Sperm storage and viability in Photinus fireflies

In many species females mate with and store sperm from multiple males, and some female insects have evolved multiple compartments for sperm storage. Sperm storage and sperm viability were investigated in two firefly species, Photinus greeni and P. ignitus, which differ in the morphology of the female reproductive tract. Although the primary spermatheca is similar in both species, P. greeni females have an additional, conspicuous outpocketing within the bursa copulatrix whose potential role in sperm storage was investigated in this study. An assay that distinguishes between live and dead sperm was used to examine sperm viability in male seminal vesicles and sperm storage sites within the female reproductive tract. For both Photinus species, sperm from male seminal vesicles showed significantly higher viability compared to sperm from the primary spermatheca of single mated females. In single mated P. greeni females, sperm taken from the channel outpocketing (secondary spermatheca) showed significantly higher viability compared to sperm from the primary spermatheca. This sperm viability difference was not evident in double mated females. There were no significant differences between P. greeni and P. ignitus females in the viability of sperm from the primary spermatheca. These studies contribute to our understanding of post-mating processes that may influence paternity success, and suggest that sexual conflict over control of fertilizations may occur in multiply mated firefly females.     

Copulation patterns in the golden orb-web spider Nephila madagascariensis

A consequence of multiple mating by females can be that the sperm of two or more males directly compete for the fertilisation of ova inside the female reproductive tract. Selection through sperm-competition favours males that protect their sperm against that of rivals and strategically allocate their sperm, e.g., according to the mating status of the female and the morphology of the spermatheca. In the majority of spiders, we encounter the otherwise unusual situation that females possess two independent insemination ducts, both ending in their own sperm storage organ, the spermatheca. Males have paired mating organs, but generally can only fill one spermatheca at a time. We investigated whether males of the African golden orb-web spider Nephila madagascariensis can prevent rival males from mating into the same spermatheca and whether the mating status of the female and/or the spermatheca causes differences in male mating behaviour. There was no significant difference in the duration of copulations into unused spermathecae of virgin and mated females. We found that copulations into previously inseminated spermathecae were generally possible, but shorter than copulations into the unused side of mated females or with virgins. Thus, male N. madagascariensis may have an advantage when they mate with virgins, but cannot prevent future males from mating. However, in rare instances, parts of the male genitals can completely obstruct a female genital opening.     

Sperm storage and viability within females of Euscepes postfasciatus: Effect of irradiation on sperm abundance and viability within female

Irradiation has been widely used in suppression or eradication programs that use the sterile insect technique (SIT) or in studying sperm competition. Although it is well known that irradiation has negative impacts on reproductive (sperm) cells, previous studies have assumed that sperm from irradiated males behave identically to normal sperm in the female reproductive tract after mating. In this study, we used the West Indian sweetpotato weevil Euscepes postfasciatus to investigate the effect of irradiation on the abundance and viability of sperm in female spermatheca for 14 days after copulation. The abundance of sperm in females did not change throughout the experimental period, and sperm viability gradually decreased regardless of irradiation. In this weevil, irradiated sperm appear to behave identically to normal sperm in females for 14 days following irradiation/copulation. Therefore, the effect of irradiation on sperm viability within the female spermatheca is considered to be insignificant.     

Increased fecundity, as a function of multiple mating, in an arctiid moth, Utetheisa ornatrix

1. Female Utetheisa ornatrix mate multiply and may receive up to thirteen spermatophores. Spermatophores provide the female not only with sperm but also with a nuptial gift of pyrrolizidine alkaloid that she transmits to the eggs, protecting them against predation. Thus, through multiple mating the female accrues nuptial gifts that add to the defence of her offspring.
2. Multiple mating in female U. ornatrix brings about increases in fecundity, but not in longevity or egg mass. These increases occur through the third mating; the fourth and fifth matings, however, have no additional effect on fecundity.
3. These results, taken with the fact that spermatophores are sizeable and are digested within the female, suggest that they bear nutritive gifts that the female uses in egg construction. Multiple mating thus allows females to accrue the resources to build additional eggs.
4. These results are discussed in relation to the potential benefits accruing to donating such gifts and to male and female mating strategies.     

