NV  100%/NV  100%  Second sire  10  NV  0%/NV  0%  20  First sire  17  NV  100%/0%  73.9%  Second sire  16  NV  0%/100%  26.1%  21  First sire  16  NV  100%/0%  46.8%  Second sire  20  NV  0%/100%  53.2%  22  First sire  16  100%  100%/0%  28.7%  Second sire  17  0%  0%/100%  71.3%  23  First sire  55  0%  100%/0%  50.7%  Second sire  14  100%  0%/100%  49.3%  24  First sire  35  0%  0%/0%  0%    Second sire  11  100%  100%/100%  100%  25  First sire  13  0%  100%/0%  59.4%  Second sire  31  100%  0%/100%  40.6%  26  First sire  17  NV  100%/0%  67.8%  Second sire  24  NV  0%/100%  32.2%  27  First sire  19  0%  100%/0%  53.8%  Second sire  22  100%%  0%/100%  46.2%  28  First sire  20  NV  100%/0%  36.7%  Second sire  17  NV  0%/100%  63.3%  29  First sire  9  NV  0%/NV  0%  Second sire  35  NV  100%/NV  100%  30  First sire  31  NV  100%/0%  32.9%  Second sire  15  NV  0%/100%  67.1%  31  First sire  10  NV  100%/0%  100%  Second sire  5  NV  0%/NV  0%  32  First sire  14  0%  100%/0%  74.3%  Second sire  38  100%  0%/100%  25.7%  33  First sire  12  NV  0%/0%  0%  Second sire  12  NV  100%/100%  100%  34  First sire  12  NV  100%/0%  MD    Second sire  39  NV  0%/100%  MD  35  First sire  NV  0%  100%  MD    Second sire  NV  100%  0%  MD  36  First sire  NV  100%  0  MD    Second sire  NV  0%  100%  MD  No. of females (N = 36)  Sire  CD (min)  SP  DBL  VR  1  First sire  13  0%  NV/NV  0%  Second sire  45  100%  NV/NV  100%  2  First sire  50  NV  100%/0  100%  Second sire  20  NV  0%/NV  0%  3  First sire  11  NV  0%/NV  0%  Second sire  9  NV  100%/NV  100%  4  First sire  5  NV  0%/NV  0  Second sire  8  NV  100%/NV  100%  5  First sire  19  0%  NV/100%  38.2%  Second sire  17  100%  NV/0  61.8%  6  First sire  16  NV  100%/0%  46.2%  Second sire  15  NV  0%/100%  53.82%  7  First sire  15  NV  100%/NV  100%  Second sire  10  NV  0%/NV  0%  8  First sire  10  0%  0%/NV  0%  Second sire  38  100%  100%/NV  100%  9  First sire  7  NV  0%/0%  0%  Second sire  35  NV  100%/100%  100%  10  First sire  14  NV  0%/NV  0%  Second sire  30  NV  100%/NV  100%  11  First sire  12  0%  0%/0%  0%  Second sire  32  100%  100%/100%  100%  12  First sire  10  0%  0%/0%  0 %  Second sire  16  100%  100%/100%  100%  13  First sire  20  NV  100%/NV  100%  Second sire  7  NV  0%/NV  0%  14  First sire  9  NV  0%/NV  0%  Second sire  10  NV  100%/NV  100%  15  First sire  16  0%  100%/0%  50.3%  Second sire  21  100%%  0%/100%  49.7%  16  First sire  10  NV  0%/NV  0%  Second sire  20  NV  100%/NV  100%  17  First sire  21  0%  0%/0%  0%  Second sire  17  100%  100%/100%  100%  18  First sire  35  100%  100%/100%  100%  Second sire  11  0%  0%/0%  0%  19  First sire  40  NV  100%/NV  100%  Second sire  10  NV  0%/NV  0%  20  First sire  17  NV  100%/0%  73.9%  Second sire  16  NV  0%/100%  26.1%  21  First sire  16  NV  100%/0%  46.8%  Second sire  20  NV  0%/100%  53.2%  22  First sire  16  100%  100%/0%  28.7%  Second sire  17  0%  0%/100%  71.3%  23  First sire  55  0%  100%/0%  50.7%  Second sire  14  100%  0%/100%  49.3%  24  First sire  35  0%  0%/0%  0%    Second sire  11  100%  100%/100%  100%  25  First sire  13  0%  100%/0%  59.4%  Second sire  31  100%  0%/100%  40.6%  26  First sire  17  NV  100%/0%  67.8%  Second sire  24  NV  0%/100%  32.2%  27  First sire  19  0%  100%/0%  53.8%  Second sire  22  100%%  0%/100%  46.2%  28  First sire  20  NV  100%/0%  36.7%  Second sire  17  NV  0%/100%  63.3%  29  First sire  9  NV  0%/NV  0%  Second sire  35  NV  100%/NV  100%  30  First sire  31  NV  100%/0%  32.9%  Second sire  15  NV  0%/100%  67.1%  31  First sire  10  NV  100%/0%  100%  Second sire  5  NV  0%/NV  0%  32  First sire  14  0%  100%/0%  74.3%  Second sire  38  100%  0%/100%  25.7%  33  First sire  12  NV  0%/0%  0%  Second sire  12  NV  100%/100%  100%  34  First sire  12  NV  100%/0%  MD    Second sire  39  NV  0%/100%  MD  35  First sire  NV  0%  100%  MD    Second sire  NV  100%  0%  MD  36  First sire  NV  100%  0  MD    Second sire  NV  0%  100%  MD  CD, copulation duration; DBL, doublet spermathecae; SP, singlet spermatheca; VR, ventral receptacle. Bold numbers are ventral receptacles with sperm from two males. View Large Amplicon peak areas generated by microsatellite PCR output on a DNA sequencer can be used to estimate the relative proportions of sperm contributed by each male storing sperm in a particular sperm storage organ (Fritz et al. 2010). Since male sperm is haploid, the ratio of the peak area of one allele (of one male) to that of another male provides a relative measure of sperm quantity. If a sperm storage organ contained the sperm from two different males (as was the case for the