Protein-Level Interactions as Mediators of Sexual Conflict in Ants.

All social insects with obligate reproductive division of labor evolved from strictly monogamous ancestors, but multiple queen-mating (polyandry) arose de novo, in several evolutionarily derived lineages. Polyandrous ant queens are inseminated soon after hatching and store sperm mixtures for a potential reproductive life of decades. However, they cannot re-mate later in life and are thus expected to control the loss of viable sperm because their lifetime reproductive success is ultimately sperm limited. In the leaf-cutting ant Atta colombica,, the survival of newly inseminated sperm is known to be compromised by seminal fluid of rival males and to be protected by secretions of the queen sperm storage organ (spermatheca). Here we investigate the main protein-level interactions that appear to mediate sperm competition dynamics and sperm preservation. We conducted an artificial insemination experiment and DIGE-based proteomics to identify proteomic changes when seminal fluid is exposed to spermathecal fluid followed by a mass spectrometry analysis of both secretions that allowed us to identify the sex-specific origins of the proteins that had changed in abundance. We found that spermathecal fluid targets only seven (2%) of the identified seminal fluid proteins for degradation, including two proteolytic serine proteases, a SERPIN inhibitor, and a semen-liquefying acid phosphatase. In vitro, and in vivo, experiments provided further confirmation that these proteins are key molecules mediating sexual conflict over sperm competition and viability preservation during sperm storage. In vitro, exposure to spermathecal fluid reduced the capacity of seminal fluid to compromise survival of rival sperm in a matter of hours and biochemical inhibition of these seminal fluid proteins largely eliminated that adverse effect. Our findings indicate that A. colombica, queens are in control of sperm competition and sperm storage, a capacity that has not been documented in other animals but is predicted to have independently evolved in other polyandrous social insects.

Prudent sperm use by leaf-cutter ant queens.

In many species, females store sperm between copulation and egg fertilization, but the consequences of sperm storage and patterns of sperm use for female life history and reproductive success have not been investigated in great detail. In hymenopteran insect societies (ants, bees, wasps), reproduction is usually monopolized by one or relatively few queens, who mate only during a brief period early in life and store sperm for later use. The queens of some ants are particularly long-lived and have the potential to produce millions of offspring during their life. To do so, queens store many sperm cells, and this sperm must remain viable throughout the years of storage. Queens should also be under strong selection to use stored sperm prudently when fertilizing eggs. We used the leaf-cutter ant Atta colombica to investigate the dynamics of sperm use during egg fertilization. We show that queens are able to fertilize close to 100 per cent of the eggs and that the average sperm use per egg is very low, but increases with queen age. The robustness of stored sperm was found to decrease with years of storage, signifying that senescence affects sperm either directly or indirectly via the declining glandular secretions or deteriorating sperm-storage organs. We evaluate our findings with a heuristic model, which suggests that the average queen has sperm for almost 9 years of normal colony development. We discuss the extent to which leaf-cutter ant queens have been able to optimize their sperm expenditure and infer that our observed averages of sperm number, sperm robustness and sperm use are consistent with sperm depletion being a significant cause of mortality of mature colonies of Atta leaf-cutter ants.

Sperm use economy of honeybee (Apis mellifera) queens.

The queens of eusocial ants, bees, and wasps only mate during a very brief period early in life to acquire and store a lifetime supply of sperm. As sperm cannot be replenished, queens have to be highly economic when using stored sperm to fertilize eggs, especially in species with large and long-lived colonies. However, queen fertility has not been studied in detail, so that we have little understanding of how economic sperm use is in different species, and whether queens are able to influence their sperm use. This is surprising given that sperm use is a key factor of eusocial life, as it determines the fecundity and longevity of queens and therefore colony fitness. We quantified the number of sperm that honeybee (Apis mellifera) queens use to fertilize eggs. We examined sperm use in naturally mated queens of different ages and in queens artificially inseminated with different volumes of semen. We found that queens are remarkably efficient and only use a median of 2 sperm per egg fertilization, with decreasing sperm use in older queens. The number of sperm in storage was always a significant predictor for the number of sperm used per fertilization, indicating that queens use a constant ratio of spermathecal fluid relative to total spermathecal volume of 2.364 × 10(-6) to fertilize eggs. This allowed us to calculate a lifetime fecundity for honeybee queens of around 1,500,000 fertilized eggs. Our data provide the first empirical evidence that honeybee queens do not manipulate sperm use, and fertilization failures in worker-destined eggs are therefore honest signals that workers can use to time queen replacement, which is crucial for colony performance and fitness.

