Seasonal variation in genital and body size, sperm displacement ability, female mating rate, and male harassment in two calopterygid damselflies (Odonata: Calopterygidae)

Sperm competition is a pervasive force. One adaptation is the male ability to displace the rivals' sperm that females have stored from previous copulations. In the damselfly, Calopteryx haemorrhoidalis asturica , males with wider aedeagi displace more spermathecal sperm. The present study documents that the same mechanism operates in another damselfly, Hetaerina americana . However, this genital width in both species decreases along the season, but late-emerging females have more sperm displaced than early-emerging females. Because territorial males mated more and were larger in body and genital size than nonterritorial males, late-season females mated with considerably larger males with respect to female size and this produced higher sperm displacement. Assuming female benefits from storing sperm but that such benefit does not prevail if males displace sperm, it is predicted that, along the season, females will mate less and male harassment (in terms of male mating attempts and oviposition duration) will increase. These predictions were corroborated. In H. americana , it was also tested whether spermathecal sperm became less viable along the season. The results obtained did not corroborate this. This is the first evidence indicating that season affects sperm displacement ability and female mating frequency due to changes in male body and genital size.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 96 , 815–829.     

Function of multiple sperm-storage organs in female damselflies (Ischnura senegalensis): difference in amount of ejaculate stored, sperm loss, and priority in fertilization

We studied changes in the number of sperm within two kinds of female sperm-storage organ in the damselfly Ischnura senegalensis (Odonata: Coenagrionidae): the bursa copulatrix and the spermatheca. We counted the number of sperm within each storage organ and tested their viability after a single copulation in female damselflies kept for seven days with and without oviposition. We also counted sperm and tested their viability in females that underwent an interrupted second copulation after the sperm-removal stage, and after subsequent oviposition. Our results showed that the bursa copulatrix and spermatheca have different sperm storage roles. Immediately after copulation, most eggs appear to have been fertilized with bursal sperm, which were positioned near the fertilization point. By seven days after copulation, a greater proportion of spermathecal sperm were used for fertilization, as the number of bursal sperm had decreased. We hypothesize that female damselflies use the spermatheca for long-term storage and the bursa copulatrix for short-term storage: bursal sperm are more likely to be used for fertilization but may have a higher risk of mortality due to sperm removal by a competing male and/or sperm expelling by the female, whereas spermathecal sperm are safer but will be used for fertilization only after their release from the spermatheca.     

Conspecifics and Their Posture Influence Site Choice and Oviposition in the Damselfly Argia moesta

Finding a suitable oviposition site can be costly because of energy and time requirements, and ovipositioning can be dangerous because of the risk of predation and harassment by males. The damselfly Argia moesta oviposits, contact-guarded by her mate, on vegetation in streams. Oviposition aggregations are commonly observed in this species, despite their territorial nature during other behaviors. We conducted experiments in the field to test the hypothesis that aggregations are the result of conspecific attraction. In the first experiment, two oviposition sites (sycamore leaves) were provided, one with models of ovipositing pairs, and one without. In the second experiment, one leaf again had ovipositing models, while the other had models of uncoupled males and females in a resting posture. In both experiments, damselfly pairs preferred the site with ovipositing models. In general, they visited the ovipositing models first more often than expected by chance, stayed longer there, were more likely to oviposit there, and laid a greater total number of eggs there. These results support the hypothesis that conspecific attraction is responsible for ovipositing aggregations in A. moesta and that posture is an important cue for attraction. Using conspecific cues could be a beneficial strategy to save in search costs while taking advantage of the presence of ovipositing conspecifics to dilute the effects of harassment and predation.     

