In most plants and animals, a consistent relationship exists between the DNA content of a cell and its metabolic activity. The male-haploid sex determination of Hymenoptera and other arthropods may therefore impose a particular selective pressure upon males, which must evolve adaptations to cope with a genomic DNA reduced by half compared with that of females. Here, we show that a nuclear DNA content similar to that of females is restored in muscles of males in all hymenopteran lineages tested except the most basal one (Xyelidae). This doubling of DNA content in males does not occur in other haplodiploid insects, such as thrips (Thysanoptera) and whiteflies (Sternorrhyncha). These results indicate that this adaptation probably occurred early in hymenopteran history, possibly because males acquired strong flying and dispersal abilities.

Social Hymenoptera are general models for the study of parent-offspring conflict over sex ratio, because queens and workers frequently have different reproductive optima. The ant Pheidole pallidula shows a split distribution of sex ratios with most of the colonies producing reproductives of a single sex. Sex ratio specialization is tightly associated with the breeding system, with single-queen (monogynous) colonies producing malebiased brood and multiple-queen (polygynous) colonies female-biased brood. Here, we show that this sex specialization is primarily determined by the queen's influence over colony sex ratio. Queens from monogynous colonies produce a significantly more male-biased primary sex ratio than queens from polygynous colonies. Moreover, queens from monogynous colonies produce a significantly lower proportion of diploid eggs that develop into queens and this is associated with lower rate of juvenile hormone (JH) production compared to queens from polygynous colonies. These results indicate that queens regulate colony sex ratio in two complementary ways: by determining the proportion of female eggs laid and by hormonally biasing the development of female eggs into either a worker or reproductive form. This is the first time that such a dual system of queen influence over colony sex ratio is identified in an ant.

Sex ratio variations during brood development have important implications for the study of sex allocation in haplodiploid insects. So far, few studies have addressed this question because of the difficulty to determine the sex of the brood. We used flow cytometry to differentiate haploid males from diploid females in the ant Linepithema humile . Our data show that flow cytometry can be used successfully to distinguish between male and female brood on the basis of their DNA content, from the very first larval stage. Moreover, we show that flow cytometry allows sex brood determination in other ant species, as well as in nonsocial Hymenoptera.Keywords : ants, flow cytometry, haplodiploidy, Hymenoptera, sex ratio Received 28 April 2003; revision accepted 4 June 2003Sex allocation in Hymenoptera has been the focus of much theoretical and empirical works in evolutionary biology (Charnov 1982;Wrensch & Ebbert 1993;Hardy 2002). However, a major problem in the study of sex allocation in Hymenoptera is to determine sex ratio changes during brood development. Sex ratios are usually determined at oviposition (primary sex ratio) and/or the adult stage (secondary sex ratio). The difference between primary and secondary sex ratios may stem from two possible causes. First, it may reflect sex-specific differences in brood mortality during development. Males of Hymenoptera usually develop from haploid eggs and females from diploid eggs. Males being haploid, they could experience higher mortality due to deleterious recessive alleles, as compared to diploid females (Smith & Shaw 1980). Second, in eusocial Hymenoptera, sex-ratio variations during development may also result from a queen-worker conflict over sex ratio (Trivers & Hare 1976). Because of haplodiploidy, workers are more closely related to their sisters than to their brothers. They may therefore maximize their inclusive fitness by raising a female-biased sex investment ratio. By contrast, queens are equally related to their sons and daughters, and are selected to favour an equal investment in both sexes. Therefore, differences between primary and secondary sex ratio may result from workers biasing sex allocation towards their own genetic interest.In several ant species, it has been shown that workers perform sex-allocation biasing by killing male larvae (Chapuisat & Keller 1999). Elimination of male brood entails costs, because most or all of the resources invested in rearing males until their elimination are lost. Hence, the earlier in development that workers are able to identify the sex of the brood, the lower will be the costs of male elimination (Nonacs & Carlin 1990). Despite its theoretical importance, the developmental stage at which males are selectively eliminated has received surprisingly little attention. This results mainly from the difficulty in determining the sex of the brood.Here, we show that flow cytometry, a method for the determination of ploidy level based on the measurement of nuclear-DNA content, can be successfully used to discriminate acc...

In the ant Cardiocondyla obscurior, wingless males compete with nestmate males for access to female mating partners, leading to local mate competition (LMC). Queen number varies between colonies, resulting in variation in the strength of LMC. Cremer & Heinze (2002, Proceedings of the Royal Society of London, Series B, 269,[417][418][419][420][421][422] showed that colonies responded to increasing queen number by producing a less femalebiased sex ratio, as predicted by LMC theory. However, the proximate mechanisms responsible for this variation in the sex ratio could not be determined because the study was restricted to adult sex ratios. With LMC, the primary sex ratio (proportion of haploid eggs laid by the queen) is expected to be female biased, which lowers the conflict between queens and workers over sex allocation. We compared the primary sex ratios laid by queens in monogynous and in polygynous experimental colonies of C. obscurior. The proportion of haploid eggs laid by queens was significantly lower in single-queen than in multiple-queen colonies. Furthermore, queens rapidly adjusted their primary sex ratios to changes in colony queen number. This is the first report of an adaptive adjustment of the primary sex ratio in response to LMC by ant queens.

The haplodiploid sex determining system in Hymenoptera, whereby males develop from haploid eggs and females from diploid eggs, allows females to control the primary sex ratio (the proportion of each sex at oviposition) in response to ecological and /or genetic conditions. Surprisingly, primary sex ratio adjustment by queens in eusocial Hymenoptera has been poorly studied, because of difficulties in sexing the eggs laid. Here, we show that fluorescence in situ hybridization (FISH) can be used to accurately determine the sex (haploid or diploid) of eggs, and hence the primary sex ratio, in ants. We first isolated the homologue coding sequences of the abdominal-A gene from 10 species of 8 subfamilies of Formicidae. Our data show that the nucleotide sequence of this gene is highly conserved among the different subfamilies. Second, we used a sequence of 4.5 kbp from this gene as a DNA probe for primary sex ratio determination by FISH. Our results show that this DNA probe hybridizes successfully with its complementary DNA sequence in all ant species tested, and allows reliable determination of the sex of eggs. Our proposed method should greatly facilitate empirical tests of primary sex ratio in ants.