Energy metabolism in the spermatophore of the silkmoth,Bombyx mori,associated with accumulation of alanine derived from arginine

In the female silkmoth, Bombyx mori, labeled glucose-1-phosphate and pyruvate were found to be metabolized with the formation of alanine in the spermatophore, which contains various secretions from some of the glands and the seminal fluid of the male reproductive system. Moreover, in the spermatophore ¹⁴CO₂ was released not only from these radioactive glycolytic intermediates and end products, but also from 2-oxoglutarate and ornithine. The ratio of the rates of metabolic formation of carbon dioxide from pyruvate and alanine was about 1:2 at 150 min after the beginning of mating. Oxidative decarboxylation of pyruvate in the TCA-cycle was accelerated by added 2-oxoglutarate. Thus, there is a pathway of cascade reactions from arginine to 2-oxoglutarate coupled with glycolysis in the spermatophore.     

Presence of eupyrene spermatozoa in vestibulum accelerates oviposition in the silkworm moth,Bombyx mori

In the silkmoth, Bombyx mori, mating accelerates egg laying. Sterilized males did not induce females to oviposit, whereas artificial insemination was enough to accelerate oviposition. These findings showed that mechanical stimulation of the outer reproductive organs and the bursa copulatrix (BC) had no effect on oviposition. Factors included in seminal fluids also did not stimulate oviposition because neither implantation of female internal reproductive organs nor injection of the spermatophore extract into virgin females activated oviposition. Preventing sperm precedence by experimental removal of the BC or spermatheca inhibited activation of egg laying. More precise surgeries on the spermatheca showed that the main factor for eliciting oviposition was the presence of matured and fertile eupyrene spermatozoa in the vestibulum. Sperm were not effective unless they reached the vestibulum. Nerve transection and electrophysiological studies revealed that nerve 4 of the terminal abdominal ganglion was the pathway by which the mating signal was transmitted toward the higher central nervous system.     

System for supply of free arginine in the spermatophore of Bombyx mori: Arginine-liberating activities of contents of male reproductive glands

The mechanism of supply of free arginine in the Bombyx mori spermatophore was analyzed by examining the arginine-liberating activities of the contents of male reproductive glands, which are constituents of the spermatophore formed during ejaculation. On autolysis, only the contents of glandula (g.) prostatica showed significant production of only free arginine. On incubation in combination with the contents of the g. prostatica, arginine liberation from the vesicula (v.) seminalis contents was highest. Similarly, the contents of the g. spermatophorae also showed arginine liberation, but those of the g. lacteola showed generalized liberation of various amino acids. Following digestion of these preparations with bovine trypsin, the v. seminalis, g. spermatophorae and g. prostatica liberated free lysine as well as arginine, while those of the g. lacteola liberated various amino acids.These findings strongly suggest that a specific proteolytic system for production of free arginine is active in the spermatophore. That is, an endopeptidase derived from the g. prostatica specifically cleaves protein at the C-terminal side of arginine residues, and then exopeptidases derived from the v. seminalis, g. spermatophorae and g. prostatica liberate free arginine from the peptides produced by the endopeptidase.     

Eupyrene sperm migrates to spermatheca after apyrene sperm in the swallowtail butterfly, Papilio xuthus L. (Lepidoptera: Papilionidae)

When swallowtail butterflies, Papilio xuthus, are mated by the hand-pairing method, both types of sperm, eupyrene and apyrene sperm, are transferred from the male to the spermatheca via the spermatophore in the bursa copulatrix. This mechanism is demonstrated by two different kinds of experiments. The first set of experiments employed interrupted copulation, and the second set was examination of the sperm in the spermatophore and spermatheca after the termination of copulation. The sperm was transferred 30 min after the start of copulation. The eupyrene sperm was still in the bundle; the number of the bundles ranged from 9 to 108 (mean, 42.7; n = 27). The bundles were gradually released after the completion of copulation, and the free eupyrene spermatozoa then remained in the spermatophore at least 2 h before migrating to the spermatheca. On the other hand, about 160 000 apyrene spermatozoa were transferred to the spermatophore and remained there for more than 1 h. We observed 11 000 apyrene spermatozoa in the spermatheca 12 h after the completion of copulation, but most of this type of sperm disappeared shortly thereafter. In contrast, the eupyrene sperm arrived in the spermatheca more than 1 day after the completion of copulation and remained there at least 1 week. Therefore, these findings suggest that apyrene sperm migrate from the spermatophore to the spermatheca earlier than eupyrene sperm. Accordingly, if females mated multiply, the time difference might avoid the mixing of sperm. In addition, the predominance of sperm from the last mating session may occur not in the bursa copulatrix but in the spermatheca. Received: January 7, 2000 / Accepted: May 24, 2000     