ventral receptacles) then the ratio of amplicon peak areas was used as a relative quantification of sperm contributed by each male (Fritz et al. 2010). PCR products were resolved on a Beckman Coulter (Fullerton, CA) CEQ 2000XL Sequence Analyser using the Beckman Coulter CEQ DNA Size Standard-400 and the CEQ Fragment Analysis Module Program (Beckman Coulter). Statistical Analysis One-way analysis of variance (ANOVA) was used to estimate the effect of the amount of time spent copulating with the first male on the amount of time spent mating with a second male (for three copulation duration categories: 1–15, 16–25, and >25 min). Tukey’s post hoc test was used for multiple comparisons of the means of the duration of copulation of female flies. Paired t-tests were used to compare the mean durations of copulation with first males with the mean time spent copulating with the second males. Homogeneity chi-square was used to test for frequency differences in the number of females copulating with their first male for a particular period of time and their propensity to mate with a second male. This statistic was also used to test for differences (for doubly mated females) in the frequency of females storing sperm from one versus two males at the three different time periods of copulation, and frequencies of sperm storage in the three spermathecae. Pearson’s ‘r’ correlation was used to examine the relationship between the durations of first and second copulations of doubly mated females. Data were analyzed using the software package R 3.0.2 (R Core Team 2013). Results Copulation Duration In total, 180 female flies copulated with at least one male. Approximately half (93) of these females subsequently mated again within 24 h. The possible effect of the duration of copulation on the propensity of females to mate again was assessed for all 180 females in this study in three first-copulation time categories (Fig. 1). The percent of females falling into all three copulation time categories showed an inverse relationship between singly mated versus doubly mated female flies (Fig. 1). Females that copulated with a second male had significantly shorter mean copulation times (t = 2.89, df = 165, P = 0.004) with their first sire (18.64 ± 10.09 min) than did females mating only once (23.43 ± 11.23 min). Almost half (44/93) of all females that mated twice had their first copulation last ≤15 min compared with about a quarter of females that only mated once (21/87; Fig. 1). Females whose first mating was 15 min or less were significantly more likely to mate a second time compared with females that had mated for 16–25 min (Fig. 1; homogeneity chi-square, χ2 = 5.75, df = 1, P < 0.02) or mated ≥26 min (homogeneity chi-square, χ2 = 8.46, df = 1, P < 0.01). Alternatively, females that had mated 16–25 min versus females mating ≥26 min with their first male did not differ significantly in their propensity to mate with a second male (homogeneity chi-square, χ2 = 0.42, df = 1, P = 0.52). Fig. 1. View largeDownload slide Percent female A. suspensa copulating once (open) or twice (stippled) grouped in three time periods according to the duration of the first copulation. Numbers within columns are sample sizes. Fig. 1. View largeDownload slide Percent female A. suspensa copulating once (open) or twice (stippled) grouped in three time periods according to the duration of the first copulation. Numbers within columns are sample sizes. Of 180 female flies, 93 copulated with a second male within the following 24 h and were, therefore, assessed further in this study. Singly mated flies were not examined in this study since sperm storage patterns for singly mated females has already been described by Fritz (2004). For doubly mated females, the duration of copulation from 3 to 55 min and the mean (± SD) for all copulation durations was 19.20 ± 9.90 min (N = 93). The duration of copulation was, however, significantly different between first versus second copulations (e.g., mean 20.90 ± 9.69 min vs 17.49 ± 9.86 min, respectively; t = 2.03, df = 92, P = 0.04). The mean copulation durations with the second males, for females in the three time categories mentioned above, were 1–15 min = 25.02 ± 11.69, 16–25 min = 19.97 ± 3.51 and ≥26 min = 12 ± 4.06 min, respectively (Fig. 2). One-way ANOVA showed an overall significant difference in mean copulation durations with the second males among the three time categories (F = 14.14, df = 2, P < 0.01). Post-ANOVA pair-wise comparison (Tukey’s HSD test) showed that the mean copulation duration of the first time category was not significantly different from the mean copulation duration of the second time category (P > 0.05). But, the mean copulation duration of first and second time categories were significantly different from the third time category (P < 0.01). Fig. 2. View largeDownload slide Mean (±SD) copulation duration with first sires (open) versus second sires (stippled) of doubly mated females grouped in three time periods according to the females’ first copulation duration (N = 93). Fig. 2. View largeDownload slide Mean (±SD) copulation duration with first sires (open) versus second sires (stippled) of doubly mated females grouped in three time periods according to the females’ first copulation duration (N = 93). For the 93 females that copulated twice and were categorized in the first and second time categories (1–15 min and 16–25 min), the mean duration of copulation with first males was shorter than the mean time spent copulating with second males and this pattern was reversed in the third time category (≥26 min). This apparent inverse relationship between the time females spent in copulation with first versus second males was congruent with correlation analysis (Pearson correlation r = −0.37691, P < 0.01, N = 93, Fig. 3). Fig. 3. View largeDownload slide Pearson correlation between females’ first copulation duration versus females’ second copulation duration (r = −0.37691, P = 0.00001, N = 93). Fig. 3. View largeDownload slide Pearson correlation between females’ first copulation duration versus females’ second copulation duration (r = −0.37691, P = 0.00001, N = 93). Microsatellite Genotypes of Males and Females The genotypes of 93 doubly mated females and the 186 males with which they mated were determined for three polymorphic microsatellite loci. Only 52 of these females (along with their 104 sires), however, were genotypically distinguishable allowing for the genetic discrimination of sperm from both males in a particular female’s sperm storage organs. For inclusion in this study, sperm had to be distinguishable between males and from the female genotype by a minimum of two microsatellite loci. The four sperm storage organs of each of the 52 females (N = 208 sperm storage organs) mentioned above were dissected and visually inspected for the presence of sperm before proceeding with the isolation of DNA using DAPI stain. Although all copulations of these 52 females lasted for at least 3 min, 16 females had no stored sperm or had an insignificant quantity (<10). Thus, 36 females (69.23% of the 52 doubly mated females) of the original 180 mated flies were found suitable for discerning the pattern of sperm storage by molecular analysis (Table 1). Sperm Storage and Copulation Duration Ninety-two percent of females that mated twice (N = 36) and copulated relatively short periods of time with the first sire (1–15 min) stored sperm from one male only (Table 1, Fig. 4): this sperm came from the second sire almost exclusively (12 out of 13 females). This pattern was reversed for females that mated with their first sire for ≥15 min (Table 1, Fig. 4); when copulation time with the first sire was greater than 15 min and the female fly stored the sperm from only one male, then it was almost exclusively sperm from the first male (5/6 = 83%; Fig. 4). In summary, short copulations with the first sire generally led to storage of sperm from a single male, the second sire. Longer copulations were less likely to lead to females storing sperm from single males (only 20%, Fig. 4), but if they did, the sperm was almost always from the first male. The percent of females storing sperm from both males was significantly different for short copulations (1–15 min) versus those lasting 16–25 min (homogeneity chi-square, χ2 = 9.31, df = 1, P < 0.01). Although inconclusive due to sample size (N = 5), 60% of females that initially copulated for ≥26 min stored the sperm from a single male (Fig. 4). Fig. 4. View largeDownload slide Number of doubly mated females (N = 36) storing sperm from one or both males’ with respect to the duration of their copulation with the first male (N = 36). Hatched columns = number of females storing sperm from the second male only; open bars = number of females storing sperm from the first male only; stippled bars = females storing sperm from both males. Fig. 4. View largeDownload slide Number of doubly mated females (N = 36) storing sperm from one or both males’ with respect to the duration of their copulation with the first male (N = 36). Hatched columns = number of females storing sperm from the second male only; open bars = number of females storing sperm from the first male only; stippled bars = females storing sperm from both males. Storage Patterns in the Ventral Receptacle and Spermathecae All 36 