Male Seminal Fluid Substances Affect Sperm Competition Success and Female Reproductive Behavior in a Seed Beetle

Male seminal fluid proteins are known to affect female reproductive behavior and physiology by reducing mating receptivity and by increasing egg production rates. Such substances are also though to increase the competitive fertilization success of males, but the empirical foundation for this tenet is restricted. Here, we examined the effects of injections of size-fractioned protein extracts from male reproductive organs on both male competitive fertilization success (i.e., P2 in double mating experiments) and female reproduction in the seed beetle Callosobruchus maculatus. We found that extracts of male seminal vesicles and ejaculatory ducts increased competitive fertilization success when males mated with females 1 day after the females' initial mating, while extracts from accessory glands and testes increased competitive fertilization success when males mated with females 2 days after the females' initial mating. Moreover, different size fractions of seminal fluid proteins had distinct and partly antagonistic effects on male competitive fertilization success. Collectively, our experiments show that several different seminal fluid proteins, deriving from different parts in the male reproductive tract and of different molecular weight, affect male competitive fertilization success in C. maculatus. Our results highlight the diverse effects of seminal fluid proteins and show that the function of such proteins can be contingent upon female mating status. We also document effects of different size fractions on female mating receptivity and egg laying rates, which can serve as a basis for future efforts to identify the molecular identity of seminal fluid proteins and their function in this model species

Mating data Callosobruchus subinnotatus

Excel file with mating data of 163 mating observations of Callosobruchus subinnotatus. There is one treatment group: AJ (ablated jaws), and three control groups: APy (ablated pygidium), APa (ablated paramere), and Non (no treatment). Every row is an observation of one couple mating twice. The female lays eggs in between (Total Eggs Between) and after the second mating (Total Eggs after Second). 'Scars old' and 'Scars new' refer to scar data gained by two different methods. 'Scars old' is count data of the number of scars, 'Scars new' is the surface area in pixels that is occupied by scars

An experimental test of temperature-dependent selection on mitochondrial haplotypes in Callosobruchus maculatus seed beetles

Mitochondrial DNA (mtDNA) consists of few but vital maternally inherited genes that interact closely with nuclear genes to produce cellular energy. How important mtDNA polymorphism is for adaptation is still unclear. The assumption in population genetic studies is often that segregating mtDNA variation is selectively neutral. This contrasts with empirical observations of mtDNA haplotypes affecting fitness-related traits and thermal sensitivity, and latitudinal clines in mtDNA haplotype frequencies. Here, we experimentally test whether ambient temperature affects selection on mtDNA variation, and whether such thermal effects are influenced by intergenomic epistasis due to interactions between mitochondrial and nuclear genes, using replicated experimental evolution inCallosobruchus maculatusseed beetle populations seeded with a mixture of different mtDNA haplotypes. We also test for sex-specific consequences of mtDNA evolution on reproductive success, given that mtDNA mutations can have sexually antagonistic fitness effects. Our results demonstrate natural selection on mtDNA haplotypes, with some support for thermal environment influencing mtDNA evolution through mitonuclear epistasis. The changes in male and female reproductive fitness were both aligned with changes in mtDNA haplotype frequencies, suggesting that natural selection on mtDNA is sexually concordant in stressful thermal environments. We discuss the implications of our findings for the evolution of mtDNA

Data from: Complex mitonuclear interactions and metabolic costs of mating in male seed beetles

The lack of evolutionary response to selection on mitochondrial genes through males predicts the evolution of nuclear genetic influence on male-specific mitochondrial function, for example by gene duplication and evolution of sex-specific expression of paralogs involved in metabolic pathways. Intergenomic epistasis may therefore be a prevalent feature of the genetic architecture of male-specific organismal function. Here, we assess the role of mitonuclear genetic variation for male metabolic phenotypes [metabolic rate and respiratory quotient (RQ)] associated with ejaculate renewal, in the seed beetle Callosobruchus maculatus, by assaying lines with crossed combinations of distinct mitochondrial haplotypes and nuclear lineages. We found a significant increase in metabolic rate following mating relative to virgin males. Moreover, processes associated with ejaculate renewal showed variation in metabolic rate that was affected by mitonuclear interactions. Mitochondrial haplotype influenced mating-related changes in RQ, but this pattern varied over time. Mitonuclear genotype and the energy spent during ejaculate production affected the weight of the ejaculate, but the strength of this effect varied across mitochondrial haplotypes showing that the genetic architecture of male-specific reproductive function is complex. Our findings unveil hitherto underappreciated metabolic costs of mating and ejaculate renewal, and provide the first empirical demonstration of mitonuclear epistasis on male reproductive metabolic processes

