Why it is difficult to model sperm displacement in Drosophila melanogaster: the relation between sperm transfer and copulation duration

Abstract.— We have investigated the effects of experimental manipulation of copulation duration on sperm displacement in Drosophila melanogaster . Both spermless and normal males were used as second (displacing) males in the experiments. Displacement induced in the absence of sperm, that is, by males that pass accessory gland fluid alone, was a relatively inefficient process and produced much lower levels of displacement than normal males. Therefore, the presence of second-male sperm is necessary (but unlikely sufficient) for the high levels of displacement commonly observed in D. melanogaster . Furthermore, when second matings were interrupted at various times after the initiation of copulation, the distribution of displacement was strongly bimodal. We conclude that sperm transfer is relatively rapid, beginning shortly after the initiation of copulation, and is essentially complete before the midpoint of copulation. Therefore, sperm transfer bears no simple relation to copulation duration. Because it would be difficult to manipulate the numbers of sperm transferred by manipulating copulation duration, methods used to study sperm displacement in other insect species are unlikely to be appropriate for D. melanogaster . We also investigated why males mate for more than twice the duration that appears to be necessary to complete sperm transfer. Experimental interruption of first matings indicated that the extra copulation time serves to delay female remating, rather than to increase that rate at which of offspring are sired before remating.     

Males do not prolong copulation in response to competitor males in the polyandrous fly Drosophila bifasciata

Males in many taxa exhibit behavioural plasticity in response to the perceived threat of sperm competition. Drosophila males prolong mating in response to the presence of competitor males before copulation. The benefits of this behaviour to males are evident in Drosophila melanogaster. However, the adaptive nature of the trait is challenged by the observation that it is present in four other Drosophila species, two of which are largely monandrous, raising the possibility that this plasticity is not evolutionarily labile. In the present study, behavioural plasticity and the mating system in Drosophila bifasciata Pominini (Diptera, Drosophilidae) are analyzed. By contrast to other Drosophila species, male D. bifasciata do not exhibit plasticity in copulation duration when competitor males are present before mating. Thus, plasticity in mating duration is not fixed in the genus Drosophila. The mating system of D. bifasciata is also examined. The species is polyandrous but, uncommonly for the genus Drosophila, males transfer a mating plug composed of sperm to females, which forms very shortly after copulation and fills the female uterus. The absence of plasticity observed in D. bifasciata may arise from the sperm plug.     

Heritability of sperm competition success in the bulb mite,Rhizoglyphus robini

Multiple mating by females has been proposed to function as a form of mate-choice, which implies that males should show heritable variation in sperm-competitive abilities. In this study, repeatability and heritability of sperm competition success was estimated in the bulb mite, Rhizoglyphus robini. Fertilization success of males was estimated in competition with sperm of two other males. Males differed consistently in their sperm competition success, with repeatability estimated at 0.22. The heritability of sperm competition success was estimated using parent-offspring regression, with the mean fertilization success from two matings used as a measure of each male's competitive ability. There was a significant association between the sperm competition success of fathers and sons. Narrow sense heritability (h²) was 0.284. This result supports the hypotheses proposing the multiple mating is selectively maintained in females by enhancing the reproductive success of their progeny.     

Numbers and size of sperm storage tubules and the duration of sperm storage in birds: a comparative study

Estimates of the numbers of sperm storage tubules (SSTs) in the utero-vaginal junction of 11 bird species are presented. Numbers of SSTs varied by a factor of 40 between species, and ranged from 500 to 20000. Body mass accounted for over 50% of the variation in SST mumbers. SST length was positively correlated with the length of spermatozoa across species. The duration of sperm storage was not correlated with the number of SSTs or the volume of sperm storage tissue. However, the number of 'active' SSTs appears to vary between species and it was not possible to make allowance for this. Sperm storage duration was weakly, positively correlated wth clutch size, but showed a significant positive relationship with the number of days over which laying occurred. The number of SSTs was also positively correlated with the number of sperm per ejaculate. The best predictor of sperm storage duration was a multiple regression equation using the spread of laying and the length of sperm storage tubules. The duration of sperm storage in birds which remain together during the pre-laying period is such that a single insemination immediately before the start of laying could fertilize the entire clutch.     

