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.

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.

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.

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.

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.

Comparative developmental analysis of Drosophila and Tribolium reveals conserved and diverged roles of abrupt in insect wing evolution.

Morphological innovation is a fundamental process in evolution, yet its molecular basis is still elusive. Acquisition of elytra, highly modified beetle forewings, is an important innovation that has driven the successful radiation of beetles. Our RNAi screening for candidate genes has identified abrupt (ab) as a potential key player in elytron evolution. In this study, we performed a series of RNA interference (RNAi) experiments in both Tribolium and Drosophila to understand the contributions of ab to the evolution of beetle elytra. We found that (i) ab is essential for proper wing vein patterning both in Tribolium and Drosophila, (ii) ab has gained a novel function in determining the unique elytron shape in the beetle lineage, (iii) unlike Hippo and Insulin, other shape determining pathways, the shape determining function of ab is specific to the elytron and not required in the hindwing, (iv) ab has a previously undescribed role in the Notch signal-associated wing formation processes, which appears to be conserved between beetles and flies. These data suggest that ab has gained a new function during elytron evolution in beetles without compromising the conserved wing-related functions. Gaining a new function without losing evolutionarily conserved functions may be a key theme in the evolution of morphologically novel structures.

Neofunctionalization of embryonic head patterning genes facilitates the positioning of novel traits on the dorsal head of adult beetles.

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.

Insights into insect wing origin provided by functional analysis of vestigial in the red flour beetle, Tribolium castaneum.

Despite accumulating efforts to unveil the origin of insect wings, it remains one of the principal mysteries in evolution. Currently, there are two prominent models regarding insect wing origin: one connecting the origin to the paranotal lobe and the other to the proximodorsal leg branch (exite). However, neither hypothesis has been able to surpass the other. To approach this conundrum, we focused our analysis on vestigial (vg), a critical wing gene initially identified in Drosophila. Our investigation in Tribolium (Coleoptera) has revealed that, despite the well-accepted view of vg as an essential wing gene, there are two groups of vg-dependent tissues in the "wingless" first thoracic segment (T1). We show that one of these tissues, the carinated margin, also depends on other factors essential for wing development (such as Wingless signal and apterous), and has nubbin enhancer activity. In addition, our homeotic mutant analysis shows that wing transformation in T1 originates from both the carinated margin and the other vg-dependent tissue, the pleural structures (trochantin and epimeron). Intriguingly, these two tissues may actually be homologous to the two proposed wing origins (paranotal lobes and exite bearing proximal leg segments). Therefore, our findings suggest that the vg-dependent tissues in T1 could be wing serial homologs present in a more ancestral state, thus providing compelling functional evidence for the dual origin of insect wings.

RNAi screening of developmental toolkit genes: a search for novel wing genes in the red flour beetle, Tribolium castaneum.

The amazing array of diversity among insect wings offers a powerful opportunity to study the mechanisms guiding morphological evolution. Studies in Drosophila (the fruit fly) have identified dozens of genes important for wing development. These genes are often called candidate genes, serving as an ideal starting point to study wing development in other insects. However, we also need to explore beyond the candidate genes to gain a more comprehensive view of insect wing evolution. As a first step away from the traditional candidate genes, we utilized Tribolium (the red flour beetle) as a model and assessed the potential involvement of a group of developmental toolkit genes (embryonic patterning genes) in beetle wing development. We hypothesized that the highly pleiotropic nature of these developmental genes would increase the likelihood of finding novel wing genes in Tribolium. Through the RNA interference screening, we found that Tc-cactus has a less characterized (but potentially evolutionarily conserved) role in wing development. We also found that the odd-skipped family genes are essential for the formation of the thoracic pleural plates, including the recently discovered wing serial homologs in Tribolium. In addition, we obtained several novel insights into the function of these developmental genes, such as the involvement of mille-pattes and Tc-odd-paired in metamorphosis. Despite these findings, no gene we examined was found to have novel wing-related roles unique in Tribolium. These results suggest a relatively conserved nature of developmental toolkit genes and highlight the limited degree to which these genes are co-opted during insect wing evolution.

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.

Genome sequences of the human body louse and its primary endosymbiont provide insights into the permanent parasitic lifestyle.

