Evolutionary reduction in testes size and competitive fertilization success in response to the experimental removal of sexual selection in dung beetles

Sexual selection is thought to favor the evolution of secondary sexual traits in males that contribute to mating success. In species where females mate with more than one male, sexual selection also continues after copulation in the form of sperm competition and cryptic female choice. Theory suggests that sperm competition should favor traits such as testes size and sperm production that increase a male's competitive fertilization success. Studies of experimental evolution offer a powerful approach for assessing evolutionary responses to variation in sexual selection pressures. Here we removed sexual selection by enforcing monogamy on replicate lines of a naturally polygamous horned beetle, Onthophagus taurus, and monitoring male investment in their testes for 21 generations. Testes size decreased in monogamous lines relative to lines in which sexual selection was allowed to continue. Differences in testes size were dependent on selection history and not breeding regime. Males from polygamous lines also had a competitive fertilization advantage when in sperm competition with males from monogamous lines. Females from polygamous lines produced sons in better condition, and those from monogamous lines increased their sons condition by mating polygamously. Rather than being costly for females, multiple mating appears to provide females with direct and/or indirect benefits. Neither body size nor horn size diverged between our monogamous and polygamous lines. Our data show that sperm competition does drive the evolution of testes size in onthophagine beetles, and provide general support for sperm competition theory.     

Males' evolutionary responses to experimental removal of sexual selection

We evaluated the influence of pre- and post-copulatory sexual selection upon male reproductive traits in a naturally promiscuous species, Drosophila melanogaster. Sexual selection was removed in two replicate populations through enforced monogamous mating with random mate assignment or retained in polyandrous controls. Monogamous mating eliminates all opportunities for mate competition, mate discrimination, sperm competition, cryptic female choice and, hence, sexual conflict. Levels of divergence between lines in sperm production and male fitness traits were quantified after 38-81 generations of selection. Three a priori predictions were tested: (i) male investment in spermatogenesis will be lower in monogamy-line males due to the absence of sperm competition selection, (ii) due to the evolution of increased male benevolence, the fitness of females paired with monogamy-line males will be higher than that of females paired with control-line males, and (iii) monogamy-line males will exhibit decreased competitive reproductive success relative to control-line males. The first two predictions were supported, whereas the third prediction was not. Monogamy males evolved a smaller body size and the size of their testes and the number of sperm within the testes were disproportionately further reduced. In contrast, the fitness of monogamous males (and their mates) was greater when reproducing in a non-competitive context: females mated once with monogamous males produced offspring at a faster rate and produced a greater total number of surviving progeny than did females mated to control males. The results indicate that sexual selection favours the production of increased numbers of sperm in D. melanogaster and that sexual selection favours some male traits conferring a direct cost to the fecundity of females.     

Sperm allocation in the three-spined stickleback

Male three-spined stickleback Gasterosteus aculeatus have a fixed amount of sperm during the breeding season because spermatogenesis is inhibited at this time. A method was developed to estimate ejaculate size in situ by removing the sperm from the male's nest. The reliability of the method was tested using known numbers of sperm. In their first mating, males ejaculated 11·64 × 10⁶ sperm (median), representing c. 5% of the male's sperm store (median 27·88 × 10⁷ sperm). The amount of sperm in the testes was significantly reduced in males that had mated several times (median 8·09 × 10⁷). Additionally, ejaculate size was smaller in these experienced males (median 8·79 × 10⁵). Heavier and larger fish invested absolutely and relatively more sperm in a mating than did lighter and smaller fish. Ejaculate size did not correlate with the mass of the egg clutch.     

Subordinate male cichlids retain reproductive competence during social suppression

Subordinate males, which are excluded from reproduction often save energy by reducing their investment in sperm production. However, if their position in a dominance hierarchy changes suddenly they should also rapidly attain fertilization capability. Here, we asked how social suppression and ascension to dominance influences sperm quality, spermatogenesis and reproductive competence in the cichlid Astatotilapia burtoni, where reproduction is tightly coupled to social status. Dominant territorial (T) males are reproductively active while subordinate non-territorial (NT) males are suppressed, but given the opportunity, NT males will perform dominance behaviours within minutes and attain T male testes size within days. Using the thymidine analogue 5-bromo-2-deoxyuridine (BrdU) to label germ cell proliferation, we found that the spermatogenic cycle takes approximately 11-12 days, and social status had no effect on proliferation, suggesting that spermatogenesis continues during reproductive suppression. Although sperm velocity did not differ among social states, NT males had reduced sperm motility. Remarkably, males ascending in status showed sperm motility equivalent to T males within 24 h. Males also successfully reproduced within hours of social opportunity, despite four to five weeks of suppression and reduced testis size. Our data suggest that NT males maintain reproductive potential during suppression possibly as a strategy to rapidly improve reproductive fitness upon social opportunity.     

