Diversification of insect wings
The impressive diversity of insect wings makes them an excellent model to study the molecular basis behind morphological evolution. We have been investigating the conserved and diverged aspects of genetic mechanisms responsible for wing development in various insects, including the fruit fly (Drosophila) and the red flour beetle (Tribolium), in order to elucidate the changes in genetic mechanisms that have contributed to the evolution and diversification of insect wings.
The two pairs of wings on extant insects have often undergone evolutionary modification. In Drosophila, the forewing is used for flight, whereas the hindwing (haltere) is highly reduced and used only for balance. The Hox gene Ultrabithorax (Ubx) modifies the hindwing by repressing various wing genes. In contrast, no Hox input is necessary for forewing formation, and therefore, the forewing is considered to be a Hox-free (or default) state. In Tribolium, modification of wings is, in a way, reversed. It is the forewing (elytron) that is modified, while the hindwing retains more ancestral wing characteristics. Using the larval RNAi technique, we revealed that the elytron is a Hox-free state despite its diverged morphology, and that Ubx cancels the modifications to maintain the rather typical insect wing morphology through activating wing genes in Tribolium. This finding suggests the intriguing possibility that the diversity of insect wing morphology might have been facilitated by the divergent use of a Hox transcription factor as an activator or a repressor in different lineages. Currently, we are further analyzing the action of Ubx through identifying Ubx response elements from the Tribolium genome by chromatin profiling.
- Tomoyasu, Y. Ultrabithorax and the evolution of insect forewing/hindwing differentiation. Current Opinion in Insect Science. 19, 8-15 (2017) pdf
- Ravisankar, P., Lai, Y., Sambrani, N., Tomoyasu, Y. * Comparative developmental analysis of Drosophila and Tribolium reveals conserved and diverged roles of abrupt in insect wing evolution. Developmental Biology. 409 (2), 518-529 (2016) pdf
- Linz, D.M., Tomoyasu, Y. RNAi screening of developmental toolkit genes: A search for novel wing genes in the red flour beetle Triboliumcastaneum. Development Genes and Evolution. 225 (1), 11-22 (2015) pdf
- Tomoyasu, Y., Arakane, Y., Kramer, K.J., Denell, R.E. Repeated co-options of exoskeleton formation during wing-to-elytron evolution in beetles. Curr Biol. 19(24):2057-65. (2009) pdf
- Tomoyasu Y., Wheeler S.R., Denell R.E. Ultrabithorax is required for membranous wing identity in the beetle Tribolium castaneum. Nature. 433(7026):643-7. (2005) pdf
A dual evolutionary origin of insect wings?
RNAi analyses for wing genes in Tribolium resulted in several interesting findings (for example, Linz and Tomoyasu 2015 DGE). Among these studies, perhaps the analysis of the vestigial (vg) gene is the most intriguing one, which provided us with new insights into the origin of insect wings. Despite the fact that 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 into two prominent hypotheses on the insect wing origin: the paranotal origin hypothesis and the exite origin hypothesis. The paranotal hypothesis connects the origin to lateral extensions of the tergum (dorsal body wall), while the exite hypothesis essentially proposes that wings formed from an outgrowth and/or branch of the pleural tissue (the most proximal leg element). Despite accumulating efforts to unveil the origin of insect wings, neither hypothesis has been able to surpass the other.
Through the functional analysis of vg, a critical wing gene, we found that there are two vg-dependent tissues in the “wingless” first thoracic segment (T1) of the Tribolium beetle. Further investigation using other wing genes has revealed that these two tissues are wing serial homologs (i.e. these tissues and wings share common ancestry) in the wingless segment. In addition, homeotic mutant analysis revealed that the merger of the two wing serial homologs is essential to form an ectopic wing on T1. Intriguingly, these two T1 wing serial homologs may actually be homologous to the two proposed wing origins (tergal/paranotal and pleural/exite). Therefore, the vg-dependent tissues in T1 could be wing serial homologs present in (or reverted to) a more ancestral state, and their merger to form a complete wing provides compelling functional evidence for the dual origin of insect wings, which may allow us to unify two prominent wing origin hypotheses. This finding provided a new and exciting direction for our research.
