Hedgehog signaling enables nutrition-responsive inhibition of an alternative morph in a polyphenic beetle

Abstract

The recruitment of modular developmental genetic components into new developmental contexts has been proposed as a central mechanism enabling the origin of novel traits and trait functions without necessitating the origin of novel pathways. Here, we investigate the function of the hedgehog (Hh) signaling pathway, a highly conserved pathway best understood for its role in patterning anterior/posterior (A/P) polarity of diverse traits, in the developmental evolution of beetle horns, an evolutionary novelty, and horn polyphenisms, a highly derived form of environment-responsive trait induction. We show that interactions among pathway members are conserved during development of Onthophagus horned beetles and have retained the ability to regulate A/P polarity in traditional appendages, such as legs. At the same time, the Hh signaling pathway has acquired a novel and highly unusual role in the nutrition-dependent regulation of horn polyphenisms by actively suppressing horn formation in low-nutrition males. Down-regulation of Hh signaling lifts this inhibition and returns a highly derived sigmoid horn body size allometry to its presumed ancestral, linear state. Our results suggest that recruitment of the Hh signaling pathway may have been a key step in the evolution of trait thresholds, such as those involved in horn polyphenisms and the corresponding origin of alternative phenotypes and complex allometries.

Keywords: allometry; co-option; developmental plasticity; modularity; threshold trait.

Fig. 1.

Effect of hh, smo, and ptc dsRNA injection on Onthophagus development. Prothorax and head of (Left) control-injected, (Center) smo^RNAi, and (Right) ptc^RNAi animals are shown. (A–C) Comparison of the prothorax from the ventral side. The differences of smo and ptc function in prothoracic development are most obvious in the sternum (stn; green), episternum (eps; blue), and epimeron (epm; yellow). Note that in situ size of the coxa (area shaded black) is reduced in smo^RNAi individuals relative to control-injected and ptc^RNAi animals. (D–F) Development of the notum is also affected by RNAi. (E) Note that the anterior edge of the pronotum (thick dashed lines in D–F) extends anteriorly in smo^RNAi animals, whereas (F) ptc^RNAi results in significant reduction of the anterior pronotal edge. (G–I) Lateral view of the same prothoraces; smo^RNAi animals exhibit prominent bulging of the pronotum, whereas ptc^RNAi animals lack the anterior region (blue arrows). (J–L) Frontal view of the same prothoraces. (K) Effect of smo^RNAi was minimal on the anterior edge, whereas (L) ptc^RNAi resulted in development of ectopic bristles. (M–O) Frontal view of the head. Compound eyes are labeled in green and highlighted by blue arrows. Head shape was only lightly affected by RNAi. (N) All but one smo^RNAi animal developed full-sized horns, whereas (O) horns in all ptc^RNAi animals were vestigial. (P–R) Lateral view of the head. (Q) Compound eyes are significantly reduced in smo^RNAi animals but (R) not reduced in ptc^RNAi animals. Images of thoraces as well as heads are taken under the same magnification. cox, coxa; fem, femur; stl, sternellum; tib, tibia.

Fig. S1.

Effect of smo^RNAi and ptc^RNAi in leg development. (Left) Ventral views of head and prothorax of control, smo^RNAi, and ptc^RNAi males. Right femur is labeled in blue. Animals’ anterior is to the top. (Right) Details of foreleg phenotypes, with coxa (cox), femur (fem), and tibia (tib) indicated. The femur is outlined to contrast differences in morphology between treatments. Note changes in bristle pattern on the femur, which was affected by RNAi, suggesting disruption of axis formation during late leg development. Arrows indicate anterior (A), posterior (Po), distal (D), and proximal (Pr) axes.

Fig. 2.

Global effects of dsRNA injection. Representative phenotypes of (Left) adults (frontal view is on the left, and lateral view is on the right) and (Right) pupae (lateral view) injected with (A) a control construct and dsRNA targeting (B) hh, (C) smo, and (D) ptc, respectively. Arrowheads indicate legs on the first thoracic segment (red) and wings on pupae (light blue).

Fig. S2.

Representative phenotypes obtained from double knockdowns. (Upper) Control-injected large male. (Lower Left) hh/ptc^RNAi, hh/smo^RNAi, and ptc/smo^RNAi from top to bottom, respectively. (Lower Right) Single-gene RNAi phenotypes for comparison (images are the same as in Fig. 2): hh^RNAi, ptc^RNAi, and smo^RNAi from top to bottom, respectively.

Fig. 3.

Effect of hh^RNAi and smo^RNAi on horn development and relative horn sizes in male O. taurus pupae. (A) Control-injected animals (gray triangles) exhibited the species-typical sigmoidal relationship between body size (x axis) and head horn size (y axis); smo^RNAi (●), in contrast, resulted in nearly all males developing full-sized horns regardless of body size and a complete linearization of the body size horn length allometry. Lastly, hh^RNAi (□) also resulted in relatively longer horns, but this effect was limited to a subset of males of intermediate body size. (B) hh^RNAi (□) modestly increased thoracic horn length in some animals, whereas smo^RNAi (●) increased horn length in all individuals, especially small, low-nutrition individuals.

Fig. 4.

Proposed models for the development of nutrition-dependent expression of alternative horned and hornless male morphs separated by a sharp body size threshold. (A) Results presented here suggest that (i) the Hh signal pathway negatively regulates horn growth in low-nutrition male beetles, whereas (ii) previous work implicated the somatic sex determination gene dsx in mediating nutrition-dependent exaggeration of horns under high nutrition. (B) Combined, dsx-mediated promotion of horns in high-nutrition individuals and Hh-mediated inhibition of horns in low-nutrition individuals have the potential to transform ancestral, linear into strongly sigmoidal scaling relationships characterized by a bimodal distribution of male phenotypes and the establishment of a sharp allometric size threshold.

Fig. S3.

Validation of RNAi by quantitative RT-PCR. Animals injected with hh, smo, or ptc dsRNA were harvested as early pupae and subjected to two-step quantitative RT-PCR. The effect of RNAi was assessed relative to each gene’s expression levels in control animals.