Larval social cues influence testicular investment in an insect

Socio-sexual environment can have critical impacts on reproduction and survival of animals. Consequently, they need to prepare themselves by allocating more resources to competitive traits that give them advantages in the particular social setting they have been perceiving. Evidence shows that a male usually raises his investment in sperm after he detects the current or future increase of sperm competition because relative sperm numbers can determine his paternity share. This leads to the wide use of testis size as an index of the sperm competition level, yet testis size does not always reflect sperm production. To date, it is not clear whether male animals fine-tune their resource allocation to sperm production and other traits as a response to social cues during their growth and development. Using a polygamous insect Ephestia kuehniella, we tested whether and how larval social environment affected sperm production, testis size, and body weight. We exposed the male larvae to different juvenile socio-sexual cues and measured these traits. We demonstrate that regardless of sex ratio, group-reared males produced more eupyrenes (fertile and nucleate sperm) but smaller testes than singly reared ones, and that body weight and apyrene (infertile and anucleate sperm) numbers remained the same across treatments. We conclude that the presence of larval social, but not sexual cues is responsible for the increase of eupyrene production and decrease of testis size. We suggest that male larvae increase investment in fertile sperm cells and reduce investment in other testicular tissues in the presence of conspecific juvenile cues.     

The Y chromosome as a battle ground for sexual selection

The Y chromosome was once thought to be devoid of genetic information. However, recent work shows that it contains numerous genes related to sperm production and dimorphic traits (such as body size and tooth development). Among mammals, these traits influence a male's competitive ability in male-male contests and in sperm competition. Therefore, sexual selection could have favoured genes on the Y chromosome that enhance male fertilization success because they spread unaltered through the male line. In contrast, female heterogamety among birds makes it possible for genes that benefit females to spread through the female line, a mechanism that could explain the prevalence of female choice.     

Sperm competition selects for increased testes mass in Australian frogs

Game theory predicts that investment in spermatogenesis will increase with the risk and intensity of sperm competition. Widespread support for this prediction has come from comparative studies of internal fertilizing species reporting positive associations between testes mass and the probability that females mate with more than one male. Data for external fertilizers have generated conflicting results. We investigated how risk of sperm competition affects testes size in two families of Australian frogs: the Myobatrachidae and the Hylidae. We also examined effects of clutch size, egg size and oviposition location as alternative factors that might influence sperm production. Species were ranked according to probability of group spawning, and hence risk of sperm competition. Controlling for body size and phylogenetic relationships, we demonstrated that within the Myobatrachidae, the risk of sperm competition explained a significant amount of variation in testes mass. Oviposition location had a weak influence, with species ovipositing into foam having smaller testes. No significant effects of clutch size or egg size were detected. In hylids, the relationship between testes mass and risk of sperm competition was positive but not significant, again with no predictable effects related to egg size or number. These data provide an important test of sperm competition theory for externally fertilizing taxa.     

The frequency of multiple paternity predicts variation in testes size among island populations of house mice

Polyandry generates selection on males through sperm competition, which has broad implications for the evolution of ejaculates and male reproductive anatomy. Comparative analyses across species and competitive mating trials within species have suggested that sperm competition can influence the evolution of testes size, sperm production and sperm form and function. Surprisingly, the intraspecific approach of comparing among population variation for investigating the selective potential of sperm competition has rarely been explored. We sampled seven island populations of house mice and determined the frequency of multiple paternity within each population. Applying the frequency of multiple paternity as an index of the risk of sperm competition, we looked for selective responses in male reproductive traits. We found that the risk of sperm competition predicted testes size across the seven island populations of house mice. However, variation in sperm traits was not explained by sperm competition risk. We discuss these findings in relation to sperm competition theory, and other intrinsic and extrinsic factors that might influence ejaculate quality.     