- Tomoyasu, Y., Ohde, T., Clark-Hachtel C.M. What serial homologs can tell us about the origin of insect wings. F1000Research. 6: 268 (2017) pdf
- Clark-Hachtel C.M., Tomoyasu Y. Exploring the origin of insect wings from an evo-devo perspective. Current Opinion in Insect Science. 13, 77-85 (2016) pdf
- Clark-Hachtel C.M., Linz D.M., Tomoyasu Y. Insights into Insect Wing Origin Provided by Functional Analysis of vestigial in the Beetle, Tribolium castaneum. Proc Natl Acad Sci U S A. 110(42):16951-6. (2013) pdf
Functional significance of beetle elytra
Coleoptera (beetles) is a massively successful order of insects, distinguished by their evolutionarily modi ed 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. We empirically tested the function of the elytra using Tribolium castaneum (the red our 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.
- Linz, D.M., Hu, A.W., Sitvarin M.I, Tomoyasu, Y. Functional value of elytra under various stresses in the red flour beetle, Tribolium castaneum. Scientific Reports. 6, Article number: 34813 (2016) pdf
Systemic RNAi in Insects
The red flour beetle, Tribolium castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully annotated genome sequence, transposon-based transgenesis, and effective RNA interference (RNAi). Among these advantages, RNAi-based gene knockdown techniques are at the core of Tribolium research. Tribolium show a robust systemic RNAi response, making it possible to perform RNAi at any life stage by simply injecting double-stranded RNA (dsRNA) into the beetle’s body cavity.
We are among the first to establish an RNAi technique that allows us to perform gene knockdown experiments during post-embryonic stages in Tribolium. Until this study, with a few exceptions (such as the transgenic-based RNAi in Drosophila), RNAi analysis was limited to studies of embryogenesis. This method enabled us to study the molecular mechanisms underlying adult morphological diversity using Tribolium as a model system. Since then, this technique has been applied to many insects and become essential to study gene function in various insects.
In addition to using RNAi as a tool, we also study RNAi itself. RNAi is a highly conserved cellular defense mechanism (in which dsRNA suppresses the translation of homologous mRNA). Interestingly, in some organisms, the RNAi response can be transmitted systemically from cell to cell, a phenomenon termed ‘systemic RNAi’. Understanding systemic RNAi will be crucial for the application of RNAi to many other insects, which will open the possibility of functional analysis in various insects and also will provide us with the route to establish RNAi-based pest management strategies.
- Linz, D.M., Clark-Hachtel, C.M., Borràs-Castells, F., Tomoyasu, Y. Larval RNA Interference in the Red Flour Beetle, Triboliumcastaneum. J. Vis. Exp. (92), e52059. (2014). link
- Miyata K., Ramaseshadri P., Zhang Y., Segers G., Bolognesi R., and Tomoyasu Y. Establishing an in vivo assay system to identify components involved in environmental RNA Interference in the Western Corn Rootworm PLoS One. 9(7): e101661. (2014) pdf
- Miller S.C., Miyata K., Brown S.J., Tomoyasu Y.* Dissecting Systemic RNA Interference in the Red Flour Beetle Tribolium castaneum: Parameters Affecting the Efficiency of RNAi. PLoS One. 7(10):e47431 (2012) pdf
- Miller, S.C., Brown, S.J., Tomoyasu, Y. Larval RNAi in Drosophila? Development Genes and Evolution. 218(9):505-10 (2008) pdf
- Tomoyasu Y., Miller, S.C., Tomita, S., Schoppmeier, M., Grossmann, D., Bucher, G. Exploring Systemic RNA Interference in Insects: a Genome-Wide Survey for RNAi Genes in Tribolium. Genome Biology. 17;9(1):R10 (2008) pdf
- Tomoyasu Y., Denell R.E. Larval RNAi in Tribolium (Coleoptera) for analyzing adult development. Development Genes and Evolution. 214(11):575-8. (2004) pdf