doubly mated females were analyzed for sperm storage patterns, all females examined stored sperm in their ventral receptacle and in at least one of the three spermathecae (Table 1). Doubly mated females that stored the sperm of only a single male, however, were more likely to have one or two empty sperm storage organs than females storing sperm from two males. Almost all females (12/13) storing sperm from two males in their ventral receptacle also had both spermathecae of the doublet (the paired spermathecae) with sperm (and from separate males). No spermathecae were observed to contain the sperm of more than one male, but 39% of all females had sperm from both males in their ventral receptacle (Table 1). If the sperm from only one male was present in the ventral receptacle of a female fly then sperm stored in any of her spermathecae were from the same male. In addition, when the sperm from a single male was stored in the ventral receptacle, they were typically from the second sire (14/20). Among the 36 doubly mated females, 61% (22/36) had no sperm in the singlet (the single spermathecae that occurs on one side of the reproductive tract) spermatheca (Table 1). If there was sperm in the singlet spermatheca then this sperm was almost always from the second male (12/14 individuals) whether the female had stored sperm from one or both males she mated with (Table 1). Considering just those 20 females with only one male’s sperm in their ventral receptacle, seven also had sperm in the singlet spermatheca; of these singlet spermathecae, all except one had the sperm of the second male (Table 1). Sperm Storage Bias If each of the three spermathecae are used equally in the storage of sperm from males, then each of the three spermathecae should have an equal frequency of sperm presence. This assumption was tested by chi-square analysis. As described previously, the three spermathecae of A. suspensa occur as a single organ (singlet spermathecae) on one side of the fly’s abdomen, whereas the other two spermathecae (doublet) occur as a pair on the opposite side of the abdomen; neither of the latter can be distinguished from one another. Thus, the null hypothesis = the singlet spermatheca accounts for 1/3 of all the spermathecae with stored sperm and the doublet accounts for 2/3 of stored sperm. Females storing sperm from single males were less likely to have sperm in the singlet spermatheca than in the doublet spermathecae (N = 36, χ2 = 11.4, df = 1, P < 0.01). Similarly, chi-square analysis of the observed and expected number of empty singlet and empty doublet spermathecae for females storing both males’ sperm showed a significantly higher number of empty singlet spermathecae (χ2 = 14.46, df = 1, P < 0.01). Although the number of females storing only one male’s sperm did have a higher frequency of empty singlet spermathecae than females with sperm from two males, this difference was not significant (χ2 = 2.68, df =1, P = 0.1). Finally, of all spermathecae storing sperm in doubly mated females, almost 2/3 stored the sperm of the second male exclusively (43/67). Relative Quantification of Sperm The only sperm storage organ that contained the sperm of more than one male was the ventral receptacle (Table 1). There were no instances where a doubly mated female was devoid of sperm in the ventral receptacle, whereas this was not the case for any of the spermathecae (Table 1). There were only 13 females, though, that stored the sperm of both males in their ventral receptacle. The sperm DNA was amplified by PCR in these 13 samples using the aforementioned microsatellite primers to generate relative peak areas of the amplicons from both males. Although the relative quantity of sperm stored within a ventral receptacle was sometimes skewed to one sire or the other, there was no discernable pattern of relative sperm quantity in the ventral receptacles of these 13 females (Table 1). Furthermore, the average relative percent of sperm in the ventral receptacle was very similar for both males—50.51% for first sires and 55.02% for second sires (Table 1). Discussion Copulation Duration Copulation durations for doubly mated A. suspensa females sampled in this study (N = 93) ranged from 3 to 55 min, consistent with copulation durations found in previous studies for singly mated females (Fritz 2001, 2004, Wallace 2005). The overall mean copulation time was consistent with the mean duration of copulation reported in previous studies for A. suspensa (Aluja et al. 2000, Fritz 2001, 2004, Wallace 2005). Previous studies on A. suspensa, however, did not consider or include the duration of copulation for doubly mated females. In this study, a