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ABSTRACT Sex‐specific trait expression represents a striking dimension of morphological variation within and across species. The mechanisms instructing sex‐specific organ development have been well studied in a small number of insect model systems, suggesting striking conservation in some parts of the somatic sex determination pathway while hinting at possible evolutionary lability in others. However, further resolution of this phenomenon necessitates additional taxon sampling, particularly in groups in which sexual dimorphisms have undergone significant elaboration and diversification. Here, we functionally investigate the somatic sex determination pathway in the gazelle dung beetleDigitonthophagus gazella, an emerging model system in the study of the development and evolution of sexual dimorphisms. We find that RNA interference (RNAi) targetingtransformer (tra)caused chromosomal females to develop morphological traits largely indistinguishable from those normally only observed in males, and thattra^RNAiis sufficient to induce splicing of the normally male‐specific isoform ofdoublesexin chromosomal females, while leaving males unaffected. Further,intersex^RNAiwas found to phenocopy previously described RNAi phenotypes ofdoublesexin female but not male beetles. These findings match predictions derived from models of the sex determination cascade as developed largely through studies inDrosophila melanogaster. In contrast, efforts to targettransformer2via RNAi resulted in high juvenile mortality but did not appear to affectdoublesexsplicing, whereas RNAi targetingSex‐lethaland two putative orthologs ofhermaphroditeyielded no obvious phenotypic modifications in either males or females, raising the possibility that the function of a subset of sex determination genes may be derived in select Diptera and thus nonrepresentative of their roles in other holometabolous orders. Our results help illuminate how the differential evolutionary lability of the somatic sex determination pathway has contributed to the extraordinary morphological diversification of sex‐specific trait expression found in nature. 

Abstract Understanding the origin of novel morphological traits is a long‐standing objective in evolutionary developmental biology. We explored the developmental genetic mechanisms that underpin the formation of a textbook example of evolutionary novelties, the cephalic horns of beetles. Previous work has implicated the gene regulatory networks associated with compound eye and ocellar development in horn formation and suggested that horns and compound eyes may influence each other's sizes. Therefore, we investigated the functional significance of genes central to visual system formation in the initiation, patterning, and size determination of head horns across three horned beetle species. We find that while the downregulation of canonical eye patterning genes reliably reduces or eliminates compound eye formation, it does not alter the position or shape of head horns yet does result in an increase in relative horn length. We discuss the implications of our results for our understanding of the genesis of cephalic horns in particular and evolutionary novelties in general. 

Abstract BackgroundNutrient availability is among the most widespread means by which environmental variability affects developmental outcomes. Because almost all cells within an individual organism share the same genome, structure-specific growth responses must result from changes in gene regulation. Earlier work suggested thathistone deacetylases(HDACs) may serve as epigenetic regulators linking nutritional conditions to trait-specific development. Here we expand on this work by assessing the function of diverseHDACsin the structure-specific growth of both sex-shared and sex-specific traits including evolutionarily novel structures in the horned dung beetleOnthophagus taurus. ResultsWe identified fiveHDACmembers whose downregulation yielded highly variable mortality depending on whichHDACmember was targeted. We then show thatHDAC1,3, and4operate in both a gene- and trait-specific manner in the regulation of nutrition-responsiveness of appendage size and shape. Specifically,HDAC 1, 3,or4knockdown diminished wing size similarly while leg development was differentially affected by RNAi targetingHDAC3andHDAC4. In addition, depletion ofHDAC3transcript resulted in a more rounded shape of genitalia at the pupal stage and decreased the length of adult aedeagus across all body sizes. Most importantly, we find thatHDAC3andHDAC4pattern the morphology and regulate the scaling of evolutionarily novel head and thoracic horns as a function of nutritional variation. ConclusionCollectively, our results suggest that both functional overlap and division of labor amongHDACmembers contribute to morphological diversification of both conventional and recently evolved appendages. More generally, our work raises the possibility thatHDAC-mediated scaling relationships and their evolution may underpin morphological diversification within and across insect species broadly. 

Phenotypic plasticity is thought to be an important driver of diversification and adaptation to environmental variation, yet the genomic mechanisms mediating plastic trait development and evolution remain poorly understood. The Scarabaeinae, or true dung beetles, are a species-rich clade of insects recognized for their highly diversified nutrition-responsive development including that of cephalic horns—evolutionarily novel, secondary sexual weapons that exhibit remarkable intra- and interspecific variation. Here, we investigate the evolutionary basis for horns as well as other key dung beetle traits via comparative genomic and developmental assays. We begin by presenting chromosome-level genome assemblies of three dung beetle species in the tribe Onthophagini (> 2500 extant species) includingOnthophagus taurus,O.sagittarius, andDigitonthophagus gazella. Comparing these assemblies to those of seven other species across the order Coleoptera identifies evolutionary changes in coding sequence associated with metabolic regulation of plasticity and metamorphosis. We then contrast chromatin accessibility in developing head horn tissues of high- and low-nutritionO.taurusmales and females and identify distinctcis-regulatory architectures underlying nutrition- compared to sex-responsive development, including a large proportion of recently evolved regulatory elements sensitive to horn morph determination. Binding motifs of known and new candidate transcription factors are enriched in these nutrition-responsive open chromatin regions. Our work highlights the importance of chromatin state regulation in mediating the development and evolution of plastic traits, demonstrates gene networks are highly evolvable transducers of environmental and genetic signals, and provides new reference-quality genomes for three species that will bolster future developmental, ecological, and evolutionary studies of this insect group. 