Sperm competition in the melon fly,Bactrocera cucurbitae (Diptera: Tephritidae): Effects of sperm “longevity” on sperm precedence

Sperm competition inBactrocera cucurbitae was studied by double matings of one female with normal and sterile males, with different intervals between the first and the second matings and with or without allowing oviposition after the first or the second mating. When the interval was less than 4 days, the last-male sperm precedence,P ₂ , was not different from 0.5, but as the interval was prolonged,P ₂ was higher than 0.5. There was no significant difference between treatments in which females were allowed to oviposit after the first mating and only after the second mating. The reason for the higherP ₂ when the interval was long was therefore attributed not to sperm usage for egg fertilization during the two matings but, possibly, to sperm mortality. ThatP ₂ was 0.5 for shorter intervals suggests that particular sperm replacement mechanisms such as removal and inactivation are absent in B. cucurbitae. Our study is the first to demonstrate a significant effect of short sperm longevity on the last-male sperm precedence.     

Determinants of sperm transfer in the scorpionfly Panorpa cognate: male variation, female condition and copulation duration

Recent studies suggest that sperm production and transfer may have significant costs to males. Male sperm investment into a current copulation may therefore influence resources available for future matings, which selects for male strategic mating investment. In addition, females may also benefit from actively or passively altering the number of sperm transferred by males. In the scorpionfly Panorpa cognata, the number of sperm transferred during copulation depended on copulation duration and males in good condition (residual weight) copulated longer and also transferred more sperm. Moreover, sperm transferred and stored per unit time was higher in copulations with females in good condition than in copulations with females in poor condition. Males varied greatly and consistently in their sperm transfer rate, indicative of costs associated with this trait. The duration of the pairing prelude also varied between males and correlated negatively with the male's sperm transfer rate, but no other male character correlated significantly with male sperm transfer rate. The results are consistent with strategic mating effort but sperm transfer could also be facilitated by the physical size of females and/or females in good condition may be more cooperative during sperm transfer.     

Pattern of sperm transfer in redback spiders: implications for sperm competition and male sacrifice

Many sperm competition studies have identified copulation durationas an important predictor of paternity. This result is ofteninterpreted as a sperm transfer effect—it is assumed thatsperm transfer is limited by copulation duration. Here we testthe assumption of duration-dependent sperm transfer in the Australianredback spider, Latrodectus hasselti, in which a correlationbetween copulation duration and paternity has been implicatedin the evolution of a rare male self-sacrifice behavior. Maleredbacks facilitate sexual cannibalism by females during copulation.Sexual cannibalism is apparently adaptive for redback males,in part because it results in longer copulations (25 versus11 min.), and copulation duration is positively correlated withpaternity. We assessed sperm transfer in normal copulationsand in copulations that we terminated at 5, 10, or 20 min. Ourresults show that the paternity advantage of sexual cannibalismis not owing to time-dependent sperm transfer, as redback malestransfer the majority of their sperm within the first 5 minof copulation. This suggests that the link between copulationduration and paternity may instead be owing to cryptic femalechoice or the transfer of nongametic ejaculatory substances.Results further indicate that the act of cannibalism itselfmight play a role in mediating sperm transfer. This study highlightsthe importance of understanding mechanisms of sperm transferwhen attempting to interpret the outcome of sperm competitionstudies.     

Adaptive modulation of sperm production rate in Drosophila bifurca, a species with giant sperm

Sperm have traditionally been regarded as energetically cheap and effectively limitless in supply, although evidence conflicting with this view has become increasingly abundant. For instance, males from a variety of taxa have been shown to strategically partition sperm across ejaculates in response to perceived sperm competition risk. It follows that males might also be predicted to adaptively modulate the rate at which sperm are produced. Here we show that, in the giant sperm producing fruitfly Drosophila bifurca, solitary males with infrequent access to females produce sperm at a much lower rate than males raised in association with females and other males. Our results support the prediction that males with little risk of sperm competition risk or few mating opportunities should divert resources away from gamete production.     

