The unexpected but understandable dynamics of mating,paternity and paternal care in the ocellated wrasse

Although theory generally predicts that males should reduce paternal care in response to cues that predict increased sperm competition and decreased paternity, empirical patterns are equivocal. Some studies have found the predicted decrease in male care with increased sperm competition, while even more studies report no effect of paternity or sperm competition on male care. Here, we report the first example, to our knowledge, of paternal care increasing with the risk and intensity of sperm competition, in the ocellated wrasse (Symphodus ocellatus). Theory also predicts that if paternal care varies and is important to female fitness, female choice among males and male indicators traits of expected paternal care should evolve. Despite a non-random distribution of mating success among nests, we found no evidence for female choice among parental males. Finally, we document the highest published levels of extra-pair paternity for a species with exclusive and obligate male care: genetic paternity analyses revealed cuckoldry at 100 per cent of nests and 28 per cent of all offspring were not sired by the male caring for them. While not predicted by any existing theory, these unexpected reproductive patterns become understandable if we consider how male and female mating and parental care interact simultaneously in this and probably many other species.     

Quantity of the male ejaculate influenced by female unreceptivity in the fly, Culicoides melleus

The number of sperm placed in the spermatophore by virgin Culicoides melleus males mated with virgin females was compared with the number deposited in matings with previously once or twice copulated females. When compared to an all-virgin mating, there was a significant reduction (P<0·001) in sperm complement (of 15 per cent) when the female had already been mated once as compared to a virgin, and a greater reduction (of 21 per cent) when the female had already been mated twice. The mean numbers (± S.E.) of sperm ejaculated with virgin, once mated, and twice mated females were 857 ± 24, 732 ± 23, and 673 ± 25, respectively.When a major activity in the unreceptive behaviour of females (kicking at the male in the early stages of copulation) was prevented by amputation of the hind legs, the mean number of sperm ejaculated (788 ± 29) was significantly larger (P<0·01) compared with twice mated females having both legs intact, and also larger, though not significantly so, than with intact once mated females. In addition to the kicking activity, there was evidence that other forms of unreceptive behaviour contributed to reducing the quantity of sperm ejaculated.     

Glucose metabolism in the cattle tick, Boophilus microplus

The contributions of the glucose catabolic pathways in different stages of the life cycle of the cattle tick, Boophilus microplus, were estimated on the basis of rates of ¹⁴CO₂ production from specifically labelled glucose. In free-living larvae the pentose cycle accounted for 20 per cent of the total glucose catabolism. In female ticks in the final 24 hr of parasitic feeding and in the pre-oviposition period the pentose cycle proportion rose to over 50 per cent and returned in the final stage of oviposition to 20 per cent. These changes are attributed to the importance of the pentose cycle in biosynthetic processes which must reach their peak during vitellogenesis in the pre-oviposition period.     

Functions of the spermathecal muscle of the boll weevil,Anthonomus grandis

In female boll weevils, Anthonomus grandis, spermathecal filling was not affected by severing the spermathecal muscles. Females whose spermathecal muscles were severed 2 to 4 weeks after mating laid infertile eggs and resumed virginal ovipositional behaviour indicating the importance of this muscle in supplying sperm for egg fertilization. The presence of normal active sperm within the spermatheca in no way influenced ovipositional behaviour. In females whose spermathecal muscles had been severed, 22 per cent sperm displacement occurred after a second mating compared with 66 per cent for normal females. The physical displacement of sperm was thus largely dependent on a functional spermathecal muscle.     

Courtship rate signals fertility in an externally fertilizing fish

Sperm limitation is widespread across many animal species. Several mechanisms of sperm allocation have been proposed, including optimal allocation according to clutch size and equal allocation across females. However, considerably less effort has been directed at investigating the behavioural signals associated with sperm limitation in males, which may include mating rate and the intensity of courtship. We investigated whether multiple successive spawnings affect individual male fertilization success, mating rates and courtship rates in Japanese medaka (Oryzias latipes). Across an average of 17 spawning events per male, fertilization success decreased from 83.7 per cent for the first spawning to 40 per cent for the last spawning while courtship rate decreased from 3.4 to 1.5 min⁻¹. Females appeared to respond to male sperm depletion by reducing clutch size. Our results suggest that male Japanese medaka are sperm-limited, and that courtship rate may be an honest indication of fertilization ability.     