Isometric patterns for male genital allometry in four damselfly species

Recent studies have found that insect genitalic traits show negative allometry, i.e., are relatively small in relation to body size. One interpretation of this is that males use their genitalia to stimulate females. Thus, given the nature of damselfly copulation in which males physically reach the rival sperm that females have stored from previous matings, male genitalic traits are not expected to show negative allometry. To test this idea, we assessed (a) the rival sperm displacement function by the mating male and (b) allometry of aedeagal length of four damselfly species (Argia anceps, Argia tezpi, Argia extranea, and Enallagma praevarum). Sperm displacement was assessed by inspecting whether the aedeagus reached the rival sperm during copulation in mating pairs for the four species. To have a standard for comparing allometric patterns, allometry of aedeagal was compared to that of two non-genital traits, tibial, and fourth abdominal segment length. In all cases, the aedeagus was found to reach the rival sperm which supports the idea that stimulation is not the mechanism for sperm displacement but physical displacement. Aedeagal length was isometric, and its slope was lower in general compared to that of tibial length and fourth abdominal segment. Given that this isometric pattern is not common for other odonate species, our interpretation of these varying aedeagal scaling patterns in this insect order is that males’ and females’ sexual interests are in conflict (males are evolving an elongated aedeagus to reach rival sperm while females are evolving unreachable sperm storage organs to prevent displacement of stored sperm). This sexual conflict scenario would favor varying scaling patterns for aedeagal length in odonates. A final interpretation is that the risk of interspecific matings in damselflies, may also explain different species-specific, aedeagal allometries.     

Territoriality and male-biased sexual size dimorphism in Argia reclusa (Odonata: Zygoptera)

In Odonata, many species present sexual size dimorphism (SSD), which can be associated with male territoriality in Zygoptera. We hypothesized that in the territorial damselfly Argia reclusa, male–male competition can favor large males, and consequently, drive selection pressures to generate male-biased SSD. The study was performed at a small stream in southeastern Brazil. Males were marked, and we measured body size and assessed the quality of territories. We tested if larger territorial males (a) defended the best territories (those with more male intrusions and visiting females), (b) won more fights, and (c) mated more. Couples were collected and measured to show the occurrence of sexual size dimorphism. Results indicated that males are larger than females, and that territorial males were larger than non-territorial males. Larger territorial males won more fights and defended the best territories. There was no difference between the mating success of large territorial and small non-territorial males. Although our findings suggest that male territoriality may play a significant role on the evolution of sexual size dimorphism in A. reclusa, we suggest that other factors should also be considered to explain the evolution of SSD in damselflies, since non-territorial males are also capable of acquiring mates.     

Copulation duration and fertilization success in a damselfly: an example of cryptic female choice?

Copulation duration is highly variable (0.5-3 h) in the damselfly, Ceriagrion tenellum (Coenagrionidae). Using laboratory experiments, we tested four adaptive hypotheses to explain this variation: the effect of time constraints, in-copula mate guarding, sperm displacement and cryptic female choice. Copulation duration was negatively correlated with time of day, as predicted by the first two hypotheses, and positively correlated with male density, as predicted by the mate-guarding hypothesis. Males prolonged copulation in response to the volume of sperm stored by females, suggesting they were able to detect and quantify the amount of sperm stored. This behaviour is not explained by mate guarding or time constraint effects. Males removed all the sperm from the bursa copulatrix in just 10 min. Our results also suggest that, because the duct is too narrow to allow male genitalia to enter, males do not remove spermathecal sperm. Therefore, direct sperm removal could not explain long copulations. Prolonged copulations could also have evolved as a result of cryptic female choice if they increase male fertilization success by female-mediated processes. Our results support this idea: male fertilization success was greater after long copulations. Apparently, male copulatory behaviour elicits female responses that increase male fertilization success. Copyright 2000 The Association for the Study of Animal Behaviour.     

Pre-oviposition mate-guarding and mating behaviour of Argia vivida (Odonata: Coenagrionidae)