Gestational exposure to ethane dimethanesulfonate permanently alters reproductive competence in the CD-1 mouse

Although the adult mouse Leydig cell (LC) has been considered refractory to cytotoxic destruction by ethane dimethanesulfonate (EDS), the potential consequences of exposure during reproductive development in this species are unknown. Herein pregnant CD-1 mice were treated with 160 mg/kg on Gestation Days 11-17, and reproductive development in male offspring was evaluated. Prenatal administration of EDS compromised fetal testosterone (T) levels, compared with controls. EDS-exposed pups recovered their steroidogenic capacities after birth because T production by hCG-stimulated testis parenchyma from prepubertal male offspring was unchanged. However, prepubertal testes from prenatally exposed males contained seminiferous tubules (STs) devoid of germ cells, indicating a delay in spermatogenesis. In adults, some STs in exposed males still contained incomplete germ cell associations corroborating observed reductions in epididymal sperm reserves, fertility ratios, and litter size. Morphometry revealed an EDS-induced increase in interstitial area and a concomitant decrease in ST area, but stereology revealed an unexpected decrease in the number and size of the LCs per testis in exposed males. Paradoxically, there was an increase in both serum LH and T production by adult testis parenchyma, indicating that the LCs were hyperstimulated. These data demonstrate permanent lesions in LC development and spermatogenesis caused by prenatal exposure in mice. Thus, although adult mouse LCs are insensitive to EDS, EDS appears to have direct action on fetal LCs, resulting in abnormal testis development.     

Differential investment into testes and sperm production in alternative male reproductive tactics of the African striped mouse (Rhabdomys pumilio)

Males that follow alternative reproductive tactics might differ in their investment into testis development and sperm production. The resource-allocation hypothesis predicts that males following a sneaker tactic should invest more into sperm production than dominant territorial males which should invest more into mate guarding. This hypothesis is supported by studies in species where individual males cannot switch between tactics (fixed tactics). Here we present the first data for a species where males can switch between tactics (plastic tactics). We studied African striped mice (Rhabdomys pumilio) in captivity, mimicking three tactics observed in the field: philopatric group-living males, singly-housed males representing roaming males, and group-living breeding males. We measured quantitative and qualitative reproductive traits, as well as serum and testis hormone concentrations. We found no support for the resource-allocation hypothesis, since breeding and singly-housed males invested similarly in testes and sperm. However, philopatric males had significantly smaller testes and epididymides, lower sperm counts, lower testosterone and higher corticosterone levels than males of the two other tactics. Philopatric males did not reach a larger body mass than singly-housed males with well developed reproductive traits, indicating that they did not trade investment in sperm production against growth. Interestingly, testis testosterone concentrations of philopatric males did not differ from those of other males. Our data suggest that philopatric males are reproductively suppressed by the breeding male, but might be ready to increase their serum testosterone levels when social and environmental conditions allow for this physiological switch accompanying the behavioral switch between tactics.     

Sperm competition, sperm numbers and sperm quality in muroid rodents

Sperm competition favors increases in relative testes mass and production efficiency, and changes in sperm phenotype that result in faster swimming speeds. However, little is known about its effects on traits that contribute to determine the quality of a whole ejaculate (i.e., proportion of motile, viable, morphologically normal and acrosome intact sperm) and that are key determinants of fertilization success. Two competing hypotheses lead to alternative predictions: (a) sperm quantity and quality traits co-evolve under sperm competition because they play complementary roles in determining ejaculate's competitive ability, or (b) energetic constraints force trade-offs between traits depending on their relevance in providing a competitive advantage. We examined relationships between sperm competition levels, sperm quantity, and traits that determine ejaculate quality, in a comparative study of 18 rodent species using phylogenetically controlled analyses. Total sperm numbers were positively correlated to proportions of normal sperm, acrosome integrity and motile sperm; the latter three were also significantly related among themselves, suggesting no trade-offs between traits. In addition, testes mass corrected for body mass (i.e., relative testes mass), showed a strong association with sperm numbers, and positive significant associations with all sperm traits that determine ejaculate quality with the exception of live sperm. An "overall sperm quality" parameter obtained by principal component analysis (which explained 85% of the variance) was more strongly associated with relative testes mass than any individual quality trait. Overall sperm quality was as strongly associated with relative testes mass as sperm numbers. Thus, sperm quality traits improve under sperm competition in an integrated manner suggesting that a combination of all traits is what makes ejaculates more competitive. In evolutionary terms this implies that a complex network of genetic and developmental pathways underlying processes of sperm formation, maturation, transport in the female reproductive tract, and preparation for fertilization must all evolve in concert.     

