Rhinoceros beetle horn development reveals deep parallels with dung beetles.
ABSTRACT: Beetle horns are attractive models for studying the evolution of novel traits, as they display diverse shapes, sizes, and numbers among closely related species within the family Scarabaeidae. Horns radiated prolifically and independently in two distant subfamilies of scarabs, the dung beetles (Scarabaeinae), and the rhinoceros beetles (Dynastinae). However, current knowledge of the mechanisms underlying horn diversification remains limited to a single genus of dung beetles, Onthophagus. Here we unveil 11 horn formation genes in a rhinoceros beetle, Trypoxylus dichotomus. These 11 genes are mostly categorized as larval head- and appendage-patterning genes that also are involved in Onthophagus horn formation, suggesting the same suite of genes was recruited in each lineage during horn evolution. Although our RNAi analyses reveal interesting differences in the functions of a few of these genes, the overwhelming conclusion is that both head and thoracic horns develop similarly in Trypoxylus and Onthophagus, originating in the same developmental regions and deploying similar portions of appendage patterning networks during their growth. Our findings highlight deep parallels in the development of rhinoceros and dung beetle horns, suggesting either that both horn types arose in the common ancestor of all scarabs, a surprising reconstruction of horn evolution that would mean the majority of scarab species (~35,000) actively repress horn growth, or that parallel origins of these extravagant structures resulted from repeated co-option of the same underlying developmental processes.
Project description:Scarab beetles exhibit an astonishing variety of rigid exo-skeletal outgrowths, known as "horns". These traits are often sexually dimorphic and vary dramatically across species in size, shape, location, and allometry with body size. In many species, the horn exhibits disproportionate growth resulting in an exaggerated allometric relationship with body size, as compared to other traits, such as wings, that grow proportionately with body size. Depending on the species, the smallest males either do not produce a horn at all, or they produce a disproportionately small horn for their body size. While the diversity of horn shapes and their behavioural ecology have been reasonably well studied, we know far less about the proximate mechanisms that regulate horn growth. Thus, using 454 pyrosequencing, we generated transcriptome profiles, during horn growth and development, in two different scarab beetle species: the Asian rhinoceros beetle, Trypoxylus dichotomus, and the dung beetle, Onthophagus nigriventris. We obtained over half a million reads for each species that were assembled into over 6,000 and 16,000 contigs respectively. We combined these data with previously published studies to look for signatures of molecular evolution. We found a small subset of genes with horn-biased expression showing evidence for recent positive selection, as is expected with sexual selection on horn size. We also found evidence of relaxed selection present in genes that demonstrated biased expression between horned and horn-less morphs, consistent with the theory of developmental decoupling of phenotypically plastic traits.
Project description:Many scarab beetles have sexually dimorphic exaggerated horns that are an evolutionary novelty. Since the shape, number, size, and location of horns are highly diverged within Scarabaeidae, beetle horns are an attractive model for studying the evolution of sexually dimorphic and novel traits. In beetles including the Japanese rhinoceros beetle Trypoxylus dichotomus, the sex differentiation gene doublesex (dsx) plays a crucial role in sexually dimorphic horn formation during larval-pupal development. However, knowledge of when and how dsx drives the gene regulatory network (GRN) for horn formation to form sexually dimorphic horns during development remains elusive. To address this issue, we identified a Trypoxylus-ortholog of the sex determination gene, transformer (tra), that regulates sex-specific splicing of the dsx pre-mRNA, and whose loss of function results in sex transformation. By knocking down tra function at multiple developmental timepoints during larval-pupal development, we estimated the onset when the sex-specific GRN for horn formation is driven. In addition, we also revealed that dsx regulates different aspects of morphogenetic activities during the prepupal and pupal developmental stages to form appropriate morphologies of pupal head and thoracic horn primordia as well as those of adult horns. Based on these findings, we discuss the evolutionary developmental background of sexually dimorphic trait growth in horned beetles.
Project description:Male-specific exaggerated horns are an evolutionary novelty and have diverged rapidly via intrasexual selection. Here, we investigated the function of the conserved sex-determination gene doublesex (dsx) in the Japanese rhinoceros beetle (Trypoxylus dichotomus) using RNA interference (RNAi). Our results show that the sex-specific T. dichotomus dsx isoforms have an antagonistic function for head horn formation and only the male isoform has a role for thoracic horn formation. These results indicate that the novel sex-specific regulation of dsx during horn morphogenesis might have been the key evolutionary developmental event at the transition from sexually monomorphic to sexually dimorphic horns.
Project description:Morphological diversity arises during development through the actions and interactions of diverse developmental pathways. Among those, the Wnt pathway is known to contribute to diverse developmental processes such as segmentation and the morphogenesis of appendages. Here, we characterize a transcription factor in the Wnt pathway, pangolin (pan), to investigate the role of Wnt signaling in the development of evolutionarily novel body structures: the horns of beetles. Beetle horns are highly diverse in size, shape, and number and develop principally from two major body regions: the head and prothorax. We investigate horns in two species of the genus Onthophagus using comparative in situ hybridization, larval RNA interference, and allometric measurements to analyze whether horn formation is regulated by pan and by extension the Wnt pathway. Our results illustrate that pan expression affects beetle horn growth in a species-, sex-, and location-specific manner in two morphologically distinct, yet closely-related, Onthophagus species.