Abstract. 1. At Halcyon Hotsprings, British Columbia, Canada, male and female Argia vivida Hagen encountered to mate in two different ways.
2. In the morning (before 12.30 hours solar time), males basked at sunspots in the forest and darted out at passing females, attempting to take them in tandem (the first method of encounter).
3. If a male was successful, the pair engaged in a 31.3±4.8 min copulation followed by an hour of tandem flight before beginning oviposition.
4. As the day progressed, unmated males moved slowly toward the water and arrived at the water at about the same time as the earliest ovipositing pairs (1131±27.5 min solar time).
5. Males retained their grasp on their mates during oviposition (contact-guarding) but since some tandems separated during oviposition, non-tandem males at the water could capture recently released, gravid females (the second method of encounter).
6. The new pairs performed a brief copulation (10.2±3.38 min) and began ovipositing immediately thereafter.
7. Some females that avoided recapture attempted to oviposit unguarded.
8. We believe the long duration of morning copulations and period of tandem constitute a male strategy, which we call 'pre-oviposition guarding', to guard females until it is warm enough at the oviposition site for the females to begin ovipositing.
9. Separation of tandems during oviposition may be initiated by either member of the pair and we suggest that one benefit to a female of leaving a guarding mate is increased efficiency of oviposition when the intensity of male harassment is low.
10. The mating system of A. vivida thus comprises a series of complementary male and female mating behaviours.     

Post-copulatory mate guarding by males of the demselfly Hetaerina vulnerata Selys (Odonata: Calopterygidae)

Males of the calopterygid damselfly Hetaerina vulnerata remain with their mates after copulating with them. The species exhibits two unusual features of post-copulatory mate guarding. First, a male will often leave his territory to accompany a female in tandem on a search for oviposition sites elsewhere. Second, a male will perch near his ovipositing female even though she completely submerges when egg-laying and cannot be captured and mated by another male while she is underwater. These activities carry two potential costs: (1) a male may miss other receptive females while guarding one mate and (2) he may lose his territory to an interloper while he is absent. These costs were low, however, because territorial males secured only one mating per 3.6 days on average. Moreover, 23 times out of 26, territorial males reclaimed their plots quickly after being away for 30–60 min. The gain from postcopulatory guarding came from being present to recapture a female should she fly up from the water after rejecting an oviposition site. There was a 40% chance that a female would leave one site to search for another during an oviposition bout. If the male were not present, his mate would be captured and mated by another individual (no female ever selected an oviposition site without being carried to it by a male). Her new partner would fertilize the remaining eggs in the female's clutch (if sperm precedence occurs in this species). The total number of eggs fertilized by a male will be affected by how well he prevents any one mate from copulating again before she lays her entire clutch and the total number of receptive females he captures. The variation in the degree of mate guarding by male odonates seems to be the evolutionary outcome of differences in fitness gains derived from these two competing activities in different ecological settings.     

The effects of exposure to seaweed on willingness to mate, oviposition, and longevity in seaweed flies

Abstract  1. Large male seaweed flies (Diptera: Coelopidae) are more likely to mate than smaller males. This is due to sexual conflict over mating, by which females physically resist male attempts to copulate. In some species, large males are simply more efficient at overpowering female resistance.
2. Female reluctance to mate is likely to have evolved due to the costs of mating to females. In many dipterans, males manipulate female behaviour through seminal proteins that have evolved through sperm competition. This behavioural manipulation can be costly to females, for example forcing females to oviposit in sub-optimal conditions and increasing their mortality.
3. Previous work has failed to identify any ubiquitous costs of mating to female coelopids. The work reported here was designed to investigate the effects of exposure to oviposition sites ( Fucus algae) on the reproductive behaviour of four species of coelopid. Algae deposition in nature is stochastic and females mate with multiple males in and around oviposition sites. Spermatogenesis is restricted to the pupal stage and there is last-male sperm precedence. It was predicted that males would avoid wasting sperm and would be more willing to mate, and to remain paired with females for longer, when exposed to oviposition material compared with control males. Females were predicted to incur longevity costs of mating if mating increased their rate of oviposition, especially in the presence of algae.
4. The behaviour of males of all four species concurred with the predictions; however mating did not affect female receptivity, oviposition behaviour, or longevity. Exposure to algae induced oviposition and increased female mortality in all species independently of mating and egg production. The evolutionary ecology of potential costs of mating to female coelopids are discussed in the light of these findings.     

