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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 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. 

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. 

Abstract Plastic responses to environmental conditions may themselves depend on other environmental conditions, but how such environment-by-environment (E×E) interactions may impact evolution remains unclear. We investigate how temperature shapes the nutritional polyphenism in horn length in a beetle and test whether “allometric plasticity” (a form of E×E) predicts latitudinal differentiation during a rapid range expansion. Rearing populations under common garden conditions demonstrates that increased temperatures reduce the body size threshold separating two male morphs in all populations but also that the magnitude of temperature-dependent changes in allometry diverged across recently established populations. Furthermore, we found a latitudinal increase in the threshold in the species’ exotic range at one of the temperatures, suggesting that allometric plasticity in response to temperature may predict evolved clinal differences. Our findings demonstrate that E×E interactions can be similar in magnitude to G×E interactions and that allometric plasticity and its evolution may impact population’s responses to environmental changes. 

The degree to which developmental systems bias the phenotypic effects of environmental and genetic variation, and how these biases affect evolution, is subject to much debate. Here, we assess whether developmental variability in beetle horn shape aligns with the phenotypic effects of plasticity and evolutionary divergence, yielding three salient results. First, we find that most pathways previously shown to regulate horn length also affect shape. Second, we find that the phenotypic effects of manipulating divergent developmental pathways are correlated with each other as well as multivariate fluctuating asymmetry—a measure of developmental variability. Third, these effects further aligned with thermal plasticity, population differences and macroevolutionary divergence between sister taxa and more distantly related species. Collectively, our results support the hypothesis that changes in horn shape—whether brought about by environmentally plastic responses, functional manipulations or evolutionary divergences—converge along ‘developmental lines of least resistance’, i.e. are biased by the developmental system underpinning horn shape. 

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. 

Abstract In this study, we explored the potential contribution of the gut microbiome to reproductive isolation in tunnelling dung beetles, usingOnthophagus taurus(Schreber, 1759) and its sister speciesO. illyricus(Scopoli, 1763) as a model system (Coleoptera: Scarabaeidae: Scarabaeinae: Onthophagini). Gut microbiota play critical roles in normative development of these beetles, and are vertically inherited via a maternally derived faecal pellet called thepedestal. We first compared the developmental outcomes of individuals reared with pedestals derived from either the same or the sister species (SelfandCrossinoculation treatments, respectively). We then crossed the resulting adultO. taurusin three combinations (Selffemale XSelfmale;Selffemale XCrossmale;Crossfemale XSelfmale). We predicted that if the vertically transmitted gut microbiome plays a role in reproductive isolation by facilitating species recognition, theSelfXSelfline would have improved reproductive outcomes compared to the lines in which partners had mismatched gut microbiomes. Instead, we found that between‐partner concordance of maternally transmitted gut microbiota resulted in fewer offspring, and that this reduction was due to partial pre‐copulatory isolation as evidenced by reduced sperm transfer in theSelfXSelfline. This pattern is consistent either with microbiome‐mediated familiarity/kin recognition, or with absence of mate choice in crosses with mismatched microbiomes. We discuss our results in the light of recent research on the influence of extracellular microbial symbionts over insects' mating preferences.