Phenotypic correlates of spermatozoon quality in the guppy, Poecilia reticulata

The phenotype-linked fertility hypothesis suggests that a femalecan benefit directly from mate choice when the cues she usesindicate the quantity and/or quality of his spermatozoa. Wetested the link between sperm quality and male body size andcoloration in the resource-free mating system of the guppy,a tropical fish characterized by strong female choice. Largermales possessed larger testes and are therefore predicted toproduce larger numbers of spermatozoa than smaller males. Largermale guppies also produced longer spermatozoa than smaller males.Degree of carotenoid coloration did not predict either the quantityor the quality of a male's spermatozoa. These results are consistentwith a previous study that showed that female guppies in thestudy population prefer larger males to brightly colored males.The male-size directed increase in spermatozoon size may bethe result of interplay between sperm competition and the coevolutionof spermatozoon traits with the female reproductive tract.     

The evolution of sperm length in moths

Sperm form and function remain poorly understood despite being of fundamental biological importance. An instructive approach has been to examine evolutionary associations across comparable taxa between sperm characters and other, potentially selective reproductive traits. We adopt this approach here in a comparative study examining how sperm lengths are associated with male and female reproductive characters across moths. Primary data have revealed Lepidoptera to be an ideal order for examination: there is profound variation in the dimensions (but not organization) of the reproductive traits between closely related species which all share a monophyletic ancestry, for example, eupyrene sperm length varies from 110 to 12,675 microm. Eupyrene (normal fertilizing) and apyrene (anucleate and non-fertile) sperm lengths are positively correlated across taxa and both sperm types show positive associations with mating pattern (as measured by the residual testis size). At fertilization, eupyrene sperm must migrate down the often elongated female spermathecal duct from storage to unite with the ovum. Across taxa, the elongation of this duct is associated with increased eupyrene sperm length, suggesting a positive female influence on sperm size since longer, more powerful sperm may be selected to migrate and/or compete successfully down greater ductal lengths. Apyrene sperm length is not associated with female reproductive tract dimensions. However, we found a positive relationship between the residual testis volume and spermathecal volume, suggesting coevolution between male investment in spermatogenesis and the extent of the female sperm storage capacity. Within males, there is a positive association between the two organs which form the ejaculate-containing spermatophore: the testes and the accessory gland. The 'trade-up' in investment to these components is discussed in relation to paternal investment and mating patterns.     

Influences of establishment and maintenance of territory on reproductive activity in the male dwarf gourami Colisa lalia

We examined the importance of establishment and maintenance of territory on reproductive activity in the male dwarf gourami, Colisa lalia. After three males had been forced to fight for territory (five sets) for three weeks, social status was divided into three classes: the territorially dominant male, which guarded the territory under the floating nest; the second male which remained near the nest and occasionally attacked the dominant male; and the third male which was non-aggressive and remained at a distance from the other two males. Comparing testicular size by gonadosomatic indices (GSI) after three weeks of aggression, GSI of the dominant male (1.19 ± 0.07) was significantly larger than that of the second (0.81 ± 0.15) and the third (0.62 ± 0.08) males, as well as the initial control (not involved in any experiments: 0.85 ± 0.10, n = 5), indicating that the testes of the dominant males enlarge during territory defense. Histological observations of testes revealed that sperm production in the dominant males was more active compared to males of other classes, although spermatogenesis was confirmed in all males examined, suggesting that dominance accelerates sperm production. Social-status dependent development of testes suggests an absence of sperm competition due to the lack of sneaking by subordinate males. Since non-territorial males do not engage in alternative tactics (e.g., sneaking) leading to emission of semen, male C. lalia must obtain and defend territory if they are to increase their reproductive success.     

Sperm competition and the coevolution of pre‐ and postcopulatory traits: Weapons evolve faster than testes among onthophagine dung beetles

Reproductive competition generates episodes of both pre‐ and postcopulatory sexual selection. Theoretical models of sperm competition predict that as the fitness gains from expenditure on the weapons of male combat increase, males should increase their expenditure on weapons and decrease their expenditure on traits that contribute to competitive fertilization success. Although traits subject to sexual selection are known to have accelerated evolutionary rates of phenotypic divergence, it is not known whether the competing demands of investment into pre‐ and postcopulatory traits affect their relative rates of evolutionary divergence. We use a comparative approach to estimate the rates of divergence in pre‐ and postcopulatory traits among onthophagine dung beetles. Weapons evolved faster than body size while testes mass and sperm length evolved more slowly than body size, suggesting that precopulatory competition is the stronger episode of sexual selection acting on these beetles. Although horns evolved faster than testes, evolutionary increases in horn length were not associated with evolutionary reductions in testes mass. Our data for onthophagines support the notion that in taxa where males are unable to monopolize paternity, expenditure on both weapons and testes should both be favored.     