significant difference was found in the amount of time females spent copulating with the first male versus the second male; more time was spent in copulation with second males. Sperm Storage Patterns Females typically use their hind legs and other body movements to dislodge males in copula as reported by Sivinski (2000), Wallace (2005), and Fritz (2009). Since females in first and second copulations were the same individuals, but the sires were different, the change in the duration of copulation suggests females are affecting this difference rather than males. The possible reason for this difference may be the significant percent of females storing little to no sperm during their first copulation and, as a consequence, mating for longer periods of time during their second copulation. Evidence in support of this hypothesis includes a study by Fritz (2004) in which sperm storage patterns in females that had copulated once were examined. Fritz (2004) reported that short copulations (e.g., <15 min) often led to insignificant storage of sperm. Furthermore, the duration of copulation correlated positively with the amount of sperm females stored (Fritz 2004). In this study, the duration of the first copulation correlated negatively with the duration of the second copulation for doubly mated females (Fig. 3, N = 93) and probably reflects a similar relationship to the quantity of sperm stored as observed by Fritz (2004). Over 30% of all females that had copulated twice, and whose sperm storage organs were visually scanned, had little or no detectable sperm in their sperm storage organs, and the majority of these females had copulated for less than 15 min. These data agree with results obtained for A. suspensa by Mazomenos et al. (1977) who reported similarly high rates of females storing few to any sperm after copulation. The molecular analysis of sperm storage patterns in 36 doubly mated females provided some insight and plausible explanations for the relationship between sperm storage and copulation duration discussed above. Over one-third (13/36) of all 36 females stored sperm from only the second male (Table 1). Moreover, most of these females were individuals that had copulated with the first male for less than 15 min. Thus, a pattern emerges whereby females that initially mate for a short period of time are more likely to have little if any sperm stored (Fritz 2004), but more likely to mate with a second male and for a longer period of time, which results in the storage of sperm primarily from the second male (Figs. 1–4). This outcome is somewhat reversed for females that had longer copulations with the first male. Females that copulated with the first male for 16–25 min copulated with the second male for only a slightly longer period of time (Fig. 2), whereas females that copulated with the first male for more than 26 min had a much shorter copulation time with the second male (Fig. 2). If these females (copulating >15 min) stored sperm from only one male, it was most likely to be sperm from the first male (Fig. 4). Finally, females copulating with the first male for more than 15 min were more likely to store sperm from both males than females copulating initially for less than 15 min; the latter generally had sperm from only one male, typically the second male. Among the spermathecae, sperm was stored significantly more often in the doublet spermathecae. The doublet spermathecae are two independent sperm storage organs held together by a muscular sheath enclosing their individual ducts for approximately one-third of their length (Fritz and Turner 2002). While previous studies on A. suspensa have documented the differential use of the paired spermathecae in singly mated females (Fritz 2004, Wallace 2005), this study is the first to document patterns of storage specific to each male in doubly mated females. For example, whereas one male’s sperm may be stored in one of the spermathecae of the doublet, the second male’s sperm may exclusively occupy the remaining doublet spermatheca. This phenomenon highlights the independent use of these two paired spermathecae and provides a unique storage site for each mate whose sperm is stored. Sexual selection theory advances two hypotheses for storage of sperm. The first, sperm competition, suggests selection has acted to promote conditions where male gametes would be stored together to provide an arena for male–male gametic competition (Parker 1970, Simmons 2001). The second, cryptic female choice, suggests that female anatomy and physiology have been selected to maintain control of different males’ sperm to set the stage for differential use in ova fertilization (Eberhard 1996, Simmons 2001). The storage patterns exhibited in the three spermathecae of A. suspensa suggest the latter as a possible hypothesis, since different male gametes are held in separate spermathecae ruling out an arena for male–male competition; in no instance were sperm from two different males found together in a spermatheca. The singlet spermathecae were less likely to house sperm than were the doublets, a finding consistent with earlier studies (Fritz 2004, Wallace 2005). The singlet spermatheca primarily stored the second males’ sperm regardless of the duration of copulation; thus, first male copulations rarely end in sperm deposition in this particular storage organ (Table 1). The spermathecae are thought to be long-term storage organs and are used to fill the ventral receptacle as it gets depleted of sperm (Twig and Yuval 2005, Pérez-Staples et al. 2007). Under-utilization of the singlet spermathecae is poorly understood and may be an artifact of the studies to date, which until now have documented single matings exclusively or may represent an anatomical or physiological difference not yet elucidated. The ventral receptacle not only disproportionately stored sperm more often than did the spermathecae (all doubly mated females had sperm in the ventral receptacle), but Fritz (2004) found this organ accounts for an average of about 45% of all sperm stored by singly mated females in A. suspensa. The ventral receptacle is considered to be the site of ova fertilization (Solinas and Nuzzaci 1984) and the site of short-term sperm storage (Twig and Yuval 2005). Scolari et al. (2014) reported that, for doubly mated females of C. capitata, sperm from two males occur in equal amounts in the ventral receptacle. Our study found that although relative quantities of sperm present in the ventral receptacle from both sires can vary, the overall average amount of sperm stored is about equal for first versus second sires. Scolari et al. (2014) investigated sperm storage patterns in the ventral receptacle of doubly mated C. capitata using fluorescently labeled sperm and reported sperm stratification in the ventral receptacle. Their data indicated a disproportionately greater number of offspring from second males, although the percent of offspring sired by the first male increased over time. Without knowledge of sperm storage patterns, this outcome might have been interpreted as a case of sperm precedence in male sperm competition. Scolari et al. (2014), however, describes sperm storage stratification in the ventral receptacle that alone can account for the preponderance of second male offspring without invoking sperm competition. Scolari et al. (2014), however, did not include spermathecae in their analysis and our sperm storage patterns provide another possible set of circumstances that can also lead to skewed paternity outcomes. For example, in our study the majority of doubly mated females storing sperm from one male represented the second sire. Furthermore, of all spermathecae storing sperm in doubly mated females (N = 36), two-third of them (43/67) stored sperm from the second sire only (Table 1). Thus, as the ventral receptacle gets depleted during oviposition, sperm remaining in the long-term storage organs (spermathecae) available to replenish the site of fertilization, are primarily of the second male. If sperm stratification occurs in the ventral receptacle of A. suspensa, as has been described for C. capitata (Scolari et al. 2014), then an overabundance of second male offspring during the initiation of oviposition would ensue simply due to the way sperm are stored. Therefore, we would further predict that as females continue to oviposit and deplete sperm from their ventral receptacle, the proportion of offspring sired by the second male would rise again, on average, as spermathecae replenished the site of fertilization, the ventral receptacle. In summary, the results of this study describe the copulatory and sperm storage outcomes when there are multiple sires and where there is no opportunity for females to oviposit (since mating trials were accomplished within 24 h). Short copulations with a male generally resulted in little transfer of sperm and predicted the length of time spent in second copulations, resulting in disproportionate sperm storage from second copulations. Sperm storage organs can fill independently with sperm from different males, but each spermatheca stores sperm from one sire only. 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Annals of the Entomological Society of AmericaOxford University Press

Published: Mar 1, 2018

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