Project description:The origin and integration of novel traits are fundamental processes during the developmental evolution of complex organisms. Yet how novel traits integrate into pre-existing contexts remains poorly understood. Beetle horns represent a spectacular evolutionary novelty integrated within the context of the adult dorsal head, a highly conserved trait complex present since the origin of insects. We investigated whether otd1/2 and six3, members of a highly conserved gene network that instructs the formation of the anterior end of most bilaterians, also play roles in patterning more recently evolved traits. Using ablation-based fate-mapping, comparative larval RNA interference (RNAi) and transcript sequencing, we found that otd1/2, but not six3, play a fundamental role in the post-embryonic formation of the adult dorsal head and head horns of Onthophagus beetles. By contrast, neither gene appears to pattern the adult head of Tribolium flour beetles even though all are expressed in the dorsal head epidermis of both Onthophagus and Tribolium We propose that, at least in beetles, the roles of otd genes during post-embryonic development are decoupled from their embryonic functions, and that potentially non-functional post-embryonic expression in the dorsal head facilitated their co-option into a novel horn-patterning network during Onthophagus evolution.
Project description:This series examines gene expression patterns in the head horns, thoracic horns, and legs of the horned beetles Onthophagus taurus. Expression in each of these tissues was compared to that in common non-appendage reference - abdominal epithelium. The series consists of three pair-wise comparisons: head horn versus abdominal epithelium, thoracic horn versus abdominal epithelium and legs versus abdominal epithelium. Each tissue sample was obtained by pooling tissue dissected from four pupae. Samples compared on the same array were derived from tissues dissected from the same four animals. Five independent biological replicates were performed for each comparison with dye flips (three in one direction and two in the opposite direction).
Project description:Research suggests dung beetles can churn, aerate, and desiccate dung in ways that influence the dung and soil microbes producing greenhouse gases (GHGs). We examined the impacts of the tunneling beetle, Onthophagus taurus (Schreber), and the dwelling beetle, Labarrus pseudolividus (Balthasar), on the carbon dioxide (CO2), methane (CH4), and nitrous oxide (N2O) emitted from pasture-laid bovine dung as well as their sum-total (CO2 + CH4 + N2O) effect on global warming, or their carbon dioxide equivalent (CO2e). Despite dung beetles potential effects on CH4 and N2O, the existing literature shows no ultimate CO2e reductions. We hypothesized that more dung beetles would degrade pats faster and reduce CO2e, and so we increased the average dung beetle biomass per dung volume 6.22× above previously published records, and visually documented any dung damage. However, the time effects were 2-5× greater for any GHG and CO2e (E = 0.27-0.77) than dung beetle effects alone (E = 0.09-0.24). This suggests that dung beetle communities cannot adequately reduce GHGs unless they can accelerate dung decomposition faster than time alone.
Project description:This series examines gene expression patterns in the head horns, thoracic horns, and legs of the horned beetles Onthophagus taurus. Expression in each of these tissues was compared to that in common non-appendage reference - abdominal epithelium. Overall design: The series consists of three pair-wise comparisons: head horn versus abdominal epithelium, thoracic horn versus abdominal epithelium and legs versus abdominal epithelium. Each tissue sample was obtained by pooling tissue dissected from four pupae. Samples compared on the same array were derived from tissues dissected from the same four animals. Five independent biological replicates were performed for each comparison with dye flips (three in one direction and two in the opposite direction).
Project description:Sex-specific trait expression is frequently associated with highly variable, condition-dependent expression within sexes and rapid divergence among closely related species. Horned beetles are an excellent example for studying the molecular basis of these phenomena because horn morphology varies markedly among species, between sexes, and among alternative, nutritionally-cued morphs within sexes. In addition, horns lack obvious homology to other insect traits and provide a good opportunity to explore the molecular basis of the rapid diversification of a novel trait within and between species. Here we show that the sex-determination gene doublesex (dsx) underlies important aspects of horn development, including differences between sexes, morphs, and species. In male Onthophagus taurus, dsx transcripts were preferentially expressed in the horns of the large, horned morph, and RNAi-mediated knockdown of dsx dramatically altered male horn allometry by massively reducing horn development in large males, but not in smaller males. Conversely, dsx RNAi induced ectopic, nutrition-sensitive horn development in otherwise hornless females. Finally, in a closely related species (Onthophagus sagittarius) that has recently evolved a rare reversed sexual dimorphism, dsx RNAi revealed reversed as well as novel dsx functions despite an overall conservation of dsx expression. This suggests that rapid evolution of dsx functions has facilitated the transition from a regular sexual dimorphism to a reversed sexual dimorphism in this species. Our findings add beetle horns to existing examples of a close relationship between dsx and sexual trait development, and suggest that dsx function has been coopted to facilitate both the evolution of environmentally-cued intrasexual dimorphisms and rapid species divergences in a novel trait.
Project description:The external organs of holometabolous insects are generated through two consecutive processes: the development of imaginal primordia and their subsequent transformation into the adult structures. During the latter process, many different phenomena at the cellular level (e.g. cell shape changes, cell migration, folding and unfolding of epithelial sheets) contribute to the drastic changes observed in size and shape. Because of this complexity, the logic behind the formation of the 3D structure of adult external organs remains largely unknown. In this report, we investigated the metamorphosis of the horn in the Japanese rhinoceros beetle Trypoxylus dichotomus. The horn primordia is essentially a 2D epithelial cell sheet with dense furrows. We experimentally unfolded these furrows using three different methods and found that the furrow pattern solely determines the 3D horn structure, indicating that horn formation in beetles occurs by two distinct processes: formation of the furrows and subsequently unfolding them. We postulate that this developmental simplicity offers an inherent advantage to understanding the principles that guide 3D morphogenesis in insects.