Pattern of sperm storage and migration in the reproductive tract of the swallowtail butterfly Papilio xuthus: cryptic female choice after second mating

Females of the swallowtail butterfly Papilio xuthus L. (Lepidoptera: Papilionidae) mate multiply during their life span and use the spermatophores transferred to increase their longevity as well as fecundity. Sperm from different males may be stored in the sperm storage organs (bursa copulatrix and spermatheca). To clarify the pattern of sperm storage and migration in the reproductive tract, mated females are dissected after various intervals subsequent to the first mating, and the type and activity of sperm in the spermatheca are observed. When virgin females are mated with virgin males, the females store sperm in the spermatheca for more than 10 days. Sperm displacement is found in females that are remated 7 days after the first mating. Immediately after remating, these females flush out the sperm of the first male from the spermatheca before sperm migration of the second male has started. However, females receiving a small spermatophore at the second mating show little sperm displacement, and the sperm derived from the small spermatophore might not be able to enter the spermatheca. Females appear to use spermatophore size to monitor male quality.     

Sperm transfer during mating, movement of sperm in the female reproductive tract, and sperm precedence in the common cutworm Spodoptera litura

Abstract. Mating behaviour, sperm transfer and sperm precedence were studied in the moth Spodoptera litura (Fabr.) (Lepidoptera: Noctuidae). There existed a rhythmic, diel pattern of mating behaviour of this moth during the scotophase, presumably set with respect to an endogenous activity rhythm. Approximately 30 min after copulation had started, the formation of the corpus of the spermatophore began in the bursa copulatrix of the female moth, but full inflation of the corpus was not completed until 45–60 min after mating had started. The mature spermatophore contained about 350 eupyrene sperm bundles and a large number of individual (loose) apyrene spermatozoa. The mating status and the age of the male insect influenced the number of sperm transferred to the female within the spermatophore, and also affected the consequent fertility. There was no evidence of sperm reflux within the male tract. Within the female, dissociation of eupyrene sperm bundles was evident within the spermatophore less than 15 min after the completion of mating. Spermatozoa began to move from the bursa (in which the spermatophore is lodged) into the spermatheca 30–45 min after the end of the copulation, and the quantity of sperm in the spermatheca reached a plateau at 90 min after mating. Apyrene sperm reached the spermatheca first, followed by eupyrene sperm. Examination of total (apyrene plus eupyrene) sperm in the female tract showed that 86% of mated females received an apparently normal amount of total sperm from the male. Examination of eupyrene sperm alone showed that 81% of matings resulted in an apparently normal transfer of eupyrene sperm. A small proportion (approximately 8%) of the matings, however, were identified as transferring a clearly subnormal quantity of eupyrene sperm to the spermatheca. The eggs produced as a result of such pairings displayed much reduced fertility (about 43%) compared to those from matings confirmed to have transferred normal quantities of sperm, which showed about 92% fertility. This shows that the availability of eupyrene sperm in the spermatheca may be an important constraint on fertility in normal populations of insects. In the laboratory, S. litura females exhibited multiple matings. Of the females, 93% mated, and the mean frequency of mating was 1.69. Mating with a fertile male led to the oviposition of an increased number of eggs. This effect continued even when the female subsequently mated with an infertile male. Displacement of sperm from previous matings is known to be an important factor in the evolution of multiple mating strategies. Our results on sperm utilization by S. litura indicated that after a second mating, the sperm utilized for subsequent fertilization were almost exclusively from the last mating with little mixing. The proportion of eggs fertilized by sperm from the second mating (P₂) was calculated as 0.95, indicating almost complete sperm precedence from the last mating.     