Size, operational sex ratio, and mate-guarding success of the carrion beetle, Necrophila americana

When male insects guard females until oviposition, the benefitsfrom last-male sperm precedence must outweigh the costs of relinquishingadditional fertilizations. The profitability of guarding isincreased when males guard large, fecund females and when femalesare scarce because fewer fertilizations are sacrificed. However,the male reproductive success is not only determined by theprofitability of guarding but also by his ability to maintainguarding. In this study, we used male carrion beetles (Necrophilaamericana) to examine the effects of sex ratio, male relativesize, and female quality on the ability to guard. First, wepresent a model of mate guarding that explores factors, suchas sperm precedence, sex ratio, male size, and female quality,that influence the profitability of postcopulatory riding. Ourmodel predicts that large N. americana males should preferentiallyguard the largest female only when the sex ratio is male biasedand sperm precedence is above 80%. In contrast, small malesgain little from guarding because they are not likely to maintainit and be the last male to mate. Then, we tested these predictionsby manipulating sex ratio, relative male size, and female quality.All males in equal sex ratio and large males in male-biasedsex ratio guarded females significantly longer than did malesin female-biased sex ratio. In male-biased sex ratio, largemales guarded significantly longer and achieved more takeoversthan small males. Large females were guarded longer. The successof guarding males in this beetle depends on their size relativeto other males and the operational sex ratio.     

Last male sperm precedence in a polygamous squid

Differential sperm usage from consecutive matings, or sperm precedence, is vital in determining male reproductive success and the outcome of sperm competition for many organisms. Sperm precedence also has significant consequences for mating system dynamics, including both male and female adaptations for increasing reproductive success and avoiding the costs of mating. Despite sexual selection being a strong driver of reproductive behaviour and morphology in cephalopods, surprisingly few studies have investigated sperm dynamics in this group. To redress this gap, we experimentally quantified sperm precedence patterns in the dumpling squid, Euprymna tasmanica, controlling for recent male mating history (first vs. second mating), mating position, and mating frequency. We found that the last male to mate gains an advantage in this system, with the second mating male siring up to 75% of offspring at the beginning of the laying period. The proportion of offspring attributable to the second mating male decreases to 54% by the end of the laying period, potentially as a result of changes in the velocity or number of sperm released from spermatangia over time. There is also significant variation among females in patterns of sperm precedence. This variation was not associated with whether it was the male's first or second mating, male mass, the duration of copulation or the number of pumps (sperm removal behaviour) by the second male. If widespread in cephalopods, last male sperm precedence could help to explain the evolution of mate guarding (or long copulation duration) and sperm removal behaviour in this group.     

Spermatophore expulsion in the carrion beetle Silpha perforata (Coleoptera: Silphidae)

Silphinae (Coleoptera: Silphidae) is an abundant decomposer that plays important roles in the ecosystem. However, there is little information about the life history of this taxon. We found sperm displacement behavior in carrion beetle Silpha perforata. Copulating males bit the female's antenna strongly and inserted the penis into the partner's genital organ more than once. We found a white substance on the tip of penis during copulation. We examined whether this white substance is a previous male's spermatophore, which was removed from the mating partner. When females were dissected just after mating, the same substance that often presents on the penis of mating males was found in the bursa copulatrix of females, although the bursa copulatrix of virgin females was empty. Male behavior during copulation with females of different mating history was also observed to confirm that the removal of spermatophores was observed only in copulation with females that have the spermatophores of previous males. Consequently, we estimated that S. perforata males removed spermatophores of previous males from mating partners. In addition, we dissected the males frozen during copulation, and inspected the penis morphology. This observation revealed that the apical part of the penis was usually hidden in the basal part of penis, but expanded and appeared during insertion. This apical part had many spines, which play an important role in sperm displacement and sexual conflict in some species. These results indicate that there is the sperm competition in S. perforata. This is the first report on sperm competition in Silphinae.     