Abstract Static allometry is a major component of morphological variation. Much of the literature on the development of allometry investigates how functional perturbations of diverse pathways affect the relationship between trait size and body size. Often, this is done with the explicit objective to identify developmental mechanisms that enable the sensing of organ size and the regulation of relative growth. However, changes in relative trait size can also be brought about by a range of other distinctly different developmental processes, such as changes in patterning or tissue folding, yet standard univariate biometric approaches are usually unable to distinguish among alternative explanations. Here, we utilize geometric morphometrics to investigate the degree to which functional genetic manipulations known to affect thesizeof dung beetle horns also recapitulate the effect of hornshapeallometry. We reasoned that the knockdown phenotypes of pathways governing relative growth should closely resemble shape variation induced by natural allometric variation. In contrast, we predicted that if genes primarily affect alternative developmental processes, knockdown effects should align poorly with shape allometry. We find that the knockdown effects of several genes (e.g.,doublesex, Foxo) indeed closely aligned with shape allometry, indicating that their corresponding pathways may indeed function primarily in the regulation of relative trait growth. In contrast, other knockdown effects (e.g.,Distal‐less,dachs) failed to align with allometry, implicating these pathways in potentially scaling‐independent processes. Our findings moderate the interpretation of studies focusing on trait length and highlight the usefulness of multivariate approaches to study allometry and phenotypic plasticity. 

Abstract Many organisms actively manipulate the environment in ways that feed back on their own development, a process referred to as developmental niche construction. Yet, the role that constructed biotic and abiotic environments play in shaping phenotypic variation and its evolution is insufficiently understood. Here, we assess whether environmental modifications made by developing dung beetles impact the environment‐sensitive expression of secondary sexual traits. Gazelle dung beetles both physically modify their ontogenetic environment and structure their biotic interactions through the vertical inheritance of microbial symbionts. By experimentally eliminating (i) physical environmental modifications and (ii) the vertical inheritance of microbes, we assess the degree to which (sym)biotic and physical environmental modifications shape the exaggeration of several traits varying in their degree and direction of sexual dimorphism. We expected the experimental reduction of a larva's ability to shape its environment to affect trait size and scaling, especially for traits that are sexually dimorphic and environmentally plastic. We find that compromised developmental niche construction indeed shapes sexual dimorphism in overall body size and the absolute sizes of male‐limited exaggerated head horns, the strongly sexually dimorphic fore tibia length and width, as well as the weakly dimorphic elytron length and width. This suggests that environmental modifications affect sex‐specific phenotypic variation in functional traits. However, most of these effects can be attributed to nutrition‐dependent plasticity in size and non‐isometric trait scaling rather than body‐size‐independent effects on the developmental regulation of trait size. Our findings suggest that the reciprocal relationship between developing organisms, their symbionts, and their environment can have considerable impacts on sexual dimorphism and functional morphology. 

Horned beetles have emerged as a powerful study system with which to investigate the developmental mechanisms underlying environment-responsive development and its evolution. We begin by reviewing key advances in our understanding of the diverse roles played by transcription factors, endocrine regulators, and signal transduction pathways in the regulation of horned beetle plasticity. We then explore recent efforts aimed at understanding how such condition-specific expression may be regulated in the first place, as well as how the differential expression of master regulators may instruct conditional expression of downstream target genes. Here, we focus on the significance of chromatin remodeling as a powerful but thus far understudied mechanism able to facilitate trait-, sex-, and species-specific responses to environmental conditions. 

Abstract Nutrition-dependent growth of sexual traits is a major contributor to phenotypic diversity, and a large body of research documents insulin signalling as a major regulator of nutritional plasticity. However, findings across studies raise the possibility that the role of individual components within the insulin signalling pathway diverges in function among traits and taxa. Here, we use RNAi-mediated transcript depletion in the gazelle dung beetle to investigate the functions of forkhead box O (Foxo) and two paralogs of the insulin receptor (InR1 and InR2) in shaping nutritional plasticity in polyphenic male head horns, exaggerated fore legs, and weakly nutrition-responsive genitalia. Our functional genetic manipulations led to three main findings: FoxoRNAi reduced the length of exaggerated head horns in large males, while neither InR1 nor InR2 knock-downs resulted in measurable horn phenotypes. These results are similar to those documented previously for another dung beetle (Onthophagus taurus), but in stark contrast to findings in rhinoceros beetles. Secondly, knockdown of Foxo, InR1, and InR2 led to an increase in the intercept or slope of the scaling relationship of genitalia size. These findings are in contrast even to results documented previously for O. taurus. Lastly, while FoxoRNAi reduces male forelegs in D. gazella and O. taurus, the effects of InR1 and InR2 knockdowns diverged across dung beetle species. Our results add to the growing body of literature indicating that despite insulin signalling's conserved role as a regulator of nutritional plasticity, the functions of its components may diversify among traits and species, potentially fuelling the evolution of scaling relationships.