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.

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.

Broad complex and wing development in cockroaches.

In hemimetabolan insects, the transcription factor Broad complex (Br-C) promotes wing growth and development during the nymphal period. We wondered whether Br-C could trigger the initiation of wing development, using the cockroach Blattella germanica as a model. We show that first instar nymphs have their unique identity of these three thoracic segments specified. During embryogenesis, the expression of Br-C and some wing-related genes show two matching waves. The first takes place before the formation of the germ band, which might be involved in the establishment of various developmental fields including a potential "wing field", and the second wave around organogenesis, possibly involved in the initiation of wing development. However, the expression of Br-C in early embryogenesis concentrates in the developing central nervous system, thus not co-localizing with the expression of the typical wing-related gene vestigial, which is expressed at the edge of the thoracic and abdominal segments. This suggests that Br-C is not specifically involved in the establishment of a potential "wing field" in early embryogenesis. Moreover, maternal RNAi for Br-C depletes the first wave of Br-C expression but does not affect the early expression of wing-related genes. As maternal Br-C RNAi did not deplete the second expression wave of Br-C, we could not evaluate if Br-C is involved in the initiation of wing development. Alternatively, using nymphal RNAi of Br-C and Sex combs reduced (Scr), we show that Br-C contributes to the formation of ectopic wing structures that develop in the prothorax when Scr is depleted. The gene most clearly influenced by Br-C RNAi is nubbin (nub), which, in nymphs is crucial for wing growth. Together, these results suggest that Br-C does not specifically contribute to the establishment of the "wing field", but it does seem important later, in the initiation of wing development, enhancing the expression of wing-related genes, especially nub. This supports the hypothesis previously proposed by the authors, whereby Br-C might have facilitated the evolution of holometaboly. However, there is no doubt that other factors have also contributed to this evolution.

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.

What crustaceans can tell us about the evolution of insect wings and other morphologically novel structures.

Acquisition of novel structures often has a profound impact on the adaptation of organisms. The wing of insects is one such example, facilitating their massive success and enabling them to become the dominant clade on this planet. However, its evolutionary origin as well as the mechanisms underpinning its evolution remain elusive. Studies in crustaceans, a wingless sister group of insects, have played a pivotal role in the wing origin debate. Three recent investigations into the genes related to insect wings and legs in crustaceans provided intriguing insights into how and where insect wings evolved. Interestingly, each study proposes a distinct mechanism as a key process underlying insect wing evolution. Here, I discuss what we can learn about the evolution of insect wings and morphological novelty in general by synthesizing the outcomes of these studies.

Tergal and pleural wing-related tissues in the German cockroach and their implication to the evolutionary origin of insect wings.

The acquisition of wings has facilitated the massive evolutionary success of pterygotes (winged insects), which now make up nearly three-quarters of described metazoans. However, our understanding of how this crucial structure has evolved remains quite elusive. Historically, two ideas have dominated in the wing origin debate, one placing the origin in the dorsal body wall (tergum) and the other in the lateral pleural plates and the branching structures associated with these plates. Through studying wing-related tissues in the wingless segments (such as wing serial homologs) of the beetle, Tribolium castaneum, we obtained several crucial pieces of evidence that support a third idea, the dual origin hypothesis, which proposes that wings evolved from a combination of tergal and pleural tissues. Here, we extended our analysis outside of the beetle lineage and sought to identify wing-related tissues from the wingless segments of the cockroach, Blattella germanica. Through detailed functional and expression analyses for a critical wing gene, vestigial (vg), along with re-evaluating the homeotic transformation of a wingless segment induced by an improved RNA interference protocol, we demonstrate that B. germanica possesses two distinct tissues in their wingless segments, one with tergal and one with pleural nature, that might be evolutionarily related to wings. This outcome appears to parallel the reports from other insects, which may further support a dual origin of insect wings. However, we also identified a vg-independent tissue that contributes to wing formation upon homeotic transformation, as well as vg-dependent tissues that do not appear to participate in wing formation, in B. germanica, indicating a more complex evolutionary history of the tissues that contributed to the emergence of insect wings.

Two sets of candidate crustacean wing homologues and their implication for the origin of insect wings.

The origin of insect wings is a biological mystery that has fascinated scientists for centuries. Identification of tissues homologous to insect wings from lineages outside of Insecta will provide pivotal information to resolve this conundrum. Here, through expression and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) functional analyses in Parhyale, we show that a gene network similar to the insect wing gene network (preWGN) operates both in the crustacean terga and in the proximal leg segments, suggesting that the evolution of a preWGN precedes the emergence of insect wings, and that from an evo-devo perspective, both of these tissues qualify as potential crustacean wing homologues. Combining these results with recent wing origin studies in insects, we discuss the possibility that both tissues are crustacean wing homologues, which supports a dual evolutionary origin of insect wings (that is, novelty through a merger of two distinct tissues). These outcomes have a crucial impact on the course of the intellectual battle between the two historically competing wing origin hypotheses.

RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points.

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is applicable to a wide variety of organisms and is highly efficient in the species-rich order Coleoptera (beetles). Here, we summarize the necessary steps for developing this technique in a novel organism and illustrate its application to the different developmental stages of the aquatic diving beetle Thermonectus marmoratus. Target gene sequences can be obtained cost-effectively through the assembly of transcriptomes against a close relative with known genomics or de novo. Candidate gene cloning utilizes a specific cloning vector (the pCR4-TOPO plasmid), which allows the synthesis of double-stranded RNA (dsRNA) for any gene with the use of a single common primer. The synthesized dsRNA can be injected into either embryos for early developmental processes or larvae for later developmental processes. We then illustrate how RNAi can be injected into aquatic larvae using immobilization in agarose. To demonstrate the technique, we provide several examples of RNAi experiments, generating specific knockdowns with predicted phenotypes. Specifically, RNAi for the tanning gene laccase2 leads to cuticle lightening in both larvae and adults, and RNAi for the eye pigmentation gene white produces a lightening/lack of pigmentation in eye tubes. In addition, the knockdown of a key lens protein leads to larvae with optical deficiencies and a reduced ability to hunt prey. Combined, these results exemplify the power of RNAi as a tool for investigating both morphological patterning and behavioral traits in organisms with only transcriptomic databases.

Sculpting new structures.

The origins of the posterior lobe, a recently evolved structure in some species of Drosophila, have become clearer.

How to study enhancers in non-traditional insect models.

Transcriptional enhancers are central to the function and evolution of genes and gene regulation. At the organismal level, enhancers play a crucial role in coordinating tissue- and context-dependent gene expression. At the population level, changes in enhancers are thought to be a major driving force that facilitates evolution of diverse traits. An amazing array of diverse traits seen in insect morphology, physiology and behavior has been the subject of research for centuries. Although enhancer studies in insects outside of Drosophila have been limited, recent advances in functional genomic approaches have begun to make such studies possible in an increasing selection of insect species. Here, instead of comprehensively reviewing currently available technologies for enhancer studies in established model organisms such as Drosophila, we focus on a subset of computational and experimental approaches that are likely applicable to non-Drosophila insects, and discuss the pros and cons of each approach. We discuss the importance of validating enhancer function and evaluate several possible validation methods, such as reporter assays and genome editing. Key points and potential pitfalls when establishing a reporter assay system in non-traditional insect models are also discussed. We close with a discussion of how to advance enhancer studies in insects, both by improving computational approaches and by expanding the genetic toolbox in various insects. Through these discussions, this Review provides a conceptual framework for studying the function and evolution of enhancers in non-traditional insect models.

Detailed analysis of the prothoracic tissues transforming into wings in the Cephalothorax mutants of the Tribolium beetle.

Despite the immense importance of the wing in the evolution and successful radiation of the insect lineages, the origin of this critical structure remains a hotly-debated mystery. Two possible tissues have been identified as an evolutionary origin of wings; the lateral expansion of the dorsal body wall (tergal edge) and structures related to an ancestral proximal leg segment (pleural tissues). Through studying wing-related tissues in the red flour beetle, Tribolium castaneum, we have previously presented evidence in support of a dual origin of insect wings, a third hypothesis proposing that wings evolved from a combination of both tergal and pleural tissues. One key finding came from the investigation of a Cephalothorax (Cx) mutant, in which the ectopic wing characteristic to this mutant was found to be formed from both tergal and pleural contributions. However, the degree of contribution of the two tissues to the wing remains elusive. Here, we took advantage of multiple Cx alleles available in Tribolium, and produced a variety of degrees and types of ectopic wing tissues in their prothoracic segments. Through detailed phenotypic scoring of the Cx phenotypes based on nine categories of mutant traits, along with comprehensive morphological analysis of the ectopic wing tissues, we found that (i) ectopic wing tissues can be formed at various locations in the prothorax, even internally, (ii) the lateral external ectopic wing tissues have tergal origin, while the internal and posterior external ectopic wing tissues appear to be of pleural origin, and (iii) the ectopic wing tissues of both tergal and pleural origin are capable of transforming into wing surface tissues. Collectively, these outcomes suggest that the evolutionary contribution of each tissue to a complete wing may be more complex than the simple binary view that is typically invoked by a dual origin model (i.e. the wing blade from the tergal contribution + musculature and articulation from the pleural contribution).

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.

Dual evolutionary origin of insect wings supported by an investigation of the abdominal wing serial homologs in Tribolium.

