The origin and evolution of insect flight is of major interest in evolutionary biology. More than half of all living species identified to date are insects making Insecta the most diverse class on Earth and arguably among the most successful animals in natural history. Flightin is a muscle protein known to be essential for flight in the fruit fly, Drosophila melanogaster. These studies will combine phylogenetic analyses with biochemical techniques to examine the rate of evolution of flightin among Drosophila lineages (aim 1) and to examine the phylogenetic distribution and tissue expression of flightin across insects and crustaceans (aim 2). The goal of the first aim is to test the hypothesis that flightin is under dual evolutionary constraints (purifying and positive selection) to fulfill a conserved muscle physiological function and a rapidly evolving behavior-associated function. This hypothesis will be tested by using Drosophila phylogeny to analyze the evolution of flightin, its propensity for multiple phosphorylations, and flight muscle biomechanical properties. The goal of the second aim is to test a key hypothesis that the assignation of flightin to the flight muscles correlates with the extensive insect radiation during the late Carboniferous (~290 Mya). This hypothesis will be examined by surveying increasingly deeper nodes in Pancrustacea phylogeny, from Holometabola to Crustacea, for expression of flightin by RNA, protein, and immunocytological analyses. The achievement of the stated goals will help to elucidate the mechanistic links between molecular physiology and cellular mechanics of muscle and the ecobehavioral aerodynamics and evolution of insects, and will pave the way towards a synthesis of the macro-and microevolutionary trends in the most prolific terrestrial metazoan. Additional broader impacts of this research are primarily in the areas of education and the generation of biological reagents for the research communities. This research will provide training opportunities for undergraduate and graduate students, and occasional high school students, and inclusiveness of students and research personnel from underrepresented communities and disadvantaged backgrounds. Knowledge generated from this research will find its way to advanced undergraduate and graduate special topic courses and to review articles and book chapters intended for the broader scientific community. Results will be broadly disseminated by undergraduate and graduate students who will present their work at scientific meetings and will publish their studies in peer-reviewed journals.
The world is full of insects; as many as 75% of all animal species identified to date are insects. The abundance of today’s insects, in terms of sheer numbers as well as in numbers of colorful and intricately varied species broadly distributed throughout most of Earth’s habitats, rests on a long evolutionary history that dates back to approximately 400 million years ago. Winged insects are widely considered to be the first organisms to have acquired flight. Evidence suggest that flight originated approximately 325 million years ago, marking a pivotal moment in the history of life as the transition to air opened up vast opportunities for colonization of new habitats and niche exploitation, which lead to adaptive radiation and origin of new insect species. Insects rely on flight muscles to propel the wings and many species of insects have evolved a special type of muscle that is fast and powerful and may have triggered the diversification of insects. In this study we found that the flight muscles, in addition to their well-established role in powering flight, are essential for the production of the mating song, a key component of the courtship ritual that males use to attract and mate with females of the same species. To gain an understanding of the evolution of this unique muscle type, and how it can fulfill its two distinct roles in powering flight and producing sound, we studied flightin, a protein that is essential for the structural integrity and normal contractile function of Drosophila flight muscles. While in Drosophila flightin is uniquely expressed in the powerful flight muscles, here we found that flightin is widely expressed among insects and other hexapods and crustaceans, but absent in two other groups of arthropods, the annelids and chelicerates. The studies suggest that flightin originated de novo in the last common ancestor to decapods (lobsters, shrimps, and the like) and branchiopods (fresh water shrimps and the like) approximately 500 million years ago, well before the origin of insects and the origin of flight. The evolution of flightin as a flight muscle-specific protein is more recent and appears to be limited to higher order dipterans (flies and mosquitoes). We used a combination of genetic approaches, muscle mechanics, imaging and biophysical techniques, and behavioral assays to determine the functional contributions of flightin sequences. We found that flightin consists of three separate regions of approximately equal length (~60 amino acids each) and that each region represents an unique protein domain. The amino-terminal domain is the least conserved phylogenetically and is highly variable even among Drosophila species. This domain is required for normal myofilament lattice order and sarcomeric structure and for optimal oscillatory power output for flight. More importantly, removal of the amino-terminal domain of flightin results in defective mating songs and lower courtship success, suggesting that this region is evolving under sexual selection and its primary role is to impart species-specific attributes to the mating song. In contrast, the second domain consists of sequences that are highly conserved in insects and crustaceans. This domain represents the most ancestral and fundamental function of flightin, that of binding to myosin filaments. The third domain, located in the carboxy-terminal region of flightin, is necessary for normal myofilament lattice organization and required to achieve high power output for flight. In summary, these studies provide evidence that separate domains of flightin are evolving in response to distinct evolutionary forces, natural selection and sexual selection, underlying the flight muscle’s ability to fulfill dual roles in flight and song production.