Impact of male mating history on the temporal sperm dynamics of Choristoneura rosaceana and C. fumiferana females

In the oblique-banded leafroller, Choristoneura rosaceana, and the spruce budworm, C. fumiferana, male reproductive performance decreases with consecutive matings. While the onset time of mating did not vary, the time spent mating was longer in mated than in virgin males. Furthermore, a decline observed in the spermatophore mass with successive matings was associated with a concomitant decline in its apyrene and eupyrene spermatozoa content. In the hours following mating, spermatozoa migrate from the spermatophore, located in the bursa copulatrix, to the spermatheca. Regardless of the male's previous mating history, the number of apyrene sperm dropped rapidly in the days following mating whereas the number of eupyrene spermatozoa declined gradually. As the temporal pattern of sperm movement was similar in all treatments, females mated with previously-mated males would suffer from sperm shortage sooner than those mated with virgins. Large C. rosaceana females stored more apyrene spermatozoa in their spermatheca than small ones, irrespective of the time after mating or male mating history, while only large females mated with once-mated males received more apyrene sperm and accessory gland secretions than small ones mated with virgin or twice-mated males. The results obtained in this study are discussed in relation with their potential impact on the reproductive success of both sexes.     

Arginine metabolism in the sheep abomasal nematode parasites Haemonchus contortus and Teladorsagia circumcincta

The ornithine urea cycle, polyamine synthesis, nitric oxide synthesis and metabolism of arginine to putrescine have been investigated in L3 and adult Haemonchus contortus and Teladorsagia circumcincta. Neither parasite had a detectable arginine deiminase/dihydrolase pathway nor a functional ornithine urea cycle. Nitric oxide synthase was present in central and peripheral nerves, but was not detected in whole parasite homogenates. Both arginase (E.C. 3.5.3.1) and agmatinase (E.C. 3.5.3.11) activities were present in both species. Arginase did not require added Mn²⁺ and had an optimal pH of 8.5. Polyamine metabolism differed in the two species and from that in mammals. Ornithine decarboxylase (E.C. 4.1.1.17) was present in both parasites, but no arginine decarboxylase (E.C. 4.1.1.19) activity was detected in T. circumcincta. The flexibility of synthesis of putrescine in H. contortus may make this pathway less useful as a target for parasite control than in T. circumcincta, in which only the ornithine decarboxylase pathway was detected.     

Adenine compounds in the male reproductive tract and the spermatophore of the silkmoth, Bombyx mori.

1. Studies were made on the levels of six adenine compounds in various glands of the male reproductive tract before and after ejaculation and their changes with time in the spermatophore of the silkworm, Bombyx mori. 2. The ductus (d.) deferens, ampulla d. deferentis and vesicular seminalis contain high concentrations of cAMP. The total amount of cAMP transferred from the male reproductive tract corresponds to its content in the spermatophore, and is sufficient to induce motility of apyrene spermatozoa. 3. The ATP content of the g. pellucida is much higher than those of the two other adenine nucleotides, indicating a high energy charge. But, in the other male glands, the AMP content is very much higher than that of ATP, indicating low energy charges.     

Structure and function of the spermathecal complex in the phlebotomine sandflyPhlebotomus papatasi Scopoli (Diptera: Psychodidae): II. Post-copulatory histophysiological changes during the gonotrophic cycle

The spermathecal complex ofPhlebotomus papatasi Scopoli (Diptera: Psychodidae) undergoes histological and physiological changes during its gonotropic cycle. The present histochemical study revealed a mucopolysaccharide secretory mass in the spermathecae of the newly emerged sandfly. Sperm competition occurs when two or more males compete to fertilize an ovum in the female reproductive tract. In this study, spermatophores of two or more competing males were deposited at the base of the spermathecal ducts, which originate from the female bursa copulatrix. This suggests that females play a role in sperm displacement, which is defined as any situation in which the last male to mate with a female fertilizes maximum number her eggs. A blood meal ingested by the female for ovary development and egg laying stimulates the release of sperm from the spermatophore. The spermatozoa then migrate to the lumen of the spermatheca. The ultrastructure of spermatozoa comprises a head with double-layered acrosomal perforatorium, an elongate nucleus, and the axoneme with a 9 + 9 + 0 flagellar pattern. This axomene differs from the aflagellate axoneme of other Psychodinae. Morphological changes, such as the casting off of the acrosomal membrane, and histological changes in the spermatophore are also described. Mating plugs that have been described previously in sandflies appear to be artefacts. Females ofP. papatasi may be inseminated more than once during each gonotrophic cycle, and additional inseminations may be necessary for each cycle. The relationships between the volumes of the sperm and the spermatheca were calculated to determine sperm utilization and fecundity ofP. papatasi. As the females ofP. papatasi mate polyandrously, the anatomical and physiological complexity of the spermathecal complex may be related to post-copulatory sexual selection.     

