Male Mating Rate Research Articles

A red flag: an experimental analysis of the function of pink colouration in males of Calopteryx haemorrhoidalis asturica (Odonata: Calopterygidae).

Males display phenotypic characteristics that may be associated with their quality, allowing non-random mating and post-copulatory female choice. In the damselfly Calopteryx haemorrhoidalis asturica, males have a conspicuous pink colouration in the underside of abdominal segments 8-10, which they exhibit during pre- and post-copulatory courtship. We hypothesized that this colouration functions to increase male mating success and/or to elicit females to oviposit. We estimated mating and oviposition success of 27 males, and on the following day, treated males had their segments 8-10 painted black and control males the seventh segment. We recorded the number of male-male fights and courtships, whether the courtship ended in copulation, and whether the female remained in the territory and laid eggs. Our results indicate that the mating success of male C. h. asturica was not significantly affected by the removal of the pink colouration of the abdominal tip, but this colouration clearly affected their success in enticing females to oviposit. Courtship frequency, fat content and muscle mass were positively correlated to male mating rate, and the number of aggressive encounters was negatively correlated. Our study yields experimental evidence for the function of pink colouration of male C. h. asturica, in the context of post-copulatory sexual selection.

Small-bodied males invest in larger testes when highly ornamented.

Sperm competition and male mating rate are two non-mutually exclusive key evolutionary pressures selecting for larger testes within and across animal taxa. A few studies have tried to test the role of mating rate in the absence of sperm competition. Under the mating rate hypothesis, particular phenotypes of a given population that are expected to gain more mates (e.g., more ornamented males) are expected to make higher investments in testes size (a proxy for sperm production). We test this prediction in Polistes simillimus, a neotropical paper wasp in which females are single mated (no sperm competition) and males can mate with multiple partners. Testes size was predicted by body size (positive association), sexual ornamentation (negative association), and their interaction (among small males, testes size was positively related to ornamentation, but the opposite pattern was observed among large males). We propose that small-bodied well-ornamented males may face the highest risk of sperm depletion. Small-bodied males make relatively higher investment in testes size when highly ornamented. This strategy might be less profitable to large males, as they have overall larger testes. Our results provide strong evidence for the mating rate hypothesis.

Thermal Plasticity in Behavioral Traits Mediates Mating and Reproductive Dynamics in an Ectotherm.

AbstractEnvironmental temperatures potentially influence reproductive performance and sexual selection by restricting opportunities for activity. However, explicit tests of the behavioral mechanisms linking thermal variation to mating and reproductive performance are rare. We address this gap in a temperate lizard by combining social network analysis with molecular pedigree reconstruction in a large-scale thermal manipulation experiment. Populations exposed to cool thermal regimes presented fewer high-activity days compared with populations exposed to a warmer regime. While plasticity in thermal activity responses in males masked overall differences in activity levels, prolonged restriction nevertheless affected the timing and consistency of male-female interactions. Females were less capable than males of compensating for lost activity time under cold stress, and less active females in this group were significantly less likely to reproduce. While sex-biased activity suppression appeared to limit male mating rates, this did not correspond to a heightened intensity of sexual selection or shifts in the targets of sexual selection. In many populations facing thermal activity restriction, sexual selection on males may play a limited role relative to other thermal performance traits in facilitating adaptation.

X-linked meiotic drive can boost population size and persistence.

X-linked meiotic drivers cause X-bearing sperm to be produced in excess by male carriers, leading to female-biased sex ratios. Here, we find general conditions for the spread and fixation of X-linked alleles. Our conditions show that the spread of X-linked alleles depends on sex-specific selection and transmission rather than the time spent in each sex. Applying this logic to meiotic drive, we show that polymorphism is heavily dependent on sperm competition induced both by female and male mating behavior and the degree of compensation to gamete loss in the ejaculate size of drive males. We extend these evolutionary models to investigate the demographic consequences of biased sex ratios. Our results suggest driving X-alleles that invade and reach polymorphism (or fix and do not bias segregation excessively) will boost population size and persistence time by increasing population productivity, demonstrating the potential for selfish genetic elements to move sex ratios closer to the population-level optimum. However, when the spread of drive causes strong sex-ratio bias, it can lead to populations with so few males that females remain unmated, cannot produce offspring, and go extinct. This outcome is exacerbated when the male mating rate is low. We suggest that researchers should consider the potential for ecologically beneficial side effects of selfish genetic elements, especially in light of proposals to use meiotic drive for biological control.