Mating biology of the leaf-cutting ants Atta colombica and A. cephalotes

Copulation behavior has often been shaped by sexually selected sperm competition or cryptic female choice. However, manipulation of previously deposited ejaculates is unknown in the social Hymenoptera and the degree to which sperm competes after insemination or is actively selected by females has remained ambiguous. We studied the mating process in the leaf-cutting ants Atta colombica and A. cephalotes, which belong to one of the few derived social insect lineages where obligate multiple mating has evolved. As copulations often occur at night and in remote places, direct observations were impossible, so we had to reconstruct the sequential copulation events by morphological analysis of the male and female genitalia and by tracking the process of sperm transfer and sperm storage. We show that Atta male genitalia have two external rows of spiny teeth, which fit into a specialized pouch organ in the female sexual tract. Reconstruction of the sperm storage process indicated that sperm is transferred to the spermatheca during or immediately after ejaculation and without being mixed with sperm and seminal fluids from other males. A convergent mechanism of direct sperm transfer to the spermatheca of queens is known from two species of dwarf honeybees. Direct sperm transfer may restrict female control over the sperm storage process and the number of males that contribute to the stored sperm.     

Male copulatory sensory stimulation induces female ejection of rival sperm in a damselfly

Male damselflies possess very specialized genitalia. Females mate multiply and store sperm in two sperm storage organs, the bursa copulatrix and the spermatheca. During copulation, males physically remove the sperm stored in these organs using their genitalia. I document a novel mechanism by which males gain access to the spermatheca in Calopteryx haemorrhoidalis asturica. The mechanism is based on male stimulation of the female sensory system that controls egg fertilization and laying. During copulation, the aedeagus (a male genitalic structure indirectly involved in sperm transfer) distorts the cuticular plates in the female genital tract that bear mechanoreceptive sensilla. This stimulation results in sperm ejection from the spermatheca. Aedeagus width is positively correlated with the amount of sperm ejected. I propose that males have exploited a pre-existing female sensory bias to gain access to otherwise physically unreachable sperm. These results shed light on the issue of the origin of female preferences in current models of sexual selection and on the evolution of genitalia via sexual selection. It is postulated that females might use this process as a form of post-copulatory sexual selection on the basis of males'' genitalia.     

Strategic Investment in Sperm Removal Behaviour in a Bushcricket

Multiple mating by females is widespread and generates sperm competition among the ejaculates of rival males over fertilization. One way in which males can avoid or reduce sperm competition is by displacing or removing previous males’ sperm from female sperm stores. An apparent example of this occurs in the bushcricket Metaplastes ornatus. Males perform a specialised sperm removal behaviour (SRB), using their highly-derived subgenital plate, with which they remove sperm from the female’s spermatheca during the early phases of mating before transferring a spermatophore of their own. Here we investigated whether males strategically invest in SRB according to the amount of previously stored sperm present in females. Each male was tested twice, once with a female containing sperm (‘filled’ condition) and once with a female from whom most previously deposited sperm had recently been removed by another male (‘emptied’ condition). For comparison, a separate group of males was paired with virgin females. Males did not discriminate between non-virgin females in the ‘emptied’ or ‘filled’ conditions in terms of their investment in SRB, suggesting they may not able to perceive the amount of sperm present in the female’s spermatheca. By contrast, male investment in SRB was significantly reduced in pairings with virgin females, indicating that males are sensitive to some aspect of a female’s mating status. Our results thus suggest that males modulate SRB in response to female-mediated cues, possibly chemical cues left by previous males, which would not be present on virgin but would be on non-virgin females.     

SPERM COMPETITION AND THE EVOLUTION OF NUPTIAL FEEDING BEHAVIOR IN THE CRICKET,GRYLLODES SUPPLICANS (WALKER)

The pattern of sperm predominance in doubly mated female crickets, Gryllodes supplicans, was investigated using a radiation-sterility technique. Female G. supplicans made significant use of sperm from both males in fertilizing eggs; overall, first males to mate enjoyed a small advantage, fertilizing about 60% of the offspring produced subsequent to the second mating. The combined use of the sperm of both males in fertilizing eggs occurred soon after the second mating; evidently, mixing of ejaculates within a female's spermatheca does occur. Male G. supplicans provide females with a nuptial gift, the spermatophylax, which influences the time at which a female removes the externally attached sperm-ampulla; this in turn determines the quantity of sperm that is transferred. Moreover, the degree of sperm precedence achieved by a male may be positively related to the time at which the female removes his sperm ampulla. Thus males, by feeding females, ensure not only that a sufficient number of sperm are transferred to fertilize all of a female's eggs, but also may increase the certainty of their paternity. In mating systems in which females control sperm transfer and paternity is influenced by numbers of sperm (i.e., numerical sperm competition), an increase in prezygotic investment in females may be an adaptive male response.     