The effect of remating on sperm number in the spermatophores of Teleogryllus commodus (Gryllidae)

Factors controlling sperm production in members of the Orthoptera have not been fully elucidated. In this study, the influence of intermating interval and ad libidum mating on sperm number was investigated in black field crickets (Teleogryllus commodus). Remating at a variety of time intervals was not characterized by a significant change in sperm number compared with the first mating. Ad libitum mating (i.e., continuous availability of unmated females) had two main effects on spermatophore production and sperm number: first, there was a trend toward increased time between copulations with each successive remating, and second, the number of spermatozoa encapsulated in the transferred spermatophore declined after most rematings, with 61.8% of the initial sperm number being produced for the second spermatophore and 51.3% of the initial sperm number being produced for the third. The decrease in mean sperm number was accompanied by increased variance in sperm number in later rematings. This study suggests that males are willing to suffer a decrease in sperm number if a mating opportunity occurs before the completion of sperm production.     

Morphological changes in eupyrene and apyrene spermatozoa in the reproductive tract of the male butterfly Atrophaneura alcinous Klug

Summary

Eupyrene and apyrene spermatozoa are contained in separate cysts in the testis of the butterfly Atrophaneura alcinous. Spermatozoa of both types from various parts of the male reproductive tract were examined with particular reference to their morphological characteristics. All spermatozoa collected from the vas deferens and the vesicula seminalis were found to be immotile under a dissecting microscope. No spermatozoa of either type were recognized in any part of the ejaculatory duct. Within the testis, eupyrene spermatozoa are present in bundles and each spermatozoon has a slender nucleus with an acrosome and a long flagellum containing mitochondrial derivatives. Two kinds of appendages, lacinate and reticular, are present on the surface of the sperm membrane. They are replaced with an extracellular sheath during passage through the vas deferens. In contrast, apyrene spermatozoa have neither nucleus nor acrosome, whereas a cup-shaped structure was found at the sperm tip instead of the acrosome. Unlike eupyrene spermatozoa, they are surrounded by a concentric sheath outside the sperm membrane in the vas deferens. Individual apyrene spermatozoa and coiled bundles of eupyrene spermatozoa were both found to accumulate in the vesicula seminalis before mating. These morphological changes during passage through the male reproductive tract suggests the occurrence of a kind of maturation and capacitation process reminiscent of mammalian spermatozoa.     

Motility and dynamics of eupyrene and apyrene sperm in the spermatheca of the monandrous swallowtail butterfly Byasa alcinous

Lepidopteran males produce two sperm types: nucleated eupyrene sperm and non‐nucleated apyrene sperm. Although apyrene sperm are infertile, both sperm types migrate from the spermatophore to the spermathecal after copulation. As a dominant adaptive explanation for migration of apyrene sperm in polyandrous species, the cheap filler hypothesis suggests that the presence of a large number of motile apyrene sperm in the spermatheca reduces female receptivity to re‐mating. However, apyrene sperm are also produced in males of the monandrous swallowtail butterfly Byasa alcinous Klug. To identify the role of apyrene sperm in these males, the present study examines the number of spermatozoa produced and transferred and the dynamics and motility of spermatozoa in the spermatheca for each type of sperm. Apyrene sperm represents approximatey 89% of the sperm produced and transferred, which is comparable to polyandrous species. Two‐day‐old males transfer approximately 17 000 eupyrene and 230 000 apyrene spermatozoa to a spermatophore; approximately 5000 eupyrene and 47 000 apyrene spermatozoa arrive at the spermatheca. Eight days after copulation, most eupyrene spermatozoa remain in the spermatheca and a quarter of them are still active. However, the number of apyrene spermatozoa decreases and those remaining lose their motility after the arriving at the spermatheca. Consequently, 8 days after copulation, no motile apyrene sperm are found. The high proportion of apyrene sperm in the spermatophore, as well as in sperm migration, suggests that the production and migration of apyrene sperm is not simply an evolutionary vestigial trait. The possible functions of apyrene sperm in monandrous species are discussed.     

