Social insect castes represent some of the most spectacular examples of phenotypic plasticity, with each caste being associated with different environmental conditions during their life. Here we examine the level of genetic variation in different castes of two polyandrous species of Acromyrmex leaf-cutting ant for the antibiotic-producing metapleural gland, which has a major role in defence against parasites. Gland size increases allometrically. The small workers that play the main role in disease defence have relatively large glands compared with larger workers, while the glands of gynes are substantially larger than those of any workers, for their body size. The gland size of large workers varies significantly between patrilines in both Acromyrmex echinatior and Acromyrmex octospinosus . We also examined small workers and gynes in A. echinatior , again finding genetic variation in gland size in these castes. There were significant positive relationships between the gland sizes of patrilines in the different castes, indicating that the genetic mechanism underpinning the patriline variation has remained similar across phenotypes. The level of expressed genetic variation decreased from small workers to large workers to gynes. This is consistent with the hypothesis that there is individual selection on disease defence in founding queens and colony-level selection on disease defence in the worker castes. Proc Biol Sci 137 procb RSPB Proceedings of the Royal Society B: Biological Sciences 0962-8452 1471-2954 The Royal Society PMC2842681 PMC2842681.1 2842681 2842681 19864289 10.1098/rspb.2009.1415 rspb20091415 1 Research articles 1001 70 Caste-specific expression of genetic variation in the size of antibiotic-producing glands of leaf-cutting ants Hughes W. O. H. 1 2 * Bot A. N. M. 3 Boomsma J. J. Here we examine the level of genetic variation in different castes of two polyandrous species of Acromyrmex leaf-cutting ant for the antibiotic-producing metapleural gland, which has a major role in defence against parasites. Gland size increases allometrically. The small workers that play the main role in disease defence have relatively large glands compared with larger workers, while the glands of gynes are substantially larger than those of any workers, for their body size. The gland size of large workers varies significantly between patrilines in both Acromyrmex echinatior and Acromyrmex octospinosus . We also examined small workers and gynes in A. Queens are faced with very high disease pressure when founding colonies (which they do independently), but are protected by workers before and after this stage. The environmental conditions experienced over the lifetime of the different castes can therefore be markedly different and they may express different disease resistance as a result. A key component of disease defence in leaf-cutting ants is the metapleural gland. This paired structure consists of bundles of secretory cells, a collection sac and a storage chamber (the bulla), which forms a visible bulge in the exterior surface ( Hölldobler & Wilson 1990 ; Bot & Boomsma 1996 ; Bot et al . 2001 ). The secretion from the gland contains at least 20 compounds ( Ortius-Lechner et al . 2000 b ), many of which have antibiotic properties ( Bot et al . 2002 ). Ants actively spread the secretion over their cuticle and mouthparts, and also actively apply it to the fungus garden, reducing the viability of parasite spores ( Hughes et al . 2002 ; Fernandez-Marin et al . 2006 , 2009 ). The secretion makes ants with functional glands substantially more resistant to parasites ( Poulsen et al . 2002 b ) and is also likely to be important in protecting the mutualistic fungus garden against competitive or pathogenic micro-organisms. Indeed, there is comparative evidence for such a trade-off, with the leaf-cutting ants having evolved substantially larger metapleural glands than all the other members of their tribe owing to a change in parasite pressure ( Hughes et al . 2008 b ) and a socially parasitic species having evolved smaller glands because it relies on its host for protection ( Sumner et al . 2003 ). Investment in the metapleural gland by leaf-cutting ants thus seems likely to vary between caste phenotypes, both in the overall mean and in terms of expressed genetic variation for the trait. In order to allow the measurements of metapleural gland diameter to be standardized for variation in body size, a photograph and measurement were also made of the width of the ventral side of the pronotum. (a) Molecular analysis To determine the genotypes of the ants sampled, a single leg (for gynes), a pair (for large workers) or all six legs (for small workers) were removed from the ants. DNA was extracted from the legs using Chelex beads (BioRad; A. echinatior ) or the CTAB procedure ( A. octospinosus ). The A. octospinosus samples were amplified at the microsatellite loci Ech1390, Ech3385 and Ech4126, while the A. 2002 a , 2006 ), they also generally spend their entire life working within the nest, so that their antibiotic defence consists not just of their own secretion but also that of the other small workers with which they interact. In this caste, the metapleural gland defence is therefore largely a collective trait, so that colony-level selection may favour the expression of a wide continuous range of phenotypes around the caste-specific mean values with the expression of patriline-specific genetic variation for metapleural gland size being unconstrained. This contrasts with the body size of workers where caste-specific optima apply and genetic variation around these means is hardly expressed. The metapleural gland may therefore have a greater role in this worker caste as an individual defence mechanism than in small workers, but less than in gynes. The seemingly strange pattern of the two female castes for whom