Frequent mating reduces male mating rate but not offspring quality or quantity in a neriid fly

Frequent mating can deplete sperm and seminal fluid, limiting male ability to sire offspring. Frequent mating could also deplete non-genetic ejaculate components that affect offspring quality. These effects of frequent mating on male reproductive success may be mediated by male condition, or by modification of subsequent male mating behaviour. Using the neriid fly Telostylinus angusticollis, we conducted two experiments to examine whether a history of frequent mating affects males’ subsequent mating rate and offspring traits. The first experiment tested whether male condition (manipulated by varying larval diet quality) and mating history affects male performance in a subsequent mating with a single novel female whereby we predicted effects of previous mating may be more prevalent in low-condition males. Prior mating resulted in a reduction in mating rate with the novel female, but we did not detect an effect of mating history or male condition on offspring quality or quantity. The second experiment tested whether costs of mating become more evident when males encounter multiple novel females. Surprisingly, while prior mating once again resulted in a reduction in mating rate with the novel females, we still did not detect an effect of condition or mating history on any offspring traits. Therefore, male neriid flies appear to be able to mate many times without suffering a reduction in offspring quality or quantity. The apparent lack of an effect of frequent mating on such traits could be mediated by a reduction in mating rate, reflecting male prudence with ejaculate expenditure.

Does sexual cannibalism secure genetic benefits of polyandry in a size-dimorphic spider?

Females mate multiply despite numerous costs. It is well established that polyandry can result in sexual conflict, favoring male adaptations that prevent sperm competition often to the disadvantage of the female. Such adaptations are extreme in spiders with one-shot genitalia of which parts break off and act as mating plugs, rendering them dysfunctional. In the spider Argiope bruennichi, mating plugs effectively prevent further males from inseminating and males that inseminate and plug both genital openings of a female secure exclusive paternity. However, females frequently prevent monopolization by attacking and cannibalizing males during their first copulation, leaving their second spermatheca free for another male. Here, we test whether the high frequency of sexual cannibalism evolved as a female adaptation to resist monopolization and secure indirect benefits of polyandry. To standardize conditions, we double-mated females either with the same or two different males and prevented male consumption. Using a split-brood design, we raised offspring to maturity under poor and rich food conditions and measured their survival, duration of juvenile phase, and adult body mass. Under low food, daughters of polyandrous mothers matured later but slightly heavier than daughters of monandrous females. Since the adaptive value of this combination is unclear, these findings lend no conclusive support to our hypothesis. We discuss the stereotypic nature of the female attack in the context of antagonistic co-evolution considering previous studies that found modest direct benefits of cannibalism as well as a potential for non-additive benefits.Significance statementSexual conflict is extreme in spiders where sexual cannibalism impairs male mating rates. Males of the spider Argiope bruennichi possess one-shot genitalia which they break off to plug female genital openings. They gain exclusive paternity with a female if two copulations are achieved and both genital openings plugged. Females, however, stereotypically attack every male at the onset of copulation, limiting most males to single copulation but retaining the option to secure potential benefits of polyandry. Previous studies revealed weak direct and non-additive indirect benefits of multiple mating. In this study, we tested for the presence of additive genetic benefits but again found only inconclusive evidence for adaptive differences in offspring quality between monandrous and polyandrous females. All results combined, we here speculate that the stereotypic female attack might be a ghost of a past antagonistic co-evolution.

An experimental test for body size-dependent effects of male harassment and an elevated copulation rate on female lifetime fecundity and offspring performance.