Male sperm expenditure under sperm competition risk and intensity in quacking frogs

In the frog Crinia georgiana, reproductive behavior comprisesa "guarding tactic," in which males defend spawn sites and attractfemales by calling, and a "sneak tactic," in which males joinspawning pairs. The aims of the present study were to (1) relateejaculate expenditure by "guarding" and "sneak" males to theirprobability of mating with other males present (sperm-competitionrisk), and (2) determine if males adjust their ejaculate expenditureaccording to the number of males involved in a spawning (sperm-competitionintensity). Theory predicts that because sneak males alwaysmate with other males present, they will experience a highersperm-competition risk and should release larger ejaculatesrelative to that of guarding males. However, as the proportionof sneaks in a population increases so does the risk of spermcompetition to guarders, so expenditure by each tactic shouldmove toward equality. Given that the incidence of sneak behavioris high in C. georgiana, guarders and sneaks were expected toexperience similar risks of sperm competition and show similarinvestment in spermatogenesis. Comparison of testes size andejaculate size showed no difference between tactics. Modelsof sperm-competition intensity predict that males should increasetheir ejaculate size when spawning in the presence of one othermale but decrease their ejaculate size when spawning in thepresence of multiple males. Here, males maintained a constantsperm number irrespective of whether a mating involved one,two, or three males. This result suggests that male C. georgianado not facultatively adjust ejaculate investment in responseto fluctuating intensities of sperm competition.     

The effects of copulation duration in the bruchid beetle Callosobruchus maculatus

Control over copulation duration is a potentially importantgenerator of sexual conflict that has received little empiricalattention. The copulatory behavior of the bruchid beetle Callosobruchusmaculatus may reflect a sexual conflict over copulation duration.Males have spines on their intromittent organs that puncturethe female reproductive tract, and females kick their matesduring copulation. If females are prevented from kicking, copulationslast longer and the injuries females sustain are more severe.Males supposedly use the spines as anchors to prolong copulationduration, and females kick to terminate copulations. We manipulatedcopulation duration experimentally and quantified its effectson male and female fitness components to test whether or notthere is a conflict over copulation duration in C. maculatus.Females did not suffer from long copulations but instead experiencedincreased lifetime fecundity. Ejaculate size increased withcopulation duration, and females apparently derive materialbenefits from the ejaculates. Males that mated first and hadlong copulations were relatively unsuccessful when competingwith sperm from other males. However, there was a trend forfemale remating propensity to decrease with long copulationdurations, and first males may therefore also benefit from longcopulations. The copulation duration of the second male to matedid not have a significant effect on sperm precedence. We concludethat even though it seems likely that the male spines have evolvedto act as an anchor during copulation, there seems to be littleconflict over copulation duration per se in C. maculatus.     

The copulatory plug delays ejaculation by rival males and affects sperm competition outcome in house mice

Females of many species mate with multiple males (polyandry), resulting in male–male competition extending to post‐copulation (sperm competition). Males adapt to such post‐copulatory sexual selection by altering features of their ejaculate that increase its competitiveness and/or by decreasing the risk of sperm competition through female manipulation or interference with rival male behaviour. At ejaculation, males of many species deposit copulatory plugs, which are commonly interpreted as a male adaptation to post‐copulatory competition and are thought to reduce or delay female remating. Here, we used a vertebrate model species, the house mouse, to study the consequences of copulatory plugs for post‐copulatory competition. We experimentally manipulated plugs after a female's first mating and investigated the consequences for rival male behaviour and paternity outcome. We found that even intact copulatory plugs were ineffective at preventing female remating, but that plugs influenced the rival male copulatory behaviour. Rivals facing intact copulatory plugs performed more but shorter copulations and ejaculated later than when the plug had been fully or partially removed. This suggests that the copulatory plug represents a considerable physical barrier to rival males. The paternity share of first males increased with a longer delay between the first and second males' ejaculations, indicative of fitness consequences of copulatory plugs. However, when males provided little copulatory stimulation, the incidence of pregnancy failure increased, representing a potential benefit of intense and repeated copulation besides plug removal. We discuss the potential mechanisms of how plugs influence sperm competition outcome and consequences for male copulatory behaviour.     

The evolution of sperm-allocation strategies and the degree of sperm competition

The prevailing viewpoint in the study of sperm competition is that male sperm-allocation strategies evolve in response to the degree of sperm competition an ejaculate can expect to experience within a given mating. If males cannot assess the degree of sperm competition their ejaculate will face and/or they are unable to facultatively adjust sperm investment in response to perceived levels of competition, high sperm allocation (per mating) is predicted to evolve in the context of high sperm competition. An implicit assumption of the framework used to derive this result is that the degree of sperm competition is unaffected by changes in sperm-allocation strategies. We present theory based on an alternative perspective, in which the degree of sperm competition and the sperm-allocation strategy are coupled traits that coevolve together. Our rationale is that the pattern of sperm allocation in the population will, in part, determine the level of sperm competition by affecting the number of ejaculates per female in the population. In this setting, evolution in sperm-allocation strategies is driven by changes in underlying environmental parameters that influence both the degree of sperm competition and sperm allocation. This change in perspective leads to predictions that are qualitatively different from those of previous theory.     

