The life history of Drosophila sperm involves molecular continuity between male and female reproductive tracts.

SignificanceIn species with internal fertilization, sperm spend an important part of their lives within the female. To examine the life history of the sperm during this time, we used semiquantitative proteomics and sex-specific isotopic labeling in fruit flies to determine the extent of molecular continuity between male and female reproductive tracts and provide a global catalog of sperm-associated proteins. Multiple seminal fluid proteins and female proteins associate with sperm immediately after mating. Few seminal fluid proteins remain after long-term sperm storage, whereas female-derived proteins constitute one-fifth of the postmating sperm proteome by then. Our data reveal a molecular "hand-off" from males to females, which we postulate to be an important component of sperm-female interactions.

Pronounced Postmating Response in the Drosophila Female Reproductive Tract Fluid Proteome.

Fertility depends on the progression of complex and coordinated postmating processes within the extracellular environment of the female reproductive tract (FRT). Molecular interactions between ejaculate and FRT proteins regulate many of these processes, including sperm motility, migration, storage, and modification, along with concurrent changes in the female. Although extensive progress has been made in the proteomic characterization of the male-derived components of sperm and seminal fluid, investigations into the FRT have remained more limited. To achieve a comparable level of knowledge regarding female-derived proteins that comprise the reproductive environment, we utilized semiquantitative MS-based proteomics to study the composition of the FRT tissue and, separately, the luminal fluid, before and after mating in Drosophila melanogaster. Our approach leveraged whole-fly isotopic labeling to delineate female proteins from transferred male ejaculate proteins. Our results revealed several characteristics that distinguish the FRT fluid proteome from the FRT tissue proteome: (1) the fluid proteome is encoded by genes with higher overall levels of FRT gene expression and tissue specificity, including many genes with enriched expression in the fat body, (2) fluid-biased proteins are enriched for metabolic functions, and (3) the fluid exhibits pronounced postmating compositional changes. The dynamic mating-induced proteomic changes in the FRT fluid inform our understanding of secretory mechanisms of the FRT, serve as a foundation for establishing female contributions to the ejaculate-female interactions that regulate fertility, and highlight the importance of applying proteomic approaches to characterize the composition and dynamics of the FRT environment.

Quantitative proteomics reveals rapid divergence in the postmating response of female reproductive tracts among sibling species.

Fertility depends, in part, on interactions between male and female reproductive proteins inside the female reproductive tract (FRT) that mediate postmating changes in female behaviour, morphology, and physiology. Coevolution between interacting proteins within species may drive reproductive incompatibilities between species, yet the mechanisms underlying postmating-prezygotic (PMPZ) isolating barriers remain poorly resolved. Here, we used quantitative proteomics in sibling Drosophila species to investigate the molecular composition of the FRT environment and its role in mediating species-specific postmating responses. We found that (i) FRT proteomes in D. simulans and D. mauritiana virgin females express unique combinations of secreted proteins and are enriched for distinct functional categories, (ii) mating induces substantial changes to the FRT proteome in D. mauritiana but not in D. simulans, and (iii) the D. simulans FRT proteome exhibits limited postmating changes irrespective of whether females mate with conspecific or heterospecific males, suggesting an active female role in mediating reproductive interactions. Comparisons with similar data in the closely related outgroup species D. melanogaster suggest that divergence is concentrated on the D. simulans lineage. Our study suggests that divergence in the FRT extracellular environment and postmating response contribute to previously described patterns of PMPZ isolation and the maintenance of species boundaries.

Exploratory Activities for Understanding Evolutionary Relationships Depicted by Phylogenetic Trees: United but Diverse.

Evolution explains both the unity and the diversity of all organisms, and developing students' ability to represent and communicate evolutionary relationships is an important component of a complete biology education. We present a series of student-centered, exploratory activities to help students develop their tree-thinking skills. In these activities, students use complementary phenotypic and molecular data to explore how to build phylogenetic trees and interpret the evolutionary relationships they represent. This learning module is designed to engage students in the process of science, provide them with active learning experiences using online bioinformatics tools, and foster their appreciation for the evolutionary connections across the tree of life.

Population density mediates the interaction between pre- and postmating sexual selection.

When females mate with more than one male, sexual selection acts both before and after mating. The interaction between pre- and postmating episodes of selection is expected to be context dependent, but few studies have investigated how total sexual selection changes under different ecological conditions. We examined how population density mediates the interaction between pre- and postmating sexual selection by establishing replicate populations of the horned dung beetle Onthophagus taurus at low, medium, and high densities, and then using microsatellite-based parentage analyses to measure male fitness. We found that mating success and fertilization success were positively correlated at all three densities, but the strength of the correlation decreased with increasing density. We also found a shift from negative to positive linear selection on testes mass as density increased, and opposing selection on weapons and testes at high densities. These patterns suggest that the importance of postmating processes increases with increasing population density, which reduces the selective advantage of weapons for premating contest competition, and increases the selective advantage of large ejaculates for postmating sperm competition. We expect that density-dependent selection on testes mass has contributed to the phenotypic variation observed between natural populations of O. taurus that differ in density.

Benefits of polyandry: Molecular evidence from field-caught dung beetles.