Morphology and function of the reproductive tract of the spider crab Libinia spinosa (Crustacea, Brachyura, Majoidea): pattern of sperm storage

Morphology and function of the male reproductive tract, female spermatheca and patterns of sperm storage were assessed in the crab Libinia spinosa using histological methods. Testes are characterized by the presence of peripheral spermatogonia and different sequences of sperm maturity. Spermatophores begin to be packed in the last portion. The vas deferens consists of three sections: anterior, with undeveloped spermatophores and free sperm; median, with well-developed spermatophores; and posterior with granular secretions. Female spermathecae are of the ventral type, with a velum separating dorsal and ventral chambers. Live individuals were kept in the laboratory and arranged in pairs. An experiment was conducted toward the end of the reproductive season, in which males with the right gonopod excised were placed with receptive females. After mating, females were killed and the spermathecae dissected for histological study and observation of the pattern of sperm storage. Spermatozoa were found forming discrete sperm packages. New ejaculates can fill the entire spermatheca or be restricted to the ventral chamber; sperm are rounded, with a distinguishable acrosomal core. Old ejaculates are restricted to the dorsal chamber and are of irregular shape and larger size; an acrosomal core was not distinguishable. The secretions produced by the glandular epithelium of the dorsal chamber of the spermathecae are likely to have a role in the removal of dead sperm.     

Possible Removal of Rival Sperm by the Elongated Genitalia of the Earwig, Euborellia plebeja

Sperm displacement is a sperm competition avoidance mechanism that reduces the paternity of males that have already mated with the female. Direct anatomical sperm removal or sperm flushing is known to occur in four insect orders: Odonata, Orthoptera, Coleoptera and Hymenoptera. In a fifth order, Dermaptera (earwigs), I found that the virga (the elongated rod of the male genitalia) of Euborellia plebeja seems to be used to remove rival sperm from the spermatheca (a fine-tubed female sperm storage organ). In this species, copulation lasted on average 4.6 minutes, during which time the male inserted the virga deep into the spermatheca, and then extracted it ejaculating semen from the opening of the virgal tip. The extraction of virgae (with its brim-like tip) appeared to cause removal of stored sperm in the spermatheca. The virga was as long as the body length of males, and the spermatheca was twice the female body length. The long length of the spermatheca and the possible removal function of the virga may select for virgal elongation.     

Seminal fluid reduces female longevity and stimulates egg production and sperm trigger oviposition in a moth

Previous studies suggest that a number of factors in relation to mating may reduce female longevity and stimulate egg production and oviposition. However, it is still not clear whether these factors act on these parameters independently or in a collective way. Here we carried out a series of experiments including mating trials and seminal fluid injection to determine the factors responsible for reducing female longevity and stimulating egg production and oviposition in relation to mating in the moth Ephestia kuehniella Zeller (Lepidoptera: Pyralidae). Results show that seminal fluid and sperm work collectively to allow females to achieve maximum realized fecundity (number of eggs laid) in E. kuehniella but these factors play different roles in the process and their actions are independent. Seminal fluid signals females to allocate resources to ova, resulting in shorter longevity and greater egg production while eupyrene (not apyrene) sperm in the spermatheca trigger females to lay maximum number of eggs. We suggest that the receptors for seminal fluid signal may be located in the female reproductive tract and haemolymph, and those for sperm signal may be in the spermatheca. Hypotheses that females prolong their longevity by oosorption, physical injuries by males reduce female longevity, and mechanical stimulation by males triggers oviposition, are not substantiated in the present study.     

Physiological basis for sterilizing effects of constant light in Lymantria dispar

ABSTRACT Females of gypsy moth, Lymantria dispar L. (Lepidoptera: Lymantriidae), mated to males kept in constant light (LL) as pharate adults fail to oviposit. In males, a rhythm of sperm release from the testis that occurs in light-dark (LD) cycles is abolished in LL, and the total amount of sperm released from the testis is approximately half of that of LD males. Moreover, any sperm that may be released from the testis of LL males tend to remain in the vasa deferentia instead of moving into the duplex as in LD males. Consequently, in LL very few sperm bundles are transferred to the bursa copulatrix during mating; furthermore, these bundles fail to disperse into spermatozoa and sperm do not reach the spermatheca. The presence of a spermatheca filled with sperm must play an important role in controlling oviposition because their removal from mated females prevents egg-laying. Our results indicate that the rhythm of sperm release from the testis is essential for the ability of sperm to migrate in male and female reproductive tracts. The rhythms may help to synchronize final stages of sperm development with the activity of phagocytic and secretory cells lining the reproductive tract.     