Many studies investigate the benefits of polyandry, but repeated interactions with males can lower female reproductive success. Interacting with males might even decrease offspring performance if it reduces a female's ability to transfer maternal resources. Male presence can be detrimental for females in two ways: by forcing females to mate at a higher rate and through costs associated with resisting male mating attempts. Teasing apart the relative costs of elevated mating rates from those of greater male harassment is critical to understand the evolution of mating strategies. Furthermore, it is important to test whether a male's phenotype, notably body size, has differential effects on female reproductive success versus the performance of offspring, and whether this is due to male body size affecting the costs of harassment or the actual mating rate. In the eastern mosquitofish Gambusia holbrooki, males vary greatly in body size and continually attempt to inseminate females. We experimentally manipulated male presence (i.e., harassment), male body size and whether males could copulate. Exposure to males had strong detrimental effects on female reproductive output, growth and immune response, independent of male size or whether males could copulate. In contrast, there was a little evidence of a cross-generational effect of male harassment or mating rate on offspring performance. Our results suggest that females housed with males pay direct costs due to reduced condition and offspring production and that these costs are not a consequence of increased mating rates. Furthermore, exposure to males does not affect offspring reproductive traits.

Effects of Two Conventional Insecticides on Male-Specific Sex Pheromone Discrimination and Mate Choice in Trichogramma chilonis (Hymenoptera: Trichogrammatidae).

Trichogramma chilonis Ishii is an important natural enemy of many lepidopterous pests on vegetables and field crops. The effects of two conventional insecticides on male-specific sex pheromone discrimination and mate choice in T. chilonis was evaluated in the laboratory. Beta-cypermethrin LC20 exposure induced decreases in male conspecific sex pheromone discrimination and mating rate in T. chilonis, and these decreases were not due to the lower locomotor activity of the surviving T. chilonis males. Spinosad LC20 exposure caused a significant decrease in male locomotor activity of T. chilonis, but did not affect male-specific sex pheromone discrimination (conspecific sex pheromone discrimination or virgin sex pheromone discrimination) or mating rate. However, there was no significant difference in specific sex pheromone discrimination, mate choice, and locomotor activity between control males and males exposed to the low concentration (LC1) of insecticide (beta-cypermethrin or spinosad). In conclusion, beta-cypermethrin LC20 exposure was harmful to male-specific sex pheromone discrimination and mate choice in T. chilonis.

The evolution of genital complexity and mating rates in sexually size dimorphic spiders.

BackgroundGenital diversity may arise through sexual conflict over polyandry, where male genital features function to manipulate female mating frequency against her interest. Correlated genital evolution across animal groups is consistent with this view, but a link between genital complexity and mating rates remains to be established. In sexually size dimorphic spiders, golden orbweaving spiders (Nephilidae) males mutilate their genitals to form genital plugs, but these plugs do not always prevent female polyandry. In a comparative framework, we test whether male and female genital complexity coevolve, and how these morphologies, as well as sexual cannibalism, relate to the evolution of mating systems.ResultsUsing a combination of comparative tests, we show that male genital complexity negatively correlates with female mating rates, and that levels of sexual cannibalism negatively correlate with male mating rates. We also confirm a positive correlation between male and female genital complexity. The macroevolutionary trajectory is consistent with a repeated evolution from polyandry to monandry coinciding with the evolution towards more complex male genitals.ConclusionsThese results are consistent with the predictions from sexual conflict theory, although sexual conflict may not be the only mechanism responsible for the evolution of genital complexity and mating systems. Nevertheless, our comparative evidence suggests that in golden orbweavers, male genital complexity limits female mating rates, and sexual cannibalism by females coincides with monogyny.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-016-0821-y) contains supplementary material, which is available to authorized users.

Sexually antagonistic selection on genetic variation underlying both male and female same-sex sexual behavior.

BackgroundIntralocus sexual conflict, arising from selection for different alleles at the same locus in males and females, imposes a constraint on sex-specific adaptation. Intralocus sexual conflict can be alleviated by the evolution of sex-limited genetic architectures and phenotypic expression, but pleiotropic constraints may hinder this process. Here, we explored putative intralocus sexual conflict and genetic (co)variance in a poorly understood behavior with near male-limited expression. Same-sex sexual behaviors (SSBs) generally do not conform to classic evolutionary models of adaptation but are common in male animals and have been hypothesized to result from perception errors and selection for high male mating rates. However, perspectives incorporating sex-specific selection on genes shared by males and females to explain the expression and evolution of SSBs have largely been neglected.ResultsWe performed two parallel sex-limited artificial selection experiments on SSB in male and female seed beetles, followed by sex-specific assays of locomotor activity and male sex recognition (two traits hypothesized to be functionally related to SSB) and adult reproductive success (allowing us to assess fitness consequences of genetic variance in SSB and its correlated components). Our experiments reveal both shared and sex-limited genetic variance for SSB. Strikingly, genetically correlated responses in locomotor activity and male sex-recognition were associated with sexually antagonistic fitness effects, but these effects differed qualitatively between male and female selection lines, implicating intralocus sexual conflict at both male- and female-specific genetic components underlying SSB.ConclusionsOur study provides experimental support for the hypothesis that widespread pleiotropy generates pervasive intralocus sexual conflict governing the expression of SSBs, suggesting that SSB in one sex can occur due to the expression of genes that carry benefits in the other sex.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-016-0658-4) contains supplementary material, which is available to authorized users.