When females mate with multiple males, they set the stage for postcopulatory sexual selection via sperm competition and/or cryptic female choice. Surprisingly little is known about the rates of multiple mating by females in the wild, despite the importance of this information in understanding the potential for postcopulatory sexual selection to drive the evolution of reproductive behaviour, morphology and physiology. Dung beetles in the genus Onthophagus have become a laboratory model for studying pre- and postcopulatory sexual selection, yet we still lack information about the reproductive behaviour of female dung beetles in natural populations. Here, we develop microsatellite markers for Onthophagus taurus and use them to genotype the offspring of wild-caught females and to estimate natural rates of multiple mating and patterns of sperm utilization. We found that O. taurus females are highly polyandrous: 88% of females produced clutches sired by at least two males, and 5% produced clutches with as many as five sires. Several females (23%) produced clutches with significant paternity skew, indicating the potential for strong postcopulatory sexual selection in natural populations. There were also strong positive correlations between the number of offspring produced and both number of fathers and paternity skew, which suggests that females benefit from mating polyandrously by inciting postcopulatory mechanisms that bias paternity towards males that can sire more viable offspring. This study evaluates the fitness consequences of polyandry for an insect in the wild and provides strong evidence that female dung beetles benefit from multiple mating under natural conditions.

Why Sexually Selected Weapons Are Not Ornaments.

The elaboration and diversification of sexually selected weapons remain poorly understood. We argue that progress in this topic has been hindered by a strong bias in sexual selection research, and a tendency for weapons to be conflated with ornaments used in mate choice. Here, we outline how male-male competition and female choice are distinct mechanisms of sexual selection, and why weapons and ornaments are fundamentally different types of traits. We call for research on the factors contributing to weapon divergence, the potential for male-male competition to drive speciation, and the specific use of weapons in the context of direct fights versus displays. Given that weapons are first and foremost fighting structures, biomechanical approaches are an especially promising direction for understanding weapon design.

Structural adaptations to diverse fighting styles in sexually selected weapons.

The shapes of sexually selected weapons differ widely among species, but the drivers of this diversity remain poorly understood. Existing explanations suggest weapon shapes reflect structural adaptations to different fighting styles, yet explicit tests of this hypothesis are lacking. We constructed finite element models of the horns of different rhinoceros beetle species to test whether functional specializations for increased performance under species-specific fighting styles could have contributed to the diversification of weapon form. We find that horns are both stronger and stiffer in response to species-typical fighting loads and that they perform more poorly under atypical fighting loads, which suggests weapons are structurally adapted to meet the functional demands of fighting. Our research establishes a critical link between weapon form and function, revealing one way male-male competition can drive the diversification of animal weapons.

Mechanical limits to maximum weapon size in a giant rhinoceros beetle.

The horns of giant rhinoceros beetles are a classic example of the elaborate morphologies that can result from sexual selection. Theory predicts that sexual traits will evolve to be increasingly exaggerated until survival costs balance the reproductive benefits of further trait elaboration. In Trypoxylus dichotomus, long horns confer a competitive advantage to males, yet previous studies have found that they do not incur survival costs. It is therefore unlikely that horn size is limited by the theoretical cost-benefit equilibrium. However, males sometimes fight vigorously enough to break their horns, so mechanical limits may set an upper bound on horn size. Here, I tested this mechanical limit hypothesis by measuring safety factors across the full range of horn sizes. Safety factors were calculated as the ratio between the force required to break a horn and the maximum force exerted on a horn during a typical fight. I found that safety factors decrease with increasing horn length, indicating that the risk of breakage is indeed highest for the longest horns. Structural failure of oversized horns may therefore oppose the continued exaggeration of horn length driven by male-male competition and set a mechanical limit on the maximum size of rhinoceros beetle horns.

Elaborate horns in a giant rhinoceros beetle incur negligible aerodynamic costs.

Sexually selected ornaments and weapons are among nature's most extravagant morphologies. Both ornaments and weapons improve a male's reproductive success; yet, unlike ornaments that need only attract females, weapons must be robust and functional structures because they are frequently tested during male-male combat. Consequently, weapons are expected to be particularly costly to bear. Here, we tested the aerodynamic costs of horns in the giant rhinoceros beetle, Trypoxylus dichotomus. We predicted that the long, forked head horn would have three main effects on flight performance: increased body mass, an anterior shift in the centre of mass and increased body drag. We found that the horns were surprisingly lightweight, and therefore had a trivial effect on the male beetles' total mass and mass distribution. Furthermore, because beetles typically fly at slow speeds and high body angles, horns had little effect on total body drag. Together, the weight and the drag of horns increased the overall force required to fly by less than 3 per cent, even in the largest males. Because low-cost structures are expected to be highly evolutionarily labile, the fact that horns incur very minor flight costs may have permitted both the elaboration and diversification of rhinoceros beetle horns.

Interpatch foraging in honeybees-rational decision making at secondary hubs based upon time and motivation.

For honeybees, Apis mellifera, the hive has been well known to function as a primary decision-making hub, a place from which foragers decide among various directions, distances, and times of day to forage efficiently. Whether foraging honeybees can make similarly complex navigational decisions from locations away from the hive is unknown. To examine whether or not such secondary decision-making hubs exist, we trained bees to forage at four different locations. Specifically, we trained honeybees to first forage to a distal site "CT" 100 m away from the hive; if food was present, they fed and then chose to go home. If food was not present, the honeybees were trained to forage to three auxiliary sites, each at a different time of the day: A in the morning, B at noon, and C in the afternoon. The foragers learned to check site CT for food first and then efficiently depart to the correct location based upon the time of day if there was no food at site CT. Thus, the honeybees were able to cognitively map motivation, time, and five different locations (Hive, CT, A, B, and C) in two spatial dimensions; these are the contents of the cognitive map used by the honeybees here. While at site CT, we verified that the honeybees could choose between 4 different directions (to A, B, C, and the Hive) and thus label it as a secondary decision-making hub. The observed decision making uncovered here is inferred to constitute genuine logical operations, involving a branched structure, based upon the premises of motivational state, and spatiotemporal knowledge.