Prior oviposition, female receptivity and last-male sperm precedence in the cosmopolitan pest Callosobruchus maculatus (Coleoptera: Bruchidae)

Sperm competition studies have shown that P₂ (the proportion of ova fertilized by the last male to mate) increases as the interval between inseminations is experimentally increased. Variation in the number of sperm in storage is associated with sperm use (or loss) from the female's sperm stores between copulations (fewer sperm from previous mates at the time of the last copulation) and with the extent of prior oviposition and female receptivity to further copulation: females that lay many eggs tend to have few remaining sperm in storage and to be more receptive to further copulation. Using the bruchid beetle Callosobruchus maculatus, we examined the effect of prior oviposition and female receptivity to further copulation on the extent of last-male sperm precedence (measured as P₂). Extent of prior oviposition was experimentally manipulated independently of the intermating interval by altering the availability of oviposition sites between inseminations. Females given few or no oviposition sites laid fewer eggs, were less receptive and had a lower P₂ than females given abundant oviposition sites. To examine the effect of female receptivity on P₂ independently of prior oviposition, we examined the outcome of sperm competition experiments using (1) females from lines that had been selected for different latencies to copulation and (2) natural variation in female latency to receptivity. Female receptivity to further copulation had no detectable effect on P₂. When oviposition resource is abundant, female receptivity may be a poor predictor of current sperm load.     

Sperm precedence in the dragonfly Erythemis simplicicollis

Females of the dragonfly Erythemis simplicicollis (Say) (Odonata, Libellulidae) store enough sperm to fertilize 6–13 clutches of eggs laid on consecutive days. Nonetheless, they usually mate one or more times per day. Males wait for females at ponds containing surface vegetation on which the females lay eggs. Some males defend vegetation while other act as satellites. After mating, both types of males attempt to guard females against takeover by other males. Sperm precedence by male E. simplicicollis was studied using sterility produced by gamma irradiation to label sperm. After a dose-response analysis, males receiving a dose of 25 kiloroentgens (>99.9% sterile) were returned to their home pond as territory residents and satellites. Both types of males fertilized an average of 99.5% (range 97.3–100%) of the female's remaining clutch. After mating with a sterile male, females were isolated in a large cage, and eggs collected for several consecutive days. These clutches revealed that sperm mixing in the bursa of the females is essentially complete after 24 to 48 h and that the last male to mate had replaced an average of more than 57–75% of the sperm stored by female from previous matings. Thus, the last sperm in is the first sperm out fertilizing essentially all of the eggs laid soon (5–6 min) after the mating. Sperm from the most recent mating competes for fertilizations with sperm stored from previous matings only if the female oviposits on the following day without remating.     

FEMALE CONTROL OF SPERM TRANSFER AND INTRASPECIFIC VARIATION IN SPERM PRECEDENCE: ANTECEDENTS TO THE EVOLUTION OF A COURTSHIP FOOD GIFT

Manipulation of ejaculates is believed to be an important avenue of female choice throughout the animal kingdom, but evidence of its importance to sexual selection remains scarce. In crickets, such manipulation is manifest in the premature removal of the externally attached spermatophore, which may afford females an important means of postcopulatory mate choice. We tested the hypothesis that premature spermatophore removal contributes significantly to intraspecific variation in sperm precedence by (1) experimentally manipulating spermatophore attachment durations of competing male Gryllodes sigillatus and (2) employing protein electrophoresis to determine the paternity of doubly mated females. The relative spermatophore attachment durations of competing males had a significant influence on male paternity, but the pattern of sperm precedence deviated significantly from the predictions of an ideal lottery. Instead, paternity data and morphological evidence accorded best with a model of partial sperm displacement derived here. Our model is similar to a displacement model of Parker et al. in that sperm of the second male mixes instantaneously with that of the first throughout the displacement process, but the novel feature of our model is that the number of sperm displaced is only a fraction of the number of sperm transferred by the second male. Regardless of the underlying mechanism, female G. sigillatus can clearly alter the paternity of their offspring through their spermatophore-removal behavior, and employ such cryptic choice in favoring larger males and those providing larger courtship food gifts. We discuss how female control of sperm transfer and intraspecific variation in sperm precedence may be important precursors to the evolution of gift giving in insects.     

