An organism’s fitness depends strongly on its age and size at maturation. Although the evolutionary forces acting on these critical life history traits have been heavily scrutinized, the developmental mechanisms underpinning intraspecific variation in adult size and development time remain much less well understood. Using RNA interference, I here show that the highly conserved sex determination gene doublesex (dsx) mediates sexual size dimorphism (SSD) in the gazelle dung beetle Digitonthophagus gazella. Because doublesex undergoes sex-specific splicing and sex-limited isoforms regulate different target genes, this suggests dsx contributes to the resolution of intralocus sexual conflict in body size. However, these results contrast with previous studies demonstrating that dsx does not affect body size or SSD in Drosophila. This indicates that intraspecific body size variation is underlain by different developmental mechanisms in different insect lineages. Furthermore, although male D. gazella have a longer development time than females, sexual bimaturism was not affected by dsx expression knockdown. In addition, and in contrast to secondary sexual morphology, dsx did not significantly affect nutritional plasticity in life history. Taken together, these findings indicate that dsx signaling contributes to intraspecific life history variation but that dsx’s function in sexual dimorphism in life history differs among traits and species. More generally, these findings suggest that genes ancestrally tasked with sex determination have been coopted into the developmental regulation of life history traits and may represent an underappreciated mechanism of life history evolution.

To investigate the developmental underpinnings of variation in body size and development time, larvae were reared under standardized laboratory conditions, crossing a nutritional manipulation with the application of RNA interference. First, 6 females were haphazardly selected from the laboratory colony and transferred into rectangular oviposition containers (27cm × 17cm × 28cm) that were filled with a sterilized sand-soil mixture and topped off with ca. 800g defrosted cow dung. Reproductively active females dig vertical tunnels (typically 10-30cm deep) immediately underneath the dung pat and, pulling dung form the surface, construct several compact spheres out of dung in which a single egg is laid. After 5 days, these so-called ‘brood balls’ were sifted from the soil. Because body size is strongly dependent on larval nutrition and maternal investment in this species (Moczek, 1998), offspring were removed from their natal brood balls and placed in standardized, artificial brood balls as described previously (Shafiei, Moczek & Nijhout, 2001). In brief, all natal brood balls were opened and eggs or newly hatched first instar larvae (L1) were transferred into separate wells of a standard 12-well tissue culture plate.

To manipulate larval nutrition, half or all animals received a full well (3.2g) of homogenized cow dung, while the other half received only 50% as much food (1.6g). These two treatments are hereafter referred to as high- and low- quality nutrition, respectively. Before the start of the experiment, cow dung was thoroughly mixed using a hand-held electric cement mixer (Nordstrand, PWT-PM0) at the start of the experiments and several aliquots were frozen and thawed for larval rearing as needed.

To assess the function of doublesex (dsx) in the regulation of life history, I applied RNA interference (RNAi). RNAi was applied in half of all individuals within a given 12-well plate including individuals subjected to both nutritional treatments. In brief, dsx template DNA was amplified by PCR using dsx-specific primers attached to a T7 promoter sequence. MEGAscript T7 transcription and MEGAclear kits (Invitrogen) were used to synthesize and purify dsRNA. dsRNA was then diluted in injection buffer to reach a concentration of 1.0μg/μl dsRNA. Using a hand-held syringe, 3μg dsRNA were consequently injected into the thorax of early L3 larvae. Control injections were performed by injecting buffer solution only. Larvae were inspected daily and the age at pupation, as well as the age at adult emergence was recorded. Pupae were weighed using a Mettler Toledo (AL54 Ohio, USA, d = 0.1mg) scale. After complete sclerotization, emerging adults were sacrificed and stored in 70% ethanol.

Calibrated pictures of the pronotum, the fore and hind legs, the elytra, as well as the head of each adult individual were obtained using a digital camera (Scion, Frederick, MD, USA) mounted on a Leica MZ-16 stereomicroscope (Bannockburn, IL, USA). Using tpsDig2, I then took eight linear measures for pronotum width, pronotum length, elytra length, elytra width, metatibia length, profemur length, profemur width, and head width.