Long-lived sperm in the geothermal bryophyte Pohlia nutans

Non-vascular plants rely on sperm to cross the distance between male and female reproductive organs for fertilization and sexual reproduction to occur. The majority of non-vascular plants have separate sexes, and thus, this distance may be a few millimetres to many metres. Because sperm need water for transport, it has been assumed that sperm lifespans are short and that this type of sexual reproduction limits the expansion of non-vascular plants in terrestrial environments. However, little data is available on the lifespan of sperm in non-vascular plants, and none is available for bryophytes, the group thought to have first colonized terrestrial habitats. Here, we documented the lifespan of sperm of Pohlia nutans, collected from a geothermal spring''s area, and tested the effects of variation under environmental conditions on this lifespan. Surprisingly, 20 per cent of the sperm were still motile after 100 h, and sperm lifespan was not significantly affected by temperature variation between 22 and 60°C. Lifespan was significantly affected by sperm dilution and temperatures above 75°C. These results suggest the need to reconsider the importance of sperm motility in bryophyte fertilization.     

REVIVAL OF MAMMALIAN SPERM AFTER IMMERSION IN LIQUID NITROGEN

1. A wide variety of procedures was used to test the motility of mammalian sperm after plunging them into liquid nitrogen at –195°C. and later rapidly warming them to 35°C. by plunging them into a suitable balanced and isotonic medium. 2. Using seminal fluid sperm from the same human donor, maximal numbers of motile sperm survived vitrification when the samples were (a) very fresh, (b) untreated with plasmolysing solutions, (c) plunged into the refrigerant in the form of a foam. The maximum yield of motile human sperm recoverable from the liquid nitrogen was 50 per cent. Since in this sample only 75 per cent of the sperm were alive before immersion, 67 per cent of the living sperm survived vitrification. 3. Experiments with sperm from 31 rabbits were made with a variety of conditions of pretreatment to obtain maximal yields of recoverable, motile sperm after vitrification by liquid nitrogen. (a) A consistent recoverable yield of about 0.5 per cent was obtained when the untreated suspension of sperm was smeared on cellophane and partially dried in air before immersing in liquid nitrogen. (b) On a few out of many occasions plasmolysis for several minutes with hypertonic Ringer solution gave a recoverable yield of 0.1 per cent as did (c) pretreatment with hypertonic Ringer and butyric acid.     

Inbreeding depresses sperm competitiveness,but not fertilization or mating success in male Tribolium castaneum

As populations decline to levels where reproduction among close genetic relatives becomes more probable, subsequent increases in homozygous recessive deleterious expression and/or loss of heterozygote advantage can lead to inbreeding depression. Here, we measure how inbreeding across replicate lines of the flour beetle Tribolium castaneum impacts on male reproductive fitness in the absence or presence of male–male competition. Effects on male evolution from mating pattern were removed by enforcing monogamous mating throughout. After inbreeding across eight generations, we found that male fertility in the absence of competition was unaffected. However, we found significant inbreeding depression of sperm competitiveness: non-inbred males won 57 per cent of fertilizations in competition, while inbred equivalents only sired 42 per cent. We also found that the P₂ ‘offence’ role in sperm competition was significantly more depressed under inbreeding than sperm ‘defence’ (P₁). Mating behaviour did not explain these differences, and there was no difference in the viability of offspring sired by inbred or non-inbred males. Sperm length variation was significantly greater in the ejaculates of inbred males. Our results show that male ability to achieve normal fertilization success was not depressed under strong inbreeding, but that inbreeding depression in these traits occurred when conditions of sperm competition were generated.     