The relationship between mating system and penis morphology in ischnuran damselflies (Odonata: Goenagrionidae)

Zygopterans belonging to the genus Ischnura are unusual amongst damselflies because of the variety of mate guarding techniques employed by males of different species. The lack of post-copulatory guarding combined with lengthy copulations in one group of ischnuran species suggest that these males guard females in copula. An examination of the accessory penes of species in this group indicates that all but one species have considerable microspination on the distal end (the flagella) of their penes that can function in sperm displacement. The flagella of these species are long and thin compared to those of other ischnurans. This is likely an adaptation to gain access to the spermatheca of the female. Two species tandem guard their mates during ovipositing. These species are the only ischnurans missing a stout pair of basal spines on the penultimate segments of their penes. They have considerable microspination over much of their penes but their flagella are of only moderate length and stout. Ischnurans that do not mate guard have short, stout flagella and most species examined from this group (5 of 7) have litde microspination on their flagella tips. It is proposed that females of these species mate only once and therefore their males do not displace sperm.     

Sperm stratification and paternity success in red flour beetles

Abstract. When females are inseminated by multiple males, male paternity success (sperm precedence) is determined by the underlying processes of sperm storage and sperm utilization. Although informative for many questions, two-male sperm competition experiments may offer limited insight into natural mating scenarios when females are likely to mate with several males. In this study, genetic markers in Tribolium castaneum are used to trace paternity for multiple sires, and to determine whether displacement of stored sperm that occurs after a third mating equally affects both previous mates, or if fertilizations are disproportionately lost by the female's most recent mate. For 20 days after triple-matings, first males retain significantly higher paternity success (relative to first male paternity in double-matings) compared with second males. These results demonstrate that when females remate before sperm mixing occurs, sperm stratification results in differential loss of sperm from the most recent mate. This study provides insight into the mechanisms underlying sperm precedence in a promiscuous mating system, and suggests that T. castaneum females could limit paternity success of particular mates by remating with more highly preferred males.     

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.     

Alternative oviposition behaviours in three New Zealand corduliid dragonflies: their adaptive significance and implications for male mating tactics

Oviposition behaviours of female, and mate acquisition and defence behaviours of male, 'Procordulia' grayi, Procordulia smithii and Hemicordulia australiae (three phylogenetically close corduliid species) are contrasted. Twelve distinct methods of oviposition occur involving different motor patterns. These different oviposition behaviours place ova into different microhabitats. Each species has a distinct repertoire of oviposition methods, with only one of the 12 methods occurring in more than one species. The oviposition behaviours differ in their susceptibility to male interference. The implications for male sperm displacement tactics and for the development of conditional male strategies are discussed. Male mating behaviour varies with the geometry of the breeding site, in a manner consistent with an ideal free distribution model where females vary in their 'value' through differential susceptibility to takeover by other males. It is shown that differential susceptibility of females to takeover would stabilize the observed mixed mating strategy among male 'P.' grayi and P. smithii .     

Copula in yellow dung flies (Scathophaga stercoraria): investigating sperm competition models by histological observation

While sperm competition has been extensively studied, the mechanisms involved are typically not well understood. Nevertheless, awareness of sperm competition mechanisms is currently recognised as being of fundamental importance for an understanding of many behavioural strategies. In the yellow dung fly, a model system for studies of sperm competition, second male sperm precedence appears to result from a combination of sperm displacement and sperm mixing. Displacement was until recently thought to be directly from the female's sperm stores, the spermathecae (i.e. males were thought to ejaculate directly into these stores), and under male control. However, recent work indicates displacement is indirect (i.e. males do not ejaculate directly into the sperm stores) and that it is female-aided, although the evidence was not based on direct observation. Here, we used histological techniques to directly determine interactions during copula and sperm transfer. Our results are consistent with inference and clearly show that males ejaculate into the bursa copulatrix. Our data are also consistent with active female involvement in sperm displacement, which is indirect, and indicate the aedeagus may remove some spermatozoa from the bursa at the end of copula. In addition, evidence suggests females aid sperm transport to and from the spermathecae, possibly by muscular movement of a spermathecal invagination.