The ejaculatory biology of leafcutter ants.

The eusocial ants are unique in that females (queens) acquire and store sperm on a single mating flight early in adult life. This event largely determines the size (possibly millions of workers), longevity (possibly decades) and genetic variation of the colonies that queens found, but our understanding of the fundamental biology of ejaculate production, transfer and physiological function remains extremely limited. We studied the ejaculation process in the leafcutter ant Atta colombica and found that it starts with the appearance of a clear pre-ejaculatory fluid (PEF) at the tip of the endophallus that is followed by the joint expulsion of the remainder of accessory gland (AG) secretion, sperm, accessory testes (AT) secretion, and a small mating plug. PEF, AG secretion and AT secretion all contribute to sperm survival, but PEF and AG secretion also reduce the survival of sperm from other males. We show that PEF is produced in the AGs and is likely identical to AG secretion because protein-banding patterns of PEF and AG secretion were similar on 1D electrophoresis gels, but differed from the protein-banding pattern of AT secretion. We show that proteins in AG secretion are responsible for the incapacitation of rival sperm and infer that transfer of AG secretion prior to sperm may allow these components to interact with rival sperm, while at the same time providing a supportive biochemical environment for the arrival of own sperm.

Patterns of paternity skew among polyandrous social insects: what can they tell us about the potential for sexual selection?

Monogamy results in high genetic relatedness among offspring and thus it is generally assumed to be favored by kin selection. Female multiple mating (polyandry) has nevertheless evolved several times in the social Hymenoptera (ants, bees, and wasps), and a substantial amount of work has been conducted to understand its costs and benefits. Relatedness and inclusive fitness benefits are, however, not only influenced by queen mating frequency but also by paternity skew, which is a quantitative measure of paternity biases among the offspring of polyandrous females. We performed a large-scale phylogenetic analysis of paternity skew across polyandrous social Hymenoptera. We found a general and significant negative association between paternity frequency and paternity skew. High paternity skew, which increases relatedness among colony members and thus maximizes inclusive fitness gains, characterized species with low paternity frequency. However, species with highly polyandrous queens had low paternity skew, with paternity equalized among potential sires. Equal paternity shares among fathers are expected to maximize fitness benefits derived from genetic diversity among offspring. We discuss the potential for postcopulatory sexual selection to influence patterns of paternity in social insects, and suggest that sexual selection may have played a key, yet overlooked role in social evolution.

Seminal fluid mediates ejaculate competition in social insects.

Queens of ants and bees normally obtain a lifetime supply of sperm on a single day of sexual activity, and sperm competition is expected to occur in lineages where queens receive sperm from multiple males. We compared singly mated (monandrous) and multiply mated (polyandrous) sister groups of ants and bees and show that seminal fluid of polyandrous species has a more positive effect on the survival of a male's own sperm than on other males' sperm. This difference was not observed in the monandrous species, suggesting that incapacitation of competing sperm may have independently evolved in both bees and ants. In Atta leafcutter ants, the negative effect of the seminal fluid of other males was negated by secretion from the queen sperm-storage organ, suggesting that queens may control ejaculate competition after sperm storage.

Honey bee males and queens use glandular secretions to enhance sperm viability before and after storage.

Internal fertilization requires live sperm to be transferred from male to female before egg fertilization. Both males and females assist the insemination process by providing sperm with glandular secretions, which have been inferred to contain subsets of proteins that maintain sperm viability. Here we show that in the honeybee (Apis mellifera) secretions of the male accessory glands, the major contributors towards seminal fluid, enhance sperm survival. We further demonstrate that the protein fraction of the male accessory gland secretion is indeed important for achieving the maximal effect on sperm survival. After sperm storage, the queens also provide sperm with secretions from spermathecal glands and we show that these secretions have a comparable positive effect on sperm viability. SDS gels show that the proteomic profiles of accessory gland secretion and spermathecal fluid secretion hardly overlap, which suggests that males and females use different proteins to enhance sperm viability during, respectively, ejaculation and final sperm storage.