Sperm numbers,their storage and usage in the fly Dryomyza anilis

In the fly Dryomyza anilis females have two kinds of sperm storage organs: one bursa copulatrix and three spermathecae (two spermathecae with a common duct form the doublet, and the third is a singlet spermathecal unit). At the beginning of a mating the male deposits his sperm in the bursa copulatrix. After sperm transfer the male taps the female''s abdomen with his claspers. This behaviour has been shown to increase the male''s fertilization success. After mating, the female discharges large quantities of sperm before oviposition. To find out where the sperm remaining in the female are stored, I counted the number of sperm in the droplet and in the female''s sperm storage organs after different types of mating. I carried out three mating experiments. In experiment 1, virgin females were mated with one male and the matings were interrupted either immediately after sperm transfer or after several tapping sequences. The results show that during male tapping more sperm moved into the singlet spermatheca. In addition, the total number of sperm correlated with sperm numbers in all sperm storage organs, and male size was positively related to the number of sperm remaining in the bursa. In experiment 2, females mated with several males. The number of sperm increased with increasing number of matings only in the doublet spermatheca. No increase in the number of sperm in the singlet spermatheca during consecutive matings suggests that sperm were replaced or did not reach this sperm storage organ. In experiment 3, virgin females were mated with a single male and half of them were allowed to lay eggs. The experiment showed that during egglaying, females primarily used sperm from their singlet spermatheca. The results from the three experiments suggest that sperm stored in the singlet spermatheca is central for male fertilization success and male tapping is related to sperm storage in the singlet spermatheca. The different female''s sperm storage organs in D. anilis may have separate functions during sperm storage as well as during sperm usage.     

Cryptic female preference for genetically unrelated males is mediated by ovarian fluid in the guppy

As inbreeding is costly, it has been suggested that polyandry may evolve as a means to reduce the negative fitness consequences of mating with genetically related males. While several studies provide support for this hypothesis, evidence of pure post-copulatory mechanisms capable of biasing paternity towards genetically unrelated males is still lacking; yet these are necessary to support inbreeding avoidance models of polyandry evolution. Here we showed, by artificially inseminating a group of female guppies with an equal number of sperm from related (full-sib) and unrelated males, that sperm competition success of the former was 10 per cent lower, on average, than that of the unrelated male. The paternity bias towards unrelated males was not due to differential embryo survival, as the size of the brood produced by control females, which were artificially inseminated with the sperm of a single male, was not influenced by their relatedness with the male. Finally, we collected ovarian fluid (OF) from virgin females. Using computer-assisted sperm analysis, we found that sperm velocity, a predictor of sperm competition success in the guppy, was significantly lower when measured in a solution containing the OF from a sister as compared with that from an unrelated female. Our results suggest that sperm-OF interaction mediates sperm competition bias towards unrelated mates and highlight the role of post-copulatory mechanisms in reducing the cost of mating with relatives in polyandrous females.     

Ovarian fluid enhances sperm velocity based on relatedness in lake trout,Salvelinus namaycush

Studying mate choice at the gamete level can provide valuable insights into proximate mechanisms that underlie the evolution of mating systems. The objective was to assess whether ovarian fluid enhances sperm performance based on relatedness of mates in lake trout, Salvelinus namaycush, an iteroparous salmonid. Twelve trios were used, each composed of a female and two male fish; one male was related (full sibling) to the female, whereas the other was unrelated. Sperm from each male was activated in hatchery water or ovarian fluid from each corresponding female. No significant difference in sperm velocity was detected between the related and unrelated male fish when activated in hatchery water. However, when sperm was activated in ovarian fluid, sperm velocity from the related male was significantly higher than that of the unrelated male fish. Overall, ovarian fluid enhanced sperm performance of related male fish and might act as part of a recognition system to select sperm of a specific genotype.     

Cryptic female choice favours sperm from major histocompatibility complex-dissimilar males

Cryptic female choice may enable polyandrous females to avoid inbreeding or bias offspring variability at key loci after mating. However, the role of these genetic benefits in cryptic female choice remains poorly understood. Female red junglefowl, Gallus gallus, bias sperm use in favour of unrelated males. Here, we experimentally investigate whether this bias is driven by relatedness per se, or by similarity at the major histocompatibility complex (MHC), genes central to vertebrate acquired immunity, where polymorphism is critical to an individual''s ability to combat pathogens. Through experimentally controlled natural matings, we confirm that selection against related males'' sperm occurs within the female reproductive tract but demonstrate that this is more accurately predicted by MHC similarity: controlling for relatedness per se, more sperm reached the eggs when partners were MHC-dissimilar. Importantly, this effect appeared largely owing to similarity at a single MHC locus (class I minor). Further, the effect of MHC similarity was lost following artificial insemination, suggesting that male phenotypic cues might be required for females to select sperm differentially. These results indicate that postmating mechanisms that reduce inbreeding may do so as a consequence of more specific strategies of cryptic female choice promoting MHC diversity in offspring.     

