The leading hypothesis for the evolution of male genital complexity proposes that genital traits evolve in response to post-insemination sexual selection; that is, via cryptic female choice or sperm competition. Here, we describe a laser ablation technique for high-precision manipulation of microscale body parts of insects, and employ it to discern the adaptive function of a rapidly evolving and taxonomically important genital trait: the intromittent claw-like genital spines of maleDrosophila bipectinataDuda. We demonstrate experimentally and unambiguously that the genital spines of this species function to mechanically couple the genitalia together. The excision of the spines by laser ablation sharply reduced the ability of males both to copulate and to compete against rival males for mates. When spineless males did succeed to copulate, their insemination success and fertilization rate were not statistically different from controls, at odds with the post-insemination sexual selection hypothesis of genital function and evolution. The results provide direct experimental support for the hypothesis that genital traits evolve in response to sexual selection occurring prior to insemination.

Well-established causal links exist between maternal nutritional deficits and embryo health and viability. By contrast, environmental effects operating through the father that could influence embryo mortality have seldom been examined. Yet, ejaculates can require non-trivial resource allocation, and seminal plasma components are increasingly recognized to exert wide-ranging effects on females and offspring, so paternal dietary effects on the embryo should be expected. We test for effects of varying levels of protein (P), carbohydrate (C) and caloric load in adult male diet on embryo mortality inDrosophila melanogaster. We demonstrate that macronutrient balance and caloric restriction exert significant effects, and that nutritional effects are more impactful when a prior mating has occurred. Once-mated males produced embryos with marginally elevated mortality under high-caloric densities and a 1 : 8 P : C ratio. In contrast, embryos produced by twice-mated males were significantly more likely to die under male caloric restriction, an outcome that may have resulted from shifts in ejaculate quality and/or epigenetic paternal effects. Body nutrient reserves were strongly and predictably altered by diet, and body condition, in turn, was negatively related to embryo mortality. Thus, sire nutritional history and resultant shifts in metabolic state predict embryo viability and post-fertilization fitness outcomes.

Developmental selection, the non-random elimination of offspring during development, is hypothesized to alter the opportunity for selection on a given trait at later stages of the life cycle. Here, we provide a direct demonstration of developmental selection against developmental instability, assessed as the incidence of minor, discrete phenotypic abnormalities in the male sex comb, a condition-dependent secondary sexual trait in Drosophila bipectinata . We exposed developing flies from two geographically separate populations to increasing levels of temperature stress, and recovered the males that died during development by teasing them out of their pupal cases. These dead males, the so-called ‘invisible fraction’ of the population, were more developmentally unstable than their surviving counterparts, and dramatically so under conditions of relatively high temperature stress. We illustrate that had these dead juvenile flies actually survived and entered the pool of sexually mature adult individuals, their mating success would have been significantly reduced, thus intensifying sexual selection in the adult cohort for reducing developmental instability. The data suggest that without accounting for developmental selection, a study focusing exclusively on the adult cohort may unwittingly underestimate the net force of selection operating on a given phenotypic trait.