DOI: 10.5061/dryad.prr4xgxmd
title: A role for sex determination genes in life history evolution? Doublesex mediates sexual size dimorphism in the gazelle dung beetle

description: 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.
description: 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 (<i>dsx)</i> 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, <i>dsx</i>
 template DNA was amplified by PCR using <i>dsx</i>-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.