Female genotype affects male success in sperm competition

The central question addressed by most studies of sperm competition is: ''what determines which male''s sperm are used at fertilization?'' Empirical and theoretical studies that address this question have traditionally focused on adaptations which enhance male fertilization success while treating the female as a receptacle in which sperm competition is played out. Here we provide evidence which suggests that female genotype strongly influences the outcome of sperm competition. When the sperm of two males are in competition the proportion of offspring fathered by the second male to mate (P₂) was found to be highly repeatable only if the male pair were mated to three different, but genetically similar females (full-sisters to each other; unrelated to either of the males). In contrast, if a male pair were mated to three females that were unrelated then P₂ was either non-repeatable or marginally repeatable. We also show that male success in sperm competition is determined, to a large extent, by gamete and/or male–female compatibility. This conclusion is derived from the observation that P₂ was repeatable among full-sisters mated to different, yet genetically similar male pairings, whilst P₂ was non-repeatable among full-sisters mated to different, genetically distinct male pairings.     

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.     

Performance of non-motile male gametes in the sea: analysis of paternity and fertilization success in a natural population of a red seaweed,Gracilaria gracilis

In haploid–diploid red seaweeds, the dispersal of male gametes is presumed limited due to their lack of flagella. It has been suggested that this group suffers from sperm limitation and, consequently, that fertilization is relatively inefficient. Fertilization in most floridean rhodophytes results in the formation a cystocarp, a swelling on the haploid female thallus housing the diploid zygote and its thousands of diploid daughter spores. To study the performance of non-motile male gametes in the sea, we evaluated both female and male fertilization success in a natural population of the red marine alga Gracilaria gracilis. Female fertilization success, estimated by cystocarp yield per unit female thallus, was evaluated with respect to the availability of male gametes. Male fertilization success, estimated by the individual contribution of different males to zygotes, was assessed by paternity analyses on 350 cystocarps produced in one reproductive season using two microsatellite loci. The results show that cystocarp yield is not sperm limited and that the large variation in male fertilization success cannot be solely explained by the distance travelled by the male gamete to find a mate. Taken together, the results suggest that, not only is fertilization efficient, but that male–male competition and/or female choice may play a role in shaping population mating patterns.     

The Effect of Female Quality on Male Ejaculatory Expenditure and Reproductive Success in a Praying Mantid

Strategic ejaculation is a behavioural strategy shown by many animals as a response to sperm competition and/or as a potential mechanism of cryptic male choice. Males invest more mating resources when the risk of sperm competition increases or they invest more in high quality females to maximize their reproductive output. We tested this hypothesis in the false garden mantid Pseudomantis albofimbriata, where females are capable of multiply mating and body condition is an indicator of potential reproductive fitness. We predicted male mantids would ejaculate strategically by allocating more sperm to high quality females. To determine if and how males alter their ejaculate in response to mate quality, we manipulated female food quantity so that females were either in good condition with many eggs (i.e. high quality) or poor condition with few eggs (i.e. low quality). Half of the females from each treatment were used in mating trials in which transferred sperm was counted before fertilisation occurred and the other half of females were used in mating trials where fertilisation occurred and ootheca mass and total eggs in the ootheca were recorded. Opposed to our predictions, the total number of sperm and the proportion of viable sperm transferred did not vary significantly between female treatments. Male reproductive success was entirely dependent on female quality/fecundity, rather than on the number of sperm transferred. These results suggest that female quality is not a major factor influencing postcopulatory male mating strategies in P. albofimbriata, and that sperm number has little effect on male reproductive success in a single mating scenario.     

A comparison of nuclear and cytoplasmic genetic effects on sperm competitiveness and female remating in a seed beetle

It is widely assumed that male sperm competitiveness evolves adaptively. However, recent studies have found a cytoplasmic genetic component to phenotypic variation in some sperm traits presumed important in sperm competition. As cytoplasmic genes are maternally transmitted, they cannot respond to selection on sperm and this constraint may affect the scope in which sperm competitiveness can evolve adaptively. We examined nuclear and cytoplasmic genetic contributions to sperm competitiveness, using populations of Callosobruchus maculatus carrying orthogonal combinations of nuclear and cytoplasmic lineages. Our design also enabled us to examine genetic contributions to female remating. We found that sperm competitiveness and remating are primarily encoded by nuclear genes. In particular, a male's sperm competitiveness phenotype was contingent on an interaction between the competing male genotypes. Furthermore, cytoplasmic effects were detected on remating but not sperm competitiveness, suggesting that cytoplasmic genes do not generally play a profound evolutionary role in sperm competition.     