Sperm storage by spermatodoses in the spermatheca of Trioza alacris (Flor, 1861) hemiptera,psylloidea, triozidae: A structural and ultrastructural study

Female insects generally store sperm received during mating in specific organs of their reproductive tract, i.e., the spermathecae, which keep the sperm alive for a long time until fertilization occurs. We investigated spermatheca morphology and ultrastructure in the psylloidean insect Trioza alacris (Flor, 1861 ) in which spheroidal sperm packets that we refer to as ‘spermatodoses’ are found after mating. The ectoderm‐derived epithelium of the sac‐shaped spermatheca that has a proximal neck, consists of large secretory and flat cuticle‐forming cells. Secretory cells are characterized by a wide extracellular cavity, bordered by microvilli, in which electron‐dense secretion accumulates before discharge into the spermathecal lumen. The cuticle‐forming cells produce the cuticular intima of the organ and a peculiar specialized apical structure, through which secretion flows into the lumen. At mating, the male transfers bundles of sperm cells embedded in seminal fluid into the spermathecal neck. Sperm cells proceed towards the spermathecal sac lumen, where they are progressively compacted and surrounded with an envelope that also encloses secretions of both male and female origin. We describe the formation of these sperm containing structures and document the contribution of the female secretion to spermatodose or female‐determined spermatophore construction. We also discuss the choice of the term ‘spermatodose’ for T. alacris and suggest it be used to refer to sperm masses constructed in the female reproductive organs, at least when they involve the contribution of female secretion. © 2011 Wiley Periodicals, Inc.     

Paternity analysis of wild‐caught females shows that sperm package size and placement influence fertilization success in the bushcricket Pholidoptera griseoaptera

In species where females store sperm, males may try to influence paternity by the strategic placement of sperm within the female's sperm storage organ. Sperm may be mixed or layered in storage organs, and this can influence sperm use beyond a ‘fair raffle’. In some insects, sperm from different matings is packaged into discrete packets (spermatodoses), which retain their integrity in the female's sperm storage organ (spermatheca), but little is known about how these may influence patterns of sperm use under natural mating conditions in wild populations. We examined the effect of the size and position of spermatodoses within the spermatheca and number of competing ejaculates on sperm use in female dark bushcrickets (Pholidoptera griseoaptera) that had mated under unmanipulated field conditions. Females were collected near the end of the mating season, and seven hypervariable microsatellite loci were used to assign paternity of eggs laid in the laboratory. Females contained a median of three spermatodoses (range 1–6), and only six of the 36 females contained more than one spermatodose of the same genotype. Both the size and relative placement of the spermatodoses within the spermatheca had a significant effect on paternity, with a bias against smaller spermatodoses and those further from the single entrance/exit of the spermatheca. A higher number of competing males reduced the chances of siring offspring for each male. Hence, both spermatodose size and relative placement in the spermatheca influence paternity success.     

Production of polymorphic sperm by anopheline mosquitoes and their fate within the female genital tract

The males of two mosquito species within the Anopheles gambiae complex, An. gambiae s.s. and An. quadriannulatus, as well as males of An. darlingi, produced sperm of significantly varying lengths, while a sperm polymorphism was absent in Aedes aegypti and other anophelines not suspected of belonging to species complexes. The polymorphic distribution of these sperm lengths was not significantly different in smaller adult males that were reared on a low larval diet. The reproductive tract of the female was more likely to contain larger sperm, but overall sperm retention varied depending on the size of the female and the volume of the spermatheca she contained. The presence of a sperm polymorphism may be a factor that has promoted speciation, as well as providing an indication that females may mate multiply.     

Competitive PCR reveals the complexity of postcopulatory sexual selection in Teleogryllus commodus

The outcome of mate choice depends on complex interactions between males and females both before and after copulation. Although the competition between males for access to mates and premating choice by females are relatively well understood, the nature of interactions between cryptic female choice and male sperm competition within the female reproductive tract is less clear. Understanding the complexity of postcopulatory sexual selection requires an understanding of how anatomy, physiology and behaviour mediate sperm transfer and storage within multiply mated females. Here we use a newly developed molecular technique to directly quantify mixed sperm stores in multiple mating females of the black field cricket, Teleogryllus commodus. In this species, female postcopulatory choice is easily observed and manipulated as females delay the removal of the spermatophore in favour of preferred males. Using twice‐mated females, we find that the proportion of sperm in the spermatheca attributed to the second male to mate with a female (S₂) increases linearly with the time of spermatophore attachment. Moreover, we show that the insemination success of a male increases with its attractiveness and decreases with the size of the female. The effect of male attractiveness in this context suggests a previously unknown episode of mate choice in this species that reinforces the sexual selection imposed by premating choice and conflicts with the outcome of postmating male harassment. Our results provide some of the clearest evidence yet for how sperm transfer and displacement in multiply mated females can lead directly to cryptic female choice, and that three distinct periods of sexual selection operate in black field crickets.     