Data from: Postmating sexual selection and the enigmatic jawed genitalia of Callosobruchus subinnotatus

Insect genitalia exhibit rapid divergent evolution. Truly extraordinary structures have evolved in some groups, presumably as a result of post-mating sexual selection. To increase our understanding of this phenomenon, we studied the function of one such structure. The male genitalia of Callosobruchus subinnotatus (Coleoptera: Bruchinae) contain a pair of jaw-like structures with unknown function. Here, we used phenotypic engineering to ablate the teeth on these jaws. We then experimentally assessed the effects of ablation of the genital jaws on mating duration, ejaculate weight, male fertilization success and female fecundity, using a double-mating experimental design. We predicted that copulatory wounding in females should be positively related to male fertilization success. However, we found no significant correlation between genital tract scarring in females and male fertilization success. Male fertilization success was, however, positively related to the amount of ejaculate transferred by males and negatively related to female ejaculate dumping. Ablation of male genital jaws did not affect male relative fertilization success but resulted in a reduction in female egg production. Our results suggest that postmating sexual selection in males indeed favors these genital jaws, but not primarily through an elevated relative success in sperm competition but by increasing female egg production

Male Seminal Fluid Substances Affect Sperm Competition Success and Female Reproductive Behavior in a Seed Beetle

Male seminal fluid proteins are known to affect female reproductive behavior and physiology by reducing mating receptivity and by increasing egg production rates. Such substances are also though to increase the competitive fertilization success of males, but the empirical foundation for this tenet is restricted. Here, we examined the effects of injections of size-fractioned protein extracts from male reproductive organs on both male competitive fertilization success (i.e., P2 in double mating experiments) and female reproduction in the seed beetle Callosobruchus maculatus. We found that extracts of male seminal vesicles and ejaculatory ducts increased competitive fertilization success when males mated with females 1 day after the females' initial mating, while extracts from accessory glands and testes increased competitive fertilization success when males mated with females 2 days after the females' initial mating. Moreover, different size fractions of seminal fluid proteins had distinct and partly antagonistic effects on male competitive fertilization success. Collectively, our experiments show that several different seminal fluid proteins, deriving from different parts in the male reproductive tract and of different molecular weight, affect male competitive fertilization success in C. maculatus. Our results highlight the diverse effects of seminal fluid proteins and show that the function of such proteins can be contingent upon female mating status. We also document effects of different size fractions on female mating receptivity and egg laying rates, which can serve as a basis for future efforts to identify the molecular identity of seminal fluid proteins and their function in this model species

Receptivity to mating in virgin females (N = 20–36 per block and treatment group) injected with a fraction of male accessory gland extract (Fraction I [molecular weight <25 kDa], Fraction II [MW, 25–50 kDa], or Fraction III [MW > 50 kDa]) or with Milli-Q water only (control), at 3–5 h, 1, 2, and 3 d after injection.

<p>The two panels and represent the two experimental blocks.</p

Generalized linear model of the effects of injection of molecular-weight fractions of male accessory gland extract (treatment), the day at which females were offered a mating (days), and experimental block (block) on the number of dead females by day 3 after the injection of extracts (N = 20–36 females per day, block and treatment group).

<p>Non-significant interactions were removed to avoid over-parameterization.</p><p>Generalized linear model of the effects of injection of molecular-weight fractions of male accessory gland extract (treatment), the day at which females were offered a mating (days), and experimental block (block) on the number of dead females by day 3 after the injection of extracts (N = 20–36 females per day, block and treatment group).</p

Generalized linear model of the effects of different male reproductive organ—derived extracts (treatment) and the number of days in between matings (days) on the sperm competitive success of the second male in double mating experiments (N = 29–42 females per day and treatment group).

<p>The number of eggs laid in between matings was used as a covariate.</p><p>Generalized linear model of the effects of different male reproductive organ—derived extracts (treatment) and the number of days in between matings (days) on the sperm competitive success of the second male in double mating experiments (N = 29–42 females per day and treatment group).</p