Factors Affecting Sperm Quality Before and After Mating of Calopterygid Damselflies

Damselflies (Odonata: Zygoptera) have a more complex sperm transfer system than other internally ejaculating insects. Males translocate sperm from the internal reproductive organs to the specific sperm vesicles, a small cavity on the body surface, and then transfer them into the female. To examine how the additional steps of sperm transfer contribute to decreases in sperm quality, we assessed sperm viability (the proportion of live sperm) at each stage of mating and after different storage times in male and female reproductive organs in two damselfly species, Mnais pruinosa and Calopteryx cornelia. Viability of stored sperm in females was lower than that of male stores even just after copulation. Male sperm vesicles were not equipped to maintain sperm quality for longer periods than the internal reproductive organs. However, the sperm vesicles were only used for short-term storage; therefore, this process appeared unlikely to reduce sperm viability when transferred to the female. Males remove rival sperm prior to transfer of their own ejaculate using a peculiar-shaped aedeagus, but sperm removal by males is not always complete. Thus, dilution occurs between newly received sperm and aged sperm already stored in the female, causing lower viability of sperm inside the female than that of sperm transferred by males. If females do not remate, sperm viability gradually decreases with the duration of storage. Frequent mating of females may therefore contribute to the maintenance of high sperm quality.     

Variation in the number of sperm transferred during mating among males of the Colorado potato beetle (Coleoptera: Chrysomelidae)

We suggest an explanation for the bimodal distribution in the number of sperm transferred into the female during mating by males of the Colorado potato beetle (Leptinotarsa decemlineata). While in most matings females receive 20,000 sperm, in others they receive less than 1000. A variety of potential proximate causes for this bimodal distribution including experimental procedures and several physiological and behavioral causes were examined: male body size, female body size, male mating history, socio-sexual environments, duration and number of mounts, and duration and number of copulations. Surprisingly, none of these explained the observed distribution. However, we did find that the total time spent in copulation was significantly less variable among males that transferred more than 20,000 sperm than among males that passed less than 1000 sperm. In addition, males transferring large amounts of sperm resulted in proportionally more sperm inside the female’s spermatheca within a short period of time than males passing less than 1000 sperm. The lack of pattern in male sperm ejaculate suggests definite differences in sperm delivery tactics and may be related to the female condition rather than the male.     

Large Neurological Component to Genetic Differences Underlying Biased Sperm Use in Drosophila

Sperm competition arises as a result of complex interactions among male and female factors. While the roles of some male factors are known, little is known of the molecules or mechanisms that underlie the female contribution to sperm competition. The genetic tools available for Drosophila allow us to identify, in an unbiased manner, candidate female genes that are critical for mediating sperm competition outcomes. We first screened for differences in female sperm storage and use patterns by characterizing the natural variation in sperm competition in a set of 39 lines from the sequenced Drosophila Genetic Reference Panel (DGRP) of wild-derived inbred lines. We found extensive female variation in sperm competition outcomes. To generate a list of candidate female genes for functional studies, we performed a genome-wide association mapping, utilizing the common single-nucleotide polymorphisms (SNPs) segregating in the DGRP lines. Surprisingly, SNPs within ion channel genes and other genes with roles in the nervous system were among the top associated SNPs. Knockdown studies of three candidate genes (para, Rab2, and Rim) in sensory neurons innervating the female reproductive tract indicate that some of these candidate female genes may affect sperm competition by modulating the neural input of these sensory neurons to the female reproductive tract. More extensive functional studies are needed to elucidate the exact role of all these candidate female genes in sperm competition. Nevertheless, the female nervous system appears to have a previously unappreciated role in sperm competition. Our results indicate that the study of female control of sperm competition should not be limited to female reproductive tract-specific genes, but should focus also on diverse biological pathways.