Last–male sperm precedence in the bushcricket Poecilimon veluchianus (Orthoptera, Tettigonioidea) demonstrated by DNA fingerprinting

Males of the bushcricket Poecilimon veluchianus pass a large spermatophore to the female during mating. The spermatophore is eaten by the female after copulation. Because females mate with several males during their reproductive life, the competition between spermatozoa of different males affects a male's reproductive success. In order to determine the outcome of sperm competition, the paternity of the progeny of double–mated females was established by DNA fingerprinting with the oligonucleotide (GATA)₄. Typical P. veluchianus DNA fingerprints consisted of 15 scoreable fragments per individual. The proportion of bands shared between presumably unrelated bushcrickets was 17%. After the second copulation the second mating male clearly predominated at fertilization. The mean proportion of eggs fertilized by the second male was 90.1%. There was no significant relationship between the level of sperm precedence and the time of ovipositions after the second mating. If female P. veluchianus increase the fitness of their offspring by the incorporation of spermatophore–derived substances in developing eggs, there is little chance for the feeding male to fertilize eggs containing his nutrients, because of the very short mating intervals of females and the observed high level of last–male sperm precedence in this species. Under such conditions the last mating male would fertilize many eggs containing nutrients from a prior male. Because nuptial gifts, like the tettigoniid spermatophore, function only as paternal investment if the donating male's progeny benefit from the gift, a paternal investment function of the P. veluchianus spermatophore seems to be unlikely.     

Trehalase in the spermatophore from the bean-shaped accessory gland of the male mealworm beetle,Tenebrio molitor: purification,kinetic properties and localization of the enzyme

Trehalase from the bean-shaped accessory glands of the male mealworm beetle, Tenebrio molitor, was purified by acid treatment, with subsequent chromatography on columns of DEAE-cellulofine and Sephacryl S-300. The molecular masses of the native and the denatured forms were estimated to be 43 and 62 kDa by gel filtration and SDS-PAGE, respectively, an indication that the trehalase may be composed of a single polypeptide. The optimum pH of the reaction catalyzed by trehalase was 5.6–5.8. The K _m for trehalose was 4.4 mmol·l^–1. Immunohistochemical experiments with trehalase-specific antiserum showed that the enzyme was localized in one specific type of secretory cell in the bean-shaped accessory gland epithelium and within the semisolid secretory mass that was a precursor to the wall of spermatophore. SDS-PAGE and immunoblotting analysis revealed the presence of a polypeptide of about 62 kDa in the spermatophore, Immunohistochemical observations showed that the trehalase was located at the outgrowth in the anterior portion of the spermatophore. When a fresh spermatophore was immersed in phosphate-buffered saline it discharged sperm in the same manner as in the bursa copulatrix of the female. Before the rupture of the expanded bulb of the spermatophore, almost all of the trehalase had dissolved in the phosphate-buffered saline. The addition of validoxylamine A to the saline, a specific inhibitor of trehalase, did not affect the expansion and evacuation of the spermatophore. These results demonstrate that trehalase, synthesized by a specific type of secretory cell in the bean-shaped accessory gland epithelium, is actively passed into the lumen of the bean-shaped accessory gland and then incorporated into the spermatophore. Trehalase appears to be one of the structural proteins of the spermatophore, although the possibility can not yet be completely ruled out that the trehalase-trehalose system functions for the nourishment and/or activation of the sperm in the bursa copulatrix of the female.Abbreviations BAG bean-shaped accessory gland(s) - DEAE diethylaminoethyl - Kpi buffer K₂HPO₄/KH₂PO₄ buffer (pH 7.0) - PAGE polyacrylamide gel electrophoresis - PBS phosphate-buffered saline - SDS sodium dodecy sulphate - Spph spermatophore(s) - TAG tubular accessory gland(s)     

The functional significance of the spermatophore and the fate of spermatozoa in the genital tract of Helix pomatia (Gastropoda: Stylommatophora)

In the Helicidae and in some other Stylommatophora the sperm are transferred in a spermatophore, even though there appears to be no need for protection of the sperm during the transfer. The function of the spermatophore and related problems concerning the genital organs of Helix pomatia were studied by means of morphological and experimental methods.
The spermatophore is necessary to ensure the functioning of the female system at copulation. Its structure allows some of the spermatozoa it contains to escape through its tail canal, pass from the stalk of the bursa and reach the spermatheca by way of the oviduct; but most of the sperm pass into the bursa copulatrix and are destroyed, as is also the fate of the spermatophore. Only foreign sperm are stored in the spermatheca.
Spermiogenesis was found to take place throughout the whole summer. At intervals some sperm are released from the hermaphrodite duct and are conducted via the spermoviduct and oviduct to the bursa, where they are digested. The two grooves of the spermoviduct are functionally separated only for a few minutes at actual copulation, when sperm are conducted to the copulatory organs, where the spermatophore is being formed.     