Evolution of mating behavior between two populations adapting to common environmental conditions.

Populations from the same species may be differentiated across contrasting environments, potentially affecting reproductive isolation among them. When such populations meet in a novel common environment, this isolation may be modified by biotic or abiotic factors. Curiously, the latter have been overlooked. We filled this gap by performing experimental evolution of three replicates of two populations of Drosophila subobscura adapting to a common laboratorial environment, and simulated encounters at three time points during this process. Previous studies showed that these populations were highly differentiated for several life-history traits and chromosomal inversions. First, we show initial differentiation for some mating traits, such as assortative mating and male mating rate, but not others (e.g., female mating latency). Mating frequency increased during experimental evolution in both sets of populations. The assortative mating found in one population remained constant throughout the adaptation process, while disassortative mating of the other population diminished across generations. Additionally, differences in male mating rate were sustained across generations. This study shows that mating behavior evolves rapidly in response to adaptation to a common abiotic environment, although with a complex pattern that does not correspond to the quick convergence seen for life-history traits.

Rapid range expansion is not restricted by inbreeding in a sexually cannibalistic spider.

Few studies investigated whether rapid range expansion is associated with an individual's short-term fitness costs due to an increased risk of inbred mating at the front of expansion. In mating systems with low male mating rates both sexes share potential inbreeding costs and general mechanisms to avoid or reduce these costs are expected. The spider Argiope bruennichi expanded its range recently and we asked whether rapid settlement of new sites exposes individuals to a risk of inbreeding. We sampled four geographically separated subpopulations, genotyped individuals, arranged matings and monitored hatching success. Hatching success was lowest in egg-sacs derived from sibling pairs and highest in egg-sacs derived from among-population crosses, while within-population crosses were intermediate. This indicates that inbreeding might affect hatching success in the wild. Unlike expected, differential hatching success of within- and among-population crosses did not correlate with genetic distance of mating pairs. In contrast, we found high genetic diversity based on 16 microsatellite markers and a fragment of the mitochondrial COI gene in all populations. Our results suggest that even a very recent settlement secures the presence of genetically different mating partners. This leads to costs of inbreeding since the population is not inbred.

Large donors transfer more sperm, but depletion is faster in a promiscuous hermaphrodite

Males are limited in sperm and seminal fluid, which can lead to prudent sperm allocation across consecutive matings. While sperm depletion is usually considered a population characteristic, we investigated an individual size-related variation in sperm depletion in the promiscuous sea slug Chelidonura sandrana. We found that sperm counts substantially declined across four successive copulations, indicating sperm depletion. Sperm depletion occurred at a disproportionally fast rate in larger sperm donors. Based on our finding of body size-dependent sperm depletion, we further investigated whether this translates into prudent sperm allocation across ejaculates. We found no evidence that slugs adjusted the number of delivered sperm in response to partner body weight, a tight proxy of fecundity. Instead, sperm counts increased with sperm donor body weight. Sperm donation independent of partner body size and—as found in previous work—partner novelty leaves scope for currently unidentified traits like the sperm competition environment to affect male sperm allocation. Alternatively, we propose that, in C. sandrana, a low optimal male mating rate close to the female optimum as indicated by our data may ultimately render indiscriminate sperm allocation the most beneficial male mating strategy.