The origin of insect wings is still a highly debated mystery in biology, despite the importance of this evolutionary innovation. There are currently two prominent, but contrasting wing origin hypotheses (the tergal origin hypothesis and the pleural origin hypothesis). Through studies in the Tribolium beetle, we have previously obtained functional evidence supporting a third hypothesis, the dual origin hypothesis. Although this hypothesis can potentially unify the two competing hypotheses, it requires further testing from various fields. Here, we investigated the genetic regulation of the tissues serially homologous to wings in the abdomen, outside of the appendage-bearing segments, in Tribolium We found that the formation of ectopic wings in the abdomen upon homeotic transformation relies not only on the previously identified abdominal wing serial homolog (gin-trap), but also on a secondary tissue in the pleural location. Using an enhancer trap line of nubbin (a wing lineage marker), we were able to visualize both of these two tissues (of tergal and pleural nature) contributing to form a complete wing. These results support the idea that the presence of two distinct sets of wing serial homologs per segment represents an ancestral state of the wing serial homologs, and can therefore further support a dual evolutionary origin of insect wings. Our analyses also uncovered detailed Hox regulation of abdominal wing serial homologs, which can be used as a foundation to elucidate the molecular mechanisms that have facilitated the evolution of bona fide insect wings, as well as the diversification of other wing serial homologs.

Evo-Devo: The Double Identity of Insect Wings.

Sometime in the Devonian, perhaps about 400 million years ago, insects became the first clade to conquer the sky. Recent evo-devo studies have begun to unravel the mysterious origin of the flight structure that made insects into extraordinary six-legged fliers.

Ultrabithorax and the evolution of insect forewing/hindwing differentiation.

Decades have passed since the stunning four-winged phenotype of the Drosophila Ultrabithorax (Ubx) mutant was reported, and accumulating knowledge obtained from studies on Ubx in Drosophila has provided a framework to investigate the role of Ubx during insect wing evolution. With several new insights emerging from recent studies in non-Drosophila insects, along with the outcomes of genomic studies focused on identifying Ubx targets, it appears to be an appropriate time to revisit the Drosophila paradigm regarding insect wing development and evolution. Here, I review the recent findings related to Ubx during wing development, and discuss the impact of these findings on the current view of how Ubx came to regulate wing differentiation in the evolution of insect flight structures.

What serial homologs can tell us about the origin of insect wings.

Although the insect wing is a textbook example of morphological novelty, the origin of insect wings remains a mystery and is regarded as a chief conundrum in biology. Centuries of debates have culminated into two prominent hypotheses: the tergal origin hypothesis and the pleural origin hypothesis. However, between these two hypotheses, there is little consensus in regard to the origin tissue of the wing as well as the evolutionary route from the origin tissue to the functional flight device. Recent evolutionary developmental (evo-devo) studies have shed new light on the origin of insect wings. A key concept in these studies is "serial homology". In this review, we discuss how the wing serial homologs identified in recent evo-devo studies have provided a new angle through which this century-old conundrum can be explored. We also review what we have learned so far from wing serial homologs and discuss what we can do to go beyond simply identifying wing serial homologs and delve further into the developmental and genetic mechanisms that have facilitated the evolution of insect wings.

Functional value of elytra under various stresses in the red flour beetle, Tribolium castaneum.

Coleoptera (beetles) is a massively successful order of insects, distinguished by their evolutionarily modified forewings called elytra. These structures are often presumed to have been a major driving force for the successful radiation of this taxon, by providing beetles with protection against a variety of harsh environmental factors. However, few studies have directly demonstrated the functional significance of the elytra against diverse environmental challenges. Here, we sought to empirically test the function of the elytra using Tribolium castaneum (the red flour beetle) as a model. We tested four categories of stress on the beetles: physical damage to hindwings, predation, desiccation, and cold shock. We found that, in all categories, the presence of elytra conferred a significant advantage compared to those beetles with their elytra experimentally removed. This work provides compelling quantitative evidence supporting the importance of beetle forewings in tolerating a variety of environmental stresses, and gives insight into how the evolution of elytra have facilitated the remarkable success of beetle radiation.

Exploring the origin of insect wings from an evo-devo perspective.

Although insect wings are often used as an example of morphological novelty, the origin of insect wings remains a mystery and is regarded as a major conundrum in biology. Over a century of debates and observations have culminated in two prominent hypotheses on the origin of insect wings: the tergal hypothesis and the pleural hypothesis. However, despite accumulating efforts to unveil the origin of insect wings, neither hypothesis has been able to surpass the other. Recent investigations using the evolutionary developmental biology (evo-devo) approach have started shedding new light on this century-long debate. Here, we review these evo-devo studies and discuss how their findings may support a dual origin of insect wings, which could unify the two major hypotheses.