A new suite of reporter vectors and a novel landing site survey system to study cis-regulatory elements in diverse insect species.

Comparative analyses between traditional model organisms, such as the fruit fly Drosophila melanogaster, and more recent model organisms, such as the red flour beetle Tribolium castaneum, have provided a wealth of insight into conserved and diverged aspects of gene regulation. While the study of trans-regulatory components is relatively straightforward, the study of cis-regulatory elements (CREs, or enhancers) remains challenging outside of Drosophila. A central component of this challenge has been finding a core promoter suitable for enhancer-reporter assays in diverse insect species. Previously, we demonstrated that a Drosophila Synthetic Core Promoter (DSCP) functions in a cross-species manner in Drosophila and Tribolium. Given the over 300 million years of divergence between the Diptera and Coleoptera, we reasoned that DSCP-based reporter constructs will be useful when studying cis-regulation in a variety of insect models across the holometabola and possibly beyond. To this end, we sought to create a suite of new DSCP-based reporter vectors, leveraging dual compatibility with piggyBac and PhiC31-integration, the 3xP3 universal eye marker, GATEWAY cloning, different colors of reporters and markers, as well as Gal4-UAS binary expression. While all constructs functioned properly with a Tc-nub enhancer in Drosophila, complications arose with tissue-specific Gal4-UAS binary expression in Tribolium. Nevertheless, the functionality of these constructs across multiple holometabolous orders suggests a high potential compatibility with a variety of other insects. In addition, we present the piggyLANDR (piggyBac-LoxP AttP Neutralizable Destination Reporter) platform for the establishment of proper PhiC31 landing sites free from position effects. As a proof-of-principle, we demonstrated the workflow for piggyLANDR in Drosophila. The potential utility of these tools ranges from molecular biology research to pest and disease-vector management, and will help advance the study of gene regulation beyond traditional insect models.

Problems with paralogs: the promise and challenges of gene duplicates in evo-devo research.

Gene duplicates, or paralogs, serve as a major source of new genetic material and comprise seeds for evolutionary innovation. While originally thought to be quickly lost or non-functionalized following duplication, now a vast number of paralogs are known to be retained in a functional state. Daughter paralogs can provide robustness through redundancy, specialize via sub-functionalization, or neo-functionalize to play new roles. Indeed, the duplication and divergence of developmental genes have played a monumental role in the evolution of animal forms (e.g. Hox genes). Still, despite their prevalence and evolutionary importance, the precise detection of gene duplicates in newly sequenced genomes remains technically challenging and often overlooked. This presents an especially pertinent problem for evolutionary developmental biology (evo-devo), where hypothesis testing requires accurate detection of changes in gene expression and function, often in non-traditional model species. Frequently, these analyses rely on molecular reagents designed within coding sequences that may be highly similar in recently duplicated paralogs, leading to cross-reactivity and spurious results. Thus, care is needed to avoid erroneously assigning diverged functions of paralogs to a single gene, and potentially misinterpreting evolutionary history. This perspective aims to overview the prevalence and importance of paralogs and to shed light on the difficulty of their detection and analysis while offering potential solutions.

Evolution and molecular mechanisms of wing plasticity in aphids.

Aphids present a fascinating example of phenotypic plasticity, in which a single genotype can produce dramatically different winged and wingless phenotypes that are specialized for dispersal versus reproduction, respectively. Recent work has examined many aspects of this plasticity, including its evolution, molecular control mechanisms, and genetic variation underlying the trait. In particular, exciting discoveries have been made about the signaling pathways that are responsible for controlling the production of winged versus wingless morphs, including ecdysone, dopamine, and insulin signaling, and about how specific genes such as REPTOR2 and vestigial are regulated to control winglessness. Future work will likely focus on the role of epigenetic mechanisms, as well as developing transgenic tools for more thoroughly dissecting the role of candidate plasticity-related genes.

Transcriptomic exploration of the Coleopteran wings reveals insight into the evolution of novel structures associated with the beetle elytron.

The acquisition of novel traits is central to organismal evolution, yet the molecular mechanisms underlying this process are elusive. The beetle forewings (elytra) are evolutionarily modified to serve as a protective shield, providing a unique opportunity to study these mechanisms. In the past, the orthologs of genes within the wing gene network from Drosophila studies served as the starting point when studying the evolution of elytra (candidate genes). Although effective, candidate gene lists are finite and only explore genes conserved across species. To go beyond candidate genes, we used RNA sequencing and explored the wing transcriptomes of two Coleopteran species, the red flour beetle (Tribolium castaneum) and the Japanese stag beetle (Dorcus hopei). Our analysis revealed sets of genes enriched in Tribolium elytra (57 genes) and genes unique to the hindwings, which possess more "typical" insect wing morphologies (29 genes). Over a third of the hindwing-enriched genes were "candidate genes" whose functions were previously analyzed in Tribolium, demonstrating the robustness of our sequencing. Although the overlap was limited, transcriptomic comparison between the beetle species found a common set of genes, including key wing genes, enriched in either elytra or hindwings. Our RNA interference analysis for elytron-enriched genes in Tribolium uncovered novel genes with roles in forming various aspects of morphology that are unique to elytra, such as pigmentation, hardening, sensory development, and vein formation. Our analyses deepen our understanding of how gene network evolution facilitated the emergence of the elytron, a unique structure critical to the evolutionary success of beetles.

Butterfly eyespots evolved via cooption of an ancestral gene-regulatory network that also patterns antennae, legs, and wings.

Butterfly eyespots are beautiful novel traits with an unknown developmental origin. Here we show that eyespots likely originated via cooption of parts of an ancestral appendage gene-regulatory network (GRN) to novel locations on the wing. Using comparative transcriptome analysis, we show that eyespots cluster most closely with antennae, relative to multiple other tissues. Furthermore, three genes essential for eyespot development, Distal-less (Dll), spalt (sal), and Antennapedia (Antp), share similar regulatory connections as those observed in the antennal GRN. CRISPR knockout of cis-regulatory elements (CREs) for Dll and sal led to the loss of eyespots, antennae, legs, and also wings, demonstrating that these CREs are highly pleiotropic. We conclude that eyespots likely reused an ancient GRN for their development, a network also previously implicated in the development of antennae, legs, and wings.

Enhancer identification and activity evaluation in the red flour beetle, Tribolium castaneum.

Evolution of cis-regulatory elements (such as enhancers) plays an important role in the production of diverse morphology. However, a mechanistic understanding is often limited by the absence of methods for studying enhancers in species other than established model systems. Here, we sought to establish methods to identify and test enhancer activity in the red flour beetle, Tribolium castaneum To identify possible enhancer regions, we first obtained genome-wide chromatin profiles from various tissues and stages of Tribolium using FAIRE (formaldehyde-assisted isolation of regulatory elements)-sequencing. Comparison of these profiles revealed a distinct set of open chromatin regions in each tissue and at each stage. In addition, comparison of the FAIRE data with sets of computationally predicted (i.e. supervised cis-regulatory module-predicted) enhancers revealed a very high overlap between the two datasets. Second, using nubbin in the wing and hunchback in the embryo as case studies, we established the first universal reporter assay system that works in various contexts in Tribolium, and in a cross-species context. Together, these advances will facilitate investigation of cis-evolution and morphological diversity in Tribolium and other insects.