The impact ofWolbachia, male age and mating history on cytoplasmic incompatibility and sperm transfer inDrosophila simulans

Most insects harbour a variety of maternally inherited endosymbionts, the most widespread being Wolbachia pipientis that commonly induce cytoplasmic incompatibility (CI) and reduced hatching success in crosses between infected males and uninfected females. High temperature and increasing male age are known to reduce the level of CI in a variety of insects. In Drosophila simulans, infected males have been shown to mate at a higher rate than uninfected males. By examining the impact of mating rate independent of age, this study investigates whether a high mating rate confers an advantage to infected males through restoring their compatibility with uninfected females over and above the effect of age. The impact of Wolbachia infection, male mating rate and age on the number of sperm transferred to females during copulation and how it relates to CI expression was also assessed. As predicted, we found that reproductive compatibility was restored faster in males that mate at higher rate than that of low mating and virgin males, and that the effect of mating history was over and above the effect of male age. Nonvirgin infected males transferred fewer sperm than uninfected males during copulation, and mating at a high rate resulted in the transfer of fewer sperm per mating irrespective of infection status. These results indicate that the advantage to infected males of mating at a high rate is through restoration of reproductive compatibility with uninfected females, whereas uninfected males appear to trade off the number of sperm transferred per mating with female encounter rate and success in sperm competition. This study highlights the importance Wolbachia may play in sexual selection by affecting male reproductive strategies.

Coevolving parasites and population size shape the evolution of mating behaviour

BackgroundCoevolution with parasites and population size are both expected to influence the evolution of mating rates. To gain insights into the interaction between these dual selective factors, we used populations from a coevolution experiment with the red flour beetle, Tribolium castaneum, and its microsporidian parasite, Nosema whitei. We maintained each experimental population at two different population sizes. We assayed the mating behaviour of both males and females from coevolved and paired non-coevolved control populations after 24 generations of coevolution with parasites.ResultsMales from large, coevolved populations (i.e. ancestors were exposed to parasites) showed a reduced eagerness to mate compared to males from large, non-coevolved populations. But in small populations, coevolution did not lead to decreased male mating rates. Coevolved females from both large and small populations appeared to be more willing to accept mating than non-coevolved females.ConclusionsThis study provides unique, experimental insights into the combined roles of coevolving parasites and population size on the evolution of mating rate. Furthermore, we find that males and females respond differently to the same environmental conditions. Our results show that parasites can be key determinants of the sexual behaviour of their hosts.

The Role of Sexual Selection and Conflict in Mediating Among-Population Variation in Mating Strategies and Sexually Dimorphic Traits in Sepsis punctum

The black scavenger fly Sepsis punctum exhibits striking among-population variation in the direction and magnitude of sexual size dimorphism, modification to the male forelimb and pre-copulatory behaviour. In some populations, male-biased sexual size dimorphism is observed; in other, less dimorphic, populations males court prior to mating. Such variation in reproductive traits is of interest to evolutionary biologists because it has the potential to limit gene flow among populations, contributing to speciation. Here, we investigate whether large male body size and modified forefemur are associated with higher male mating success within populations, whether these traits are associated with higher mating success among populations, and if these traits carry viability costs that could constrain their response to sexual selection. Flies from five distinct populations were reared at high or low food, generating high and low quality males. The expression of body size, forelimb morphology and courtship rate were each greater at high food, but high food males experienced higher mating success or reduced latency to first copulation in only one of the populations. Among populations, overall mating success increased with the degree of male-bias in overall body size and forelimb modification, suggesting that these traits have evolved as a means of increasing male mating rate. The increased mating success observed in large-male populations raises the question of why variation in magnitude of dimorphism persists among populations. One reason may be that costs of producing a large size constrain the evolution of ever-larger males. We found no evidence that juvenile mortality under food stress was greater for large-male populations, but development time was considerably longer and may represent an important constraint in an ephemeral and competitive growth environment.

Natural genetic variation in male reproductive genes contributes to nontransitivity of sperm competitive ability in Drosophila melanogaster

Female Drosophila melanogaster frequently mate with multiple males, and the success of a given male depends not only on his genotype but also on the genotype of his competitor. Here, we assess how natural genetic variation affects male-male interactions for traits influencing pre- and postcopulatory sexual selection. Males from a set of 66 chromosome substitution lines were competed against each other in a 'round-robin' design, and paternity was scored using bulk genotyping. We observed significant effects of the genotype of the first male to mate, the second male to mate and an interaction between the males for measures of male mating rate and sperm utilization. We also identified specific combinations of males who show nontransitive patterns of reproductive success and engage in 'rock-paper-scissors' games. We then tested for associations between 245 polymorphisms in 32 candidate male reproductive genes and male reproductive success. We identified eight polymorphisms in six reproductive genes that associate with male reproductive success independent of the competitor (experimentwise P < 0.05). We also identified four SNPs in four different genes where the relative reproductive success of the alternative alleles changes depending on the competing males' genetic background (experimentwise P < 0.05); two of these associations include premature stop codons. This may be the first study that identifies the genes contributing to nontransitivity among males and further highlights that 'rock-paper-scissors' games could be an important evolutionary force maintaining genetic variation in natural populations.