Are secondary sex traits, parasites and immunity related to variation in primary sex traits in the Arctic charr?

Primary and secondary sex traits are influenced by the same sex hormones, and the expression of secondary sex traits may consequently signal males' capacity for sperm production. Sperm quality may also be influenced by immune activity, as sperm are non-self to the male. Parasite infections alter immune activity and may thus reduce ejaculate quality. In the Arctic charr (Salvelinus alpinus) the red abdominal colour is considered an ornament that signals important information in mate choice. We captured and individually caged sexually mature male Arctic charr during the spawning period. Afterwards we estimated abdominal colour, parasite infections, gonad mass and several spermatological and immunological variables. Intensity of abdominal colour was positively correlated to testes mass, milt mass and sperm cell numbers produced. Additionally, males with low parasite intensities had high testes mass and produced milt with high sperm density, indicating a trade-off between parasite resistance and development of primary sex traits. Our measures of immunity were, however, not related to primary sex traits. We conclude that females evaluating male abdominal coloration may obtain information about differences between males in fertilization potential and parasite resistance.     

Sexual selection drives the coevolution of male and female reproductive traits in Peromyscus mice

When females mate with multiple partners within a single reproductive cycle, sperm from rival males may compete for fertilization of a limited number of ova, and females may bias the fertilization of their ova by particular sperm. Over evolutionary timescales, these two forms of selection shape both male and female reproductive physiology when females mate multiply, yet in monogamous systems, post-copulatory sexual selection is weak or absent. Here, we examine how divergent mating strategies within a genus of closely related mice, Peromyscus, have shaped the evolution of reproductive traits. We show that in promiscuous species, males exhibit traits associated with increased sperm production and sperm swimming performance, and females exhibit traits that are predicted to limit sperm access to their ova including increased oviduct length and a larger cumulus cell mass surrounding the ova, compared to monogamous species. Importantly, we found that across species, oviduct length and cumulus cell density are significantly correlated with sperm velocity, but not sperm count or relative testes size, suggesting that these female traits may have coevolved with increased sperm quality rather than quantity. Taken together, our results highlight how male and female traits evolve in concert and respond to changes in the level of post-copulatory sexual selection.     

The evolutionary ecology of testicular function: size isn't everything

Larger testes are considered the quintessential adaptation to sperm competition. However, the strong focus on testis size in evolutionary research risks ignoring other potentially adaptive features of testicular function, many of which will also be shaped by post‐mating sexual selection. Here we advocate a more integrated research programme that simultaneously takes into account the developmental machinery of spermatogenesis and the various selection pressures that act on this machinery and its products. The testis is a complex organ, and so we begin by outlining how we can think about the evolution of testicular function both in terms of the composition and spatial organisation of the testis (‘testicular histology’), as well as in terms of the logical organisation of cell division during spermatogenesis (‘testicular architecture’). We then apply these concepts to ask which aspects of testicular function we can expect to be shaped by post‐mating sexual selection. We first assess the impact of selection on those traits most strongly associated with sperm competition, namely the number and kind of sperm produced. A broad range of studies now support our contention that post‐mating sexual selection affects many aspects of testicular function besides gross testis size, for example, to maximise spermatogenic efficiency or to enable the production of particular sperm morphologies. We then broaden our focus to ask how testicular function is affected by fluctuation in sperm demand. Such fluctuation can occur over an individual's lifetime (for example due to seasonality in reproduction) and may select for particular types of testicular histology and architecture depending on the particular reproductive ecology of the species in question. Fluctuation in sperm demand also occurs over evolutionary time, due to shifts in the mating system, and this may have various consequences for testicular function, for example on rates of proliferation‐induced mutation and for dealing with intragenomic conflict. We end by suggesting additional approaches that could be applied to study testicular function, and conclude that simultaneously considering the machinery, products and scheduling of spermatogenesis will be crucial as we seek to understand more fully the evolution of this most fundamental of male reproductive traits.