As an obligatory parasite of humans, the body louse (Pediculus humanus humanus) is an important vector for human diseases, including epidemic typhus, relapsing fever, and trench fever. Here, we present genome sequences of the body louse and its primary bacterial endosymbiont Candidatus Riesia pediculicola. The body louse has the smallest known insect genome, spanning 108 Mb. Despite its status as an obligate parasite, it retains a remarkably complete basal insect repertoire of 10,773 protein-coding genes and 57 microRNAs. Representing hemimetabolous insects, the genome of the body louse thus provides a reference for studies of holometabolous insects. Compared with other insect genomes, the body louse genome contains significantly fewer genes associated with environmental sensing and response, including odorant and gustatory receptors and detoxifying enzymes. The unique architecture of the 18 minicircular mitochondrial chromosomes of the body louse may be linked to the loss of the gene encoding the mitochondrial single-stranded DNA binding protein. The genome of the obligatory louse endosymbiont Candidatus Riesia pediculicola encodes less than 600 genes on a short, linear chromosome and a circular plasmid. The plasmid harbors a unique arrangement of genes required for the synthesis of pantothenate, an essential vitamin deficient in the louse diet. The human body louse, its primary endosymbiont, and the bacterial pathogens that it vectors all possess genomes reduced in size compared with their free-living close relatives. Thus, the body louse genome project offers unique information and tools to use in advancing understanding of coevolution among vectors, symbionts, and pathogens.

Oenocyte development in the red flour beetle Tribolium castaneum.

Oenocytes are a specialized cell type required for lipid processing, pheromone secretion, and developmental signaling. Their development has been well characterized in Drosophila melanogaster, but it remains unknown whether the developmental program is conserved in other insect species. In this study, we compare and contrast the specification and development of larval oenocytes between Drosophila and the red flour beetle, Tribolium castaneum. First, we identify several useful reagents to label larval oenocytes, including both a Tribolium GFP enhancer trap line and a simple flurophore-conjugated streptavidin staining method that recognizes oenocytes across insect species. Second, we use these tools to describe oenocyte development in Tribolium embryos, and our findings provide evidence for conserved roles of MAP kinase signaling as well as the Spalt, Engrailed, hepatocyte nuclear factor-4, and ventral veins lacking factors in producing abdominal-specific oenocyte cells. However, Tribolium embryos produce four times as many oenocytes per abdominal segment as Drosophila, and unlike in Drosophila, these cells rapidly downregulate the expression of the Spalt transcription factor. Thus, these results provide new insight into the molecular pathways regulating oenocyte specification across insect species.

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.

Ultrabithorax is required for membranous wing identity in the beetle Tribolium castaneum.

The two pairs of wings that are characteristic of ancestral pterygotes (winged insects) have often undergone evolutionary modification. In the fruitfly, Drosophila melanogaster, differences between the membranous forewings and the modified hindwings (halteres) depend on the Hox gene Ultrabithorax (Ubx). The Drosophila forewings develop without Hox input, while Ubx represses genes that are important for wing development, promoting haltere identity. However, the idea that Hox input is important to the morphologically specialized wing derivatives such as halteres, and not the more ancestral wings, requires examination in other insect orders. In beetles, such as Tribolium castaneum, it is the forewings that are modified (to form elytra), while the hindwings retain a morphologically more ancestral identity. Here we show that in this beetle Ubx 'de-specializes' the hindwings, which are transformed to elytra when the gene is knocked down. We also show evidence that elytra result from a Hox-free state, despite their diverged morphology. Ubx function in the hindwing seems necessary for a change in the expression of spalt, iroquois and achaete-scute homologues from elytron-like to more typical wing-like patterns. This counteracting effect of Ubx in beetle hindwings represents a previously unknown mode of wing diversification in insects.

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.

Sculpting new structures.

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

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.

Development of a wingless morph in the ladybird beetle, Adalia bipunctata.

Many taxa of winged insects have independently lost the ability to fly and often possess reduced wings. Species exhibiting natural variation in wing morphology provide opportunities to investigate the genetics and developmental processes underlying the evolution of alternative wing morphs. Although many wing dimorphic species of beetles are known, the underlying mechanisms of variation are not well understood in this insect order. Here, we examine wing development of wild type and natural wingless morphs of the two-spot ladybird beetle, Adalia bipunctata. We show that both pairs of wings are distally truncated in the wingless adults. A laboratory population of the wingless morph displays heritable variation in the degree of wing truncation, reflecting reduced growth of the larval wing discs. The coexistence of variable wingless morphs supports the idea that typical monomorphic wingless insects may be the result of a gradual evolution of wing loss. Gene expression patterns in wing discs suggest that the conserved gene network controlling wing development in wild-type Adalia is disrupted in the dorsoventral patterning pathway in the wingless morphs. Previous research on several species of ant has revealed that the anteroposterior wing patterning pathway is disrupted in wingless workers. Future investigations should confirm whether interruptions in both taxa are limited to the patterning pathways found thus far, or whether there are also shared interruption points. Nevertheless, our results highlight that diverse mechanisms of development are likely to underlie the evolution of wingless insects.