Determinants of Natural Mating Success in the Cannibalistic Orb-Web Spider Argiope bruennichi

Monogynous mating systems (low male mating rates) occur in various taxa and have evolved several times independently in spiders. Monogyny is associated with remarkable male mating strategies and predicted to evolve under a male-biased sex ratio. While male reproductive strategies are well documented and male mating rates are easy to quantify, especially in sexually cannibalistic species, female reproductive strategies, the optimal female mating rate, and the factors that affect the evolution of female mating rates are still unclear. In this study, we examined natural female mating rates and tested the assumption of a male-biased sex ratio and female polyandry in a natural population of Argiope bruennichi in which we controlled female mating status prior to observations. We predicted variation in female mating frequencies as a result of spatial and temporal heterogeneity in the distribution of mature females and males. Females had a low average mating rate of 1.3 and the majority copulated only once. Polyandry did not entirely result from a male-biased sex-ratio but closely matched the rate of male bigamy. Male activity and the probability of polyandry correlated with factors affecting pheromone presence such as virgin females' density. We conclude that a strong sex ratio bias and high female mating rates are not necessary components of monogynous mating systems as long as males protect their paternity effectively and certain frequencies of bigyny stabilise the mating system.

Genetic and nutritional effects on male traits and reproductive performance in Tribolium flour beetles

In Tribolium flour beetles and other organisms, individuals migrate between heterogeneous environments where they often encounter markedly different nutritional conditions. Under these circumstances, theory suggests that genotype-by-environment interactions (GEI) may be important in facilitating adaptation to new environments and maintaining genetic variation for male traits subject to directional selection. Here, we used a nested half-sib breeding design with Tribolium castaneum to partition the separate and joint effects of male genotype and nutritional environment on phenotypic variation in a comprehensive suite of life-history traits, reproductive performance measures across three sequential sexual selection episodes, and fitness. When male genotypes were tested across three nutritional environments, considerable phenotypic plasticity was found for male mating and insemination success, longevity and traits related to larval development. Our results also revealed significant additive genetic variation for male mating rate, sperm offence ability (P(2)), longevity and total fitness and for several traits reflecting both larval and adult resource use. In addition, we found evidence supporting GEI for sperm defence ability (P(1)), adult longevity and larval development; thus, no single male genotype outperforms others in every nutritional environment. These results provide insight into the potential roles of phenotypic plasticity and GEI in facilitating Tribolium adaptation to new environments in ecological and evolutionary time.

The Evolution of Large Testes: Sperm Competition or Male Mating Rate?

AbstractA positive relationship across species between the extent to which females mate with more than one male and relative testes mass has been demonstrated in a wide range of vertebrate taxa and certain insects. At least two hypotheses, which are not necessarily mutually exclusive, could account for this pattern: (1) the numerical sperm competition hypothesis, which assumes that larger testes enable the male to transfer more sperm to each female, giving the male an advantage in sperm competition and (2) the male mating rate hypothesis, which proposes that larger testes allow the male to produce a greater number of (potentially smaller) ejaculates to engage in frequent copulations with different females. Of these hypotheses, the former has won broad acceptance, while the latter has tended to be dismissed. Here, we argue that the lines of evidence commonly used to support the numerical sperm competition hypothesis in favour of the male mating rate hypothesis are not as clear cut or generally applicable as they are purported to be and that, consequently, the male mating rate hypothesis cannot be excluded with confidence on the basis of the current evidence. Furthermore, some evidence, such as the finding that ejaculate mass and/or sperm number is negatively correlated with testes mass across species in some insects and that larger testes in Drosophila can evolve in response to an increase in the number of females available for mating in the laboratory, provides support for the male mating rate hypothesis. Further work is needed to disentangle the relative effects of these selective pressures on the evolution of testes size.