Regulation of long-term oviposition in the house cricket,Acheta domesticus: Roles of prostaglandin and factors associated with sperm

The transfer of spermatophore contents derived from testes during mating greatly stimulates ovipositional activity for long periods of time in the house cricket, Acheta domesticus (L.). Since prostaglandins appear to play a role in reproduction in several insect species, and since prostaglandin synthesis enzymes occur in cricket testes and spermatophores, we investigated the role of prostaglandins in the regulation of long-term oviposition. Inactivation of prostaglandin synthesis enzymes in males or females using specific inhibitors failed to block mating-induced increases in egg laying. However, males lacking sperm because of X-irradiation were unable to induce oviposition even though they mated, transferred spermatophores, and had high levels of prostaglandins in both testes and spermatophores. X-irradiation was also used to generate males with nonfunctional sperm. Females mated to these animals readily laid eggs, which failed to develop. It appeared that sperm or a factor associated with sperm induced long-term oviposition in female house crickets. Prostaglandin synthesis enzymes transferred from the male to females may have other roles in the female, for example, in sperm maintenance in the spermatheca. Previous observations strongly suggest that prostaglandins induce egg laying behavior and activity; they may be synthesized by female enzymes that are regulated by male-derived factors.     

Longer exaggerated male genitalia confer defensive sperm-competitive benefits in an earwig

As evidence mounts that male genitalia can affect relative fertilisation success, the role that sexual selection has played in the rapid and divergent evolution of genitalia is becoming increasingly recognized. Unfortunately, the limited functional understanding of these complex structures and their interactions with the female reproductive tract often limit interpretation regarding their evolution. Here, we address this issue using the earwig Euborellia brunneri, where both the male intromittent organ and the female spermatheca are highly exaggerated in length yet structurally simple. In a double mating design, we use the sterile male technique to study how sperm precedence patterns are affected by male genital length, male age, and the size of the male sperm storage organ, the seminal vesicle. Relative fertilisation success exhibited considerable variation around modest last-male paternity. Only an interaction between first and second male genital length affected paternity, where males gained reduced paternity when preceded by rivals with longer genitalia. Longer genitalia confer defensive benefits in sperm competition by apparently depositing ejaculate deeper in the tubular spermatheca, safe from removal by rivals. Paternity similarly depended on an interaction between the ages of both males, likely mediated by sperm traits as testes size decreased with age. Seminal vesicle size showed positive allometry but did not affect paternity; instead, greater seminal vesicle size in last males expedited oviposition. The exaggerated yet relatively simple genitalia of E. brunneri facilitate an unusually clear example of post-copulatory selection on phenotypic variation in multiple reproductive traits.     

Mating behavior,insemination and sperm transfer in the ground beetle Carabus insulicola

Mating behavior and the processes of insemination and sperm transfer in the ground beetle Carabus insulicola were analyzed. C. insulicola has elaborate genitalia, in which the strongly sclerotized male copulatory piece is inserted into the female vaginal appendix in copula. During mating, I observed pre-copulatory struggles of males and females, as well as delays in ejaculation, suggesting the presence of intersexual conflicts. Insemination was achieved with a spermatophore, which strongly adhered to the openings of the spermatheca, common oviduct, and vaginal appendix. The spermatophore dissolved after copulation, and sperm were transferred into the spermatheca within three hours after copulation. Sperm bundles were contained within the testes and spermatophores, but free spermatozoa were found in the spermatheca.     

Microsatellite analysis of sperm-use patterns in the bushcricket Requena verticalis

Abstract.— The proportion of offspring sired by the second male to mate with a doubly mated female, P2, is a ubiquitously measured statistic in the study of insect sperm competition. Nevertheless, the underlying mechanisms of sperm transfer, storage, and use that determine P2 are poorly understood. Typically the second male to mate gains moderate to high paternity. More rarely, the first male to mate gains the majority of fertilizations. Here we examine the transfer, storage, and use of sperm in the bushcricket Requena verticalis , a species with male parental investment and almost complete first male paternity. Sperm drain from an externally attached spermatophore into the female's reproductive tract, where they are transported to the sperm store or spermatheca. We find that only sperm from the first male to mate are transported to the spermatheca. We provide some data that address a number of different mechanisms that might account for the lack of storage of second-male sperm. DNA microsatellite markers are developed to assign paternity. By manipulating the numbers of sperm transferred by first and second males, we show that the size of the ejaculate transferred by the first male has a major impact on paternity; increasing ejaculate size of the first male assures his paternity. Paternity assurance in R. verticalis holds significant implications for the evolution of paternal investment via the male's nuptial gift.