Cellular organization in the synganglion of the mite Macrocheles muscaedomesticae (Acarina: Macrochelidae)

Summary A large DNA containing body is found in oocytes of the house cricket, Acheta domesticus. Little or no RNA synthesis is associated with the DNA body during the leptotene, zygotene, and pachytene stages of meiotic prophase I. During the early diplotene stage of development, large masses of nucleolar material begin to accumulate at the periphery of the DNA body. The onset of RNA synthesis correlates with a change in the histochemically detectable histone proteins associated with the DNA body. In ovaries of animals injected with uridine-H³, most of the label accumulates in ribosomal RNA. Autoradiographic studies show that the cytoplasm of late diplotene stage cells accumulates uridine label to a greater extent than does the cytoplasm of early diplotene stage cells. Increased transport of nucleolar material through the nuclear envelope of late diplotene stage cells accounts for the increased cytoplasmic labeling.This investigation was supported by PHS Research Grant No. GM 16440 from the Institute of General Medical Sciences, and by Grants No. L-16 and J-1 from the Health Research and Services Foundation.The authors gratefully acknowledge the technical assistance of Mrs. Marcia Andrews and Miss Celeste Malinoski.     

Male mating behavior and ejaculate expenditure under sperm competition risk in the eastern mosquitofish

Theory predicts that males should tailor the size of their ejaculatesaccording to temporal changes in the risk of sperm competition.Specifically, males are predicted to allocate more sperm toeach mating event with increasing risk (i.e., the probabilitythat the sperm from two males will compete for fertilization).We tested this hypothesis by using the eastern mosquitofish,a freshwater species of fish exhibiting a coercive mating systemand internal fertilization. We manipulated the perception ofsperm competition risk by adjusting the sex ratio under whichmales were maintained over 8 days. Males were housed eitherwith three females and one male (simulating high sperm competitionrisk) or with four females (low risk). After the treatment,we presented each test male individually to an unfamiliar male-deprivedfemale for 30 minutes and observed his mating behavior. We thenartificially stripped the test males of sperm and recoveredthe ejaculates from the females. Our results revealed that malesin the high-risk group performed higher levels of mating activityand sperm expenditure (i.e., used up more of their sperm reserves)than did low-risk males. A control experiment, in which testmales were treated but did not participate in the mating trials,revealed no significant difference in the number of sperm strippedfrom high- and low-risk males, indicating that sperm productionwas not affected by the treatment. We did not detect a differencein the number of sperm retrieved from females among the groups,raising the possibility that some sperm are lost during matingactivity, either through ejaculation with incomplete or interruptedpenetration, or via female ejection.     

Male-derived cuticular hydrocarbons signal sperm competition intensity and affect ejaculate expenditure in crickets

Female sexual promiscuity can have significant effects on male mating decisions because it increases the intensity of competition between ejaculates for fertilization. Because sperm production is costly, males that can detect multiple matings by females and allocate sperm strategically will have an obvious fitness advantage. The presence of rival males is widely recognized as a cue used by males to assess sperm competition. However, for species in which males neither congregate around nor guard females, other more cryptic cues might be involved. Here, we demonstrate unprecedented levels of sperm competition assessment by males, which is mediated via the use of chemical cues. Using the cricket Teleogryllus oceanicus, we manipulated male perception of sperm competition by experimentally coating live unmated females with cuticular compounds extracted from males. We found that males adjusted their ejaculate allocation in response to these compounds: the viability of sperm contained within a male's ejaculate decreased as the number of male extracts applied to his virgin female partner was increased. We further show that males do not respond to the relative concentration of male compounds present on females, but rather to the number of distinct signature odours of individual males. Our results conform to sperm competition theory, and show for the first time, to our knowledge, that males can detect different intensities of sperm competition by using distinct chemical cues of individual males present on females.