Courtship and Mating in the Giant Hairy Desert Scorpion, Hadrurus arizonensis (Scorpionida, Iuridae)

Hadrurus arizonensis is a large, long-lived species of North American desert scorpion with lengthy, stereotyped courtship behaviors that lead to sperm transfer via an external spermatophore. Courtship and mating behaviors in H. arizonensis and other members of the Iuridae family have not been described. H. arizonensis has reproductive behavior similar to that of other scorpions, including the promenade a deux, but with some unique components described here for the first time. Courtship and mating behaviors of H. arizonensis are presented in a flowchart to emphasize its stereotypical nature and suitability for experimental manipulation in field and laboratory studies.     

Dynamics of eupyrene and apyrene sperm storage in ovipositing females of the swallowtail butterfly Papilio xuthus (Lepidoptera: Papilionidae)

A male swallowtail butterfly, Papilio xuthus, transfers both eupyrene and apyrene sperm during copulation, both of which migrate to the spermatheca via the spermatophore in the bursa copulatrix of the female. Because the spermatheca seems to remain constant in size during the female lifespan, the excess sperm migration may cause the spermatheca to overflow. Approximately 9000 eupyrene and 265 000 apyrene spermatozoa were transferred during a single copulation, and approximately 1000 eupyrene and 1100 apyrene spermatozoa successfully arrived in the spermatheca. The number of both types of spermatozoon decreased in the spermatheca after the onset of oviposition, and no eupyrene spermatozoa were found by 7 days after copulation, partly due to insemination. The spermathecal gland leading from the distal end of the spermatheca was gradually filled by eupyrene spermatozoa. Although the function of the gland remains unclear, the final destination of the sperm is likely to be the gland.     

Effects of different male remating intervals on the reproductive success of Choristoneura rosaceana males and females

The mass of the spermatophore transferred by a previously mated Choristoneura rosaceana male increases with time elapsed since the last mating but, even after 4 days, it never reaches the mass of the spermatophore of a virgin male. However, spermatophore mass is clearly not a good indicator of the male reproductive investment as the quantity of sperm in the second ejaculate of a previously mated male is the same as that of his first, if he is allowed a 2 (eupyrene sperm) to 3 day (apyrene sperm) recovery period. The interval between the first two matings had no influence on female fecundity or longevity but significantly affected fertility if the male had only 1 day to recover. The length of the post-copulatory refractory period was also shorter in females mated with previously mated males than in those mated with virgins, regardless of the male's remating interval. Furthermore, a significant variation in the eupyrene sperm content of the spermatophore transferred by virgin males had no influence on the length of the female refractory period. Globally, these results support the hypothesis that a factor, other than sperm numbers in the spermatheca, is responsible for maintaining the inhibition of pheromone production in this species.     

Multiple matings increase the fecundity of the yellow swallowtail butterfly,Papilio xuthus L., in summer generations

Females of the yellow swallowtail butterfly, Papilio xuthus,were reared in the laboratory. They were divided into four groups held under different mating conditions: nonmating (virgin) and mated once, twice, and three times. The number of eggs in the ovaries was counted by dissection. Virgin females produced increasing numbers of mature eggs, up to about 30, in the week following emergence. When the female had mated once, the number of mature eggs was significantly higher than that of virgin females by the second day after emergence. However, the double- and triplemated females did not increase the number of eggs in each state further than the singlemated females. The double-mated females deposited significantly more eggs than the singlemated females in the laboratory. The triplemated females also deposited more eggs on the day after the third mating than the doublemated females. Thus, multiple matings increased the number of eggs deposited. The change in the hatchability and the morphology of the spermatophore in the bursa copulatrix suggested that the sperm from the last mating had precedence.